（两个多月前，我收到了来自美国胡步根的一封电子邮件。信中他对我细述了他自己在耶鲁工作时的学术不端举报遭遇。耶鲁对他的报复和对造假的掩盖包庇如此似曾相识。 在后来和胡步根的多次交流中，他给我发来了大量的文字，数据和图象。我认为，这又是一起发生在美国顶级名校耶鲁的恶劣原始造假案。案件涉及的文章都发在顶级杂志里，如Cell, Science等。 我现将他的第一封邮件译成中文，放在科学网上。为方便普通读者了解此事，我基本上采用意译，并对译文做了删节简化。） 郭磊博士： 我叫胡步根，原来在耶鲁大学干细胞中心从事科研工作。2009年10月，我在文学城上读到了关于你揭露耶鲁，哈佛等美国一流大学学术造假与腐败的文章。 在揭露学术造假腐败的过程中，我经历了和你非常相似的境遇。直到现在，我仍在为此而抗争。我在中国的朋友帮我找到了你的邮箱地址。因此，我冒昧写信给你，希望你能帮助我将我的经历发表你的博客上。我将自己负责由此而引起的一切法律和学术后果。 作为一名科研人员，我在美国的学术界和生物技术产业有20多年的研究工作经历，我分别在免疫学，细胞生物学、分子生物学以及动物模型等领域工作过。2006年10月我加入耶鲁大学黛安.克劳斯的研究组，进行成年胚胎干细胞研究。 多年来黛安的实验室一直使用以下实验系统进行成年干细胞研究：采用性别错配的小鼠供受体模型，以Y-FISH技术（Y染色体FISH技术）在雌性受体小鼠身上鉴别移入的雄性成年干细胞来源的各类组织细胞。采用Y-FISH技术结合特殊胞浆及膜蛋白质作为组织型标记物的免疫荧光法来鉴别来源于雄性供体干细胞的特异组织细胞类型。 应该说，这一实验系统听起来是简洁有效的。 在我加入黛安的实验室之前，她的实验室已经利用此实验系统在成年干细胞方面发表多篇非常有影响的科学论文，如2001年的《细胞》文章等 (是指这篇文章 《细胞》 2001 May 4;105(3):369-77. Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. 起源于单个造血干细胞的多器官多细胞系寻址和嫁接。克劳斯 等 . 摘要如下： Purification of rare hematopoietic stem cell(s) (HSC) to homogeneity is required to study their self-renewal, differentiation, phenotype, and homing. Long-term repopulation (LTR) of irradiated hosts and serial transplantation to secondary hosts represent the gold standard for demonstrating self-renewal and differentiation, the defining properties of HSC. We show that rare cells that home to bone marrow can LTR primary and secondary recipients. During the homing, CD34 and SCA-1 expression increases uniquely on cells that home to marrow. These adult bone marrow cells have tremendous differentiative capacity as they can also differentiate into epithelial cells of the liver, lung, GI tract, and skin. This finding may contribute to clinical treatment of genetic disease or tissue repair) 。正是这些文章奠定了黛安在成年干细胞领域学术地位的基础。 然而我在她的实验室里，做了无数次的实验，却未能重复她们以前发表的那些结果。 在2007年初，我发现了她们在这些文章里用的实验方案里的问题。我发现，用她的这种方法，根本不能得出如她们在论文中所述的 结果和数据。她们发表的图像都是伪造的，数字也是伪造的！ 这样的发现令我异常震惊！ 因此，我在实验室内部从未承认过她们以前论文上的那些数据和结论。在黛安要求我开口对她的文章进行评价时，我保持沉默。 黛安娜给了我相当的压力，让我重复或相信她们的数据。甚至有时候，在她需要一些阳性的结果来证实她的结论或理论时，她会直接强迫我改变我的实验判断结果，将某些阴性结果改为阳性。在2008年2月末到3月初，在每周一对一的会议上，她又强迫我改变判读结果，让我将实验结果从阴性改为阳性。我明确拒绝。 她竟对我大声骂：FXXX UP。 她的这种行为，给了我巨大的心理压力。我向当局（耶鲁，ORI――美国卫生部科研诚信办公室）报告了她的这种不端行为。 我报告后，你可以想象，耶鲁和ORI玩弄的是多么肮脏、丑陋的政治游戏。他们最终以可能的诚实错误作为对这起严重科研造假案的掩盖。 我的指控仅针对她们文章中的一组数据，即我认为一组对WT雄性鼠BM细胞离心涂片的Y FISH结果完全是假的。只要承认这一点，她们的所有系列文章就会象多米诺骨牌一样倒下。原因如下： 1、现在没有一个人的Y FISH技术方案能在做雄性BM细胞染色时达到 90%的敏感度。黛安娜建立的方法在做雄性BM细胞染色时的敏感度不要说她们宣称的99%，甚至连50%都很难达到。 2、获得Y FISH数据的方法很简单明了：用你的眼睛去观察每一个视野并逐行计数每一个细胞是Y+或Y-。这并不需要很高的科技：Y信号非常清楚和明显，所谓诚实的错误的可能性是不存在的。 3、因此，我认为她们论文中由结合免疫荧光法的Y FISH技术所产生的结果或数据都是造出来的。以此类推，我可以断定她们的所有相关论文（如Science，2004；Cell，2001等），都完全是造出来的。 4，很多年来，黛安娜在干细胞研究中仅使用相同的实验系统-性别错配模型，结合免疫荧光法的Y FISH技术。所以证明了这一点，她们的论文就会像多米诺骨牌一样被推翻。 2009年2月27日，我被黛安从耶鲁解雇了。 到现在为止，我一直努力地向各界揭露这一丑恶的政治事件。我已以邮件的形式将相关的信息递交给主流媒体。 我一定要揭露这一肮脏的丑闻！ 我对这些信息、证据和事实以及我所发的电子邮件持有科学和法律责任。现在，我附上一些文件让你仔细了解我的案件。 谢谢你的关注，并在此对你的意见和帮助表示感谢。 胡步根 附注：更详细报道请见胡步根的科学网博客： http://www.sciencenet.cn/u/bugenhu/ 。
Bugen Hu August 7, 2009 Legend for the lung images by using my unpublished protocols of Y FISH and immunofluorescent staining contained in the CD All the images are coming from the original images when I examined the slides under fluorescent microscope, I did not do any art work or edit on any of such images. Amplification: 40X Wild type male mouse lung paraffin section labeled with Y FISH, anti-SPC (in-house guinea pig anti-spc serum from Jeffs lab), and anti-CD45/F4/80. Signals labeled as the following: Rhodamine for Y (intense pink dots in the DAPI counter stained blue nucleus), Alexafluor 488 for SPC (green in cytoplasmic part), Texas-Red for CD45 and F4/80 (orange red on cell membrane). If the authors could really get experimental protocols of Y FISH and immunofluorescent staining (anti-SPC, and anti-CD45) work, this image should be positive control image for the FASEB paper Fig 2A. This image can show the people, including the peer-reviewers, and readers how well the experimental methods (protocols) are, and what should be the positive markers for each labeling (Y/SPC/CD45), and these positive markers right locations on the slide and in the cell. The most important information of this image should show is that after all labeling is finished (Y FISH, and immunofluorescent staining), as a whole picture what really looks like (Y signal: what percentage of cells is labeled as Y positive, SPC signal: in the physiological locations of lung type II cells (on the alveoli) how well the type II cells are labeled as SPC positive, CD45 signal: whether or not membrane of leukocyte can be labeled as CD45 positive). In this image: a. The whole structure of alveoli is clearly shown up at normal exposure time due to autofluorescence of the tissue itself, b. Most of nuclei are labeled as Y positive, but there is still a small percentage of nuclei being labeled as Y negative (detection sensitivity of Y FISH protocol, but this image is still extremely good image in terms of Y FISH and SPC staining detection sensitivity if you have doubt about it, you can do literature search on internet to see if there is any image of lung paraffin section labeled with Y FISH and immunofluorescent staining in any published papers is better than this image). c. At right physiological locations of type II cells there are a lot of SPC and Y doubt positive cells (definitely certain percentage of type II cells must be stained as SPC negative because I dont think that my protocols can reach 100% detection sensitivity for anti-SPC immunofluorescent staining). d. There is no cell being SPC and CD45 double positive (in this field there is no obvious CD45 positive cell). spcko male mouse lung paraffin section labeled with Y FISH, anti-spc, anti-CD45, and anti-F4/80. Compared with the image of wt male control, overall spcko has much stronger autofluorescence (the whole alveoli structure is clearly shown up as very strong green autofluorescence even exposure conditions I used is the same as I used for wt male control section). For Formalin or PFA fixed tissues, lung is one of the tissues with the strongest autofluorescence, another one is GI tissue), and the alveoli have more fibers than the alveoli of wt male mouse. In this image: a. Most of nuclei are labeled as Y positive, but there is still a small percentage of nuclei being labeled as Y negative. b. No single cell is labeled as SPC positive, no single cell even really looks like the SPC positive cell in the wt male control image (even there are some cells with green in their cytosplasmic part look-like positive, but this so-called green signal is the same as that of alveoli fibers but different from the real SPC positive staining in the image of wt male slide, it is very easy to tell difference between signals of SPC specific staining and green autofluorescence when compared with the real positive cells in the wt male control). c. In this field there is obvious CD45 positive cell. If the authors protocols for Y FISH and anti-SPC really work, at least double-labeling image should be presented as the image of spcko male control Fig 2B for the paper FASEB 2007. Only using above mentioned wt male and spcko male multiple labeled sections can really function as controls to show: how well Y FISH protocol works in both wt male and spcko male tissues (whether or not Y can achieve similar detection sensitivity in both tissues), and clear-cut difference between wt male and spcko male sections in terms of SPC staining, and physiological distribution of SPC positive labeled type II cells in the lung. Paraffin section from the lung of spcko male recipient of wt female bone marrow transplantation (model mouse ID: spc-14) labeled with Y FISH, anti-SPC, anti-CD45, and anti-F4/80. This image shows chimera characteristics of such model. In this image: a. Whole structure of alveoli is clearly shown up (as green autofluorescence of thick fibers). b. Most nuclei of CD45 negative cells are labeled as Y positive, but there is still a small percentage of such cells being labeled as Y negative. c. There are several obvious CD45 positive and Y negative cells (leukocytes derived from donor bone marrow cells, that is, so-called engraftment). d. There is no single cell being SPC positive, in terms of SPC specific staining the situation is the same as I mentioned for the image of spcko male control mouse. If the authors actually did such model, and their published protocols of Y FISH and immunofluorescent staining really work, by using such model to identify and prove that in vivo donor bone marrow cell can derive into type II cell by the mechanism of fusion with recipient cell, this is the image the authors should present as the Fig 2C in the paper FASEB 2007 (at least to show people that the image is really coming from lung of such model, their image Fig 2C logically can come from any mouse lung, and so-called SPC positive could be anything including autofluorescence of cytoplasmic part). This is the image (spc-14) containing the two look-like positive cells with double positive Dr. Diane Krause really wanted and put huge pressure on me trying to force me change my own judgment call on the two cells from negative to positive cells (Y and spc double positive). When I put the images of wt male and spc-14 side by side, and asked her: Even when you compare image of the two cells with image of positive control (wt male), the color and staining pattern is same or not? She said: not same, but stem cell derived type II cell is not as same as real type II cell. And I repeated my principle by saying: As PI you can make your own judgment call on my slides any way you want, and it is perfectly fine with me, but I only hold responsibility for my own judgment call. Then she forced me to trust and accept the results form the paper (FASEB 2007), after I firmly refused she became so emotional and angry with me. Finally she sent an email to Dr. Erica Herzog to ask her as blinder to examine my slides under microscope and ask Dr. Erica Herzog to show us electronic images of anti-spB the authors used for the paper FASEB 2007 (the facts are: a. Erica never showed us any images of anti-spB she only said that she needs to find such images of anti-spB from the computer., but she never showed us any anti-spB image (I dont think that she has such anti-spB images). b. No single image of anti-spB was presented in the paper. c. In the inventory boxes for immunofluorescent reagents in Dr. Krauses lab I had never seen any vials of anti-spB antibody before February, 2008. I ordered my anti-spB from Chemicon in February 2008. I have the email to prove). On March 6, 2008 after looking at my slides under fluorescent microscope, Dr. Erica Herzog sent me that famous email. Normal wt male mouse lung cytospins slide labeled with Y FISH, anti-SPC, and anti-CD45. In this image: a. Most of nuclei of cells are labeled as Y positive, but a few cells being Y negative, for example, one cell at the bottom showing most part of its nucleus. b. There are three cells being labeled as Y and SPC double positive (one is located at left of the image, two are located in the middle). c. At the left of the image adjacent to the Y SPC double positive cell there are several cells being labeled as Y positive and with yellow-green autofluorescence in their cytosplamic parts (such Y positive cells with yellow-green autofluorescence in cytoplasmic parts are common on the male mouse lung cytospins slides labeled with just Y FISH without any other immunofluorescent staining, such cells with yellow-green autofluorescence in their cytoplasmic parts are common on the mouse lung cytospins slides without any labeling, that is, blank control for immunofluorescent staining if the samples are fixed with Formalin or PFA). d. There is a cell labeled as Y positive and look-like CD45 positive located in up-right area of the image. e. There is no single cell being labeled as SPC and CD45 double positive. This image can show real clear-cut difference between signal of real cytoplasmic marker (here is SPC) immunofluorescent staining and signal of cytoplasmic autofluorescence on the mouse lung cytospins. If the images of Fig 4A, 4B (paper FASEB 2007) are really coming from the lung of the model spcko female recipient of male marrow, and the authors published protocols of Y FISH and anti-CK staining really work, for Fig 4A, and 4B, the authors should present the images like this one showing Y and cytoplasmic marker positive cells plus information of signal of X FISH in nuclei, and cells adjacent to the identified positive cells. Normal spcko male mouse lung cytospins slide labeled with Y FISH, anti-SPC, and anti-CD45. In this image: a. Most of nuclei of cells are labeled as Y positive, but a few nuclei are labeled as Y negative, located at the bottom of the image. b. No single cell is stained as SPC positive, but there are a lot of cells with strong green autofluorescence in their cytoplasmic parts. c. There are several cells containing multiple dots of Y signal in its nucleus. And scientific fact is that every cell on the slide can only contain one molecule of Y chromosomal DNA, so this image can clearly demonstrate this scientific fact that one molecule of Y DNA can be detected as Y negative, one dot of Y signal, or multiple dots of Y signal in the nucleus by interphase Y FISH. Summary: These five images of mouse lung samples (paraffin sections and cytospins slides) labeled with Y FISH and immunofluorescent staining show: For Formalin or PFA fixed mouse lung samples, cytoplasmic autofluorescence is common and strong on both paraffin section and cytospins slide, but there is clear-cut difference or manifestation between specific immunofluorescent staining signal of cytosplasmic marker and cytosplasmic autofluorescence. Without showing the real image of real positively labeled control, people not having direct experience in this technique can be easily fooled by cytoplasmic autofluorescence especially when the authors just present images with a single cell or a few cells without reference system. For both paraffin section and cytospins slide, my unpublished Y FISH protocols can achieve much higher detection sensitivity. But I can say here that even using my own protocols I have never achieved 99% detection sensitivity for either paraffin or cytospins slide, even not close to 99% positive (my standard is whole slide, not just one chosen image). By using my protocols or protocols that can achieve similar detection sensitivity of Y, dishonest people can easily make fake image (containing 20, 30, or 50 cells) showing 100% cells being labeled as Y positive by just cutting off single cell or a few cells that are labeled as Y negative from the image. For example, by using my protocols of Y FISH and immunofluorescent staining (edited image), for male sample I can generate the double-labeled images (at 40x amplification, containing 20, 30, 50, or even 100 cells) showing 100% Y detection sensitivity, and beautiful immunofluorescent staining with preserved tissue and cell morphology by just cutting off a cell or a few cells labeled as Y negative from the image. If I was dishonest, and I need such logic premise: 100% detection sensitivity of Y FISH, I submit such images as results and claim that my Y FISH protocol can reach 100% detection sensitivity, definitely based on manifestation of the edited image this statement is true, but scientifically this image does not truthfully reflect the real experimental phenomenon of the slide because the Y negative cell or cells in the real image or whole picture were intentionally cutting off from the image presented (edited image). What the real images of mouse lung sample should look like after multiple labeling of Y FISH and immunofluorescent staining. In the nucleus of one normal male cell, one molecule of Y chromosomal DNA can be detected as Y negative, one dot of Y signal, or multiple dots of Y signal. Physiological distribution of type II cells labeled as Y and SPC double positive on alveoli of the normal male mouse lung paraffin section. For any people if their protocols of Y FISH and anti-SPC immunofluorescent staining really work, and the primary antibody of anti-SPC really works on Formalin or PFA fixed samples, they should be able to produce the similar images from the normal male mouse lung paraffin section as real wt positive control image if they want to use Y and anti-SPC dual markers to identify donor stem cell derived type II cell from the tissue of spc knockout sex-mismatched model. How important it is to have both positive and negative control slides and reference system on the same slide when experimental methods of interphase Y FISH and immunofluorescent staining are used to identify donor stem cell derived tissue type cells in the recipient tissues, that is, without the controls or reference system to compare with, the so-called identified positive cell in the electronic image could be anything but real positive cell. Because in this situation two different kinds of experimental methods, in situ DNA hybridization and antigen- antibody interaction (immune staining) are used, and none of the methods can achieve 100% detection sensitivity and none of immunofluorescent staining can achieve 100% detection accuracy. The authors, Dr. Diane Krause, and Dr. Erica Herzog should know this principle pretty well because they have been using such methods to identify donor stem cell derived tissue type cells from the tissues of sex-mismatched models for years, and published multiple papers. Feb. 4, 2009 Technical Information and Explanation of Erica Herzogs Experimental Protocol for Y FISH on Bone Marrow Cytospins Published in the FASEB Paper (2007) Here the most important step is missing: Denature the target DNA, i.e. Y chromosomal DNA (the size of this DNA is 97.5Mb, for C57 background mouse) to open the double stranded target DNA. The larger the size of the target DNA, the harsher condition is required to open the target DNA. To open the double stranded target DNA is precondition for any DNA hybridization experiment, otherwise the specific probe can not bind to the complement sequence on the target. So-called Denature step in the authors protocol is 73C for 5 mins. Actually the major function of this step is not to denature the target DNA, but the probe, just like for normal DNA hybridization or classic FISH some people do denature the probe 1 st at 70-80C for about 10 mins before apply the probe to the target. Under this denature condition (73C, 5 mins) there is only certain percentage of cells their Y chromosomal DNA getting partial denature, i.e. double stranded DNA opened in some sections of the Y chromosomal DNA. That is, sacrifice the detection sensitivity for retaining the cells on the slide and cell morphology. Here the probe we used for Y FISH is a mixture of DNA, sizes from 100 to 500 nucleotides, its complement sequences on the target will cover the whole Y chromosomal DNA except for super high repeated sequences that will also appear in other chromosomes (the other name of Y FISH is whole Y chromosome painting). When utilize Y FISH (identify the origin of donor) combined with immunofluorescent staining (identify the specific tissue marker) technique to identify the donor stem cell derived specific tissue cell in sex-mismatched models, the authors encounter technical dilemma: Y FISH requires very harsh condition which will destroy the tissue physiological structure and cells morphology including cytoplasmic and cell membrane component (like CD45) , while immunofluorescent staining is just antigen antibody interaction (here is protein and protein interaction) which requires mild condition. If just add these two different kinds of experimental methods together it will not work, regardless doing immunofluorescent staining 1 st or Y FISH 1 st because in order to identify donor stem cell (Y as marker of origin for donor or recipient) derived specific tissue type cell, after both experimental procedures the both signals need to be present on the same slide otherwise the mission is impossible. If the authors use the classic denature condition for Y FISH on tissue paraffin sections as they published, 1M sodium thiocyanate at 80C for 20 mins, then neutralized with 0.2N HCl at room temperature for 12 mins, after Y FISH for most cells only nuclei left. If the authors use such denature condition on cytospins, after Y FISH probably only a few nuclei left on the slide, or nothing left on the slide. And imagine even there is such biological event- bone marrow stem cell derives into specific tissue type cell (here is from BM to type II pneumocyte) happening in vivo, the frequency of such event is very low even based on the authors calculation (less than 1 per 1000). Since no immunofluorescent staining is 100% specific and accurate, the universal accepted standard to identify donor stem cell derived specific tissue cell is triple labeling: Y, tissue specific marker and CD45 in sex-mismatched models to exclude the leukocytes that can be present in any tissue. How to solve this technical dilemma, it took me about 6 months to solve the technical dilemma above mentioned: interphase Y FISH combined with immunofluorescent staining while keeping the tissue and cell morphology normal. The strategy I used is to find the common window of experimental conditions that will fit for both Y FISH and immunofluorescent staining, then hard working of error and try. Even I say that I solved the technical dilemma, but for Y FISH my protocols cannot get 99% detection sensitivity on either paraffin section or cytospin slide. Definition of detection sensitivity needs to be defined by thousands of cells on the same slide, but not by a few cells of electronic image of very small field. In order to test the detection sensitivity of Y FISH on bone marrow cytospins, and verify the authors experimental results generated by using such protocol, I just did Y FISH on bone marrow cytospins from the perfectly normal young wild type mouse by strictly following Dr Erica Herzog and Dr Diane Krause published protocol in their paper, FASEB vol.21 August 2007, p2592-2601. I will send the slide and a CD that contains the electronic images of such slides to the Deans Office, Yale School of Medicine. Here are technical parameters: Channel 1: DAPI to stain for DNA, blue color. Channel 2: FITC, green color. Channel 3: Rhodamine for Y signal, pink color (intense dot in nuclei). Merged image: besides the Y signal, some yellow, green signals are coming from autofluorescence. Amplification: 40x. Rhodamine-conjugated anti-digoxigenin antibody (Roche), the same as Dianes lab always used. Digoxigenin labeled Y probe, the same as Dianes lab always used. IPLAB software is used to capture images. After examine the whole slide under fluorescent microscope, my conclusion is: there is no way for the authors to obtain their experimental results as they claimed in their paper (FASEB 2007) because the Y FISH detection sensitivity is too low. So their results of Y FISH on bone marrow are just intentionally fabricated numbers, not scientific experimental results at all. Bugen Hu
所有 图像均为 40 倍物 镜下所拍摄。 图 1 到 图 5 (Fig.1-Fig.5) 是用我自己的 Y/SPC/CD45 多重免疫 标记所作的野生型和 SPCKO 公鼠肺切片和 细胞片的全景图像。 图 6 到 图 10 (Fig.6-Fig.10) 是我 2009 年 2 月份 实验验证作者文章 FASEB 2007 中 Y FISH 方法在正常野生型公鼠骨髓 细胞片子的全景图像。 信号或 标记: SPC 为 Alexafluor 所 标记 - 胞 浆中绿色荧光。 Y 为 罗丹明所标记 - 在 DAPI 所染成的 兰色胞核中的芝麻大小的很强的红点。 CD45 为 Texas-Red 所 标 记 - 胞膜上桔 红色。 图 1. 野生型公鼠, 可 见完整的肺泡结构, 大部分细胞被标记为 Y 阳性, 但也有很少 细胞为 Y 阴性 - 漏 检, 许多 Y/SPC 双阳性 细胞 - 肺 II 型 细胞相隔分布于肺泡上。如果作者的 Y/SPC 双 标记方法真能工作, 则其文章 FASEB 2007 中 图 2A 应该为此图像, 作 为双阳性对照。 图 2. SPCKO 公鼠, 可 见完整的肺泡结构, 但整个结构有更强的绿色自发荧光, 大部分细胞被标记为 Y 阳性, 但也有很少 细胞为 Y 阴性 - 漏 检, 无 SPC 阳性 细胞, 但有不少细胞胞浆中有很强的绿色自发荧光 - 与真正的 SPC 所 标记的绿色荧光并不相同。如果作者 Y/SPC 双 标记真能工作, 则其文章 FASEB 2007 中 图 2B 因 该为此图像。 图 3. 模型 - 接受了野生型母鼠骨髓移植的 SPCKO 公鼠的肺, 有不少 Y 阴性但 CD45 阳性的白 细胞 - 来自于供体 ( 母鼠)的干 细胞变成即所谓的 engraftment 。其他方面与 图 2 相似。如果作者的 Y/SPC 双 标记方法真能工作, 则其文章 FASEB 2007 图 2C 因 该为此图。这就是我的模型鼠14号的图像, 老板非要两个阳性细胞以证明她的融 合机理。 因 为作者的技术瓶颈: Y FISH 方法的高漏 检率, 用于石蜡切片的 Y FISH 方法 对组织和细胞结构的破坏, 抗体 - 抗 SPC (Chemicon, Ab3428) 不工作,只要具有任何一条所指缺陷, 则作者无法显示我所显示的以上图像。况且作者的方法具有以上所有的缺陷。事实上作者根本无法显示任何公鼠Y FISH的全景图像, 因为那将暴露其最大的秘密-其YFISH 方法的高漏检率。 图4. 野生型公鼠肺细胞片子, 大多数细胞为Y阳性, 但也有少量漏检。有3个Y/SPC双阳性细胞, 不少细胞的胞浆中有黄到绿的自发荧光。如果作者的用于细胞片 (cytospins) 的Y/荧光免疫多重标记方法真能工作, 则其文章 FASEB 2007 中图4A, 4B 应该显示此类全景多重标记图像。还是因为其方法上的缺陷, 所以作者只能显 点 而 无法显面, 因为只要显面则露陷。文中的图像是假图像, 即并非作者在图解中所说 (Figure legend)。更祥细的说明请看英文版的图解说明。 图5. SPCKO公鼠肺细胞片子, 大多数细胞为Y阳性, 但也有少量漏检。许多细胞胞浆中有很强的绿色自法荧光, 但无真正的SPC阳性细胞。此图最重要之点是显示有些细胞胞核中含有多点Y信号, 这就明确无误的说明: 任何一个公鼠细胞在Y FISH以后, 或为Y阴性-漏检, 或为含一点Y, 或为含多点Y信号。而这是由其实验原理所决定了的。 图6到图10均为正常野生型公鼠骨髓细胞片子, 严格按照文章 FASEB 2007 中作者所发表的Y FISH方法所做的Y FISH实验-即实验验证作者的Y FISH方法。这里只有兰色胞核中的红色点-芝麻大小为Y信号, 其他颜色的均为自发荧光。核中有芝麻 大小红点的即为Y阳性, 无此Y信号的则为Y阴性-漏检(在这里每一个细胞核均含有 一条Y染色体, 只是该Y FISH方法未能检测出而已)! 所有的图像中大部分公鼠骨髓细胞核被标记成Y阴性-即漏检, 这就证明作者的Y FISH方法具有很高的漏检率, 也就是很低的检测敏感度！决非象作者长期以来所吹嘘的超高检测敏感度。这就在逻辑上实验技术上证明作者无法得出其文章中公鼠骨髓细胞Y FISH实验结果的数据。此数据只能是有意伪造的数字。 Bugen Hu August 7, 2009 Legend for the lung images by using my unpublished protocols of Y FISH and immunofluorescent staining contained in the CD All the images are coming from the original images when I examined the slides under fluorescent microscope, I did not do any art work or edit on any of such images. Amplification: 40X Wild type male mouse lung paraffin section labeled with Y FISH, anti-SPC (in-house guinea pig anti-spc serum from Jeffs lab), and anti-CD45/F4/80. Signals labeled as the following: Rhodamine for Y (intense pink dots in the DAPI counter stained blue nucleus), Alexafluor 488 for SPC (green in cytoplasmic part), Texas-Red for CD45 and F4/80 (orange red on cell membrane). If the authors could really get experimental protocols of Y FISH and immunofluorescent staining (anti-SPC, and anti-CD45) work, this image should be positive control image for the FASEB paper Fig 2A. This image can show the people, including the peer-reviewers, and readers how well the experimental methods (protocols) are, and what should be the positive markers for each labeling (Y/SPC/CD45), and these positive markers right locations on the slide and in the cell. The most important information of this image should show is that after all labeling is finished (Y FISH, and immunofluorescent staining), as a whole picture what really looks like (Y signal: what percentage of cells is labeled as Y positive, SPC signal: in the physiological locations of lung type II cells (on the alveoli) how well the type II cells are labeled as SPC positive, CD45 signal: whether or not membrane of leukocyte can be labeled as CD45 positive). In this image: a. The whole structure of alveoli is clearly shown up at normal exposure time due to autofluorescence of the tissue itself, b. Most of nuclei are labeled as Y positive, but there is still a small percentage of nuclei being labeled as Y negative (detection sensitivity of Y FISH protocol, but this image is still extremely good image in terms of Y FISH and SPC staining detection sensitivity if you have doubt about it, you can do literature search on internet to see if there is any image of lung paraffin section labeled with Y FISH and immunofluorescent staining in any published papers is better than this image). c. At right physiological locations of type II cells there are a lot of SPC and Y doubt positive cells (definitely certain percentage of type II cells must be stained as SPC negative because I dont think that my protocols can reach 100% detection sensitivity for anti-SPC immunofluorescent staining). d. There is no cell being SPC and CD45 double positive (in this field there is no obvious CD45 positive cell). spcko male mouse lung paraffin section labeled with Y FISH, anti-spc, anti-CD45, and anti-F4/80. Compared with the image of wt male control, overall spcko has much stronger autofluorescence (the whole alveoli structure is clearly shown up as very strong green autofluorescence even exposure conditions I used is the same as I used for wt male control section). For Formalin or PFA fixed tissues, lung is one of the tissues with the strongest autofluorescence, another one is GI tissue), and the alveoli have more fibers than the alveoli of wt male mouse. In this image: a. Most of nuclei are labeled as Y positive, but there is still a small percentage of nuclei being labeled as Y negative. b. No single cell is labeled as SPC positive, no single cell even really looks like the SPC positive cell in the wt male control image (even there are some cells with green in their cytosplasmic part look-like positive, but this so-called green signal is the same as that of alveoli fibers but different from the real SPC positive staining in the image of wt male slide, it is very easy to tell difference between signals of SPC specific staining and green autofluorescence when compared with the real positive cells in the wt male control). c. In this field there is obvious CD45 positive cell. If the authors protocols for Y FISH and anti-SPC really work, at least double-labeling image should be presented as the image of spcko male control Fig 2B for the paper FASEB 2007. Only using above mentioned wt male and spcko male multiple labeled sections can really function as controls to show: how well Y FISH protocol works in both wt male and spcko male tissues (whether or not Y can achieve similar detection sensitivity in both tissues), and clear-cut difference between wt male and spcko male sections in terms of SPC staining, and physiological distribution of SPC positive labeled type II cells in the lung. Paraffin section from the lung of spcko male recipient of wt female bone marrow transplantation (model mouse ID: spc-14) labeled with Y FISH, anti-SPC, anti-CD45, and anti-F4/80. This image shows chimera characteristics of such model. In this image: a. Whole structure of alveoli is clearly shown up (as green autofluorescence of thick fibers). b. Most nuclei of CD45 negative cells are labeled as Y positive, but there is still a small percentage of such cells being labeled as Y negative. c. There are several obvious CD45 positive and Y negative cells (leukocytes derived from donor bone marrow cells, that is, so-called engraftment). d. There is no single cell being SPC positive, in terms of SPC specific staining the situation is the same as I mentioned for the image of spcko male control mouse. If the authors actually did such model, and their published protocols of Y FISH and immunofluorescent staining really work, by using such model to identify and prove that in vivo donor bone marrow cell can derive into type II cell by the mechanism of fusion with recipient cell, this is the image the authors should present as the Fig 2C in the paper FASEB 2007 (at least to show people that the image is really coming from lung of such model, their image Fig 2C logically can come from any mouse lung, and so-called SPC positive could be anything including autofluorescence of cytoplasmic part). This is the image (spc-14) containing the two look-like positive cells with double positive Dr. Diane Krause really wanted and put huge pressure on me trying to force me change my own judgment call on the two cells from negative to positive cells (Y and spc double positive). When I put the images of wt male and spc-14 side by side, and asked her: Even when you compare image of the two cells with image of positive control (wt male), the color and staining pattern is same or not? She said: not same, but stem cell derived type II cell is not as same as real type II cell. And I repeated my principle by saying: As PI you can make your own judgment call on my slides any way you want, and it is perfectly fine with me, but I only hold responsibility for my own judgment call. Then she forced me to trust and accept the results form the paper (FASEB 2007), after I firmly refused she became so emotional and angry with me. Finally she sent an email to Dr. Erica Herzog to ask her as blinder to examine my slides under microscope and ask Dr. Erica Herzog to show us electronic images of anti-spB the authors used for the paper FASEB 2007 (the facts are: a. Erica never showed us any images of anti-spB she only said that she needs to find such images of anti-spB from the computer., but she never showed us any anti-spB image (I dont think that she has such anti-spB images). b. No single image of anti-spB was presented in the paper. c. In the inventory boxes for immunofluorescent reagents in Dr. Krauses lab I had never seen any vials of anti-spB antibody before February, 2008. I ordered my anti-spB from Chemicon in February 2008. I have the email to prove). On March 6, 2008 after looking at my slides under fluorescent microscope, Dr. Erica Herzog sent me that famous email. Normal wt male mouse lung cytospins slide labeled with Y FISH, anti-SPC, and anti-CD45. In this image: a. Most of nuclei of cells are labeled as Y positive, but a few cells being Y negative, for example, one cell at the bottom showing most part of its nucleus. b. There are three cells being labeled as Y and SPC double positive (one is located at left of the image, two are located in the middle). c. At the left of the image adjacent to the Y SPC double positive cell there are several cells being labeled as Y positive and with yellow-green autofluorescence in their cytosplamic parts (such Y positive cells with yellow-green autofluorescence in cytoplasmic parts are common on the male mouse lung cytospins slides labeled with just Y FISH without any other immunofluorescent staining, such cells with yellow-green autofluorescence in their cytoplasmic parts are common on the mouse lung cytospins slides without any labeling, that is, blank control for immunofluorescent staining if the samples are fixed with Formalin or PFA). d. There is a cell labeled as Y positive and look-like CD45 positive located in up-right area of the image. e. There is no single cell being labeled as SPC and CD45 double positive. This image can show real clear-cut difference between signal of real cytoplasmic marker (here is SPC) immunofluorescent staining and signal of cytoplasmic autofluorescence on the mouse lung cytospins. If the images of Fig 4A, 4B (paper FASEB 2007) are really coming from the lung of the model spcko female recipient of male marrow, and the authors published protocols of Y FISH and anti-CK staining really work, for Fig 4A, and 4B, the authors should present the images like this one showing Y and cytoplasmic marker positive cells plus information of signal of X FISH in nuclei, and cells adjacent to the identified positive cells. Normal spcko male mouse lung cytospins slide labeled with Y FISH, anti-SPC, and anti-CD45. In this image: a. Most of nuclei of cells are labeled as Y positive, but a few nuclei are labeled as Y negative, located at the bottom of the image. b. No single cell is stained as SPC positive, but there are a lot of cells with strong green autofluorescence in their cytoplasmic parts. c. There are several cells containing multiple dots of Y signal in its nucleus. And scientific fact is that every cell on the slide can only contain one molecule of Y chromosomal DNA, so this image can clearly demonstrate this scientific fact that one molecule of Y DNA can be detected as Y negative, one dot of Y signal, or multiple dots of Y signal in the nucleus by interphase Y FISH. Summary: These five images of mouse lung samples (paraffin sections and cytospins slides) labeled with Y FISH and immunofluorescent staining show: For Formalin or PFA fixed mouse lung samples, cytoplasmic autofluorescence is common and strong on both paraffin section and cytospins slide, but there is clear-cut difference or manifestation between specific immunofluorescent staining signal of cytosplasmic marker and cytosplasmic autofluorescence. Without showing the real image of real positively labeled control, people not having direct experience in this technique can be easily fooled by cytoplasmic autofluorescence especially when the authors just present images with a single cell or a few cells without reference system. For both paraffin section and cytospins slide, my unpublished Y FISH protocols can achieve much higher detection sensitivity. But I can say here that even using my own protocols I have never achieved 99% detection sensitivity for either paraffin or cytospins slide, even not close to 99% positive (my standard is whole slide, not just one chosen image). By using my protocols or protocols that can achieve similar detection sensitivity of Y, dishonest people can easily make fake image (containing 20, 30, or 50 cells) showing 100% cells being labeled as Y positive by just cutting off single cell or a few cells that are labeled as Y negative from the image. For example, by using my protocols of Y FISH and immunofluorescent staining (edited image), for male sample I can generate the double-labeled images (at 40x amplification, containing 20, 30, 50, or even 100 cells) showing 100% Y detection sensitivity, and beautiful immunofluorescent staining with preserved tissue and cell morphology by just cutting off a cell or a few cells labeled as Y negative from the image. If I was dishonest, and I need such logic premise: 100% detection sensitivity of Y FISH, I submit such images as results and claim that my Y FISH protocol can reach 100% detection sensitivity, definitely based on manifestation of the edited image this statement is true, but scientifically this image does not truthfully reflect the real experimental phenomenon of the slide because the Y negative cell or cells in the real image or whole picture were intentionally cutting off from the image presented (edited image). What the real images of mouse lung sample should look like after multiple labeling of Y FISH and immunofluorescent staining. In the nucleus of one normal male cell, one molecule of Y chromosomal DNA can be detected as Y negative, one dot of Y signal, or multiple dots of Y signal. Physiological distribution of type II cells labeled as Y and SPC double positive on alveoli of the normal male mouse lung paraffin section. For any people if their protocols of Y FISH and anti-SPC immunofluorescent staining really work, and the primary antibody of anti-SPC really works on Formalin or PFA fixed samples, they should be able to produce the similar images from the normal male mouse lung paraffin section as real wt positive control image if they want to use Y and anti-SPC dual markers to identify donor stem cell derived type II cell from the tissue of spc knockout sex-mismatched model. How important it is to have both positive and negative control slides and reference system on the same slide when experimental methods of interphase Y FISH and immunofluorescent staining are used to identify donor stem cell derived tissue type cells in the recipient tissues, that is, without the controls or reference system to compare with, the so-called identified positive cell in the electronic image could be anything but real positive cell. Because in this situation two different kinds of experimental methods, in situ DNA hybridization and antigen- antibody interaction (immune staining) are used, and none of the methods can achieve 100% detection sensitivity and none of immunofluorescent staining can achieve 100% detection accuracy. The authors, Dr. Diane Krause, and Dr. Erica Herzog should know this principle pretty well because they have been using such methods to identify donor stem cell derived tissue type cells from the tissues of sex-mismatched models for years, and publi shed multiple papers.
这篇博文专门解释与实验系统和实验方法相关的技术细节问题, 尤其是为何作者 Y FISH 方法漏 检率高就一定是作者有意伪造由该方法所得的实验数据？为何其 Y FISH 方法的高漏 检率的秘密对我老板团队如此重要, 一旦暴光就会象多米诺骨牌较 应等等问题？这里有一个事实: 在成年干细胞可塑性研究领域中使用 Y FISH 方法和 荧光免疫多重标记来鉴定由供体干细胞在受体鼠体内变成的组织特化细胞的实验 室并不多（其实是一个纯技术的原因, 请见我以下的技术解释), 我老板的实验室是 这方面的大老级实验室。 其 实整个成年干细胞发育可塑性研究的大命题就是一个: 体内是否还存在此类干细 胞？如有此类干细胞, 则此类干细胞能否在体内分化成某些组织特化细胞, 如有的话, 这个生物学事件也是一个很低概率事件。所以此领域研究的核心就是如何用实 验 的方法来证明这个生物学过程: 成年干细胞在体内分化为某种组织 特化细胞。 我在博文-事件回放中所说, 老板团队一直所用的实验系统就是: 供, 受体鼠性别相 反的模型(sex mismatched model) 即以公鼠作为骨髓干细胞的来源, 受体鼠则为致死 剂量照射的母鼠。即在宿主(母鼠) 组织中以Y染色体作为供体 (公鼠)干细胞来源的 识别标志。实验方法或手段就是: Y FISH 以标记Y作为来源于供体干细胞的证据, 和荧光免疫标记组织细胞特化蛋白, 如胞浆中的角质蛋白-CK就代表上皮细胞, 包 膜上的CD45代表白细胞。逻辑上和理论上这种实验系统非常简单明了, 是典型的 反推证明模式: 以Y/荧光免疫多重标记方式证明受体 (母鼠) 组织中此阳性细胞-Y阳性和细胞标志蛋白阳性但CD45阴性只能由供体 (公鼠) 细胞变成, 即中间过 程无需也无法证明。但这就要求在技术上多重标记的每个标志的真实性得以确证-在阳性和阴性甚至空白对照上。理论上Y FISH外加荧光免疫的多重标记是最直接和确实的鉴定方法。但当时世界上大多数本领域有名的实验室并不用此种Y /荧光 免疫多重标记方法以鉴定受体鼠组织中来源于供体鼠干细胞变成的组织特化细胞, 而最多使用的是绿荧光蛋白(GFP)作为供体干细胞来源的标志。其实原因 非常简 单, 纯技术上的原因。Y FISH其实就是DNA原位杂交, 而荧光 免疫标记就是抗原-抗体的反应, 这里是蛋白质-蛋白质的相互作用, 如CK和其相应抗体的结合- 这种结合力并不强 (与DNA分子杂交相比), 一旦遇上剧烈的实验条件如Y FISH 实验中的 DNA变性条件（DNA denature, Y DNA is 97.5Mbp, it requires very harsh conditions to open its double strands, which is precondition for the Y probes to bind to its complementary sequences on the target- Y DNA) 既使已结合在目标蛋白上的抗体也会解离 (dissociate), 即达不到双标记的目地。如果按传统的Y FSIH方法 (用于石蜡 切片的方法-硫氰酸 钠 80度处理, 再用盐酸室温下处理) 先作Y标记, Y FISH后则造成了组织和细胞结构 的很大破坏而使胞浆, 胞膜目标蛋白甚至不存在于片子上。即还是 达不到双标记目地。也就是这是两类实验条件不兼容的实验方法, 即技术上的两难 (dilemma)。 况且Y FISH的检测敏感度也不见得很高, 一般而论, 在公小鼠 组织石蜡切片上传统 的Y FISH方法标记后可达40%的细胞漏检而被标记成Y阴性。 如用更温和的条件下来作Y FISH以换取对组织和细胞结构的较轻破坏, 则Y FISH的检测敏感度会进一步 降低。所以即使有人应用Y FISH方法在母鼠组织中鉴定出了 Y 阳性细胞, 也只说该细胞来源于公鼠的供体细胞。没人会作如此反推理: 在公鼠样本中或公鼠细胞中未检测到Y就等于Y染色体丢式, 因为所有应用Y FISH方法的人均知道该方法有百分之几十的漏检率而将公鼠细胞标记成Y阴性 (除非他的Y FISH方法具有100% 的检测敏感度或检出率, 他才能作以上反推理。这在技术上是不可能的-技术瓶颈)。 所以老板的团队在其文章FASEB 2007 中得有意伪造一组Y FISH阳性对照组的完美的实验结果或数据以向审稿人证明作者在该文章中所用的逻辑推理前提是真实的: 即她们用于标记小鼠骨髓细胞片子 (bone marrow cytospins ) 的Y FISH方法 (protocol) 具有100%的检测敏感度-也就是漏检率为零, 所以在该文章中对于她们在实验样本中 (含公, 母鼠细胞-sex mismatched model) 的Y FISH结果作者可以做以上所讲的反 推理。该文章中作者所用的Y FISH阳性对照组就是正常的野生型公鼠骨髓细胞的Y FISH结果: 0.8 +/- 0.8 %的细胞缺乏Y染色体。这里作者还对这个假数据进行了偷梁 换柱的解读 (这 0.8 +/- 0.8% 的正常公鼠骨髓细胞是缺乏Y染色体-即作者所言的 Y 染色体丢失而不是其Y FISH方法的漏检) 以给审稿人送去信息: 即作者的Y FISH方法的检出率本身仍为100%, 而那 0.8 +/- 0.8 % 的正常公鼠骨髓细胞是由于Y染色体丢失导致作者未能测到。这可是实验方法学上一场彻头彻尾的骗局, 因为作者完全知道她们用了多年的Y FISH方法（protocol) 在公鼠细胞片子 (肺细胞, 骨髓细胞)上具有很低的检出率也就是很高的漏检率, 即Y FSIH标记完后, 片子上仍会有很大比例的公鼠细胞因漏检而被标记成Y 阴性。这就是为何作者从来不在其文章中显示 Y FISH实验阳性对照组全景图像 (包含有几十个公鼠 细胞在Y FISH以后的图像) 的根本原因。 作者Y FISH方法的实验原理如下: 这种方法的全称是间期细胞Y FISH（间期细胞中所谓染色体是成染色质状态-chromatin), Y探针是去与Y DNA上的互补序列杂交。探针的制备是以正常公鼠的Y DNA 为模板用 DOR-PCR先制备此探针的材料: 为500bp到几个Kb大小的DNA片段 , 涵盖几乎整个Y DNA序列 (极高重复序列除外-有可能出现在其他染色体DNA序列中), 尔后用商品试剂合 Dig-Nick(其实就是 nick-translation) 反应将 Dig (digoxigenin) 标记到 DOR-PCR 所制备的 DNA 片段上, Dig 标记完后其产物就为100-500bp（Dig 标记的Y探针) 大小的混合物, 即Y探针混合物分段杂交到Y DNA 上的相应互补序列, 信号则来自于罗丹明标记的抗-Dig(Roche公司）。所以其实验原理已决定: 任何一个公鼠细胞在Y FISH标记以后, 或为Y阴性 (漏检, 因为无人的Y FISH 能达 100%的检出率且相差甚远）或含一个Y信号 (核中芝麻大小的粉红点) 或含多个 Y 信号, 即根本就不存在胞核中一个Y信号就代表一条Y染色体的对应关系-这是其实验原理已决定了！这也正是任何做Y FISH实验的人会在荧光显微镜下看到的现象。 那怕就是用同样的Y探针和同样的抗-Dig, 不同的Y FISH方法 (protocol) 当然会在同样的阳性对照样本上 (正常公鼠组织细胞片或切片) 产生不同的检出率。因为不同 的方法(protocol) 对实验步骤规定了不同的实验条件（温度, 时间, pH 等等), 也就是说每一个不同的Y FISH方法 (protocol) 所具有的检出率已被其本身所决定, 例如我的Y FISH方法的检出率就远高于作者的Y FISH方法（因为我的 protocol 所规定的实验条件不同于作者的 protocol), 这就是科学技术。作者用于小鼠细胞片子 (cytospins) Y FISH 方法的硬伤是: 其 Y DNA 变性条件太差, 73度 5分钟, 此条件不足以打开所有细胞核中的 Y DNA 双链, 而这正是 Y 探针杂交的先决条件！而按照任何一个已建立的Y FISH方法 (protocol) 去做Y FISH实验则很简单, 即操作过程 并不复杂。 作者 Y FISH 方法 (protocol) 的高漏 检率与由这方法所得出的实验结果的关系: 仍以作者文章 FASEB 2007 该方法的阳性对照组为例: 正常野生型公鼠骨髓细胞片子 (bone marrow cytospins)Y FISH 结果 ; 平均 值正负标准差或标准误为: 0.8 +/- 0.8 % 的骨髓 细胞缺乏 Y 染色体 。 这里只要知道两个任何人也无法改变和否认的 事实, 则这组阳性对照组的 Y FISH 实验数据的命运就已被决定了。 1. 作者 Y FISH 方法本身所决定的高漏 检率, 而得到这个证据或事实的最确切和唯一的办法就是实验验证其方法本身, 即严格按照作者的方法在正常公鼠骨髓细胞片子上作 Y FISH 实验, 标记完片子后, 在荧光显微镜下则真相一目了然: 所看到的只能是大百分几十 的细胞因漏检而被标记成 Y 阴性细胞 - 即所称的假 阴性, 因为在片子上每一个细胞均含有一条 Y 染色体, 而只是作者的 Y FISH 方法检测不出而已。这正是由其方法 (protocol) 本身所具有的高漏 检率所决定的, 不管何人包括作者本人, 何时用这方法 (protocol) 在阳性 对照上都会得到同样的结果: 即大百分之几十的公鼠骨髓细胞因漏检而被标记成 Y 阴性细胞 , 即作者的 Y FISH 方法只能 产生高 比例的假 阴性而决非 小于 1% 的公鼠 细胞为 Y 阴性。此仍实践是检验真理的唯一标准, 而不是作者说或某 人说其 Y FISH 方法 (protocol) 能达 100% 的 检出率, 这方法就能真达 100% 检出 率！ 这 里所遵循的是实验科学的一个基本原则：可重复性, 即任何实验方法必须在同 样的对照上产生同一趋式的结果。这里还有一个特点: 被检测目标为 Y 染色体 DNA, 这是所有正常公鼠细胞中的常态恒定成分 ( 这也是遗传学的基本原则, 所以 作者不能说其 Y FISH 方法在 2007 年以前能在公鼠 细胞上测出 100% 检出率, 现在不行了, 因被测目标 - Y DNA 与以前不同了）。2. 获 得 这种 Y FISH 实验结 果数据的唯一方式(data acquisition)就是作者必 须 用自已的双眼在 荧 光 显 微 镜 下去看由她 们 自已的 Y FISH方法所 标记 的正常公鼠骨髓 细 胞片子, 根据一个非常 简单 和明 显 的依据- 在被DAPI染成 兰 色的胞核中有或无Y信号(粉 红 色点)而 对每 一个 细 胞判 别为 Y+ 或 Y- 并分 类计 数, 一个 视 野接着一个 视 野(一 张 骨髓 细 胞片子包含几十个 视 野),而 获 得那 组 阳性 对 照的Y FISH 实验 数据(0.8 +/- 0.8 % 细 胞缺乏Y染色体)作者必 须 看,判 别 , 计 数几百个 这样 的 视 野。整个 过 程无高科技,而只需要正确的科研精神-即 诚实 或事 实 求是: 将自已所看到的真 实记录 下来以采集到真正的 Y FISH 实验结 果的数据。作者Y FISH方法的高漏 检 率就决定了在此Y FISH 实验结 果的数据采集 过 程中, 每 个 视 野她 们 只能看到大百分之几十的公鼠骨髓 细 胞因漏 检 而被 标记为 Y 阴 性 细 胞而决非小于1%的 细 胞 为 Y 阴 性,而任何一双 诚实 的眼睛是无法将 每 个 视 野下大百分之几十的 Y 阴 性 细 胞判 别 , 计 数成小于1%的Y 阴 性 细 胞的。 证 明完 毕 : 作者的 这组 Y FISH 实验 数据只能是有意 伪 造的一 组 数字。因 为 文章需要此完美的数据, 否 则 无法通 过审 稿(文章中的 逻辑 推理前提), 即作者以欺 诈 的方式而 获 得 这组 完美的Y FISH 实验 数据: 一 则 可能根本就没做此 实验 , 因 为 她 们 知道其Y FISH方法的高漏 检 率是无法真正得到那 种 她 们 所需的完美数据的,有 谁 会有意去做无用功呢？其二作者有意将 每 个 视 野下所看到的大百分之几十的Y 阴 性 细 胞写成 为 小于1% 的Y 阴 性 细 胞。不管是那 种 情况,作者都是有意 伪 造 实验 数据,在本案中 连 诚实 的 错误 (honest error)也失 较 ! 就象我再次 举报此案时反复对官方强调的那样, 此案可算史无前例。这是由作者的造假方式所决定的, 作者胆大妄为, 其有意伪造的 Y FISH 实验数据不但远远 超出其方法的检测敏感度, 而且远超出该种实验技术的技术瓶颈。这种实验方法学上的 造假就决定了, 这种造假极易用逻辑上的反证法将其一步证死。作为举报人我可以 用对照样本证明在逻辑上和科学技术上用作者的实验方法是不可能得出其文章中相应的实验数据的并相差几十倍: 这里是公鼠骨髓细胞 Y FISH 方法的漏 检率只能是大百分之几十, 但文章中作者白纸黑字写下了 0.8 +/- 0.8 % 细 胞缺乏 Y 染色体！ 所以此案的 关键点就只有一个且非常简单: 到底作者 Y FISH 方法的漏 检率是零还 是大百分之几十？ 此真相一出则此案大白。所以我于 2009 年 2 月再次 举报此案时, 只打击一个实验方法和一组阳性对照组的 Y FISH 实验结果数据 0.8 +/- 0.8 % 公 鼠骨髓 细胞缺乏 Y 染色体 。并将自已 实验验证的片子本身 (物证), 所照的全景图像, 技术解释等等给了耶鲁和 ORI 让官方眼见为实。而官方对我提供的铁证只能刻意回避, 因为对方无法面对这两难处境。官方可以说我作为举报人所提供的物证没公信力 (credential) ，但你可以 对 我所提供的最直接的物 证进行交互验证吧 (cross-examination), 你耶 鲁, ORI ,作者自己去 验证总可以吧。这正是我一直所要求的, 但 ORI 告 诉我: 他 们看了也没用, 因为他们没裁决权 ! 对方拿着我的物证是两难: 如说他们已看了我所做的片子, 则必须承认大多数 (majority) 公鼠骨髓 细胞漏检而被 标记成 Y 阴性, 即假阴性 , 这就必须承认作者有意伪造 Y FISH 实验数据。如对方想挑战我的实验验证, 则他们须实验证明作者的方法可以产生漏检率为零或小于 1% 的公鼠骨髓片子。我也明确无 误 地告 诉 官方, 尽管我只打 击 一个 实验 方法和一 组 数据, 但只要其Y FISH方法的高漏 检 率暴光 则为 多米 诺 骨牌 较应 。 作者 Y FISH 方法的高漏 检率和多米诺较应: 一旦其方法的高漏 检率暴光, 例如漏检率为 50% ,只 须用同理可得的道理则作者 FASEB 2007 文章中的下列 结果的命运会如何: SPCKO 公鼠 组骨髓细胞片子 Y FISH 的 结果数据 : 0.92 +/- 1.03 % 细胞缺乏 Y 染色体, 即 Y 阴性 p.2594 ；接受了野生型公鼠骨髓移植的 SPCKO 母鼠 组骨髓细胞片子 Y FISH 的 结果数据: 94.18 +/- 2.48 % 细胞含有 Y 染色体 , 即 Y 阳性; 接受了 SPCKO 公鼠骨髓移植的 SPCKO 母鼠 组骨髓细胞片子的 Y FISH 的 结果数据: 93.28 +/- 1.78 % 细胞含有 Y 染色体,即 Y 阳性 p.2595 ; 图 4A , 图 4B 的 结果和作者所用的逻辑推理前提; 再验证作者用于石蜡切片的 Y FISH 方法的漏 检率和 Y/SPC 双 标记方法, 则文中 8 只野生型公鼠肺切片的 Y/SPC 双 标记的结果数据: 1000 分之 1000 的双阳性 ( 请见第一作者 2008 年 3 月 6 日 给我 的电子邮件: 她从来没有将 Y/SPC 双 标记搞工作过, 她的实验室看来连 FISH 也不工作了 !) 和其他 Y/SPC 双 标记的实验结果的命运会如何呢 ? 作者的其他 Y/ 荧光免疫多重标记方法和其相应结果的 命运会如何呢？目前我得先推倒第一 张骨牌, 所以在 此对后续反应不作太多祥细分析。 老板团队在期刊Science上反击 Standford 团队对其文章 Cell 2001的挑战时所用的最大实验依据就是其Y FISH方法的超高检测敏感度(请见我的博文-事件回放), 在接 受了公鼠骨髓移植的母鼠的脾中她的团队检测到大于90%的脾细胞为Y阳性-所谓的 engraftment (意即她的Y FISH方法的检测敏感度本身仍为100%)。 这是一个双重谎言, 其一她的Y FISH方法的检测敏感度离90%还差的远, 漏检率为大百分之几十, 所以她无法在以上所说的脾中测出大于90%的脾细胞为Y阳性, 其二在这种小鼠模型中受体鼠的脾不可能发生大于90%的 engraftment (即大于90%的宿主脾细胞被来自于由供体干细胞变成的脾细胞所替代)。以下是我的实验证据: 因我的专业是免疫学, 出于好奇心我想看看在这种致死照射后接受骨髓移植, 会有多少脾细胞 (T, B淋巴细 胞)被替代, 所以在我做的模型收获时我也同时将脾收获并用我自己的Y FISH方法检测脾。在接受母鼠骨髓移植的公鼠脾中Y阳性 (宿主) 与Y阴性脾细胞成分隔区域 分布, 并且Y阳性细胞并不少, 应该有大百分之几十, 这与我在同一个体的骨髓细胞Y FISH所看的 engraftment不一致, 骨髓片子上只有很小比例的细胞为Y阳性-即绝大部分骨髓细胞已被来自于供体干细胞生成的骨髓细胞所替代。而且所有的模型鼠均是这种现象, 即受体鼠脾细胞被替带的比例并不是很大(我的Y FISH方法也有一定的漏 检率, 但我在受体鼠脾中仍能看到大比例的Y阳性细胞-宿主细胞)。仔细思考后我得出了自认为合理的解释: 致死照射杀死了绝大部分的骨髓细胞, 但并没有杀死大部分相对处于静态的脾细胞, 所以宿主骨髓细胞必须大量快速被供体来源的骨髓细胞所替代, 而其脾中被替代区域应该为生发中心(germinal center) 那里有活跃的淋 巴细胞的死亡和增殖, 但脾中其他相对静态的区域则没理由被替代。 以上的例子也说明其Y FISH方法的高漏检率秘密对老板团队的重要性。她在期刊细胞 (Cell 2001)上发表那篇大作后, 因无人能重复其结果, 所以该领域的主流实验室均质疑其结果。Stanford团队则是在期刊科学 (Science 2002) 上发表文章直接挑战。 老板团队反击 (Science 2003) 的档箭牌就是其Y FISH方法的超高检测敏感度, 即用你们的方法测不到, 但我的方法能测到。Stanford团队在老板团队的反击后, 又在期刊科学 (Science 2003)上对老板的反击进行了回击, 但还是没捉到要点, 因为外人并不知道其Y FISH方法的高漏检率。谁也没有想到其实老板团队一直在她的Y FISH 方法学上唱的是空城计！我作为继任她们研究项目的人当然会知此秘密, 2007年6月我用自己的Y/CK多重标记方法检测正常公鼠肺细胞片子上(Y丢失研究）CK阳性和Y阳性，CK阳性但Y阴性的肺上皮细胞(参见我博文-事件回放)。我有意让老板看我的片子本身, 让她明白在Y FISH方法的漏检率上与我玩把戏没意思并且也没用, 还是留着去骗外人吧 (我的方法检出率本就高于她的方法) 别逼我会将Y FISH 的漏检而结论为Y丢失！她看了我的片子后又与 Erica 一起演双簧给我看, 好象她们从未见过Y FISH在公鼠细胞上的漏检似的 (请见两人2007年6月18, 19日的电子邮 件 )。 成像系统和组织样本细胞上的自发荧光: 在福尔马林或PFA固定过的样本上自发荧光非常普遍而且可以很强。不同的组织可以不同, 肺自发荧光很强, 同一组织中不同的细胞或同一种细胞的不同个体均可以表现不同。自发荧光可以是广谱的, 从短光谱的兰光到长光谱的红光, 红外光, 红, 绿荧光极为常见而且可以很强(主要在细胞浆部分，胞膜上也可有)。老板实验室的成像系统为: 荧光显微镜上配有多个滤光片。滤片1: 允许兰光通过, 用于DAPI所染 的胞核, 滤片2: 允许绿光通过, 用于FITC标记, Alexafluor488等, 滤片3: 允许红光通过, 用于罗丹明-Y的信号, 滤片4: 允许红外光如Cy5通过-肉眼不可见, 滤片5: 允许红, 绿荧光通过, 看片子时一般先用此滤片看红, 绿荧光的标记。照像则为: 从滤片1开 始, 设定暴光时间后OK, 依次重复操作到滤片4, 最后OK后, 则一张彩色电子图像形成, 和各个滤片下所摄的一张黑白图像形成并自动贮存于所联的机算机。 也就是说在各个滤片下可以设定不同的暴光时间以改变不同颜色光在最后所形成的图像中的强度。所以这种实验手段和获取图像的方式为制作假电子图像留下了巨大的空 间。例如在小鼠肺石蜡切片或细胞片(cytospins)一个具有红, 绿自发荧光的细胞 (在 滤片5下, 肉眼可见为黄色), 只需调整滤片2和滤片3下的不同暴光比例 , 则这个细胞在最后形成的彩色图像中即可以是红色荧光标记, 也可以是绿色荧光标记。也就是说将胞浆中的自发荧光做假成特异性抗体标记的信号在这种电子图像中并不难。辩别这种假图像的最好方法就是与真正的阳性对照细胞图像作比较并且用全景图像-含有内参照系统。因为真正由抗体所产生的荧光信号来自于一种纯的荧光分子所发射, 如Alexafluor488, 这种荧光的光谱很窄, 所以颜色看起来很纯而与广谱的自发绿荧光不同。请见我上传的Y/荧光免疫多重标记的全景图像和图解, 尤其是英 文的图解 (Fig.legend)。作假的图像往往只能显点而不能显面, 即有意裁 切掉内参照系统-片子上其他细胞的信息。 ----- Forwarded message from Erica Herzog erica.lyndrup@yale.edu ----- Date: Thu, 06 Mar 2008 14:51:55 -0500 From: Erica Herzog erica.lyndrup@yale.edu Reply-To: Erica Herzog erica.lyndrup@yale.edu Subject: slides To: bugen.hu@yale.edu hi bugen i left the slides and results for you on the bench. were they in any way correct? i never was able to get pro-spc to work with fish because of the autofluor but if you are able to that's great. i always did prospc first and detected that way so the signal was really bright and then did fish after doing confocal. your control looked nice and like real signal. the few experimental cells that i saw that were possibly spc+ didn't look exactly like the control. anyway let me know how this compared with your results also since my lab cannot seem to get fish to work diane said you guys would be willing to collaborate if i gave you some slides for staining - if so can i bring you the slides sometime soon? best, erica Erica Herzog MD, PhD Assistant Professor Yale University School of Medicine Internal Medicine - Pulmonary and Critical Care Division 333 Cedar St TAC 441-S New Haven CT 06511 (203)785-3207 ----- End forwarded message ----- 注： 问我她的判片结果怎样？她不能将 Y/SPC 双 标记搞工作的原因是其 Y FISH 方法本身和抗体本身不工作（ Chemicon, AB3428). autofluor( 自 发荧光)总是存在的, 这 根本不是她的 Y/SPC 双 标记不工作的原因！在实验方法学上此人一贯狡辩。 其 Y/SPC 双 标记的方法都不工作, 但敢在文章 FASEB 2007 中将此双 标记结果写成 1000 分之 1000 的双阳性（ 8 只野生型公鼠肺石蜡切片 表 -2 p.2596). 她所 说其 lab 不能将 FISH 搞工作, 应该是指很高的漏检率。当时她已不敢再对我说她们的 Y FISH 是 100% 的 检出率了, 因为她还等着我帮她一把。请见她 2007 年 6 月 18 , 19 日的 电邮, 她是怎样说其 Y FISH 方法的 检出率。 当 时她应该还不知道我老板已将我逼到死角, 我已别无选择, 准备打假了, 否则她不会给我送这电子邮件。 ----- Forwarded message from Diane Krause diane.krause@yale.edu ----- Date: Tue, 19 Jun 2007 06:32:54 -0400 From: Diane Krause diane.krause@yale.edu Reply-To: Diane Krause diane.krause@yale.edu Subject: Re: Y loss in males To: Erica Herzog erica.lyndrup@yale.edu What % of the male into male transplants were Y-? At this point Bugen is using cytospins. So far, he's only done older transplanted and untransplanted SPC-/- and untransplanted WT males. So far, with cytospins, we never see 20% without a Y. Diane On Jun 18, 2007, at 9:29 PM, Erica Herzog wrote: age matched but not irradiated truly less than 1% were Y neg unlike the male into male transplants how old are his mice - are they the old ones for the Y loss project also depending on fixation, size of probe, length of permeablization etc there can be issues.depending on the PFA strength, age of reagents there can be big issues. how is his X staining? Hi Erica In rereading the FASEB paper, I see that we don't have the % of SPC+ cells in males that are Y- by confocal. Did you do this with any males? If so, were they age-matched or younger? I am concerned that we don't know the sensitivity of the assay to detect for Y loss after cell fusion . Bugen is seeing epithelial cells have no Y on lung cytospins from male mice. Thanks for any info that you have. Diane -- Diane Krause MD, PhD Yale University School of Medicine Associate Professor, Department of Laboratory Medicine PO Box 208035 New Haven, CT 06520-8035 Phone: (203) 688-4829 Fax: (203) 688-2748 Office: BML 462 Administrative Associate, Pat Sember: (203) 688-3265 Pager: (203) 412-0805 Krauselab website: http://info.med.yale.edu/labmed/faculty/labs/krauselab/index.html Erica Herzog MD, PhD Assistant Professor Yale University School of Medicine Internal Medicine - Pulmonary and Critical Care Division 333 Cedar St TAC 441-S New Haven CT 06511 (203)785-3207 ----- End forwarded message ----- 注： 这是老板看了我用自己的 Y/CK 多重 标记方法所做的公小鼠肺细胞片子后, 她与 Erica 之 间就所谓 Y FISH 方法的 电子邮件, 但有意转给我。她两人在 Y FISH 方法漏 检率的问题上给我演双簧, 其实就是想逼我将我所做的公鼠肺细胞上的 Y 漏 检当 Y 丢失的结论。老板已知道我当时刚建好的多重标记方法已突破了她们文章 FASEB 2007 中方法的技 术瓶颈, 想让我用自己的方法做出 Y 丢失的结果去掩盖文章中谎谬 的 Y 丢失结果和结论。两人装着一副好象不知道她们的 Y FISH 方法有漏 检率似的 (因她们总是说其 Y FISH 方法具有 100% 的 检出率）。我当然是装糊涂, 对这种双簧毫无反应, 所以老板也拿我没办法。请见 Erica 于 2008 年 3 月 6 日 给我的电子邮件, 看她是如何说她们的 Y FISH 方法。 老板 说步根在公鼠肺细胞片子上已看到 CK 阳性但 Y 阴性的细胞（其实就是 Y 漏 检）也应该是警示 Erica : 其 实我已知道她们的秘密只是不说而已。 这里 Erica 的所 谓解释在技术上均是一派胡言。
标签: 耶鲁
