大多数跟我当年一样是身处科研、实验一线的研究生、博士后或刚刚毕业希望继续搞科研的年轻人,除了课题 设计外,还要日以继夜的查看文献、学方法、做试验、分析结果。只有这些一线的“战士”才最了解自己的研究领域的各方面的现状和需要解决的问题,也只有这些“战士”才最需要论著。那么,写论著的素材(题目和数据等)从哪里来呢?其实,从上面的“查看文献、学方法、做试验、分析结果” 所有过程中都可以激发灵感,“查看文献”不仅可以优化课题设计,为出好结果、发好文章奠定坚实基础, 还可以引发些英文综述的激情(已在其他帖中讲解)。“学方法、做试验”不仅可以加强理论知识、掌握 实验技能、更能了解目前方法的不足和需改进的地方,从中思考是否可以改进、改良现有方法,甚至发 明新的方法。“分析结果”更是极其重要。任何相关的结果都不应放过,如果实验材料和方法本身没有问 题,所有结果都应该受到尊重和重视。很多新发现都是从看似无关的结果中得到的。 下面谈谈我发论著的经验。 我在博士及博士后的5 年里,主要从事幽门螺杆菌的研究。以第一作者发了10 多篇论著。这些论文大 致可分三类。 第1章:既定实验 的完美结局 我主要的课题分两个方面,幽门螺杆菌耐药性和幽门螺杆菌感染“根除治疗”后复发。 前一课题所发的主要论文有: 1. Prevalence of metronidazole-resistantHelicobacter pylori in dyspeptic patients. Xia HX, Daw MA, Beattie S, Keane CT,O'Morain CA. Ir J Med Sci. 1993 Mar;162(3):91-4. PMID: 8473130 2. Clarithromycin resistance inHelicobacter pylori: prevalence in untreated dyspeptic patients and stabilityin vitro. Xia HX, Buckley M, Keane CT, O'Morain CA. J Antimicrob Chemother.1996 Mar;37(3):473-81. PMID: 9182104 后一课题所发的主要论文有: 1、Recrudescence ofHelicobacter pylori after apparently successful eradication: novel applicationof randomly amplified polymorphic DNA fingerprinting. Xia HX, Windle HJ,Marshall DG, Smyth CJ, Keane CT, O'Morain CA. Gut. 1995 Jul;37(1):30-4. PMID:7672675 2、Recrudescence ofHelicobacter pylori infection in patients with healed duodenal ulcer aftertreatment with different regimens. Xia HX, Gilvarry J, Beattie S, Hamilton H,Keane CT, Sweeney EC, O'Morain CA. Am J Gastroenterol. 1995 Aug;90 :1221-5.PMID: 7639218 这些结果应该说是比较理想的,特别是后一课题,结果已足够博士毕业(在国外,不一定要发SCI论文才可毕业,但实验结果必须要达到Internal and External Examiners的要求)。所以我94年博士毕业,论文结果部分在95-96年发表,最后一篇在1999年才发。 第2章:仔细观察试验意外现象 幽门螺杆菌很“娇嫩”,但我无意中发现有些菌株是可以经受“锻炼”的,即从微需氧‘trained”到需氧。为此,我做了些验证试验,发了篇短文Culture of Helicobacter pylori under aerobic conditions on solidmedia. Xia HX, Keane CT, O'Morain CA. Eur J Clin Microbiol Infect Dis. 1994May;13(5):406-9. PMID: 8070454 这只是我“意外观察、意外收获”的开端,真正有给我惊喜的还是我到悉尼大学任高级研究人员(Senior Research Officer)意外发现的结果。这里先埋个伏笔,以后会为大家详述。 刚到Prof. NJ Talley位于悉尼西郊的悉尼大学教学医院Nepen Hospital的实验室时,除了一张床,一套测胃肠道压力Barostat装置和一台给病人解闷的电视机外,几乎没有任何实验室所需的东西,包括试剂、移液管、冰箱、离心机等,更不用说我需要的细菌培养箱。而且,试验人员仅我一人,Prof. Talley是功能性疾病专家,但基本没做过基础研究。所以,对我来说,很基础的研究课题根本没有条件开展。因此,我只好利用我熟悉的幽门螺杆菌的培养、药敏和PCR技术来设计课题。 一般认为,幽门螺杆菌菌株在人体有排他性,即所谓“一人一菌”。我设计的课题就是要验证这一现象。因此就有了一项称之为“Mapping study”的研究项目,即从胃的不同部位(底、体、角、窦)取多个活检标本作幽门螺杆菌培养,病理、快速尿素酶试验、并采血备用。同时将临床、内镜诊断、病理等资料收集在设计好地CRF(临床记录表,或临床报告表)上。 澳洲做内镜病人不多,而且,进入课题的病人事先必须签Consent Form,采血必须由注册的有资格的医务人员执行。所以,近三年才收集200多例。而幽门螺杆菌感染阳性率仅在40%左右(国内为60%左右)。我充分利用这些病人的活检标本和血清做的许多工作,发表了些“副产品”: 1、Metronidazole- and clarithromycin-resistant Helicobacter pylori indyspeptic patients in western Sydney as determined by testing multiple isolatesfrom different gastric sites. Xia HH, Kalantar J, Talley NJ.J GastroenterolHepatol. 1998 Oct;13(10):1044-9; 2、High sensitivity andspecificity of a laboratory-based serological test, pylori DTect ELISA, fordetection of Helicobacter pylori infection. Xia HH, Kalantar JS, Wyatt JM,Adams S, Cheung K, Eslick GD, Talley NJ. Diagn Microbiol Infect Dis. 2000Feb;36(2):69-74.; 3、Can helicobacterpylori serology still be applied as a surrogate marker to identify peptic ulcerdisease in dyspepsia? Xia HH, Kalantar JS, Mitchell HM, Talley NJ. AlimentPharmacol Ther. 2000 May;14(5):615-24; 4、Demographic andendoscopic characteristics of patients with Helicobacter pylori positive andnegative peptic ulcer disease. Xia HH, Phung N, Kalantar JS, Talley NJ. Med JAust. 2000 Nov 20;173(10):515-9. 然而,我原本设计的验证实验(genotyping)却因条件所限一直未能开展。正当我为此感到沮丧的时候,我在分析由病理科发出的病理报告结果时意外发现,绝大多数幽门螺杆菌感染的病人其胃角黏膜为胃窦型,而未感染的病人胃角黏膜为胃体型。这一发现使我欣喜若狂。因此,我和Prof. Talley决定在不告诉我们的意图的情况下让病理科同事严格按国际公的“Sydney标准”对所有病例重新阅片。 结果,不但证实了我的原来的观察,还显示这一现象与胃萎缩性胃炎、胃肠化生(癌前病变)有关。为此,我“发明”了“Antralization”这一概念,发表在Am J Gastroenterol上(Antral-type mucosa in the gastric incisura, body, and fundus(antralization): a link between Helicobacter pylori infection and intestinalmetaplasia? Xia HH, Kalantar JS, Talley NJ, Wyatt JM, Adams S, Chueng K,Mitchell HM. Am J Gastroenterol. 2000 Jan;95(1):114-21.) 此后,我完全放弃了原来设计的课题,全力投入在“Antralization”之中。这一“项目”成为我进入香港大学医学院的“敲门砖”,近500万港币的基金也由此而得。并相继发表了数篇有关论著: 1、Topographic association of gastric epithelial expression of Ki-67,Bax, and Bcl-2 with antralization in the gastric incisura, body, and fundus..Xia HH,Zhang GS, Talley NJ, Wong BC, Yang Y, Henwood C, Wyatt JM, Adams S, Cheung K,Xia B, Zhu YQ, Lam SK.Am J Gastroenterol. 2002 Dec;97(12):3023-31; 2、Antralization at theedge of proximal gastric ulcers: does Helicobacter pylori infection play arole?Xia HH, Lam SK, Wong WM, Hu WH, Lai KC, Wong SH, Leung SY, Yuen ST,Wright NA, Wong BC.World J Gastroenterol. 2003 Jun;9 :1265-9. 3、Aberrant epithelialexpression of trefoil family factor 2 and mucin 6 in Helicobacter pyloriinfected gastric antrum, incisura, and body and its association withantralisation.Xia HH, Yang Y, Lam SK, Wong WM, Leung SY, Yuen ST, Elia G, WrightNA, Wong BC.J Clin Pathol. 2004 Aug;57 :861-6. 4、Antralization ofgastric incisura is topographically associated with increased gastricepithelial apoptosis and proliferation, but not with CagA seropositivity.Xia HH, WongBC, Zhang GS, Yang Y, Wyatt JM, Adams S, Cheung K, Lam SK, Talley NJ.JGastroenterol Hepatol. 2004 Nov;19(11):1257-63; 5、Pancreatic duodenalhomeobox-1 (PDX1) functions as a tumor suppressor in gastric cancer.Ma J, ChenM, Wang J, Xia HH, Zhu S, Liang Y, Gu Q, Qiao L, Dai Y, Zou B, Li Z, Zhang Y,Lan H, Wong BC.Carcinogenesis. 2008 Jul;29(7):1327-33. Epub 2008 May 13; 6、Alterations ofGastric Homeoprotein Expression in Helicobacter pylori Infection, IncisuralAntralisation, and Intestinal Metaplasia.Zhu S, Xia HH, YangY, Ma J, Chen M, Hu P, Gu Q, Liang Y, Lin H, Wong BC.Dig Dis Sci.2008 Aug 27. 目前,“Antralization or Antralisation”这一概念已被许多学者应用。我希望也相信将来在这方面会有更大的突破(我虽离开科研一线,但会时时关注)。 第3章:副产品 我在博士期间最大的收获莫过于“副”产品了。尤其是微生物方法学方面。另一实验室中国学者的课题需要大量幽门螺杆菌细胞作抗原(该学者老板每月根据细菌量付给我一定的报酬)。幽门螺杆菌固体培养生长缓慢,产量极低,而当时没有成熟的液体培养法。这就催生了我的第一篇方法学论著Enhanced cultivation of Helicobacter pylori in liquid media. Xia HX,English L, Keane CT, O'Morain CA. J Clin Pathol. 1993 Aug;46 :750-3. PMID:8408702 由于我们在幽门螺杆菌研究方面做得比较“有名”,许多国家的学者向我们要菌株。这就涉及到幽门螺杆菌菌株转运问题。幽门螺杆菌是一种微需氧,很难生存的细菌,没有合适的环境,很快就会死亡。为此,我在同事的启发和帮助下在实验室做了幽门螺杆菌生存试验,找到了合适的转运培养基。然后付诸实施。由此发热量偏方法学论著: 1、Determination of the optimal transport system for Helicobacterpylori cultures. Xia HX, Keane CT, O'Morain CA. J Med Microbiol. 1993 Nov;39(5):334-7.PMID: 8246249 ; 2、Transportation ofHelicobacter pylori cultures by optimal systems. Xia HX, Keane CT, Chen J,Zhang J, Walsh EJ, Moran AP, Hua JS, Megraud F, O'Morain CA. J Clin Microbiol.1994 Dec;32(12):3075-7. PMID: 7883907 与此同时,我也大胆“标准化”幽门螺杆菌药敏试验方法,发了论著Standardization of disk diffusion test and its clinical significancefor susceptibility testing of metronidazole against Helicobacter pylori. Xia H,Keane CT, Beattie S, O'Morain CA. Antimicrob Agents Chemother. 1994Oct;38(10):2357-61. PMID: 7840570 。该“标准化”被多次引用。 此外,我也根据一时的想法做了一些小的试验(这些试验成功与否完全影响不到课题大局和毕业),发了两篇小文章: 1、Pre-formed urease activity of Helicobacter pylori as determined by aviable cell count technique--clinical implications. Xia HX, Keane CT, O'MorainCA. J Med Microbiol. 1994 Jun;40 :435-9. PMID: 8006937 ; 2、Comparison betweenMcCoy cell line and HeLa cell line for detecting Helicobacter pyloricytotoxicity: clinical and pathological relevance. Xia HH, Gallagher C, Hyde D,Talley NJ, Keane CT, O'Morain CA. Ital J Gastroenterol Hepatol. 1999 Nov;31:663-8. PMID: 10730556
How to draft a research hypothesis orresearch opening report? My first chatter is about researchhypothesis (rationale, justification of a given study, or opening report inChinese), which is the first step for us to conduct high impact research. If you have had a right and feasiblehypothesis, your research could be more than half way done. It seems very sillyto discuss it since we are all scientists by training. It is true that during our training, we asstudents usually spend months and months of time to search literature andprepare or write a review in the field before performing any real experiments.We all know that the research hypothesis is sole important and directly determinesour research impact. But often time, we do ignore the importanceof research rationale before and during experiments, data collection andsummary, or manuscript preparation. For biomedical research, we can propose ourresearch hypothesis in the following ways, which doesn't matter whether you area basic, translational, or clinic researcher: 1) Focus on the risk factor-mediated genealterations. For example, lung cancer is associated with tobacco smoke; thus,we may hypothesize tobacco carcinogen-caused gene alterations (gene mutations,methylation, and other epigenetic changes); 2) Gene pathway-related gene alterations,e.g., p53 isoften mutated in lung cancer; how about the role of the p53 up- ordown-streamgenes in lung cancer progression; 3) Mechanism of gene or drug action inaparticular disease or organ site. For example, a gene is mutated or adrugworked well in a disease, so we will rationalize how and why. This kind ofstudy specifically suits for GWAS data to provide hypothesis for further studyof a particular gene in a given disease. Taking home message is whatever we like todo needs a justification. In other words, we will rationalize why we perform astudy and what implication we will expect to be reached in a given study. But we will not rationalize that A gene was abnormally expressed inone cancer but its expression is unknown in another cancer; thus, we performedthis study; or A gene plays an important role inregulation of apoptosis in a cancer, but it is unknown whether this gene alsoplays a role in another cancer; thus, we performed the current study; or A gene is mutated in a cancer;thus, we assessed this gene mutation in another cancer. These rationales were wrongly proposedbecause different diseases or cancers have the own risk factor, etiology, andpathogenesis. We can't just fish something for novelty. Medjaden editor 译文: 如何起草实验假设或开题报告 如果您有了正确的方向和可行的假设,您的研究就成功了一半。现在讨论这个似乎比较可笑,因为我们都是已经受过良好训练的“科研人员”。是的,在学生时代,我们在真正开始实验之前,就被训练着花费数月的时间寻找相关参考文献,用以准备一篇本研究领域的综述。 我们都明白,一个好的实验前提,对文稿最终的发表影响力有多么重要,但是在实验、数据收集整理和论文的撰写中,大家仍旧经常忽略研究的立意。 对于生物医学类的研究,不论涉及到基础研究还是临床研究,以下几点意见都可供您在提出实验假设时进行参考: 1)关注风险因子介导的基因突变。例如:吸烟与肺癌有关,我们就可以假设烟草致癌物会导致基因突变(序列的变异,甲基化和其他表观遗传的改变); 2)关注信号通路上的基因改变。例如:肺癌患者中 p53 经常有变化,那么肺癌的致病过程中,在 p53 上游或者下游的基因是如何变化的? 3)关注基因或者药物在特异疾病或组织中的作用机制。例如:我们知道一个基因突变或者一个药物对某种疾病有效,那么我们就需要了解为什么有效和如何生效。而这个理由也特别适合对 GWAS 分析文章后续的深入研究。 总之大家要记住:不论写哪方面的文章,都需要有坚实的研究依据。换句话说,我们需要指出我们做这个实验的原因,希望得到什么样的结论。以下是作者叙述实验依据中不合格的例子: 因为这个基因在某种癌症中表达正常,但是在另一种癌症中还尚无研究,所以我们进行了如下实验。不合格! 一个基因在某种癌细胞凋亡的调控中起到重要作用,但在其他的癌症中作用尚不知,所以我们进行了这个研究。不合格! 在某种癌症中一个基因突变了,但我们想研究一下在其他癌症中,这个基因是否会突变。不合格! 由于不同的疾病都有特异的风险因子,病因和发病机制,所以以上理由都是站不住脚的,大家不能用钓鱼策略来寻找文章的 “Novelty” 。 原文:美捷登编辑 翻译:美捷登学术部 美捷登版权www.medjaden.com欢迎转载,转载请勿修改内容 欢迎关注我们的微信