巧妙思路可以弥补技术的缺乏

开展科研工作时，我们经常苦于实验条件无法满足需要，难以清楚回答自己选择的问题，也就是说存在许多难以实现的科研思路，但如果能在巧妙上多下一些功夫，也能够开展很不错的研究，甚至对解决实际问题有更大帮助。

我一直觉得，开展转化医学研究的人总是把眼睛盯着细胞学机制的研究不是正确的道路，要解决实际问题不见得一定采用特别高的技术，越是简单的技术手段，如果发挥地好，距离解决问题越近，因为简单的技术在应用上更容易被应用。下面我以一篇刚看到发表在Journal of Neurotrauma上的文章，尝试说明这个问题。

这是一个关于脑创伤的研究，研究的目的是看血液中的非细胞成分DNA水平和脑创伤的关系。DNA在正常情况下只存在细胞内，当身体有细胞发生坏死的时候，由于细胞内的DNA发生断裂，变成小片断，细胞内的小片断DNA可以扩散到组织周围细胞外，也有一些扩散到血管内进入循环系统。

作者用了39只大鼠（够少的），将动物分成假手术和模型两组（够少够简单的），用打击法，就是用重物击打脑组织，制备成弥散性脑损伤模型（经典但粗糙的模型）。模型作好后1小时、24小时、48小时进行神经行为学评价，就是根据动物的运动和感觉反射等方面的行为表现来判断神经系统功能受到伤害的程度，这是非常常用的不是非常稳定的方法，但不影响其他研究内容。一般开展神经系统损伤的实验室都玩这个东西。在模型0小时（模型前为基数）、12、24、48、72、120小时（5天）分别取动物血液进行非细胞成分DNA水平检测。在48小时的时候，11只动物（每组？）处死后取脑组织，进行脑水肿检测，怎么检测？就是把脑组织称重量，然后用100度烤箱烤成干，再称重量，两者差就是水的重量，水重在脑组织中的比例就是脑组织含水量，当发生脑水肿的时候，可以从78%左右上升到82-83%左右。这个是经典的脑水肿检测方法（够简单）。检测的结果发现，脑创伤后24和48小时，血液中的非细胞成分DNA水平明显增加，其中24小时的增加和48小时脑水肿以及其他时间的行为学改变存在相关性。这个研究给临床上提供了一种快速简单有效的可以用血液中成分分析脑损伤的方法。总之，作者用了很少的材料，很简单的技术，完成了一个很不错的研究。尽管初步，但新颖且具有启发性。

后评。1）我们可以借助这个思路开展其他类型疾病的研究，当然思路没有那么巧妙了，当仍有意义。2）更值得的是，可以用这个方法结合临床，采用这个指标研究是否真的具有诊断价值，建立诊断标准，推广到临床上。3）胡乱猜想的，不同类型组织是否存在不同的DNA，似乎不是，因为理论上所有细胞的DNA序列都一样的，不过不同寿命的细胞可能存在不同程度的DNA修饰或突变。如果能开展不同组织类型DNA特点的研究，可以在将来把这种手段开发成靶器官的诊断。4）当然这种研究仍存在很大不足，这种非细胞DNA也可以从炎症细胞释放，不一定从损伤的组织。5）细胞外DNA已经被认为属于损伤模式分子，也就是说可以引起免疫反应，这种改变也类似于炎症反应分子的一种特殊类型。那么本类研究更应该检测炎症反应的程度，并进行相关分析。

Journal of Neurotrauma

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Cell-Free DNA as a Marker for Prediction of Brain Damage in Traumatic Brain Injury in Rats

To cite this article:

Sharon Ohayon, Matthew Boyko, Amit Saad, Amos Douvdevani, Benjamin F. Gruenbaum, Israel Melamed, Yoram Shapira, Vivian I. Teichberg, and Alexander Zlotnik. Journal of Neurotrauma. January 20, 2012, 29(2): 261-267. doi:10.1089/neu.2011.1938.

Published in Volume: 29 Issue 2: January 30, 2012

Online Ahead of Print: December 7, 2011

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Sharon Ohayon,1*

*The first two authors contributed equally to this article.

Abstract

Traumatic brain injury (TBI) is a major cause of morbidity and mortality, and early predictors of neurological outcomes are of great clinical importance. Cell free DNA (CFD), a biomarker used for the diagnosis and monitoring of several diseases, has been implicated as a possible prognostic indicator after TBI. The purpose of this study was to determine the pattern and timing of CFD levels after TBI, and whether a relationship exists between the level of CFD and brain edema and neurological outcomes. Thirty-nine Sprague–Dawley rats were randomly assigned to two groups: rats in group 1 (sham group) were anesthetized and had a scalp incision without TBI, and rats in group 2 were anesthetized and had a scalp incision with TBI, which was induced by using a weight drop model that causes diffuse brain injury. A neurological severity score (NSS) was assessed at 1, 24, and 48 h after TBI. CFD was measured via blood samples drawn at t=0 (baseline), 12, 24, 48, 72, and 120 h after TBI. At 48 h after TBI, brain edema was determined in a subgroup of 11 rats by calculating the difference between rats' wet and dry brain weight. The significance of comparisons between and within groups (CFD levels, brain water content, and NSS) were determined using the Kruskal–Wallis, Mann–Whitney and Student t test. The correlation between CFD levels and the NSS, as well as between CFD levels and the extent of brain edema, was calculated using the Spearman and Pearson tests, respectively. Compared with baseline levels, the CFD levels in rats subjected to TBI were significantly increased at 24 and 48 h after TBI (p<0.01 and p<0.05, respectively). A positive correlation was demonstrated between CFD levels 24 h following TBI and the extent of brain edema (r=0.63, p<0.05), as well as between CFD levels and the NSS (r=0.79, p<0.005). In this study, we demonstrated an increase in CFD levels after TBI, as well as a correlation between CFD levels and brain edema and NSS. CFD levels may provide a quick, reliable, and simple prognostic indicator of neurological outcome in animals after TBI. Its role in humans has not been clearly elucidated, but has potentially significant clinical implications.