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轻松指导研究生查文献：类风湿实例分析
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我们在3、4年前开始介入类风湿性关节炎（RA）的分子病理学研究，创立并验证了“炎症诱发一氧化氮驱动缺氧致血管形成与滑膜增生”的RA病因学说，最近打算进一步探讨自身抗体的形成及其在RA发生中的作用。
既然要立项做研究，就要先查文献。根据我们的经验，先由学生"独立查阅"后由师生"共同查阅"的方式效果较好。在学生查阅阶段，应注重“面”，不要局限在国内或国外某个特定的数据库，以防错过有用的文献，同时也不会因为太难而使学生看不懂；而在师生查阅阶段，则应强调“点”，就是针对一个或几个重点、难点问题去国际知名数据库查找最新文献，以收到以点带面、追踪前沿的效果。
第一步，全面覆盖，快速入门
在这个阶段，学生已经花了很多时间查阅大量国内外文献，算是在RA的基础理论方面入了门，并对有关病理和药理知识有了初步了解。比如，已经了解到在RA的免疫学检测中抗环瓜氨酸肽（CCP）抗体是特异性强的指标之一，而且还知道RA患者体内某些蛋白质中的精氨酸被脱掉氨基生成了瓜氨酸，被称为“瓜氨酸化”(citrullination)，导致人体免疫系统将这种瓜氨酸化蛋白质视为外来抗原而诱发形成抗体，被称为“自身抗体”（autoantibody），也查到瓜氨酸化反应是由肽酰精氨酸脱亚胺酶4（PAD4）催化的。
第二步，以点带面，各个击破
但是，学生对RA患者体内PAD4是如何活化的却不清楚。针对这个问题，我们一起登入NCBI的PubMed检索栏中，键入PAD4进行检索，结果在一篇综述文章中看到下面一段话：
Live bacteria, the Gram-negative bacterial cell wall component lipopolysaccharide (LPS), the Gram-positive bacterial cell wall component lipoteichoic acid (LTA),the fungal cell wall component zymosan,the proinflammatory cytokine TNFα, and H2O2 have been shown to induce PAD4 activity.
也就是说，PAD4的激活因素有多种，但都与感染和炎症有关。有趣的是，RA的发病原因同样是感染和炎症。于是，我立即做出大胆假设：RA的发作与PAD4的激活是平行发生的，而感染性炎症是二者的共同诱因，但RA与PAD4之间没有因果关系！
第三步，寻找反例，声东击西
要验证以上假说，必须找到涉及二者关系的文献，尤其要得到基因敲除实验结果的支持。于是，我们以关键词PAD4 & RA继续查找，竟然恰好发现一篇文献使用的正是高效表达肿瘤坏死因子基因的野生型PAD4(+/+)小鼠与基因敲除PAD4(-/-)小鼠所做的研究，其结论是：
TNFα-overexpressing mice had increased levels of autoantibodies reactive against native and citrullinated antigens. PAD4 (-/-) mice with TNFα-induced arthritis had lower levels of autoantibodies reactive against native and citrullinated antigens, decreased T cell activation and total IgG levels, and reduced inflammation and arthritis compared to PAD4 (+/+) TNFα-overexpressing mice. PAD4 mediates autoantibody production and inflammatory arthritis downstream of TNFα.
从这个结果来看，似乎不太支持我的假说，因为PAD4的活性与RA的发病程度存在一定相关性，有PAD4的小鼠比无PAD4的小鼠关节炎症更为严重。
第四步，寻找正例，左右逢源
我们不愿就此善罢甘休，于是继续搜索又发现另一篇文献，同样是采用野生型小鼠与基因敲除小鼠探讨PAD4与RA的因果关系，从它的标题就得出了结论：
PAD4 is not essential for disease in the K/BxN murine autoantibody-mediated model of arthritis
其详细的结论如下：
PAD4 activity is readily detectable in the inflamed synovium of WT but not PAD4 deficient animals, as demonstrated by histone citrullination and NET formation. However, PAD4 WT and KO animals develop K/BxN serum transfer disease with comparable severity and kinetics, with no statistically significant differences noted in clinical scores, swelling, joint erosion or joint invasion. PAD4 WT and KO mice develop disease in the K/BxN serum transfer model of arthritis with similar severity and kinetics, indicating that PAD4 is dispensable in this effector phase model of disease.
这意味着我前面的假设得到该结论支持，即PAD4与RA不存在因果关系！比较这两篇文献，前一篇是通过高效表达肿瘤坏死因子诱发RA，后一篇则通过注射RA小鼠血清诱发RA，但两者为何得出不同结论，正好形成了我们即将启动的类似研究的“创新点”！
第五步，深入分析，融会贯通
至于PAD4是如何被激活的，有一篇文章这么说：在正常情况下，PAD4在高浓度钙离子作用下活性被阻遏，当它与PAD3结合后，其钙离子结合位点被封闭，结果因钙离子无法结合PAD3-PAD4复合体而使PAD4被激活。因此，实际上应该解决为什么PAD3会与PAD4交联的问题。PAD4原本负责染色体组蛋白的瓜氨酸化修饰，像甲基化、乙酰化那样调节基因表达，但由于瓜氨酸修饰组蛋白位于细胞核内，不会接触免疫细胞，因此不会诱导产生自身抗体。
瓜氨酸化蛋白如角蛋白（keratin）、丝聚合蛋白（filaggrin）、波形蛋白（vimentin）等因细胞破裂而释放入血诱发的自身抗体称为“抗瓜氨酸化蛋白抗体”（ACPA）。在RA患者中，可以用人工合成的环瓜氨酸肽（CCP）抗原检测血清中的ACPA，称为抗CCP抗体。在基质蛋白酶水解作用下，软骨及骨骼细胞受损而将瓜氨酸化蛋白释放出来，从而在血液循环中诱导相应的抗体。
第六步，引经据典，举一反三
既然抗CCP抗体是RA特有的，那么谁解决了抗CCP抗体的来源问题，谁就能找到RA的真正病因。不久前，有人发现口腔细菌本身就有一种PAD，可以促进RA的发生和发展。论文发表在2013年9月12日的《国家图书馆——病原体》（PLoS Pathogens）上，以下是该论文的题目及摘要：
牙龈卟啉单胞菌通过其独特的细菌肽酰精氨酸脱亚胺酶（PAD）促进破坏性关节炎的发生和发展
Rheumatoid arthritis and periodontitis are two prevalent chronic inflammatory diseases in humans and are associated with each other both clinically and epidemiologically. Recent findings suggest a causative link between periodontal infection and rheumatoid arthritis via bacteria-dependent induction of a pathogenic autoimmune response to citrullinated epitopes. Here we showed that infection with viable periodontal pathogen Porphyromonas gingivalis strain W83 exacerbated collagen-induced arthritis (CIA) in a mouse model, as manifested by earlier onset, accelerated progression and enhanced severity of the disease, including significantly increased bone and cartilage destruction. The ability of P. gingivalis to augment CIA was dependent on the expression of a unique P. gingivalis peptidylarginine deiminase (PPAD), which converts arginine residues in proteins to citrulline. Infection with wild type P. gingivaliswas responsible for significantly increased levels of autoantibodies to collagen type II and citrullinated epitopes as a PPAD-null mutant did not elicit similar host response. High level of citrullinated proteins was also detected at the site of infection with wild-type P. gingivalis. Together, these results suggest bacterial PAD as the mechanistic link between P. gingivalisperiodontal infection and rheumatoid arthritis.
Clinical and epidemiological data indicates that chronic periodontal disease (PD), one of the most prevalent infectious inflammatory disease of mankind, is linked to systemic inflammatory diseases such as cardiovascular diseases (CVD), rheumatoid arthritis (RA) and chronic obstructive pulmonary disease (COPD). Nevertheless, the causative mechanisms of association between PD and chronic inflammatory diseases are very poorly understood. Recent findings suggest a causative link between periodontal infection and rheumatoid arthritis viabacteria-dependent induction of a pathogenic response to citrullinated epitopes. In present study we show that infection with viable periodontal pathogen Porphyromonas gingivalis but not another oral bacterium (Prevotella intermedia), exacerbated CIA, as manifested by earlier onset, accelerated progression and enhanced severity of the disease, including significantly increased bone and cartilage destruction. The ability of P. gingivalis to augment CIA was dependent on the expression of a unique enzyme peptidylarginine deiminase, which converts arginine residues in proteins to citrulline. This knowledge may create new perspectives in the treatment and prevention of RA in susceptible individuals.
现在留下的问题是：细菌的PAD是导致RA的病因？还是PAD催化的瓜氨酸化仅仅是RA发生过程中平行发生的伴随现象？值得我们好好深入研究。
https://blog.sciencenet.cn/blog-281238-802468.html
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