我們在3、4年前開始介入類風(fēng)濕性關(guān)節(jié)炎（RA）的分子病理學(xué)研究，創(chuàng)立并驗(yàn)證了“炎癥誘發(fā)一氧化氮驅(qū)動(dòng)缺氧致血管形成與滑膜增生”的RA病因?qū)W說，最近打算進(jìn)一步探討自身抗體的形成及其在RA發(fā)生中的作用。
既然要立項(xiàng)做研究，就要先查文獻(xiàn)。根據(jù)我們的經(jīng)驗(yàn)，先由學(xué)生"獨(dú)立查閱"后由師生"共同查閱"的方式效果較好。在學(xué)生查閱階段，應(yīng)注重“面”，不要局限在國內(nèi)或國外某個(gè)特定的數(shù)據(jù)庫，以防錯(cuò)過有用的文獻(xiàn)，同時(shí)也不會(huì)因?yàn)樘y而使學(xué)生看不懂；而在師生查閱階段，則應(yīng)強(qiáng)調(diào)“點(diǎn)”，就是針對一個(gè)或幾個(gè)重點(diǎn)、難點(diǎn)問題去國際知名數(shù)據(jù)庫查找最新文獻(xiàn)，以收到以點(diǎn)帶面、追蹤前沿的效果。
第一步，全面覆蓋，快速入門
在這個(gè)階段，學(xué)生已經(jīng)花了很多時(shí)間查閱大量國內(nèi)外文獻(xiàn)，算是在RA的基礎(chǔ)理論方面入了門，并對有關(guān)病理和藥理知識(shí)有了初步了解。比如，已經(jīng)了解到在RA的免疫學(xué)檢測中抗環(huán)瓜氨酸肽（CCP）抗體是特異性強(qiáng)的指標(biāo)之一，而且還知道RA患者體內(nèi)某些蛋白質(zhì)中的精氨酸被脫掉氨基生成了瓜氨酸，被稱為“瓜氨酸化”(citrullination)，導(dǎo)致人體免疫系統(tǒng)將這種瓜氨酸化蛋白質(zhì)視為外來抗原而誘發(fā)形成抗體，被稱為“自身抗體”（autoantibody），也查到瓜氨酸化反應(yīng)是由肽酰精氨酸脫亞胺酶4（PAD4）催化的。
第二步，以點(diǎn)帶面，各個(gè)擊破
但是，學(xué)生對RA患者體內(nèi)PAD4是如何活化的卻不清楚。針對這個(gè)問題，我們一起登入NCBI的PubMed檢索欄中，鍵入PAD4進(jìn)行檢索，結(jié)果在一篇綜述文章中看到下面一段話：
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.
也就是說，PAD4的激活因素有多種，但都與感染和炎癥有關(guān)。有趣的是，RA的發(fā)病原因同樣是感染和炎癥。于是，我立即做出大膽假設(shè)：RA的發(fā)作與PAD4的激活是平行發(fā)生的，而感染性炎癥是二者的共同誘因，但RA與PAD4之間沒有因果關(guān)系！
第三步，尋找反例，聲東擊西
要驗(yàn)證以上假說，必須找到涉及二者關(guān)系的文獻(xiàn)，尤其要得到基因敲除實(shí)驗(yàn)結(jié)果的支持。于是，我們以關(guān)鍵詞PAD4 & RA繼續(xù)查找，竟然恰好發(fā)現(xiàn)一篇文獻(xiàn)使用的正是高效表達(dá)腫瘤壞死因子基因的野生型PAD4(+/+)小鼠與基因敲除PAD4(-/-)小鼠所做的研究，其結(jié)論是：
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.
從這個(gè)結(jié)果來看，似乎不太支持我的假說，因?yàn)镻AD4的活性與RA的發(fā)病程度存在一定相關(guān)性，有PAD4的小鼠比無PAD4的小鼠關(guān)節(jié)炎癥更為嚴(yán)重。
第四步，尋找正例，左右逢源
我們不愿就此善罷甘休，于是繼續(xù)搜索又發(fā)現(xiàn)另一篇文獻(xiàn)，同樣是采用野生型小鼠與基因敲除小鼠探討PAD4與RA的因果關(guān)系，從它的標(biāo)題就得出了結(jié)論：
PAD4 is not essential for disease in the K/BxN murine autoantibody-mediated model of arthritis
其詳細(xì)的結(jié)論如下：
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.
這意味著我前面的假設(shè)得到該結(jié)論支持，即PAD4與RA不存在因果關(guān)系！比較這兩篇文獻(xiàn)，前一篇是通過高效表達(dá)腫瘤壞死因子誘發(fā)RA，后一篇?jiǎng)t通過注射RA小鼠血清誘發(fā)RA，但兩者為何得出不同結(jié)論，正好形成了我們即將啟動(dòng)的類似研究的“創(chuàng)新點(diǎn)”！
第五步，深入分析，融會(huì)貫通
至于PAD4是如何被激活的，有一篇文章這么說：在正常情況下，PAD4在高濃度鈣離子作用下活性被阻遏，當(dāng)它與PAD3結(jié)合后，其鈣離子結(jié)合位點(diǎn)被封閉，結(jié)果因鈣離子無法結(jié)合PAD3-PAD4復(fù)合體而使PAD4被激活。因此，實(shí)際上應(yīng)該解決為什么PAD3會(huì)與PAD4交聯(lián)的問題。PAD4原本負(fù)責(zé)染色體組蛋白的瓜氨酸化修飾，像甲基化、乙?；菢诱{(diào)節(jié)基因表達(dá)，但由于瓜氨酸修飾組蛋白位于細(xì)胞核內(nèi)，不會(huì)接觸免疫細(xì)胞，因此不會(huì)誘導(dǎo)產(chǎn)生自身抗體。
瓜氨酸化蛋白如角蛋白（keratin）、絲聚合蛋白（filaggrin）、波形蛋白（vimentin）等因細(xì)胞破裂而釋放入血誘發(fā)的自身抗體稱為“抗瓜氨酸化蛋白抗體”（ACPA）。在RA患者中，可以用人工合成的環(huán)瓜氨酸肽（CCP）抗原檢測血清中的ACPA，稱為抗CCP抗體。在基質(zhì)蛋白酶水解作用下，軟骨及骨骼細(xì)胞受損而將瓜氨酸化蛋白釋放出來，從而在血液循環(huán)中誘導(dǎo)相應(yīng)的抗體。
第六步，引經(jīng)據(jù)典，舉一反三
既然抗CCP抗體是RA特有的，那么誰解決了抗CCP抗體的來源問題，誰就能找到RA的真正病因。不久前，有人發(fā)現(xiàn)口腔細(xì)菌本身就有一種PAD，可以促進(jìn)RA的發(fā)生和發(fā)展。論文發(fā)表在2013年9月12日的《國家圖書館——病原體》（PLoS Pathogens）上，以下是該論文的題目及摘要：
芽孢卟啉單胞菌通過其獨(dú)特的細(xì)菌肽酰精氨酸脫亞胺酶（PAD）促進(jìn)破壞性關(guān)節(jié)炎的發(fā)生和發(fā)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.
現(xiàn)在留下的問題是：細(xì)菌的PAD是導(dǎo)致RA的病因？還是PAD催化的瓜氨酸化僅僅是RA發(fā)生過程中平行發(fā)生的伴隨現(xiàn)象？值得我們好好深入研究。
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