本文關(guān)鍵詞：輔助性T細(xì)胞17（Th17）與調(diào)節(jié)性T細(xì)胞（Treg）的平衡關(guān)系在全身炎癥反應(yīng)綜合征（SIRS）中的作用及機(jī)制研究，由筆耕文化傳播整理發(fā)布。

        多器官功能障礙綜合征（MODS）是導(dǎo)致急危重患者死亡的主要原因。其死亡率高，治療周期長，家庭及社會(huì)負(fù)擔(dān)沉重，是危重醫(yī)學(xué)亟待解決的重要問題之。近三十年來，盡管國內(nèi)外學(xué)者為此做了大量的探索，但是MODS的發(fā)病率和病死率仍居高不下。全身炎癥反應(yīng)（SIRS）被認(rèn)為是MODS的共同通路。目前認(rèn)為，抗炎與促炎因素的失平衡導(dǎo)致“炎癥反應(yīng)失控”是SIRS的主要發(fā)生機(jī)制，但具體的調(diào)控機(jī)制目前尚不清楚。CD4+Th細(xì)胞在免疫反應(yīng)和炎癥疾病中均起著重要的作用。其中特異性表達(dá)轉(zhuǎn)錄因子RORγ t的Th17細(xì)胞通過分泌IL-17促使下游的炎性因子大量釋放,在多種免疫炎性疾病中起到關(guān)鍵的促炎作用；而特異性表達(dá)轉(zhuǎn)錄因子Foxp3的Treg細(xì)胞通過直接或者間接地方式對(duì)Th17細(xì)胞進(jìn)行拮抗，起到抑炎和抑制免疫反應(yīng)的作用。已有大量研究證明Th17/Treg細(xì)胞失平衡在誘導(dǎo)多種自身免疫疾病中扮演重要角色。如風(fēng)濕性關(guān)節(jié)炎（RA），實(shí)驗(yàn)性自身免疫性腦脊髓炎（EAE），哮喘及系統(tǒng)性紅斑狼瘡（SLE）等。在SIRS、MODS的發(fā)生發(fā)展過程中，肺臟是最先受損傷的靶器官，而SIRS在肺臟中的表現(xiàn)就是急性肺損傷（ALI）。同時(shí)在CLP引起的SIRS模型血清中以及LPS誘導(dǎo)的急性肺損傷模型中均檢測到IL-17的參與。所以本研究提出以下推測：(1) Th17細(xì)胞與Treg細(xì)胞可能參與了SIRS的發(fā)生，并且兩者變化過程之間及相關(guān)炎性因子的變化可能均存在相關(guān)性。(2)通過干預(yù)、拮抗Th17細(xì)胞或者Treg細(xì)胞的分化和功能，之后SIRS的嚴(yán)重程度會(huì)發(fā)生相應(yīng)的變化。進(jìn)步闡明兩者在SIRS的發(fā)病過程中所起的作用。本研究主要是對(duì)上述推測的第部分進(jìn)行研究，同時(shí)，也是2010年國家自然科學(xué)基金面上項(xiàng)目資助項(xiàng)目（No.30972854)目的：本實(shí)驗(yàn)研究以小鼠腹腔注射酵母多糖制造MODS為模型，并以肺臟作為觀察Th17細(xì)胞與Treg細(xì)胞在SIRS模型中發(fā)生發(fā)展的靶器官，在基因，蛋白及相關(guān)炎性因子水平進(jìn)行多時(shí)間點(diǎn)、多層面、系統(tǒng)的觀察兩者的變化過程及相關(guān)關(guān)系。方法：120只昆明小鼠隨機(jī)分為模型組（n=90）、對(duì)照組（n=20）及死亡率觀察組（10），模型組又分為6h、1d、2d、3d、5d和7d六個(gè)亞組（n=15/亞組）。腹腔注射酵母多糖制造SIRS模型；觀察小鼠死亡率及小鼠中毒癥狀（結(jié)膜炎、毛發(fā)凌亂、腹瀉、嗜睡）的嚴(yán)重程度并對(duì)小鼠進(jìn)行評(píng)估；免疫熒光染色觀察Th17細(xì)胞與Treg細(xì)胞在肺臟組織中的浸潤情況；并采用Elisa、Real-time PCR及Western blotting檢測特異性轉(zhuǎn)錄因子RORγ t和Foxp3基因和蛋白的表達(dá)以及相關(guān)炎性因子的變化。結(jié)果：全身炎癥反應(yīng)發(fā)生后，肺臟1d時(shí)組織中即有炎性細(xì)胞浸潤，以后逐漸加重并有大量淋巴細(xì)胞浸潤，肺泡水腫、出血、肺泡壁逐漸加厚并逐漸斷裂。小鼠死亡率在兩天時(shí)達(dá)到高峰；Elisa結(jié)果顯示肺組織中的IL-17在炎癥6h (393.30±38.78)、1d (364.72±64.57)和7d (306.86±2.65)均較對(duì)照組（219.67±8.50）明顯增高，在肺臟中的變化是6h和1d時(shí)濃度最高，2d時(shí)開始下降，5d時(shí)又開始濃度升高，第7天又接近初始狀態(tài)。血清中的IL-17在6h(113.39±5.76)時(shí)開始增高，1d時(shí)(272.71±58.61)達(dá)到高峰。Real-time PCR結(jié)果顯示Th17細(xì)胞的轉(zhuǎn)錄因子RORγ t與對(duì)照組(0.25±0.02)相比表達(dá)量是1d (0.47±0.02)開始升高、2d (0.63±0.06)和3d (0.63±0.07)達(dá)到高峰，5d(0.23±0.02)時(shí)又開始下降。而Treg細(xì)胞轉(zhuǎn)錄因子Foxp3與對(duì)照組(0.99±0.04)相比，1d (0.49±0.07)時(shí)表達(dá)量下降、2d (0.58±0.03)時(shí)開始升高，5d (1.41±0.07)達(dá)到最高峰，7d(0.64±0.08)又開始下降。RORγ t與Foxp3蛋白表達(dá)與基因表達(dá)情況相致，無論在基因還是蛋白層面兩者的關(guān)系均呈負(fù)相關(guān)關(guān)系(R=-0.4540，R=-0.2272)；而促炎細(xì)胞因子IL-6、TNF-和抗炎細(xì)胞因子IL-10的表達(dá)變化均呈負(fù)相關(guān)(R=-0.7455，R=-0.6323)；另外，通過免疫熒光雙標(biāo)染色我們觀察到肺臟組織中有少量的Th17細(xì)胞的浸潤。結(jié)論：SIRS發(fā)生以后， Th17細(xì)胞與Treg細(xì)胞的轉(zhuǎn)錄因子及蛋白均發(fā)生了變化，并且存在負(fù)性相關(guān)關(guān)系，相關(guān)炎性因子同樣呈現(xiàn)出此起彼伏的相互拮抗現(xiàn)象，而血清與肺臟中的IL-17后期卻呈現(xiàn)不樣的表現(xiàn)，，同時(shí)我們發(fā)現(xiàn)IL-17的表達(dá)情況并不與Th17細(xì)胞相致，也不與Treg細(xì)胞存在相關(guān)關(guān)系。我們可以初步得出這樣的結(jié)論：Th17細(xì)胞與Treg細(xì)胞參與了SIRS的發(fā)生發(fā)展過程，并發(fā)生失平衡；SIRS早期，IL-17升高伴有Treg細(xì)胞功能的缺失；后期，IL-17的升高可能與RORγ t中后期升高有關(guān)；后期肺臟局部釋放了不同于全身的IL-17；IL-17與Th17不存在負(fù)性相關(guān)關(guān)系，可能在此過程還有其它分泌IL-17的細(xì)胞；后期Treg細(xì)胞的功能逐漸恢復(fù)，小鼠的死亡率及中毒癥狀得到改善，，兩者變化過程是與小鼠癥狀嚴(yán)重程度密切相關(guān)的。糾正和維持促炎和抗炎因素的平衡關(guān)系有望成為治療SIRS的新靶點(diǎn)。

    Multiple organ dysfunction syndrome (MODS) is the main reason for deathin patients with Intensive. With high mortality, long treatment cycle and theheavy burden on the family and society, it is one of the important issues to besolved in critical care medicine.In the recent three decades，although domesticand foreign scholars have done a lot of exploration, the morbidity and mortalityof MODS remains high.Systemic inflammatory response (SIRS) is considered tobe the common pathway for MODS.Currently considered,“the loss of balancebetween anti-inflammatory and pro-inflammatory factors leading to inflammatoryresponse out of control "is the main mechanism of SIRS, but the specificmechanism of regulation is not clear.CD4+Th cells play an important role in immune response and inflammatorydiseases. Which specificly expressing the transcription factor RORγt and secreting IL-17,prompted a large number of downstream inflammatory factors torelease, Th17cells play a key pro-inflammatory role in a variety of immuneinflammatory diseases；Treg cells specificlt expressing the transcription factorFoxp3directly or indirectly antagonized Th17cells, they suppress inflammationand inhibit the immune response.A lot of research has proved that the imbalance of Th17/Treg cells play animportant role in the induction of a variety of autoimmune diseases. Such asrheumatoid arthritis (RA), experimental autoimmune encephalomyelitis (EAE),asthma and systemic lupus erythematosus (SLE). During the development ofSIRS and MODS,the lung is the primary target organ to suffer injury, theperformance of SIRS in the lungs is acute lung injury (ALI). The participation ofIL-17were detected in the SIRS animal models and patients with acute lunginjury. Therefore, this study asked the following speculations:Th17cells and Treg cells may be involved in the occurrence of SIRS，andchanges between the both and the related inflammatory factors change processesmay be correlated. Through intervention, the antagonistic Th17cells, observedthe change of SIRS severity and differentiation and function of Treg cells. Furtherelucidate the role of two cells in the pathogenesis of SIRS.This study is the first part of the above conjecture.At the same time，it isalso the National Natural Science Foundation funded project in2010（No.30972854)Aim：In this study, we make a model of SIRS by intraperitoneal injectingzymosan to the mouse, And make lung as a window to observe the occurrenceand development of Th17cells and Treg cells in the SIRS model.In terms ofgenes, proteins and related inflammatory factors, multiple points in time,multi-level, to observ both the process of change systematicly. Method：120mice were randomly divided into model group（n=90）, controlgroup（n=20）and Mortality observed group（n=10）.The model group was dividedinto6h,1d,2d,3d,5d and7d six subgroups（n=15/subgroups）. Make model ofSIRS by intraperitoneal injecting zymosan to the mouse；Assess the severity ofmice through observing the mortality and poisoning symptoms (conjunctivitis,mao faling disorder, diarrhea, lethargy)；To observe the infiltration of Th17cellsand Treg cells in the lung tissue by immunofluorescence staining；And analysethe expression changes of specific transcription factor RORγt and Foxp3in bothgene and protein levels and related inflammatory factors by Elisa, real-time PCRand Western blotting.Results：After the occurrence of systemic inflammatory response, theinflammatory cells infiltrated in the lungs on1d, and then gradually increased andmassive lymphocytes infiltrated in the lung， alveolar edema, hemorrhage,alveolar wall thickened and gradually fractured.The mortality of mice reached apeak on2d； Results of Elisa showed that the IL-17in lung tissue on6h(393.30±38.78),1d (364.72±64.57) and7d (306.86±2.65) were significantlyhigher than the control group (219.67±8.50), In the lung the changes of IL-17is:it has the highest concentration at6h and1d, it started to decline on2d (86.75±7.12), on5d (129.39±5.97) the concentration increases again and on7d itbecome close to the initial state. The IL-17in the serum begin to increse on6h(113.39±5.76) and to a peak on1d(272.71±58.61). Real-time PCR resultsshowed that the expression level of transcription factor RORγt of Th17cells,began to increase on1d (0.47±0.02), to the peaked on2d (0.63±0.06) and3d(0.63±0.07) and begin to decline on5d (0.23±0.02). compared with the controlgroup (0.25±0.02). The expression levels of transcription factor Foxp3of Tregcell declined on1d(0.49±0.07)and rised on2d(0.58±0.03),then to the peak at 5d (1.41±0.07) and at last it have begun to drop at7d (0.64±0.08) comparedwith the control group (0.99±0.04). RORγt and Foxp3protein expression isconsistent with the gene expression, the two had a negative correlation no matteron gene or protein level(R=-0.4540，R=-0.2272); Expression of proinflammatorycytokines IL-6, TNF-alpha was negatively correlated with anti-inflammatorycytokines IL-10(R=-0.7455，R=-0.6323); In addition, we observed that a smallamount of Th17cells infiltrated in lung tissue by immunofluorescence doublestaining.Conclusion：After SIRS，transcription factors and protein of Th17cells andTreg cells were changed, and showed a negative relationship, RelatedInflammatory factors also showed one after another the mutual antagonizedphenomenon.But we also found that IL-17expression is not consistent with theTh17cells, nor a negative correlation with Treg cells. We can conclude thatpreliminarily: Th17cells and Treg cells involved in the occurrence anddevelopment of SIRS, In the Early stage, function of Treg cells was not onlyinhibited by Th17cells, there may be other lymphocytes which secrete IL-17.The function of Treg cells gradually recovered later, and the mortality andpoisoning symptoms improved in mice. The process of change between the two isclosely related to the severity of symptoms of mice. Correct and maintain thebalance between pro-inflammatory and anti-inflammatory factors is expected tobecome a new target for the treatment of SIRS.

        輔助性T細(xì)胞17（Th17）與調(diào)節(jié)性T細(xì)胞（Treg）的平衡關(guān)系在全身炎癥反應(yīng)綜合征（SIRS）中的作用及機(jī)制研究

縮略語表5-8中文摘要8-11Abstract11-14前言15-17文獻(xiàn)回顧17-311 材料31-32    1.1 主要試劑31-32    1.2 主要儀器322 方法32-35    2.1 動(dòng)物分組32-33    2.2 建立 MODS 模型33    2.3 組織標(biāo)本制備33    2.4 小鼠中毒癥狀評(píng)估33    2.5 肺臟的免疫組織化學(xué)染色和干濕重比值33    2.6 免疫熒光雙標(biāo)33-34    2.7 Real-time PCR 檢測轉(zhuǎn)錄因子 RORγ tmRNA 和 Foxp3mRNA 的表達(dá)34    2.8 Western Blotting 檢測轉(zhuǎn)錄因子 RORγ t 和 Foxp3 蛋白表達(dá)34-35    2.9 Elisa 檢測血清及腦組織中的 IL-17 濃度35    2.10 統(tǒng)計(jì)分析353 結(jié)果35-42    3.1 小鼠中毒癥狀35    3.2 肺臟組織的病理和干濕重比值的變化35-37    3.3 肺臟中的 Foxp3 與 RORγ t 無論在基因還是蛋白水平都存在相關(guān)性37-38    3.4 肺臟中的炎性因子也呈現(xiàn)出相應(yīng)變化38-40    3.5 血清和肺臟中的 IL-17 表達(dá)趨勢竟存在明顯差異40    3.6 Th17 細(xì)胞侵入到了受損傷肺臟中40-424. 討論42-45小結(jié)45-46參考文獻(xiàn)46-59個(gè)人簡歷和研究成果59-60致謝60

  本文關(guān)鍵詞：輔助性T細(xì)胞17（Th17）與調(diào)節(jié)性T細(xì)胞（Treg）的平衡關(guān)系在全身炎癥反應(yīng)綜合征（SIRS）中的作用及機(jī)制研究，由筆耕文化傳播整理發(fā)布。