辅助性T细胞17（Th17）与调节性T细胞（Treg）的平衡关系在全身炎症反应综合征（SIRS）中的作用及机制研究

  本文关键词：辅助性T细胞17（Th17）与调节性T细胞（Treg）的平衡关系在全身炎症反应综合征（SIRS）中的作用及机制研究，由笔耕文化传播整理发布。

        多器官功能障碍综合征（MODS）是导致急危重患者死亡的主要原因。其死亡率高，治疗周期长，家庭及社会负担沉重，是危重医学亟待解决的重要问题之。近三十年来，尽管国内外学者为此做了大量的探索，但是MODS的发病率和病死率仍居高不下。全身炎症反应（SIRS）被认为是MODS的共同通路。目前认为，抗炎与促炎因素的失平衡导致“炎症反应失控”是SIRS的主要发生机制，但具体的调控机制目前尚不清楚。CD4+Th细胞在免疫反应和炎症疾病中均起着重要的作用。其中特异性表达转录因子RORγ t的Th17细胞通过分泌IL-17促使下游的炎性因子大量释放,在多种免疫炎性疾病中起到关键的促炎作用；而特异性表达转录因子Foxp3的Treg细胞通过直接或者间接地方式对Th17细胞进行拮抗，起到抑炎和抑制免疫反应的作用。已有大量研究证明Th17/Treg细胞失平衡在诱导多种自身免疫疾病中扮演重要角色。如风湿性关节炎（RA），实验性自身免疫性脑脊髓炎（EAE），哮喘及系统性红斑狼疮（SLE）等。在SIRS、MODS的发生发展过程中，肺脏是最先受损伤的靶器官，而SIRS在肺脏中的表现就是急性肺损伤（ALI）。同时在CLP引起的SIRS模型血清中以及LPS诱导的急性肺损伤模型中均检测到IL-17的参与。所以本研究提出以下推测：(1) Th17细胞与Treg细胞可能参与了SIRS的发生，并且两者变化过程之间及相关炎性因子的变化可能均存在相关性。(2)通过干预、拮抗Th17细胞或者Treg细胞的分化和功能，之后SIRS的严重程度会发生相应的变化。进步阐明两者在SIRS的发病过程中所起的作用。本研究主要是对上述推测的第部分进行研究，同时，也是2010年国家自然科学基金面上项目资助项目（No.30972854)目的：本实验研究以小鼠腹腔注射酵母多糖制造MODS为模型，并以肺脏作为观察Th17细胞与Treg细胞在SIRS模型中发生发展的靶器官，在基因，蛋白及相关炎性因子水平进行多时间点、多层面、系统的观察两者的变化过程及相关关系。方法：120只昆明小鼠随机分为模型组（n=90）、对照组（n=20）及死亡率观察组（10），模型组又分为6h、1d、2d、3d、5d和7d六个亚组（n=15/亚组）。腹腔注射酵母多糖制造SIRS模型；观察小鼠死亡率及小鼠中毒症状（结膜炎、毛发凌乱、腹泻、嗜睡）的严重程度并对小鼠进行评估；免疫荧光染色观察Th17细胞与Treg细胞在肺脏组织中的浸润情况；并采用Elisa、Real-time PCR及Western blotting检测特异性转录因子RORγ t和Foxp3基因和蛋白的表达以及相关炎性因子的变化。结果：全身炎症反应发生后，肺脏1d时组织中即有炎性细胞浸润，以后逐渐加重并有大量淋巴细胞浸润，肺泡水肿、出血、肺泡壁逐渐加厚并逐渐断裂。小鼠死亡率在两天时达到高峰；Elisa结果显示肺组织中的IL-17在炎症6h (393.30±38.78)、1d (364.72±64.57)和7d (306.86±2.65)均较对照组（219.67±8.50）明显增高，在肺脏中的变化是6h和1d时浓度最高，2d时开始下降，5d时又开始浓度升高，第7天又接近初始状态。血清中的IL-17在6h(113.39±5.76)时开始增高，1d时(272.71±58.61)达到高峰。Real-time PCR结果显示Th17细胞的转录因子RORγ t与对照组(0.25±0.02)相比表达量是1d (0.47±0.02)开始升高、2d (0.63±0.06)和3d (0.63±0.07)达到高峰，5d(0.23±0.02)时又开始下降。而Treg细胞转录因子Foxp3与对照组(0.99±0.04)相比，1d (0.49±0.07)时表达量下降、2d (0.58±0.03)时开始升高，5d (1.41±0.07)达到最高峰，7d(0.64±0.08)又开始下降。RORγ t与Foxp3蛋白表达与基因表达情况相致，无论在基因还是蛋白层面两者的关系均呈负相关关系(R=-0.4540，R=-0.2272)；而促炎细胞因子IL-6、TNF-和抗炎细胞因子IL-10的表达变化均呈负相关(R=-0.7455，R=-0.6323)；另外，通过免疫荧光双标染色我们观察到肺脏组织中有少量的Th17细胞的浸润。结论：SIRS发生以后， Th17细胞与Treg细胞的转录因子及蛋白均发生了变化，并且存在负性相关关系，相关炎性因子同样呈现出此起彼伏的相互拮抗现象，而血清与肺脏中的IL-17后期却呈现不样的表现，，同时我们发现IL-17的表达情况并不与Th17细胞相致，也不与Treg细胞存在相关关系。我们可以初步得出这样的结论：Th17细胞与Treg细胞参与了SIRS的发生发展过程，并发生失平衡；SIRS早期，IL-17升高伴有Treg细胞功能的缺失；后期，IL-17的升高可能与RORγ t中后期升高有关；后期肺脏局部释放了不同于全身的IL-17；IL-17与Th17不存在负性相关关系，可能在此过程还有其它分泌IL-17的细胞；后期Treg细胞的功能逐渐恢复，小鼠的死亡率及中毒症状得到改善，，两者变化过程是与小鼠症状严重程度密切相关的。纠正和维持促炎和抗炎因素的平衡关系有望成为治疗SIRS的新靶点。

    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.

        辅助性T细胞17（Th17）与调节性T细胞（Treg）的平衡关系在全身炎症反应综合征（SIRS）中的作用及机制研究

缩略语表5-8中文摘要8-11Abstract11-14前言15-17文献回顾17-311 材料31-32    1.1 主要试剂31-32    1.2 主要仪器322 方法32-35    2.1 动物分组32-33    2.2 建立 MODS 模型33    2.3 组织标本制备33    2.4 小鼠中毒症状评估33    2.5 肺脏的免疫组织化学染色和干湿重比值33    2.6 免疫荧光双标33-34    2.7 Real-time PCR 检测转录因子 RORγ tmRNA 和 Foxp3mRNA 的表达34    2.8 Western Blotting 检测转录因子 RORγ t 和 Foxp3 蛋白表达34-35    2.9 Elisa 检测血清及脑组织中的 IL-17 浓度35    2.10 统计分析353 结果35-42    3.1 小鼠中毒症状35    3.2 肺脏组织的病理和干湿重比值的变化35-37    3.3 肺脏中的 Foxp3 与 RORγ t 无论在基因还是蛋白水平都存在相关性37-38    3.4 肺脏中的炎性因子也呈现出相应变化38-40    3.5 血清和肺脏中的 IL-17 表达趋势竟存在明显差异40    3.6 Th17 细胞侵入到了受损伤肺脏中40-424. 讨论42-45小结45-46参考文献46-59个人简历和研究成果59-60致谢60

  本文关键词：辅助性T细胞17（Th17）与调节性T细胞（Treg）的平衡关系在全身炎症反应综合征（SIRS）中的作用及机制研究，由笔耕文化传播整理发布。