1、Spred2在ConA诱导的小鼠肝炎中的保护作用Spred-2在ConA诱导的小鼠肝炎中的保护作用大连医科大学硕士学位论文Spred-2在ConA诱导的小鼠肝炎中的保护作用姓名:曹晨申请学位级别:硕士专业:病理学与病理生理学指导教师:关宏伟201106Spred-2在ConA诱导的小鼠肝炎中的保护作用硕士生姓名:曹晨 指导教师:关宏伟教授 专业名称:病理学与病理生理学摘要引言:T细胞在多种肝脏疾病的发病过程中具有重要作用,如病毒性肝炎,自 身免疫性肝炎,酒精性肝病和非酒精性脂肪肝病等,但是T细胞如何发挥作用及 其相关细胞因子的信号调控机制仍不清楚。其中急性乙肝病毒(HBV)的清除和肝 细胞的损
2、伤是两个不同过程,HBV清除在肝细胞损伤前就已经完成,当特异性杀伤 性T细胞(CTL)的缺乏导致病毒复制不能得到有效控制时,可引起大量非特异性 炎性细胞,特别是非特异性CD8+T细胞在多种细胞因子的趋化作用下对肝组织的 广泛浸润,这是造成肝组织大面积损伤的基础。为了深入研究T细胞应答在肝脏 疾病中的作用机制,选择和应用与人类肝炎发病特征相类似的动物肝炎模型十分 必要。注射刀豆蛋白A(Concanaval in A,ConA)诱发动物肝炎是一个已经建立完 善的模型,这种模型的病理特征与人类病毒性肝炎,自身免疫性肝炎和药物性肝 炎相似。ConA诱导的肝炎主要通过快速活化多克隆T细胞包括为CD4+T
3、细胞、CD8+T 细胞和NKT细胞来实现自身免疫损伤。其特征是血清谷丙转氨酶水平迅速升高, 血清中含有大量细胞因子,肝脏出现白细胞浸润,肝细胞坏死和凋亡。在这种由 ConA诱导的免疫系统过度活化所引起的肝脏急性损伤模型中,T细胞的激活依赖 于Thl细胞因子IFN y及其信号传导通路。研究表明丝裂原活化蛋白激酶(mitogenactivated protein kinase,MAPK) 信号传递途径在肝脏先天抗病毒防御系统、急性期反应、肝脏损伤与修复中均起 着重要作用。Spred(Sprouty相关的蛋白激酶异构体一1(EVH_1)domain)蛋白家 族是一组与Sprouty相关的酪氨酸激酶结
4、合蛋白,是重要的负调控因子,能够抑 制生长因子诱导的胞外信号调节激酶(extracellular signa卜regulated kinase, ERK)的活化从而下调RasERK一姒PK信号通路。鉴于MAPK通路在肝脏生物学中的 作用,由此推断Spred可能在T细胞介导的肝脏损伤中发挥重要作用。通过 Northern杂交检测小鼠各组织中的mRNA,发现Spred-2在全身各处均有表达, Spred一1仅分布于心、脑、肾、结肠等,而Spred一3特异地表达于脑组织中。课题组之前利用DNA同源重组技术和胚胎干细胞(ES细胞)发育全能性的原理,定点改变小鼠基因组的序列和结构,获得特定基因(基因组)
5、即Spred-2被修饰的 遗传性小鼠突变模型。因此本课题将应用基因敲除技术构建的动物模型对Spred-2 在T细胞介导的肝脏损伤的调控机制进行深入研究,为肝炎的治疗提供新的思路 和启发。目的:探讨Spred-2在ConA诱导的小鼠肝炎中的保护作用。 方法:雌性68周C57BL6J(wT)小鼠和Spred一2基因敲除(Spred一2 KO)小鼠,单次静脉注射ConA(15 mgkg体重)后,6h、12h、24h麻醉后处死。血 浆液氮冻存,肝脏生理盐水冲洗后,一部分液氮冻存,一部分10中性甲醛固定, 另一部分025胶原酶消化后分离肝脏白细胞。生化法测定血清谷丙转氨酶浓度; 试剂盒比色法测定casp
6、ase一3,8,9含量,ELISA法测定血清IFN Y浓度;应用流 式细胞仪测定肝脏T细胞和NKT细胞数目;Real time PCR测定CXCL9IO,CXCR3 受体,Perforin,Fas,TNFQ,IL-17 mRNA水平。结果:ConA注射12小时后,Spred一2 K0小鼠肝脏损伤较wT小鼠严重,血清 谷丙转氨酶(747620138754 IUL vs37526380336 IUL,P005)浓度升高;Caspase-3,-8,-9相对值为(63781190 YS1735448,P005; 4821762 VS889192,P005;4807739 YS823236,P005)活
7、性增加;血清IFN Y(189018 ngml VS081022 ngm1,P005)浓 度升高,肝脏IFN Y相对值(257049 VS140025,P005)水平提高: 流式细胞术发现,Spred-2 KO小鼠肝脏CDS+T细胞数量显著增加(841213 105ml VS515118 105m1,P005),CD4+T细胞和NKT细胞数量与WT小 鼠无显著差异;CXCL9IO,CXCR3受体,Perforin,Fas表达水平提高。结论:1、肝细胞损伤程度在具有Spred-2基因的野生型小鼠中较轻,肝细胞 凋亡较少,提示Spred-2基因可能发挥肝细胞保护作用。2、在小鼠急性肝炎过程中,包含
8、Spred-2基因的野生型小鼠可能通过抑制Thl 细胞的反应,降低炎症反应和趋化因子的水平来发挥肝细胞保护作用的。3、Spred一2基因在ConA诱导的小鼠肝炎中的起到一定的保护作用,可能成为 T细胞诱导肝脏损伤新的治疗靶点。关键词:Spred一2刀豆蛋白A肝炎CD8+T细胞2Protection of Spred2 in the ConA-induced Liver Inj uryMaster degree candidate:Cao Chen Supervisor:Professor Guan Hongwei Major:Pathology and Pathological physiol
9、ogyAbstractBackground:T cells play central roles in the pathogenesis of liver diseases, including viral hepatitis,autoimmune hepatitis,alcoholic liver disease and fatty liver diseaseAlthough T cell response is crucial in the liver pathology,the underlying regulatory mechanism by cytokine signaling r
10、emains unclearThere are two different processes for acute hepatitis B virus(HBV)clearance and liver cell damage,the clearance of HBV is completed before the liver cell injury,and the lack of specific CTL activity could not make HBV replication under control,it could cause a large number of nonspecif
11、ic inflammatory cells,particularly non-specific CD8+T cells chemotactic cytokines under the action of a large invasion,which cause extensive damage in liver tissueTo address this,studies嘶tll animal models of hepatitis ale necessary Concanavalin A(ConA)induced hepatitis is a wellestablished animal mo
12、del of hepatitis and its histological features resemble those of viral-,autoimmune-ordruginduced hepatitis in humansConAinduced hepatitis,mainly through the rapidactivation of polyclonal T cells wele mainly CD4+T cells,CD8+T cells and NKT cells to realize their own immune injuryCharacterized by the
13、rapid serum alanine aminotransferase levels increased and serum contains large amounts of cytokines,leukocyte infiltration in the liver,liver cell necrosis and apoptosisIn that ConA induced by the excessive activation of the immune system caused by acute liver injury model, As hepatic T cells are ra
14、pidly activated following ConA injection and mice lacking T cells were resistant to the hepatotoxicityActivation of the cells is dependent on Thl cytokine IFM and its signaling pathwayEvidence indicates that MAP kinase cascade plays critical roles in the liver innatesystem during antiviral defense,a
15、cute phase response,hepatic injury and regeneration Suppressor of cytokine signaling(Spred)proteins are intracellular inhibitors of cytokine signaling pathways,mainly MAP kinase cascadeSpredl Spred2 and Spred-3 could inhibit growth factor induced-ERK activated and downregulate Ras-ERK single,3Considering the involvement of MAPK cascade in the liver biology,it is reasonable to speculate that Spred proteins may play a role in T-cell-mediated liver injuryIn the present study,we have focused on Spred-2 in T cells and investigated the regulatory mechanism behi
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