小胶质细胞介导青光眼视神经保护性自身免疫机制的研究

发布时间：2018-04-03 05:34

  本文选题：多聚物-1(Cop-1)　切入点：Th1细胞　出处：《复旦大学》2011年硕士论文

【摘要】：青光眼致盲后视力不可逆,严重影响了人类生活和生产。目前青光眼治疗方法主要以药物或手术手段控制眼压,辅以视神经保护治疗。部分患者虽然成功地控制了眼压,但视功能损害仍继续发展。 近年来的研究表明视神经损伤后发生的自身免疫反应作为双刃剑发挥作用。一旦视神经系统受损害,针对损伤局部的自身免疫即刻启动,自身免疫反应过强或过弱会进一步加重视神经的损伤,只有适度的自身免疫反应才会对抗损伤因素、发挥神经保护作用。 这种保护性自身免疫是机体针对神经损伤的内在抗损伤力量,其作用比较综合,接近生理状态,副作用少,是神经保护比较理想的方法。近年来有研究发现在脑、脊髓和视神经损伤中,髓磷脂碱性蛋白(myelin basic protein,MBP)诱导的自身免疫能促进神经组织形态和功能的恢复即有保护性自身免疫的作用,但MBP同时不可避免地诱发自身免疫性疾病。 多聚物-1(Copolymer-1, Cop-1)是髓磷脂碱性蛋白(myelinbasicprotein, MBP)的人工合成拟似物,研究发现Cop-1对于损伤的中枢神经系统,可以通过自身免疫反应发挥一定的神经保护作用,且没有致自身免疫性疾病的副作用。 本研究通过用Cop-1诱导自身免疫,使高眼压大鼠体内产生Cop-1反应性T细胞,探讨了视网膜上Thl/Th2细胞形态及表达的变化；分离出Cop-1反应性T细胞,体外实验观察Cop-1反应性T细胞与小胶质细胞相互作用后对培养的视网膜神经节细胞的作用,并进一步检测几种细胞因子的变化,探讨了Cop-1对视网膜神经节细胞的保护机制。 第一部分 多聚物-1免疫高眼压大鼠视网膜中Th1与Th2细胞表达的变化 目的：探讨T细胞亚型Thl与Th2在多聚物-1(Copolymer-1, Cop-1)免疫的高眼压大鼠视网膜中表达的变化,推测不同T细胞亚型在Cop-1对高眼压大鼠视网膜神经节细胞的保护中可能发挥的作用。 方法：随机对照实验设计方法。结扎3根巩膜上静脉,建立高眼压模型,取96只高眼压大鼠,随机分为Cop-1免疫组48只,PBS免疫组48只,另取10只正常大鼠作对照。后肢足垫注射Cop-1或PBS与完全性弗氏佐剂混合液0.4m1,于免疫后3d、7d、10d、17d、24d、31d,用免疫组化染色法观察视网膜冰冻切片中Thl、Th2细胞数量的变化,根据免疫组化结果用免疫蛋白印迹法检测不同组别大鼠视网膜中IL-4蛋白是否有相应的变化。统计采用成组资料t检验或近似t检验。 结果：免疫组化染色结果显示,免疫后3d,Cop-1组视网膜上Thl数量,PBS组视网膜上Thl数量,与正常大鼠无明显差异,7d时,Cop-1组与PBS组视网膜上Thl数量增多达高峰,分别为216±21/mm2与194±27/mm2,此后两组的Thl数量随时间延长而减少,Cop-1组和PBS组视网膜上Thl数量在各个时间点均无统计学差异；免疫后3d,Cop-1组、PBS组视网膜上Th2数量,较正常大鼠明显增多,但两组间无统计学差异,7d时Cop-1组视网膜上Th2数量达高峰为300±28/mm2,远大于PBS组为129±27/mm2(P0.01),此后Cop-1组的Th2数量随时间延长而减少,但仍超过PBS组的Th2数量。免疫蛋白印迹结果表明,免疫后3d,Cop-1组视网膜上IL-4相对蛋白含量已明显高于PBS组,7d时,Cop-1组视网膜上IL-4相对蛋白量达到高峰为2.11±0.06,高于PBS组的0.57±±0.05,此后,Cop-1组视网膜上IL-4蛋白含量逐渐减少,但每个时间点均高于PBS组,且差别有统计学意义(均p0.05),与免疫组化结果相符。 结论：Cop-1可以引起高眼压大鼠视网膜中Th2细胞的聚集,提示Cop-1对视网膜神经节细胞的保护可能是通过特异性Th2细胞发挥效应。 第二部分多聚物-1反应性T细胞与小胶质细胞相互作用对视网膜神经节细胞的保护 目的：将多聚物-1反应性T细胞(TCop-1)与体外纯化培养的小胶质细胞共培养,将共培养上清液作用于体外培养的视网膜神经节细胞(RGCs, Retinal ganglion cells,),观察RGCs凋亡变化。 方法：体外纯化培养的T细胞分别置于含15μg/mL Cop-1的刺激性培养液,与不含Cop-1的增殖性培养液中,反复刺激扩增,备用；出生2-3dWistar大鼠,取视网膜,培养小胶质细胞；成年Wistar大鼠100-150g,取视网膜,体外纯化培养RGCs.分别将T细胞与小胶质细胞共培养组、Tcop-1与小胶质细胞共培养组、小胶质细胞组,Tcop-1细胞组上清液分别加入体外纯化培养的RGCs细胞中,72h后用Tunel法检测各组RGCs的凋亡率,RT-PCR法检测RGCs中凋亡蛋白Caspase-3, Caspase-8的mRNA的变化。结果采用单因素方差分析和Boffiironni法进行统计分析。 结果：Tcop-1与小胶质细胞共培养48h后上清液加入体外培养72h的RGCs中,TUNEL法测得Tcop-1与小胶质细胞共培养48h的上清液可使RGCs细胞凋亡数(25.36%)较TCop-1组(31.03%),小胶质细胞组(61.54%)明显减少；RT-PCR结果显示Tcop-1与小胶质细胞共培养组RGCs细胞凋亡蛋白Caspase-3、Caspase-8的mRNA的表达(6.22×10-3、1.26×10-5)较小胶质细胞组(6.86×10-3、1.53×10-5)(P=0.136,P=0.001)与T细胞与小胶质细胞共培养组(9.04×10-、3.12×10-5)(P=0.04,P0.001)均有所下调。 结论：Tcop-1与小胶质细胞相互作用后,共培养的上清液可使体外培养的RGCs凋亡延缓,凋亡蛋白Caspase-3、Caspase-8的mRNA表达减少,证实了Tcop-1与小胶质细胞作用后对RGCs细胞具有保护作用的。 第三部分 多聚物-1反应性T细胞与小胶质细胞对视网膜神经节细胞的保护机制 目的：检测多聚物-1反应性T细胞与体外纯化培养的小胶质细胞共培养上清液中细胞因子的变化,探讨Cop-1反应性T细胞与小胶质细胞的相互作用产生效应的可能机制。 方法：体外纯化培养的T细胞分别置于含15μg/mL的Cop-1的刺激性培养液,与不含Cop-1的增殖性培养液中,反复刺激扩增,备用。出生2-3d Wistar大鼠,取视网膜,培养小胶质细胞。T细胞与小胶质细胞共培养组、Tcop-1与小胶质细胞共培养组、小胶质细胞组、Tcop-1组、T细胞组培养12h、24h、48h后,离心取上清液,酶联免疫吸附试验(Elisa法)检测上清液中的IGF-1、BDNF、TNF-α、IL-10分泌量的变化。结果采用单因素方差分析和Boffironni法进行统计分析。 结果：Tcop-1与小胶质细胞共培养12h后,上清液中IGF-1、BDNF、TNF-α、IL-10分泌量(281.66 pg/ml、437.52 pg/ml、50.16 pg/ml、67.12 pg/ml)开始增多,较单纯T细胞(171.50 pg/ml、198.11 pg/ml、18.25 pg/ml、18.09 pg/ml)与小胶质细胞培养组(219.58 pg/ml、31.14 pg/ml、15.03 pg/ml、10.87 pg/ml)均升高；24h后,Tcop-1与小胶质细胞共培养组上清液中的BDNF含量最高(495.52 pg/ml),较对照组均有统计学意义；48h后,Tcop-1与小胶质细胞共培养组上清液中IGF-1, IL-10的含量最高(1005.574 pg/ml、184.88 pg/ml),较单纯Tcop-1组(343.54pg/ml、57.04 pg/ml),单纯小胶质细胞组(233.45 pg/ml、16.11 pg/ml),单纯TPBS与小胶质细胞共培养组(61.90 pg/ml、51.99±3.96pg/ml)均有统计学差异。 结论：检测到Tcop-1与小胶质细胞后局部微环境中的细胞因子含量发生了变化,揭示了Tcop-1与小胶质细胞相互作用后对RGCs保护作用的可能机制为,神经营养因子的分泌的增多,以及一些抗炎因子及促炎因子对免疫反应的调控。
[Abstract]:The blindness of vision after blindness in glaucoma affects human life and production seriously . At present , glaucoma treatment method controls intraocular pressure mainly by means of medicine or surgery , and is treated with optic nerve protection . Some patients have successfully controlled intraocular pressure , but the impairment of visual function continues to develop .

In recent years , the self - immune response after optic nerve injury has been shown to play a role in the double - edged sword .

In recent years , it has been found that the autoimmunity induced by myelin basic protein ( MBP ) in brain , spinal cord and optic nerve injury can promote the restoration of nerve tissue morphology and function , that is , protective self - immunity , but MBP inevitably induces autoimmune diseases .

Polymer - 1 ( cop - 1 , cop - 1 ) is a synthetic peptidomimetic of myelin basic protein ( MBP ) , and it is found that cop - 1 plays a role in protecting the central nervous system of the injured central nervous system and has no side effect on autoimmune diseases .

In this study , we studied the changes of Thl / Th2 cell morphology and expression in high intraocular pressure rats by inducing autoimmunity with Copco - 1 .
The effect of cop - 1 reactive T cells and microglial cells on cultured retinal ganglion cells was observed in vitro and the changes of several cytokines were detected in vitro , and the protective mechanism of cop - 1 on retinal ganglion cells was discussed .

the first portion

Changes of Th1 and Th2 in the retina of rats with high intraocular pressure

Objective : To investigate the changes of T cell subsets Thl and Th2 in the retina of high intraocular pressure rats immunized with poly - 1 ( cop - 1 , cop - 1 ) , and to speculate that different T cell subtypes play a role in the protection of retinal ganglion cells in rats with high intraocular pressure .

Methods : Thirty - six rats with high intraocular pressure were randomly divided into two groups : one immune group , 48 rats and 10 normal rats . The changes of Thl and Th2 cells in the retina were observed by immunohistochemical staining .

Results : The number of Thl in the retina and the number of Thl on the retina of PBS group increased to peak at 7 d , 216 卤 21 / mm2 and 194 卤 27 / mm2 , respectively .
Compared with PBS group , the amount of IL - 4 protein in the retina was significantly higher than that in PBS group ( P < 0.01 ) , but the amount of IL - 4 protein in the retina was significantly higher than that in PBS group ( P < 0.05 ) .

Conclusion : cop - 1 can induce the aggregation of Th2 cells in the retina of rats with high intraocular pressure , and suggests that the protective effect of cop - 1 on retinal ganglion cells may be mediated by specific Th2 cells .

The protection of retinal ganglion cells with the interaction between the second part of multipolymer - 1 reactive T cells and microglial cells

Objective : To co - culture multipolymer - 1 reactive T cell ( Tcop - 1 ) with cultured microglial cells in vitro , and to observe the changes of RGCs apoptosis in cultured retinal ganglion cells ( RGCs ) and cultured retinal ganglion cells ( RGCs ) .

Methods : The cultured T cells were cultured in vitro and cultured in a culture medium containing 15 渭g / mL cop - 1 , and the cultured T cells were repeatedly stimulated and amplified in a culture broth containing 15 渭g / mL , respectively .
2 - 3dwistar rats were born , retina was taken and microglial cells were cultured ;
RGCs were isolated from adult Wistar rats , and RGCs were cultured in vitro and cultured in vitro . The apoptosis rate of RGCs was detected by Tunel method after 72 h . The expression of Caspase - 3 , Caspase - 8 mRNA in RGCs was detected by RT - PCR .

Results : After cultured for 48 hours , the supernatant of Tcop - 1 and microglial cells was cultured in vitro for 72 h , and the supernatant of 48h supernatant of Tcop - 1 and microglial cells was detected by TUNEL . The number of apoptotic cells ( 25.36 % ) was lower than that in Tcop - 1 group ( 31.03 % ) , and the small glia group ( 61.54 % ) was significantly decreased .
The expression of Caspase - 3 , Caspase - 8 mRNA ( 6.22 脳 10 - 3 , 1.26 脳 10 - 5 ) ( P = 0.136 , P = 0.001 ) and T - cell and microglia co - culture group ( 9.04 脳 10 - , 3.12 脳 10 - 5 ) ( P = 0.04 , P0.001 ) decreased .

Conclusion : After the interaction of Tcop - 1 with microglial cells , the co - cultured supernatant can delay the apoptosis of RGCs in vitro , decrease the mRNA expression of Caspase - 3 and Caspase - 8 , and prove that Tcop - 1 has protective effect on RGCs cells after the action of microglial cells .

PART III

Protective mechanism of multipolymer - 1 reactive T cells and microglial cells on retinal ganglion cells

Objective : To investigate the changes of cytokines in the culture supernatant of multipolymer - 1 reactive T cells and cultured microglial cells in vitro , and to explore the possible mechanism of the interaction between the reactive T cells and microglial cells .

Methods : The cultured T cells were cultured in vitro and cultured for 12 h , 24 h and 48 h . The results were analyzed by single factor variance analysis and Boffironni method .

缁撴灉锛歍cop-1涓庡皬鑳惰川缁嗚優鍏卞煿鍏，

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