本文選題：多聚物-1(Cop-1)　切入點：Th1細(xì)胞　出處：《復(fù)旦大學(xué)》2011年碩士論文

【摘要】：青光眼致盲后視力不可逆,嚴(yán)重影響了人類生活和生產(chǎn)。目前青光眼治療方法主要以藥物或手術(shù)手段控制眼壓,輔以視神經(jīng)保護(hù)治療。部分患者雖然成功地控制了眼壓,但視功能損害仍繼續(xù)發(fā)展。 近年來的研究表明視神經(jīng)損傷后發(fā)生的自身免疫反應(yīng)作為雙刃劍發(fā)揮作用。一旦視神經(jīng)系統(tǒng)受損害,針對損傷局部的自身免疫即刻啟動,自身免疫反應(yīng)過強(qiáng)或過弱會進(jìn)一步加重視神經(jīng)的損傷,只有適度的自身免疫反應(yīng)才會對抗損傷因素、發(fā)揮神經(jīng)保護(hù)作用。 這種保護(hù)性自身免疫是機(jī)體針對神經(jīng)損傷的內(nèi)在抗損傷力量,其作用比較綜合,接近生理狀態(tài),副作用少,是神經(jīng)保護(hù)比較理想的方法。近年來有研究發(fā)現(xiàn)在腦、脊髓和視神經(jīng)損傷中,髓磷脂堿性蛋白(myelin basic protein,MBP)誘導(dǎo)的自身免疫能促進(jìn)神經(jīng)組織形態(tài)和功能的恢復(fù)即有保護(hù)性自身免疫的作用,但MBP同時不可避免地誘發(fā)自身免疫性疾病。 多聚物-1(Copolymer-1, Cop-1)是髓磷脂堿性蛋白(myelinbasicprotein, MBP)的人工合成擬似物,研究發(fā)現(xiàn)Cop-1對于損傷的中樞神經(jīng)系統(tǒng),可以通過自身免疫反應(yīng)發(fā)揮一定的神經(jīng)保護(hù)作用,且沒有致自身免疫性疾病的副作用。 本研究通過用Cop-1誘導(dǎo)自身免疫,使高眼壓大鼠體內(nèi)產(chǎn)生Cop-1反應(yīng)性T細(xì)胞,探討了視網(wǎng)膜上Thl/Th2細(xì)胞形態(tài)及表達(dá)的變化；分離出Cop-1反應(yīng)性T細(xì)胞,體外實驗觀察Cop-1反應(yīng)性T細(xì)胞與小膠質(zhì)細(xì)胞相互作用后對培養(yǎng)的視網(wǎng)膜神經(jīng)節(jié)細(xì)胞的作用,并進(jìn)一步檢測幾種細(xì)胞因子的變化,探討了Cop-1對視網(wǎng)膜神經(jīng)節(jié)細(xì)胞的保護(hù)機(jī)制。 第一部分 多聚物-1免疫高眼壓大鼠視網(wǎng)膜中Th1與Th2細(xì)胞表達(dá)的變化 目的：探討T細(xì)胞亞型Thl與Th2在多聚物-1(Copolymer-1, Cop-1)免疫的高眼壓大鼠視網(wǎng)膜中表達(dá)的變化,推測不同T細(xì)胞亞型在Cop-1對高眼壓大鼠視網(wǎng)膜神經(jīng)節(jié)細(xì)胞的保護(hù)中可能發(fā)揮的作用。 方法：隨機(jī)對照實驗設(shè)計方法。結(jié)扎3根鞏膜上靜脈,建立高眼壓模型,取96只高眼壓大鼠,隨機(jī)分為Cop-1免疫組48只,PBS免疫組48只,另取10只正常大鼠作對照。后肢足墊注射Cop-1或PBS與完全性弗氏佐劑混合液0.4m1,于免疫后3d、7d、10d、17d、24d、31d,用免疫組化染色法觀察視網(wǎng)膜冰凍切片中Thl、Th2細(xì)胞數(shù)量的變化,根據(jù)免疫組化結(jié)果用免疫蛋白印跡法檢測不同組別大鼠視網(wǎng)膜中IL-4蛋白是否有相應(yīng)的變化。統(tǒng)計采用成組資料t檢驗或近似t檢驗。 結(jié)果：免疫組化染色結(jié)果顯示,免疫后3d,Cop-1組視網(wǎng)膜上Thl數(shù)量,PBS組視網(wǎng)膜上Thl數(shù)量,與正常大鼠無明顯差異,7d時,Cop-1組與PBS組視網(wǎng)膜上Thl數(shù)量增多達(dá)高峰,分別為216±21/mm2與194±27/mm2,此后兩組的Thl數(shù)量隨時間延長而減少,Cop-1組和PBS組視網(wǎng)膜上Thl數(shù)量在各個時間點均無統(tǒng)計學(xué)差異；免疫后3d,Cop-1組、PBS組視網(wǎng)膜上Th2數(shù)量,較正常大鼠明顯增多,但兩組間無統(tǒng)計學(xué)差異,7d時Cop-1組視網(wǎng)膜上Th2數(shù)量達(dá)高峰為300±28/mm2,遠(yuǎn)大于PBS組為129±27/mm2(P0.01),此后Cop-1組的Th2數(shù)量隨時間延長而減少,但仍超過PBS組的Th2數(shù)量。免疫蛋白印跡結(jié)果表明,免疫后3d,Cop-1組視網(wǎng)膜上IL-4相對蛋白含量已明顯高于PBS組,7d時,Cop-1組視網(wǎng)膜上IL-4相對蛋白量達(dá)到高峰為2.11±0.06,高于PBS組的0.57±±0.05,此后,Cop-1組視網(wǎng)膜上IL-4蛋白含量逐漸減少,但每個時間點均高于PBS組,且差別有統(tǒng)計學(xué)意義(均p0.05),與免疫組化結(jié)果相符。 結(jié)論：Cop-1可以引起高眼壓大鼠視網(wǎng)膜中Th2細(xì)胞的聚集,提示Cop-1對視網(wǎng)膜神經(jīng)節(jié)細(xì)胞的保護(hù)可能是通過特異性Th2細(xì)胞發(fā)揮效應(yīng)。 第二部分多聚物-1反應(yīng)性T細(xì)胞與小膠質(zhì)細(xì)胞相互作用對視網(wǎng)膜神經(jīng)節(jié)細(xì)胞的保護(hù) 目的：將多聚物-1反應(yīng)性T細(xì)胞(TCop-1)與體外純化培養(yǎng)的小膠質(zhì)細(xì)胞共培養(yǎng),將共培養(yǎng)上清液作用于體外培養(yǎng)的視網(wǎng)膜神經(jīng)節(jié)細(xì)胞(RGCs, Retinal ganglion cells,),觀察RGCs凋亡變化。 方法：體外純化培養(yǎng)的T細(xì)胞分別置于含15μg/mL Cop-1的刺激性培養(yǎng)液,與不含Cop-1的增殖性培養(yǎng)液中,反復(fù)刺激擴(kuò)增,備用；出生2-3dWistar大鼠,取視網(wǎng)膜,培養(yǎng)小膠質(zhì)細(xì)胞；成年Wistar大鼠100-150g,取視網(wǎng)膜,體外純化培養(yǎng)RGCs.分別將T細(xì)胞與小膠質(zhì)細(xì)胞共培養(yǎng)組、Tcop-1與小膠質(zhì)細(xì)胞共培養(yǎng)組、小膠質(zhì)細(xì)胞組,Tcop-1細(xì)胞組上清液分別加入體外純化培養(yǎng)的RGCs細(xì)胞中,72h后用Tunel法檢測各組RGCs的凋亡率,RT-PCR法檢測RGCs中凋亡蛋白Caspase-3, Caspase-8的mRNA的變化。結(jié)果采用單因素方差分析和Boffiironni法進(jìn)行統(tǒng)計分析。 結(jié)果：Tcop-1與小膠質(zhì)細(xì)胞共培養(yǎng)48h后上清液加入體外培養(yǎng)72h的RGCs中,TUNEL法測得Tcop-1與小膠質(zhì)細(xì)胞共培養(yǎng)48h的上清液可使RGCs細(xì)胞凋亡數(shù)(25.36%)較TCop-1組(31.03%),小膠質(zhì)細(xì)胞組(61.54%)明顯減少；RT-PCR結(jié)果顯示Tcop-1與小膠質(zhì)細(xì)胞共培養(yǎng)組RGCs細(xì)胞凋亡蛋白Caspase-3、Caspase-8的mRNA的表達(dá)(6.22×10-3、1.26×10-5)較小膠質(zhì)細(xì)胞組(6.86×10-3、1.53×10-5)(P=0.136,P=0.001)與T細(xì)胞與小膠質(zhì)細(xì)胞共培養(yǎng)組(9.04×10-、3.12×10-5)(P=0.04,P0.001)均有所下調(diào)。 結(jié)論：Tcop-1與小膠質(zhì)細(xì)胞相互作用后,共培養(yǎng)的上清液可使體外培養(yǎng)的RGCs凋亡延緩,凋亡蛋白Caspase-3、Caspase-8的mRNA表達(dá)減少,證實了Tcop-1與小膠質(zhì)細(xì)胞作用后對RGCs細(xì)胞具有保護(hù)作用的。 第三部分 多聚物-1反應(yīng)性T細(xì)胞與小膠質(zhì)細(xì)胞對視網(wǎng)膜神經(jīng)節(jié)細(xì)胞的保護(hù)機(jī)制 目的：檢測多聚物-1反應(yīng)性T細(xì)胞與體外純化培養(yǎng)的小膠質(zhì)細(xì)胞共培養(yǎng)上清液中細(xì)胞因子的變化,探討Cop-1反應(yīng)性T細(xì)胞與小膠質(zhì)細(xì)胞的相互作用產(chǎn)生效應(yīng)的可能機(jī)制。 方法：體外純化培養(yǎng)的T細(xì)胞分別置于含15μg/mL的Cop-1的刺激性培養(yǎng)液,與不含Cop-1的增殖性培養(yǎng)液中,反復(fù)刺激擴(kuò)增,備用。出生2-3d Wistar大鼠,取視網(wǎng)膜,培養(yǎng)小膠質(zhì)細(xì)胞。T細(xì)胞與小膠質(zhì)細(xì)胞共培養(yǎng)組、Tcop-1與小膠質(zhì)細(xì)胞共培養(yǎng)組、小膠質(zhì)細(xì)胞組、Tcop-1組、T細(xì)胞組培養(yǎng)12h、24h、48h后,離心取上清液,酶聯(lián)免疫吸附試驗(Elisa法)檢測上清液中的IGF-1、BDNF、TNF-α、IL-10分泌量的變化。結(jié)果采用單因素方差分析和Boffironni法進(jìn)行統(tǒng)計分析。 結(jié)果：Tcop-1與小膠質(zhì)細(xì)胞共培養(yǎng)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)開始增多,較單純T細(xì)胞(171.50 pg/ml、198.11 pg/ml、18.25 pg/ml、18.09 pg/ml)與小膠質(zhì)細(xì)胞培養(yǎng)組(219.58 pg/ml、31.14 pg/ml、15.03 pg/ml、10.87 pg/ml)均升高；24h后,Tcop-1與小膠質(zhì)細(xì)胞共培養(yǎng)組上清液中的BDNF含量最高(495.52 pg/ml),較對照組均有統(tǒng)計學(xué)意義；48h后,Tcop-1與小膠質(zhì)細(xì)胞共培養(yǎng)組上清液中IGF-1, IL-10的含量最高(1005.574 pg/ml、184.88 pg/ml),較單純Tcop-1組(343.54pg/ml、57.04 pg/ml),單純小膠質(zhì)細(xì)胞組(233.45 pg/ml、16.11 pg/ml),單純TPBS與小膠質(zhì)細(xì)胞共培養(yǎng)組(61.90 pg/ml、51.99±3.96pg/ml)均有統(tǒng)計學(xué)差異。 結(jié)論：檢測到Tcop-1與小膠質(zhì)細(xì)胞后局部微環(huán)境中的細(xì)胞因子含量發(fā)生了變化,揭示了Tcop-1與小膠質(zhì)細(xì)胞相互作用后對RGCs保護(hù)作用的可能機(jī)制為,神經(jīng)營養(yǎng)因子的分泌的增多,以及一些抗炎因子及促炎因子對免疫反應(yīng)的調(diào)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 鹵 21 / mm2 and 194 鹵 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 .

緇撴灉錛歍cop-1涓庡皬鑳惰川緇嗚優(yōu)鍏卞煿鍏，

本文編號：1703880