RCS大鼠视网膜色素变性过程中视网膜免疫微环境的改变

Changes of the immune-microenvironment in retinal degeneration process of RCS rat

目的研究在英国皇家外科学院(RCS)大鼠视网膜色素变性(RP)发展过程中,介导细胞免疫的T淋巴细胞和自然杀伤(NK)细胞及其分泌的细胞因子γ干扰素(IFN-γ)的变化。方法选取出生后20日龄(P20)、P40、P60 RCS-rdy--P+大鼠(简称RCS大鼠)和相应日龄RCS-rdy+-P+大鼠(简称对照大鼠)各12只,细胞因子液相悬浮芯片检测大鼠视网膜匀浆中多种炎性细胞因子的质量浓度。实时荧光定量聚合酶链反应(real-time PCR)检测各日龄大鼠视网膜中白细胞介素-2(IL-2)、CC类趋化因子配体2(CCL2)、CXC趋化因子配体9(CXCL9)、CXCL10和IFN-γmRNA的表达水平;免疫组织化学技术检测大鼠视网膜中T淋巴细胞(CD4+、CD8+)和NK细胞(CD161+)的分布;流式细胞术检测大鼠视网膜中T淋巴细胞(CD4+、CD8+)和NK细胞(CD161+)的比例,酶联免疫吸附试验(ELISA)检测大鼠视网膜组织匀浆中IFN-γ的质量浓度。结果RCS组大鼠视网膜中淋巴细胞相关细胞因子和趋化因子mRNA表达水平均表现出随日龄增加而表达上调的趋势,P60 RCS大鼠视网膜中IL-2、CCL2、CXCL9、CXCL10、CXCL11和IFN-γmRNA表达水平均较P20 RCS大鼠和对照组大鼠表达水平明显升高,差异均有统计学意义(均P<0.05)。P60 RCS大鼠视网膜中CD4、CD8、CD161细胞阳性率分别为(9.09±0.89)%、(18.77±0.38)%和(9.41±0.38)%。P60 RCS大鼠视网膜中IFN-γ阳性细胞比例为(8.29±0.27)%,较对照大鼠的(0.28±0.02)%显著升高,差异有统计学意义(t=29.03,P=0.00)。P60 RCS大鼠视网膜中CD4+、CD8+和CD161+淋巴细胞主要分布在内层视网膜,且IFN-γ阳性标记与淋巴细胞表面标记呈共定位。不同日龄RCS大鼠和对照大鼠视网膜中IFN-γ质量浓度比较,差异均有统计学意义(F组别=16.49,P<0.01;F时间=21.05,P<0.01),其中P60 RCS大鼠视网膜中IFN-γ质量浓度较P20 RCS大鼠、P40 RCS大鼠和对照组大鼠明显升高,差异均有统计学意义(均P<0.05)。结论RCS大鼠RP改变了视网膜内相对免疫豁免的微环境,视网膜中淋巴细胞趋化因子和细胞因子表达水平逐渐升高,并在病变后期引起淋巴细胞的浸润和激活,导致IFN-γ在视网膜中浓度显著升高,表明细胞免疫参与其病理机制。

Objective To explore the immune-microenvironment of the retinas at different stages of retinal degeneration in Royal College of Surgeon(RCS)rats.Methods RCS-rdy--P+(RCS)rats at early stage(P20),middle stage(P40)and late stage(P60)were involved,12 rats at each post-natal day,RCS-rdy+-P+rats severed as control.Relative concentrations of rat cytokines in rat retina homogenate were detected by using Bio-Plex Suspension Array System.Relative expressions of interleukin-2(IL-2),C-C motif ligand 2(CCL2),chemokine(C-X-C motif)ligand 9(CXCL9),CXCL10,CXCL11 and interferon-γ(IFN-γ)mRNA in rat retina were analyzed by real-time PCR.Expressions of IFN-γand immune cells surface marker CD4,CD8 and CD161 in the retinas were detected by immunohistochemical staining.Percentage of IFN-γpositive T lymphocytes and natural killer(NK)cells in rat retina were analyzed by flow cytometry.The concentrations of IFN-γin rat retina homogenate were evaluated by enzyme-linked immunosorbent assay(ELISA).The use and care of the animals complied with Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.Results Lymphocytes related cytokines and chemokines mRNA expression levels in the RCS rat retinas showed increase trends with the extension of time.The expression levels of IL-2,CCL2,CXCL9,CXCL10,CXCL11 and IFN-γmRNA in P60 RCS rat retinas were significantly increased than those in the P20 RCS rat retinas and the control rat retinas(all at P<0.05).The positive rates of CD4,CD8 and CD161 cells in the retinas of P60 RCS rats was(9.09±0.89)%,(18.77±0.38)%and(9.41±0.38)%,respectively.The proportion of IFN-γpositive cells in the retinas of P60 RCS rats was(8.29±0.27)%,which was significantly higher than that of the control rats([0.28±0.02]%),with a significant difference between them(t=29.03,P=0.00).CD4+,CD8+and CD161+lymphocytes were mainly distributed in the retinas of P60 RCS rats,and the expressions of IFN-γwere co-located with lymphocyte surface markers.There were significant differences in the concentrations of IFN-γin the retinas of RCS rats and control rats at different day ages(F group=16.49,P<0.01;F time=21.05,P<0.01),the concentration of IFN-γin retinas of P60 RCS rats was significantly higher than that of P20 RCS rats,P40 RCS rats and control rats,and the differences were statistically significant(all at P<0.05).Conclusions Along with the process of retinal degeneration,immune privilege balance in the retinas is disrupted,the expressions of lymphocytes related chemokines and cytokines are elevated.Lymphocytes infiltration and activation are appeared in the retina highly activated at the late stage of RP,leading to the significant up-regulation of inflammatory cytokine IFN-γin microenvironment,which indicates that lymphocytes mediated immune response may take part in retinal degeneration.