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目的研究人原代Müller细胞来源的外泌体(EXO)对脂多糖(LPS)刺激人视网膜色素上皮(RPE)细胞产生炎症因子的影响。方法原代纯化培养人Müller和RPE细胞,通过免疫荧光鉴定RPE细胞,用试剂盒提取得到Müller细胞来源的EXO并通过透射电镜观察和Western印迹法进行鉴定。在无EXO培养基培养人RPE细胞后,分别采用100 ng/mL LPS刺激,不加或加入EXO(50μg/mL),以不含EXO和LPS培养细胞为对照组,分别通过酶联免疫吸附试验(ELISA)测定和实时反转录聚合酶链反应(RT-PCR)研究RPE细胞产生炎症因子的变化。结果人Müller细胞来源的提取物经透射电镜观察为圆形或半圆形囊泡,直径为30~100 nm,表达EXO特有标志物CD63和Flotillin-1。LPS组上清液中白介素1β(IL-1β)、IL-6、IL-8和肿瘤坏死因子α(TNF-α)的浓度分别为(333.89±41.51)、(404.67±13.88)、(1 557.11±317.08)、(466.78±39.34)μg/mL,EXO+LPS组分别为(214.11±18.14)、(318.12±34.73)、(1 170.37±244.15)、(319.73±41.39)μg/mL,2组比较差异有统计学意义(IL-1β:P<0.01;IL-6、IL-8:P<0.05;TNF-α:P<0.001)。与LPS组相比,EXO+LPS组的相关基因表达受到抑制,其中IL-1β基因表达差异有统计学意义(P<0.05),IL-8、CCL20和TNF-α差异有统计学意义(P<0.001),CCL2与IL-6则组间差异无统计学意义(P>0.05)。结论成功获得Müller来源的EXO,其对LPS刺激RPE细胞产生炎症因子具有一定的调节作用。
Objective To study the effect of human primary Müller cell exosomes (EXO) on the production of inflammatory cytokines by lipopolysaccharide (LPS) stimulated human retinal pigment epithelium (RPE) cells. Methods Primary cultured human Müller and RPE cells were cultured and purified. The RPE cells were identified by immunofluorescence. The EXO derived from Müller cells was extracted with a kit and identified by transmission electron microscopy and Western blotting. After culturing human RPE cells without EXO medium, the cells were stimulated with 100 ng / mL LPS, without or with EXO (50 μg / mL), with EXO and LPS cultured cells as control group, respectively by enzyme-linked immunosorbent assay (ELISA) assay and real-time reverse transcription-polymerase chain reaction (RT-PCR) were used to study the changes of inflammatory cytokines in RPE cells. Results Human Müller cells derived from the extracts were observed by transmission electron microscopy round or semi-circular vesicles, 30 ~ 100 nm in diameter, EXO unique markers CD63 expression and Flotillin-1. The concentrations of IL-1β, IL-6, IL-8 and TNF-α in the supernatant of LPS group were (333.89 ± 41.51), (404.67 ± 13.88) and (155.7.11 ± 317.08) and (466.78 ± 39.34) μg / mL, respectively. The levels in EXO + LPS group were (214.11 ± 18.14), (318.12 ± 34.73), (1 170.37 ± 244.15) and (319.73 ± 41.39) μg / The difference was statistically significant (IL-1β: P <0.01; IL-6, IL-8: P <0.05; TNF-α: P <0.001). Compared with LPS group, the expression of IL-1β gene was inhibited in EXO + LPS group (P <0.05), while there was significant difference in IL-8, CCL20 and TNF-α <0.001). There was no significant difference between CCL2 and IL-6 (P> 0.05). CONCLUSIONS: EXO obtained from Müller successfully has some regulatory effects on LPS-stimulated RPE cells to produce inflammatory cytokines.