脂多糖对小鼠视网膜Müller细胞和小胶质细胞共培养体系中炎症因子水平的影响及其机制

Effect of lipopolysaccharide on levels of inflammatory factors in retinal Müller cells and microglia co-culture system of mice and its mechanism

目的:观察脂多糖(LPS)诱导小鼠视网膜单独培养Müller细胞和Müller细胞与小胶质细胞共培养2种体系中的炎症反应,阐明Müller细胞与小胶质细胞的相互作用机制。方法:培养Müller细胞QMMuC-1和小胶质细胞BV2,免疫荧光染色方法观察2种细胞形态表现。实验分为单独培养对照组[QMMuC-1细胞单独培养,采用磷酸盐(PBS)缓冲液处理]、共培养对照组(QMMuC-1细胞和BV2细胞共培养,细胞比例1∶1,采用PBS缓冲液处理)、单独培养实验组(QMMuC-1细胞单独培养,采用10 mg·L^(-1)LPS处理)和共培养实验组(QMMuC-1细胞和BV2细胞共培养,采用10 mg·L^(-1)LPS处理)。采用免疫荧光染色法观察各组细胞中胶质纤维酸性蛋白(GFAP)水平,实时荧光定量PCR(RT-qPCR)法检测各组QMMuC-1细胞中白细胞介素1β(IL-1β)、白细胞介素6(IL-6)和肿瘤坏死因子α(TNF-α)mRNA表达水平。结果:QMMuC-1细胞中神经胶质细胞标志物谷氨酰胺合成酶(GS)和GFAP阳性,BV2细胞中小胶质细胞标志物离子钙接头蛋白分子1(Iba-1)阳性。与单独培养对照组比较,单独培养实验组QMMuC-1细胞中GFAP水平升高1.7倍(P=0.005);与共培养对照组比较,共培养实验组QMMuC-1细胞中GFAP水平升高2倍(P=0.003),细胞形态逐渐变成梭形;与单独培养实验组比较,共培养实验组QMMuC-1细胞中GFAP水平升高1.4倍(P=0.0006),大部分细胞呈梭形。与单独培养对照组比较,单独培养实验组QMMuC-1细胞中IL-1β、IL-6和TNF-αmRNA表达水平升高,但差异无统计学意义(P>0.05);与共培养对照组比较,共培养实验组QMMuC-1细胞中IL-1β、IL-6和TNF-αmRNA表达水平升高(P<0.05);与单独培养实验组比较,共培养实验组QMMuC-1细胞中IL-1β和TNF-αmRNA表达水平升高(P<0.05)。结论:LPS可能通过诱导小胶质细胞激活后释放炎症因子作用于Müller细胞,并加剧了Müller细胞的炎症反应。

Objective:To observe the changes of inflammation responses induced by lipopolysaccharide(LPS)in the single cultured Müller cells system and co-culture system of Müller cells and microglia of the mice,and to elucidate the interaction between the Müller cells and the microglia.Methods:The Müller cells QMMuC-1 and microglia BV2 were cultured,and immunofluorescence staining was used to observe the morphology of two kinds of cells.The experiment was divided into single-culture control group[QMMuC-1 cells cultured alone,treated with phosphate buffer saline(PBS)],co-culture control group(QMMuC-1 cells and BV2 cells co-cultured with the ratio of 1∶1,treated with PBS buffer),single-culture experimental group(QMMuC-1 cells cultured alone,treated with 10 mg·L^(-1) LPS),and co-culture experimental group(QMMuC-1 cells and BV2 cells co-cultured,treated with 10 mg·L^(-1) LPS).Immunofluorescence staining was used to observe the levels of glial fibrillary acidic protein(GFAP)in the cells in various groups;real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the expression levels of interleukin-1β(IL-1β),interleukin-6(IL-6),and tumor necrosis factor-α(TNF-α)mRNA in the QMMuC-1 cells in various groups.Results:The expressions of glutamine synthetase(GS)and GFAP in the QMMuC-1 cells were positive,and the expression of ionized calcium-binding adapter molecule 1(Iba-1)in the BV2 cells was positive.Compared with single-culture control group,the level of GFAP in the QMMuC-1 cells in single-culture experimental group was increased by 1.7 fold(P=0.005).Compared with co-culture control group,the level of GFAP in the QMMuC-1 cells in co-culture experimental group was increased by 2 fold(P=0.003),and the morphology of the cells gradually became fusiform.Compared with single-culture experimental group,the level of GFAP in the QMMuC-1 cells in co-culture experimental group was increased by 1.4 fold(P=0.0006),and most cells exhibited a fusiform shape.Compared with single-culture control group,the expression levels of IL-1β,IL-6,and TNF-αmRNA in the QMMuC-1 cells in single-culture experimental group were increased,but the expression levels had no significant differences(P>0.05).Compared with co-culture control group,the expression levels of IL-1β,IL-6,and TNF-αmRNA in the QMMuC-1 cells in co-culture experimental group were increased(P<0.05).Compared with single-culture experimental group,the expression levels of IL-1βand TNF-αmRNA in the QMMuC-1 cells in co-culture experimental group were increased(P<0.05).Conclusion:LPS may induce the release of inflammatory cytokines from the activated microglia,which subsequently act on the Müller cells and exacerbate the inflammatory response in the Müller cells.