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目的探讨PFOA对Sertoli细胞紧密连接的影响及分子机制。方法体外分离纯化Sertoli细胞,通过细胞毒性实验,观察PFOA对原代Sertoli细胞生长影响;利用流式细胞术anexin-Ⅴ/PI双染法检测PFOA对Sertoli细胞凋亡的影响;利用双室培养模型,监测跨上皮电阻抗(TER),观察PFOA对紧密连接功能的影响;采用Western blot法检测紧密连接相关蛋白及ERK的表达。结果 0~100μmol/L剂量的PFOA对细胞生长无明显影响(P>0.05);与对照组比,50μmol/L的PFOA未见有明显的促凋亡作用[凋亡率为(9.7%±3.2)%],P>0.05;但可明显降低Sertoli细胞的TER值[(37.35±5.2)Ωcm2],联合ERK特异性抑制剂PD98059处理后TER值升高[(49.85±6.4)Ωcm2],(P<0.05);50μmol/L的PFOA可下调紧密连接蛋白claudin-11、ZO-1和occludin的表达,并上调pERK的表达(P<0.05),且可被PD98059明显抑制(P<0.05)。结论 PFOA可能通过ERK MAPK途径损伤Sertoli细胞间的紧密连接。
Objective To investigate the effect of PFOA on Sertoli cell adhesion and its molecular mechanism. Methods Sertoli cells were isolated and purified in vitro. The effects of PFOA on the growth of primary Sertoli cells were observed by cytotoxicity assay. The effect of PFOA on the apoptosis of Sertoli cells was detected by flow cytometry. , The trans-epithelial electrical impedance (TER) was monitored to observe the effect of PFOA on the tight junctions. Western blot was used to detect the expression of tight junction related protein and ERK. Results PFOA at 0-100 μmol / L had no significant effect on the cell growth (P> 0.05). Compared with the control group, 50 μmol / L PFOA showed no obvious proapoptotic effect (apoptosis rate was 9.7% ± 3.2 (P <0.05). However, the TER value in Sertoli cells [(37.35 ± 5.2) Ωcm2] was significantly decreased, and the TER value increased in combination with PD98059 [(49.85 ± 6.4) Ωcm2] <0.05). 50μmol / L PFOA downregulated the expressions of claudin-11, ZO-1 and occludin, and up-regulated the expression of pERK (P <0.05), and was significantly inhibited by PD98059 (P <0.05). Conclusion PFOA may damage the tight junctions between Sertoli cells through the ERK MAPK pathway.