双酚A对大鼠睾丸紧密连接蛋白Occludin的体外影响
发布时间：2019-05-24 08:08
【摘要】：目的观察双酚A (BPA)对体外大鼠睾丸支持细胞紧密连接occludin蛋白表达的影响,探讨BPA对精子发生的损伤机制。 方法原代培养Wistar大鼠睾丸支持细胞(Sertoli细胞)4～5天,双室培养模型的基础上建立体外紧密连接(Tight Junction, TJ)渗透屏障。含有系列浓度(10-3,10-2,10-1,100,101,102,103μmol/L) BPA的无血清培养基(DMEM)孵育Sertoli细胞24h,采用CCK8法检测BPA对Sertoli细胞增殖活性的影响。根据CCK8结果用SPSS17.0软件计算BPA半数致死量(LD50)为150μmol/L,选定梯度浓度的100μmol/L,50μmol/L,25μmol/L BPA作为后续实验组。以0.1%二甲基亚砜(DMSO)为对照组(BPA浓度为0μmol/L),分别以0μmol/L,25μmol/L,50μmol/L,100μmol/L BPA处理Sertoli细胞24h,细胞免疫荧光染色观察occludin阳性率和表达强度；Western blot半定量检测紧密连接蛋白occludin的表达变化。 结果成功分离并培养Wistar大鼠Sertoli细胞,建立了良好的体外TJ屏障模型。CCK8结果显示,不同剂量的BPA染毒24h后,Sertoli细胞的吸光度(OD值)随染毒剂量的增加呈下降趋势。与对照组(0μmol/L BPA)相比,102μmol/L、103μmol/L组的OD值明显降低(P0.05),10-2μmol/L、10-1μmol/L、100μmol/L和101μmol/L组的OD值确无明显变化(P0.05)。细胞免疫荧光染色显示,各染毒组均有occludin表达。随着BPA染毒剂量的增加,Occludin荧光表达强度逐渐降低。25μmol/L及50μmol/L组occludin表达阳性率和表达强度均明显降低(P0.05),未见有强阳性表达；100μmol/L组只有极小部分呈弱阳性表达。Western blot结果显示,Occludin在各剂量组中均有表达,其表达随BPA染毒剂量的增加呈下降趋势。与对照组相比,-25μmol/L、50μmol/L及100μmol/L组的occludin表达明显降低(P0.05)；与25μmol/L组相比,50μmol/L及100μmol/L组的occludin表达亦明显降低(P0.05)。 结论双酚A可削减Sertoli细胞occludin的正常表达,由此可能损伤紧密连接的渗透性屏障,从而影响正常的精子发生过程。
[Abstract]:Objective to observe the effect of bisphenol A (BPA) on the expression of tight junction occludin protein in rat testicular Sertoli cells in vitro and to explore the mechanism of spermatogenesis induced by BPA. Methods Wistar rat testicular Sertoli cells (Sertoli cells) were cultured for 4 days for 5 days. The tight (Tight Junction, TJ) osmotic barrier was established on the basis of two-compartment culture model. Sertoli cells were incubated in serum-free medium (DMEM) containing a series of concentrations (10: 3, 10: 2, 10-1100101102103 渭 mol / L) BPA) for 24 h. The effect of BPA on the proliferation of Sertoli cells was detected by CCK8 assay. According to the results of CCK8, the BPA half lethal dose (LD50) was calculated to be 150 渭 mol / L by SPSS17.0 software, and the gradient concentration of 100 渭 mol / L, 50 渭 mol / L and 25 渭 mol / L BPA was selected as the follow-up experimental group. Sertoli cells were treated with 0.1% dimethyl sulfoxide (DMSO) as control group (the concentration of BPA was 0 渭 mol / L, 25 渭 mol / L, 50 渭 mol / L, 100 渭 mol / L BPA, respectively). The positive rate and expression intensity of occludin were observed by immunofluorescence staining. The expression of tight junction protein occludin was detected by Western blot semi-quantitatively. Results Wistar rat Sertoli cells were successfully isolated and cultured, and a good TJ barrier model in vitro was established. CCK8 results showed that the absorbance (OD value) of Sertoli cells decreased with the increase of exposure dose after exposure to different doses of BPA for 24 hours. Compared with the control group (0 渭 mol / L), the OD values of 102 渭 mol / L and 103 渭 mol / L groups were significantly lower than those of the control group (P 0.05). There was no significant change in the OD values of 10-1 渭 mol / L, 100 渭 mol / L and 101 渭 mol / L groups (P 0.05). Cellular immunofluorescence staining showed that occludin was expressed in all groups. With the increase of BPA exposure dose, the fluorescence expression intensity of Occludin decreased gradually. The positive rate and expression intensity of occludin expression decreased significantly in 25 渭 mol / L and 50 渭 mol / L groups (P 0.05), but no strong positive expression was found. In 100 渭 mol / L group, only a small number of them were weakly positive. Western blot results showed that Occludin was expressed in all dose groups, and its expression decreased with the increase of BPA dose. Compared with the control group, the expression of occludin in-25 渭 mol / L, 50 渭 mol / L and 100 渭 mol / L groups decreased significantly (P 0.05), and the expression of occludin in 50 渭 mol / L and 100 渭 mol / L groups was also significantly lower than that in 25 渭 mol / L group (P 0.05). Conclusion Bisphenol A can reduce the normal expression of occludin in Sertoli cells, which may damage the tightly connected permeability barrier and affect the normal spermatogenesis.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R114
，
本文编号：2484703
[Abstract]:Objective to observe the effect of bisphenol A (BPA) on the expression of tight junction occludin protein in rat testicular Sertoli cells in vitro and to explore the mechanism of spermatogenesis induced by BPA. Methods Wistar rat testicular Sertoli cells (Sertoli cells) were cultured for 4 days for 5 days. The tight (Tight Junction, TJ) osmotic barrier was established on the basis of two-compartment culture model. Sertoli cells were incubated in serum-free medium (DMEM) containing a series of concentrations (10: 3, 10: 2, 10-1100101102103 渭 mol / L) BPA) for 24 h. The effect of BPA on the proliferation of Sertoli cells was detected by CCK8 assay. According to the results of CCK8, the BPA half lethal dose (LD50) was calculated to be 150 渭 mol / L by SPSS17.0 software, and the gradient concentration of 100 渭 mol / L, 50 渭 mol / L and 25 渭 mol / L BPA was selected as the follow-up experimental group. Sertoli cells were treated with 0.1% dimethyl sulfoxide (DMSO) as control group (the concentration of BPA was 0 渭 mol / L, 25 渭 mol / L, 50 渭 mol / L, 100 渭 mol / L BPA, respectively). The positive rate and expression intensity of occludin were observed by immunofluorescence staining. The expression of tight junction protein occludin was detected by Western blot semi-quantitatively. Results Wistar rat Sertoli cells were successfully isolated and cultured, and a good TJ barrier model in vitro was established. CCK8 results showed that the absorbance (OD value) of Sertoli cells decreased with the increase of exposure dose after exposure to different doses of BPA for 24 hours. Compared with the control group (0 渭 mol / L), the OD values of 102 渭 mol / L and 103 渭 mol / L groups were significantly lower than those of the control group (P 0.05). There was no significant change in the OD values of 10-1 渭 mol / L, 100 渭 mol / L and 101 渭 mol / L groups (P 0.05). Cellular immunofluorescence staining showed that occludin was expressed in all groups. With the increase of BPA exposure dose, the fluorescence expression intensity of Occludin decreased gradually. The positive rate and expression intensity of occludin expression decreased significantly in 25 渭 mol / L and 50 渭 mol / L groups (P 0.05), but no strong positive expression was found. In 100 渭 mol / L group, only a small number of them were weakly positive. Western blot results showed that Occludin was expressed in all dose groups, and its expression decreased with the increase of BPA dose. Compared with the control group, the expression of occludin in-25 渭 mol / L, 50 渭 mol / L and 100 渭 mol / L groups decreased significantly (P 0.05), and the expression of occludin in 50 渭 mol / L and 100 渭 mol / L groups was also significantly lower than that in 25 渭 mol / L group (P 0.05). Conclusion Bisphenol A can reduce the normal expression of occludin in Sertoli cells, which may damage the tightly connected permeability barrier and affect the normal spermatogenesis.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R114
，
本文编号：2484703
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