雷帕霉素对PAN损伤足细胞的作用及其对p-P70S6K表达的影响
发布时间：2018-07-23 13:16
【摘要】：目的： 通过观察不同PAN浓度下，不同剂量的雷帕霉素对足细胞Nephrin及p-P70S6K表达的影响，探讨其对PAN损伤足细胞的保护作用及可能的机制。 方法： （1）将同步化的足细胞分成4组：对照组及20、50、100μg/mlPAN组，对照组加入无血清RPMI1640培养基培养，其余三组分别加入终浓度为20、50、100μg/ml的PAN的无血清RPMI1640培养基培养。24h后收集细胞，Western blot法检测细胞Nephrin蛋白相对表达量。 （2）将同步化的足细胞分成4组：模型对照组及10、100、1000ng/ml雷帕霉素组。所有细胞加入终浓度为50μg/ml的PAN的无血清RPMI1640培养液，培养24小时后弃去PAN培养液，模型对照组加入等量雷帕霉素溶解液，其余三组分别加入终浓度为10、100、1000ng/ml的雷帕霉素培养，24h及48h后， Western blot法检测细胞Nephrin蛋白相对表达量。 （3）将同步化的足细胞分成3组：1)正常对照组：无血清的RPMI-1640培养液培养；2)模型对照组：终浓度为50μg/ml的PAN+无血清的RPMI-1640培养液；3)雷帕霉素组：终浓度为100ng/ml的雷帕霉素+终浓度为50μg/m的PAN+无血清的RPMI-1640培养液。3组细胞分别培养24h及48h，收集细胞，，观察细胞形态、Western blot检测p-P70S6K蛋白相对表达量。 结果： （1）PAN50、100μg/ml组与空白对照组相比，nephrin蛋白表达显著减少(P0.05)；PAN50μg/ml与PAN100μg/ml组相比，nephrin蛋白表达无统计学差异，与20μg/ml组相比，nephrin蛋白表达显著减少。（2）雷帕霉素100ng/ml组与10、1000ng/ml组相比，nephrin蛋白表达明显增加(P0.05)。（3）与正常对照组、PAN模型组相比，雷帕霉素（100ng/ml）组作用PAN损伤模型24h后nephrin及p-P70S6K蛋白表达无明显变化；作用48h后，雷帕霉素（100ng/ml）组与PAN模型组相比，nephrin蛋白表达显著增加，p-P70S6K蛋白表达较对照组、PAN模型组显著减少。 结论： PAN50μg/ml的剂量即为体外PAN损伤足细胞模型的最佳浓度。100ng/ml的雷帕霉素对PAN导致的足细胞损伤有很好的保护作用。雷帕霉素的这一保护作用可能通过抑制mTOR实现的，但其具体机制仍需进一步研究。
[Abstract]:Aim: to observe the effects of different doses of rapamycin on the expression of Nephrin and p-P70S6K in podocytes at different concentrations of PAN and to explore the protective effect and possible mechanism of rapamycin on podocytes damaged by PAN. Methods: (1) synchronized podocytes were divided into four groups: control group and 2050 渭 g/mlPAN group. The control group was cultured on serum-free RPMI1640 medium. The other three groups were cultured on serum-free RPMI1640 medium containing 20 渭 g/ml PAN at the final concentration of 50100 渭 g/ml for 24 hours. The relative expression of Nephrin protein was detected by Western blot assay. (2) synchronized podocytes were divided into four groups: model control group. And 10g / ml rapamycin group. All the cells were added serum-free RPMI1640 medium of 50 渭 g/ml PAN, and the PAN culture medium was discarded after 24 hours of culture. The model control group was treated with the same amount of rapamycin solution. The other three groups were incubated with rapamycin at the final concentration of 10 ~ 100ng / ml for 24 h and 48 h, respectively. (3) synchronized podocytes were divided into three groups: 1) normal control group: serum-free RPMI-1640 culture medium; 2) the model control group: the final concentration of PAN was 50 渭 g/ml, the serum-free RPMI-1640 medium was 3) rapamycin group: the final concentration of rapamycin was 50 渭 g / m of PAN serum-free RPMI-1640. 3 cells were cultured for 24 h and 48 h, respectively, and the cells were collected from rapamycin group, the final concentration of rapamycin was 50 渭 g / m and the final concentration of rapamycin was 50 渭 g / m. The relative expression of p-P70S6K protein was detected by Western blot. Results: (1) the expression of nephrin protein in the PAN50100 渭 g/ml group was significantly lower than that in the control group (P0.05) there was no significant difference in the expression of nephrin protein between the PAN100 渭 g/ml group and the PAN50 渭 g/ml group. Compared with 20 渭 g/ml group, the expression of nephrin protein in rapamycin 100ng/ml group was significantly lower than that in the control group (P0.05). (3). Compared with the control group, the expression of nephrin and p-P70S6K in rapamycin 100ng/ml group was similar to that in the control group (P0.05). (3), but the expression of nephrin and p-P70S6K protein in rapamycin (100ng/ml) group was not changed after 24 hours of PAN injury. After 48 h treatment, the expression of nephrin protein in 100ng/ml group was significantly increased compared with that in PAN model group, and the expression of p-P70S6K protein was significantly decreased compared with that in control group. Conclusion: the dosage of PAN50 渭 g/ml is the best concentration of rapamycin, 100ng / ml, for podocyte injury induced by PAN in vitro. It has a good protective effect on podocyte injury induced by PAN. This protective effect of rapamycin may be achieved by inhibiting mTOR, but its mechanism needs further study.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R692
本文编号：2139530
[Abstract]:Aim: to observe the effects of different doses of rapamycin on the expression of Nephrin and p-P70S6K in podocytes at different concentrations of PAN and to explore the protective effect and possible mechanism of rapamycin on podocytes damaged by PAN. Methods: (1) synchronized podocytes were divided into four groups: control group and 2050 渭 g/mlPAN group. The control group was cultured on serum-free RPMI1640 medium. The other three groups were cultured on serum-free RPMI1640 medium containing 20 渭 g/ml PAN at the final concentration of 50100 渭 g/ml for 24 hours. The relative expression of Nephrin protein was detected by Western blot assay. (2) synchronized podocytes were divided into four groups: model control group. And 10g / ml rapamycin group. All the cells were added serum-free RPMI1640 medium of 50 渭 g/ml PAN, and the PAN culture medium was discarded after 24 hours of culture. The model control group was treated with the same amount of rapamycin solution. The other three groups were incubated with rapamycin at the final concentration of 10 ~ 100ng / ml for 24 h and 48 h, respectively. (3) synchronized podocytes were divided into three groups: 1) normal control group: serum-free RPMI-1640 culture medium; 2) the model control group: the final concentration of PAN was 50 渭 g/ml, the serum-free RPMI-1640 medium was 3) rapamycin group: the final concentration of rapamycin was 50 渭 g / m of PAN serum-free RPMI-1640. 3 cells were cultured for 24 h and 48 h, respectively, and the cells were collected from rapamycin group, the final concentration of rapamycin was 50 渭 g / m and the final concentration of rapamycin was 50 渭 g / m. The relative expression of p-P70S6K protein was detected by Western blot. Results: (1) the expression of nephrin protein in the PAN50100 渭 g/ml group was significantly lower than that in the control group (P0.05) there was no significant difference in the expression of nephrin protein between the PAN100 渭 g/ml group and the PAN50 渭 g/ml group. Compared with 20 渭 g/ml group, the expression of nephrin protein in rapamycin 100ng/ml group was significantly lower than that in the control group (P0.05). (3). Compared with the control group, the expression of nephrin and p-P70S6K in rapamycin 100ng/ml group was similar to that in the control group (P0.05). (3), but the expression of nephrin and p-P70S6K protein in rapamycin (100ng/ml) group was not changed after 24 hours of PAN injury. After 48 h treatment, the expression of nephrin protein in 100ng/ml group was significantly increased compared with that in PAN model group, and the expression of p-P70S6K protein was significantly decreased compared with that in control group. Conclusion: the dosage of PAN50 渭 g/ml is the best concentration of rapamycin, 100ng / ml, for podocyte injury induced by PAN in vitro. It has a good protective effect on podocyte injury induced by PAN. This protective effect of rapamycin may be achieved by inhibiting mTOR, but its mechanism needs further study.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R692
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相关期刊论文 前2条
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2 王丽华;顾乐怡;梁馨月;严玉澄;校丽芳;高嘉元;张敏芳;戴慧丽;倪兆慧;钱家麒;;雷帕霉素对PAN肾病小鼠肾脏病变和VEGF及受体表达的影响[J];上海交通大学学报(医学版);2010年04期
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