【摘要】：目的： 通過觀察不同PAN濃度下，不同劑量的雷帕霉素對足細(xì)胞Nephrin及p-P70S6K表達的影響，探討其對PAN損傷足細(xì)胞的保護作用及可能的機制。 方法： （1）將同步化的足細(xì)胞分成4組：對照組及20、50、100μg/mlPAN組，對照組加入無血清RPMI1640培養(yǎng)基培養(yǎng)，其余三組分別加入終濃度為20、50、100μg/ml的PAN的無血清RPMI1640培養(yǎng)基培養(yǎng)。24h后收集細(xì)胞，Western blot法檢測細(xì)胞Nephrin蛋白相對表達量。 （2）將同步化的足細(xì)胞分成4組：模型對照組及10、100、1000ng/ml雷帕霉素組。所有細(xì)胞加入終濃度為50μg/ml的PAN的無血清RPMI1640培養(yǎng)液，培養(yǎng)24小時后棄去PAN培養(yǎng)液，模型對照組加入等量雷帕霉素溶解液，其余三組分別加入終濃度為10、100、1000ng/ml的雷帕霉素培養(yǎng)，24h及48h后， Western blot法檢測細(xì)胞Nephrin蛋白相對表達量。 （3）將同步化的足細(xì)胞分成3組：1)正常對照組：無血清的RPMI-1640培養(yǎng)液培養(yǎng)；2)模型對照組：終濃度為50μg/ml的PAN+無血清的RPMI-1640培養(yǎng)液；3)雷帕霉素組：終濃度為100ng/ml的雷帕霉素+終濃度為50μg/m的PAN+無血清的RPMI-1640培養(yǎng)液。3組細(xì)胞分別培養(yǎng)24h及48h，收集細(xì)胞，，觀察細(xì)胞形態(tài)、Western blot檢測p-P70S6K蛋白相對表達量。 結(jié)果： （1）PAN50、100μg/ml組與空白對照組相比，nephrin蛋白表達顯著減少(P0.05)；PAN50μg/ml與PAN100μg/ml組相比，nephrin蛋白表達無統(tǒng)計學(xué)差異，與20μg/ml組相比，nephrin蛋白表達顯著減少。（2）雷帕霉素100ng/ml組與10、1000ng/ml組相比，nephrin蛋白表達明顯增加(P0.05)。（3）與正常對照組、PAN模型組相比，雷帕霉素（100ng/ml）組作用PAN損傷模型24h后nephrin及p-P70S6K蛋白表達無明顯變化；作用48h后，雷帕霉素（100ng/ml）組與PAN模型組相比，nephrin蛋白表達顯著增加，p-P70S6K蛋白表達較對照組、PAN模型組顯著減少。 結(jié)論： PAN50μg/ml的劑量即為體外PAN損傷足細(xì)胞模型的最佳濃度。100ng/ml的雷帕霉素對PAN導(dǎo)致的足細(xì)胞損傷有很好的保護作用。雷帕霉素的這一保護作用可能通過抑制mTOR實現(xiàn)的，但其具體機制仍需進一步研究。
[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.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R692

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