来氟米特对PAN损伤足细胞凋亡的保护作用及机制

发布时间：2018-08-17 19:01

【摘要】：目的： 1、建立PAN损伤足细胞模型，在不同时间点检测PAN损伤足细胞的凋亡率，来氟米特干预后，损伤足细胞凋亡率的变化，证实来氟米特对损伤足细胞有保护作用。 2、在PAN损伤足细胞模型的基础上，检测同一时间点PAN损伤足细胞表达凋亡蛋白p53、抗凋亡蛋白bcl-xl，来氟米特干预后，二者表达情况的变化，，研究来氟米特保护损伤足细胞的机制。 3、通过检测损伤足细胞及来氟米特干预后p-Akt、Akt通路蛋白的变化，进而进一步探讨来氟米特是否通过激活PI3K/Akt信号通路，减少足细胞凋亡，以达到保护足细胞的作用。方法：体外培养小鼠足细胞分成3组：（1）对照组：含无血清的RPMI-1640培养液培养；（2）PAN组：终浓度为50mg/L的PAN+无血清的RPM I-1640培养液；（3）A771726组：终浓度为10μmol/L的A771726+终浓度为50mg/L的PAN+无血清的RPM I-1640培养液。应用流式细胞仪检测各组作用24h、48h后足细胞凋亡率，选出合适的时间点进行后续蛋白的检测。各组通过Western blot检测各组48h后凋亡蛋白p53、抗凋亡蛋白bcl-xl的变化。根据p-Akt活化的时间特点，最后检测各组1h后通路蛋白p-Akt、Akt蛋白表达的情况，以二者的相对表达量表示，即p-Akt/Akt。结果：（1）流式细胞仪检测结果示：PAN损伤足细胞24h后凋亡率较对照组增高，为（14.4±1.11）％，48h凋亡最为明显，为（25.6±1.25）％；来氟米特A771726干预24h、48h后凋亡率明显减少，为（8.6±0.61）％、（13.5±1.35）％，随着时间的增加，药物抑制凋亡越明显，因此干预48h后，抑制凋亡效果最明显,差异有统计学意义(P0.05)；（2）PAN损伤足细胞48h后，PAN组p53蛋白较对照组表达增多,bcl-xl蛋白表达减少，来氟米特干预后，p53蛋白表达减少,bcl-xl蛋白表达增加，差异有统计学意义(均P0.05)；（3）各组作用1h后，总蛋白Akt基本无变化，差异无统计学意义（P0.05）；所测PAN组p-Akt/Akt表达显著减少，A771726可明显增加p-Akt/Akt的表达，接近正常，差异有统计学意义(均P0.05) 结论：来氟米特活性代谢产物A771726可有效保护PAN所致的足细胞凋亡，其机制可能与激活PI3K/Akt信号转导通路有关。
[Abstract]:Objective:
1. Establish the podocyte injury model of PAN, and detect the apoptosis rate of podocytes injured by PAN at different time points. The changes of apoptosis rate of injured podocytes after leflunomide intervention proved that leflunomide has protective effect on injured podocytes.
2. On the basis of the podocyte injury model induced by PAN, the expression of apoptotic protein p53, anti-apoptotic protein Bcl-xL and leflunomide in podocytes injured by PAN were detected at the same time point to study the protective mechanism of leflunomide on injured podocytes.
3. To explore whether leflunomide can protect podocytes by activating PI3K/Akt signaling pathway and reducing apoptosis of podocytes, the changes of p-Akt and Akt pathway proteins after leflunomide intervention were detected.
Method:
Mouse podocytes cultured in vitro were divided into 3 groups.
(1) control group: cultured with serum free RPMI-1640 medium.
(2) group PAN: PAN+ serum free RPM I-1640 culture medium with a final concentration of 50mg/L;
(3) A771726 group: the final concentration of A771726 + A771726 + final concentration of 50 mg / L PAN + serum-free RPM I-1640 medium.
Flow cytometry was used to detect the apoptotic rate of podocytes 24 hours and 48 hours after treatment, and the appropriate time point was selected for subsequent protein detection. The changes of apoptotic protein p53 and anti-apoptotic protein Bcl-xL were detected by Western blot in each group after 48 hours. According to the time characteristics of p-Akt activation, the expression of p-Akt and Akt protein in each group after 1 hour was detected. The situation is expressed by the relative expression of the two party, that is, p-Akt/Akt..
Result:
(1) The results of flow cytometry showed that the apoptosis rate of podocytes injured by PAN was higher than that of the control group at 24 hours (14.4 1.11)%, and the apoptosis was most obvious at 48 hours (25.6 1.25)%. The apoptosis rate of podocytes injured by leflunomide A771726 decreased significantly after 24 hours (8.6 0.61)%, (13.5 1.35)%. With the increase of time, the drug INH After intervention 48h, the effect of inhibiting apoptosis was the most obvious, and the difference was statistically significant (P0.05).
(2) after PAN injury to podocyte 48h, the expression of p53 protein in PAN group was higher than that in control group, and the expression of Bcl-xL protein decreased.
After leflunomide intervention, the expression of p53 protein decreased and the expression of Bcl-xL protein increased, the difference was statistically significant (all P 0.05).
(3) After 1 hour, the total protein Akt had no significant change (P 0.05); the expression of p-Akt/Akt in PAN group decreased significantly, and the expression of p-Akt/Akt in A771726 group increased significantly, which was close to normal (P 0.05).
Conclusion:
Leflunomide active metabolite A771726 can effectively protect PAN-induced apoptosis of podocytes, which may be related to activation of PI3K/Akt signal transduction pathway.
【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】：R692.6

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