【摘要】：目的： 1、建立PAN損傷足細(xì)胞模型，在不同時(shí)間點(diǎn)檢測PAN損傷足細(xì)胞的凋亡率，來氟米特干預(yù)后，損傷足細(xì)胞凋亡率的變化，證實(shí)來氟米特對(duì)損傷足細(xì)胞有保護(hù)作用。 2、在PAN損傷足細(xì)胞模型的基礎(chǔ)上，檢測同一時(shí)間點(diǎn)PAN損傷足細(xì)胞表達(dá)凋亡蛋白p53、抗凋亡蛋白bcl-xl，來氟米特干預(yù)后，二者表達(dá)情況的變化，，研究來氟米特保護(hù)損傷足細(xì)胞的機(jī)制。 3、通過檢測損傷足細(xì)胞及來氟米特干預(yù)后p-Akt、Akt通路蛋白的變化，進(jìn)而進(jìn)一步探討來氟米特是否通過激活PI3K/Akt信號(hào)通路，減少足細(xì)胞凋亡，以達(dá)到保護(hù)足細(xì)胞的作用。 方法： 體外培養(yǎng)小鼠足細(xì)胞分成3組： （1）對(duì)照組：含無血清的RPMI-1640培養(yǎng)液培養(yǎng)； （2）PAN組：終濃度為50mg/L的PAN+無血清的RPM I-1640培養(yǎng)液； （3）A771726組：終濃度為10μmol/L的A771726+終濃度為50mg/L的PAN+無血清的RPM I-1640培養(yǎng)液。 應(yīng)用流式細(xì)胞儀檢測各組作用24h、48h后足細(xì)胞凋亡率，選出合適的時(shí)間點(diǎn)進(jìn)行后續(xù)蛋白的檢測。各組通過Western blot檢測各組48h后凋亡蛋白p53、抗凋亡蛋白bcl-xl的變化。根據(jù)p-Akt活化的時(shí)間特點(diǎn)，最后檢測各組1h后通路蛋白p-Akt、Akt蛋白表達(dá)的情況，以二者的相對(duì)表達(dá)量表示，即p-Akt/Akt。 結(jié)果： （1）流式細(xì)胞儀檢測結(jié)果示：PAN損傷足細(xì)胞24h后凋亡率較對(duì)照組增高，為（14.4±1.11）％，48h凋亡最為明顯，為（25.6±1.25）％；來氟米特A771726干預(yù)24h、48h后凋亡率明顯減少，為（8.6±0.61）％、（13.5±1.35）％，隨著時(shí)間的增加，藥物抑制凋亡越明顯，因此干預(yù)48h后，抑制凋亡效果最明顯,差異有統(tǒng)計(jì)學(xué)意義(P0.05)； （2）PAN損傷足細(xì)胞48h后，PAN組p53蛋白較對(duì)照組表達(dá)增多,bcl-xl蛋白表達(dá)減少， 來氟米特干預(yù)后，p53蛋白表達(dá)減少,bcl-xl蛋白表達(dá)增加，差異有統(tǒng)計(jì)學(xué)意義(均P0.05)； （3）各組作用1h后，總蛋白Akt基本無變化，差異無統(tǒng)計(jì)學(xué)意義（P0.05）；所測PAN組p-Akt/Akt表達(dá)顯著減少，A771726可明顯增加p-Akt/Akt的表達(dá)，接近正常，差異有統(tǒng)計(jì)學(xué)意義(均P0.05) 結(jié)論： 來氟米特活性代謝產(chǎn)物A771726可有效保護(hù)PAN所致的足細(xì)胞凋亡，其機(jī)制可能與激活PI3K/Akt信號(hào)轉(zhuǎn)導(dǎo)通路有關(guān)。
[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.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R692.6

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