【摘要】：目的： 1、建立PAN損傷足細(xì)胞模型，在不同時間點(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)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R692.6

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 朱全剛,胡晉紅,孫華君,宋紅杰;RP-HPLC法同時測定來氟米特及A771726[J];藥物分析雜志;2000年05期

2 勞志英,倪立青,張之澧,周嘉陵,陳麗華,張芳,劉大棟,楊曉凌,朱琦,張湛明,沈杰;來氟米特治療類風(fēng)濕關(guān)節(jié)炎雙盲試驗(yàn)[J];中國新藥與臨床雜志;2001年02期

3 孫巖,吳德全,李剛,齊忠權(quán),單世光;他克莫司和來氟米特合用抑制大鼠心臟移植排斥反應(yīng)及T淋巴細(xì)胞增殖[J];中華器官移植雜志;2003年03期

4 朱劍,黃烽,張江林,楊春花,劉湘源,鄧新立;來氟米特對類風(fēng)濕關(guān)節(jié)炎患者外周血Th1/Tc1細(xì)胞的影響[J];中華風(fēng)濕病學(xué)雜志;2003年09期

5 MARCD·COHEN ,高小溪;新信息:類風(fēng)濕性關(guān)節(jié)炎的治療[J];中國療養(yǎng)醫(yī)學(xué);2003年02期

6 張旗,楊西瑞,張磊,荊雪;來氟米特治療銀屑病關(guān)節(jié)炎11例療效觀察[J];山東醫(yī)藥;2005年04期

7 陳麗平;唐英;李軍平;;新型免疫抑制劑來氟米特的應(yīng)用及展望[J];河北醫(yī)藥;2006年01期

8 朱全剛!上海200433,胡晉紅!上海200433,孫華君!上海200433;反相高效液相色譜法測定來氟米特[J];中國藥學(xué)雜志;1999年09期

9 馬驥良;來氟米特和甲氨喋呤治療活動性類風(fēng)濕關(guān)節(jié)炎的對比觀察[J];北京醫(yī)學(xué);2000年06期

10 朱全剛,胡晉紅,孫華君,張立超;來氟米特脂質(zhì)體的工藝優(yōu)化[J];華西藥學(xué)雜志;2000年02期

相關(guān)會議論文 前10條

1 姜林娣;於強(qiáng);陳慧勇;王臻;;國產(chǎn)來氟米特治療類風(fēng)濕關(guān)節(jié)炎臨床療效和安全性評價[A];第十屆全國風(fēng)濕病學(xué)學(xué)術(shù)會議論文集[C];2005年

2 姚宏偉;李俊;;來氟米特對實(shí)驗(yàn)性肝損傷作用的研究[A];第七屆全國生化藥理學(xué)術(shù)討論會論文摘要集[C];2000年

3 彭健韞;張小如;廖益飛;楊明正;吳凌慧;李莉;;來氟米特聯(lián)合激素治療慢性進(jìn)展性IgA腎病療效分析[A];2008年浙江省腎臟病學(xué)術(shù)年會論文匯編[C];2008年

4 曹勵歐;倪兆慧;林愛武;張偉明;方煒;王琴;牟姍;陸任華;錢家麒;;來氟米特治療Ⅳ型及Ⅴ型狼瘡腎炎的中長期研究[A];2007年浙滬兩地腎臟病學(xué)術(shù)年會資料匯編[C];2007年

5 李素

本文編號：2188590