摘要
目的:探讨蛋白酶体抑制剂MG-132对体外培养的人近端肾小管上皮细胞(HK-2)转分化和凋亡的影响及其机制。方法:体外培养HK-2细胞,随机分组:对照组,TGF-β1刺激组(5μg/L),MG-132组(2.5μmol/L),MG-132+TGF-β1组(MG-1322.5μmol/L孵育30min后再加TGF-β15μg/L)。培养24h后收集细胞,RT-PCR法检测Smurf1、Smurf2和Smad7 mRNA的表达;Western免疫印迹检测Smurf1、Smurf2、Smad7和p-IκB-α和IκB-α蛋白表达;免疫组织化学检测胞浆中Fn和α-SMA的表达;Hoechst 33258染色免疫荧光检测细胞凋亡形态变化,流式细胞术检测细胞凋亡率。结果:TGF-β1刺激组HK-2Smurf1、Smurf2 mRNA及蛋白表达水平较对照组高(P<0.05),但Smad7 mRNA及蛋白表达水平较对照组低(P<0.05),且胞浆中FN和α-SMA水平较对照组高(P<0.05)。MG-132+TGF-β1组与TGF-β1刺激组比较,Smurf1、Smurf2 mRNA表达降低(P<0.05),Smad7 mRNA略升高(P>0.05),而Smad7蛋白明显升高(P<0.05),胞浆中Fn和α-SMA表达减少。MG-132组、MG-132+TGF-β1组分别与对照组及TGF-β1组比较,p-IκB-α和IκB-α蛋白浓度升高,细胞凋亡率增加,差异有统计学意义(P<0.05)。结论:MG-132可抑制TGF-β1诱导的HK-2Smurf1、Smurf2的表达,促进Smad7表达,抑制其降解,减轻FN和α-SMA的表达,从而抑制HK-2转分化;另一方面,MG132可能通过抑制TGF-β1诱导的HK-2细胞NF-κB活化促进HK-2细胞凋亡。
Objective：To approach the effects of proteasome inhibitor MG-132 on trans-differentiation and apoptosis in HK - 2 cells cultured in vitro and the molecular mechanism. Methods： Cultured HK - 2 cells were divided into 4 groups： （ 1 ） control group; （2）TGF- β1 group; （3）MG - 132 group; （4）MG 132 ＋ TGF - β1 group. The cells were collected at the 24th hour after treated. The expression of Smurfl, Smurf2 and Smad7 mRNA were tested by RT - PCR. The expression of Smurf1, Smurf2, Smad7, P-IκB-α and IκB-α protein were detected by Western blot. The expression of FN and α - SMA in the HK - 2 cytoplasm were detected by immunohischemistry and the cell morphologic changes were traced fluorescence microscope after dyed by Hoechst 33258. The cells apoptosis rate were tested by flow cytometry. Results：The expression of Smurfl, Smurf2 mRNA, protein and the secretion of FN and α- SMA in TGF- β1 group were increased, while the Smad7 mRNA and protein decreased in comparison with the control group（ P 〈 0.05）. The expression of Smurfl ,Smurf2 mR NA in MG-132 ＋ TGF- β1 group were decreased（ P 〈 0.05）, the Smad7 mRNA increased mildly in comparison with TGF- β1 group（P 〉0.05）, and the Smad7 protein increased significantly（P〈 0.05）. The secretion of FN and α - SMA in the HK-2 cytoplasm was decreased（ P 〈 0.05 ）. The concentration of P-IκB-α,IκB-α and the apoptosis rate were increased in the MG - 132 and MG- 132 ＋ TGF - β1 groups in comparison with the control group and TGF-β1 group（P〈0.05）. Conclusion： MG- 132 can inhibit the expression of Smurfl and Smurt2 induced by TGF-i31 and promote the expression of Smad7 in HK - 2, down - regulate the expression of FN and α - SMA, thus block the HK - 2 trans - differentiation. On the other hand, MG- 132 may lead HK - 2 cells to apoptosis through inhibiting the activity of NF-κB by increasing the concentration of P-IκB-α and IκB-α.
出处
《中国中西医结合肾病杂志》
2009年第5期404-408,I0003,共6页
Chinese Journal of Integrated Traditional and Western Nephrology
基金
厦门市青年创新科研基金资助项目(No.WQK0602)
厦门市学科带头人培养专项基金资助项目(No.2004353)
关键词
泛素-蛋白酶体途径
人肾小管上皮细胞
转分化
细胞凋亡
Ubiquitin- proteasome pathway Human renal tubular epithelial cell Trans- differentiation Apoptosis
