间充质干细胞旁分泌物质诱导肝星状细胞凋亡的体外研究
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江苏省医学创新团队与领军人才资助项目(LJ201121)


Human mesenchymal stem cells secreted factors inducing apoptosis of hepatic stellate cells in vitro
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    目的:研究间充质干细胞(mesenchymal stem cell,MSC)旁分泌物质对活化的肝星状细胞Lx-2的影响及作用机制-方法:应用6孔塑料细胞培养板,每孔使用半透膜,构建Transwell共培养体系;将骨髓来源的间充质干细胞(bone marrow mesenchymal stem cell,BM-MSC)和人肝星状细胞系Lx-2分别接种在体系的上下层,并把此组作为实验组,单独培养的Lx-2作为对照组,观察培养液中MSC旁分泌物质对Lx-2的影响-采用流式细胞术及Hoechst33342染色法分别检测Lx-2细胞凋亡的情况,qRT-PCR和Western blot检测Lx-2中解耦联蛋白2(uncoupling protein 2,UCP2)的mRNA和蛋白水平的表达量,荧光探针法检测Lx-2细胞内及线粒体中活性氧(reactive oxygen species,ROS)的荧光强度,采用硫代巴比妥酸(thiobarbituric acid,TBA)法检测细胞丙二醛(malondialdehyde,MDA)的含量-结果:与对照组相比,Hoechst 33342染色后可见部分Lx-2染色质固缩-颗粒状荧光等凋亡细胞典型表现,实验组Lx-2凋亡率是对照组的2.6倍(P < 0.05)-实验组Lx-2中UCP2 mRNA及蛋白表达量显著低于对照组(P < 0.05);实验组Lx-2细胞内和线粒体中ROS水平明显升高-细胞培养上清中MDA为(0.43 ± 0.47) mmol/L,显著高于对照组(0.16 ± 0.43)mmol/L(P < 0.05)-结论:MSC旁分泌物质有诱导Lx-2细胞凋亡的作用;可能与MSC旁分泌物质抑制UCP2的表达,促进细胞内及线粒体ROS过量生成有关-

    Abstract:

    Objective:To observe the paracrine effects of mesenchymal stem cells(MSC)on the activated hepatic stellate cells Lx-2,and explore the possible mechanisms. Methods:A co-culture system was established by culturing bone marrow mesenchymal stem cell(BM-MSC)in the Transwell insert and Lx-2 on the plastic plates(6 well),which were placed on the upper and lower layer repectively,and set it as the experiment group. Normal Lx-2 was cultured alone as control. Cell apoptosis was determined by Hoechst 33342 staining and flow cytometry,respectively. The expressions of uncoupling protein 2(UCP2)mRNA and protein in Lx-2 were detected by quantitative real-time PCR(qRT-PCR)and Western blot,respectively. Intracellular and mitochondrial reactive oxygen species(ROS)levels of Lx-2 were measured by fluorescent probe method. The content of MDA in co-culture supernatant was determined by thiobarbituric acid(TBA)reaction. Results:Compared with the control group,Hoechst 33342 staining showed nuclear condensation,granular fluorescence and other typical features of apoptosis in parts of Lx-2 cells. The apoptotic rate of Lx-2 in the experiment group was 2.6 times of that in the control group(P < 0.05). The expressions of UCP2 mRNA and protein in Lx-2 were significantly inhibited compared with those in the control group(P < 0.05). The intracellular and mitochondrial ROS levels of Lx-2 in the experiment group were highly enhanced. The concentration of MDA in the co-culture supernatant in the experimental group was (0.43 ± 0.47)mmol/L,which was significantly higher than the control group with (0.16 ± 0.43)mmol/L(P < 0.05). Conclusion:The paracrine effects of MSC were capable of inducing Lx-2 apoptosis,which is supposed to be the result of the suppression of UCP2 and over-expression of intracellular and mitochondrial ROS in Lx-2.

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徐 喨,施启鹏,周 晗,蔡 洁,李 军,章莉莉.间充质干细胞旁分泌物质诱导肝星状细胞凋亡的体外研究[J].南京医科大学学报(自然科学版),2014,(8):1040-1045

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  • 收稿日期:2014-03-25
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  • 在线发布日期: 2014-08-03
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