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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。
Objective: To investigate the effect of mesenchymal stem cell (MSC) parathyroid on Lx-2 activated hepatic stellate cells and its mechanism. METHODS: Transwell membrane was used to construct Transwell co-culture system with 6-hole plastic cell culture plates. Bone marrow-derived mesenchymal stem cells (BM-MSCs) and human hepatic stellate cell line Lx -2 were inoculated in the upper and lower layers of the system, and the group as an experimental group, cultured Lx-2 alone as a control group to observe the secretion of MSCs in the culture medium on the impact of Lx-2. The apoptosis of Lx-2 cells was detected by flow cytometry and Hoechst33342 staining respectively. The mRNA and protein levels of uncoupling protein 2 (UCP2) in Lx-2 were detected by qRT-PCR and Western blot. The fluorescence intensity of reactive oxygen species (ROS) in Lx-2 cells and mitochondria was detected by fluorescent probe method. The level of malondialdehyde (MDA) in cells was measured by thiobarbituric acid (TBA) MDA) content. Results: Compared with the control group, some apoptotic cells such as chromatin condensation and granular fluorescence of Lx-2 were observed after Hoechst 33342 staining. The apoptosis rate of Lx-2 in the experimental group was 2.6 times that of the control group (P <0.05 ). The expression of UCP2 mRNA and protein in Lx-2 experimental group was significantly lower than that in control group (P <0.05). The ROS level in Lx-2 cells and mitochondria in experimental group was significantly increased. MDA in the cell culture supernatant was (0.43 ± 0.47) mmol / L, which was significantly higher than that in the control group (0.16 ± 0.43) mmol / L (P <0.05). CONCLUSION: Secretion of MSC can induce the apoptosis of Lx-2 cells. It may be related to the secretion of UCP2 secreted by MSC and the excessive generation of ROS in cells and mitochondria.