间充质干细胞外泌体对体外肝星状细胞增殖和活化的影响

目的 探讨间充质干细胞来源的外泌体(MSCs-Exo)对人肝星状细胞系LX-2细胞增殖及活化的影响。方法 取MSCs-Exo,在透射电子显微镜下观察。取LX-2细胞,分为对照组、5 μg/L、10 μg/L和20 μg/L转化生长因子-β1(TGF-β1)处理组,采用RT-PCR和Western blot法检测细胞α-SMA和COL1A1 mRNA及其蛋白表达。取TGF-β1诱导活化的LX-2细胞,以50 μg/mL、100 μg/mL和200 μg/mL MSCs-Exo干预24 h,采用MTT法检测细胞增殖。结果 经电镜观察及粒径分析表明,实验所用MSCs-Exo符合外泌体的形态特征;实验所用MSCs-Exo阳性标志蛋白CD9和CD63阳性,而GM130蛋白阴性;经TGF-β1诱导活化的LX-2细胞α-SMA和COL1A1 mRNA水平及其蛋白表达均显著升高;当TGF-β1诱导浓度为20 μg/L时,细胞α-SMA和COL1A1 mRNA及其蛋白表达量最高;与对照组比,MSCs-Exo-100组和MSCs-Exo-200处理细胞增殖活力显著降低(均P<0.0001);50 μg/mL、100 μg/mL和200 μg/mL MSCs-Exo作用TGF-β1诱导活化的LX-2细胞α-SMA mRNA水平和COL1A1 mRNA水平较对照组显著降低(P<0.01),细胞α-SMA和COL1A1蛋白水平也较对照组显著降低(P<0.0001);MSCs-Exo-100和MSCs-Exo-200处理的LX-2细胞增殖活力较对照细胞显著降低(均P<0.0001)。结论 MSCs-Exo可以抑制TGF-β1诱导的LX-2细胞增殖和活化,其应用研究值得期待。

Effect of exosomes derived from mesenchymal stem cells on the proliferation and activation of hepatic stellate cells in vitro

Objective The purpose of this experiment was to investigate the effect of mesenchymal stem cells (MSC)-derived exosomes (MSCs-Exo) on the proliferation and activation of human hepatic stellate cells, e.g. LX-2 cells. Methods The MSCs-Exo were purchased and identified by electron microscopy observation and NanoFCM measurement. The LX-2 cells were stimulated with transforming growth factor-β1 (TGF-β1) at 5 μg/L, 10 μg/L and 20 μg/L concentration, and the α-SMA and COL1A1 mRNA and their proteins were detected by RT-PCR and Western blot. The activated LX-2 cells were intervened by MSCs-Exo at 50 μg/mL, 100 μg/mL at 200 μg/mL for 24 h. The LX-2 cell proliferation was detected by MTT assay. Results The morphological appearance and the particle sizes of MSCs-Exo revealed by transmission electron microscopy and NanoFCM accorded with requirement, and the MSCs-Exo positive marker proteins, CD9 and CD63 were positive, with GM130 protein negative; the α-SMA and COL1A1 mRNA and their protein expression in activated LX-2 cells by TGF-β1 stimulation were significantly increased and positively correlated with TGF-β1 concentration; the α-SMA and COL1A1 mRNA level and their protein expression were highest when the TGF-β1 induction at concentration of 20 μg/L; in the MSCs-Exo-100 and MSCs-Exo-200-intervened activated LX-2 cells, the proliferation viability of cells were significantly reduced compared with in the control (all P<0.0001); the α-SMA and COL1A1 mRNA and their protein expression in TGF-β1-activated LX-2 cells with different concentrations of MSCs-Exo intervention, e.g. at 50 μg/mL, 100 μg/mL and 200 μg/mL, were significantly decreased compared with in the control (P<0.001); the proliferation activities of LX-2 cells intervened by MSCs-Exo-100 and MSCs-Exo-200 were significantly decreased compared to that in the control cells (P<0.0001). Conclusion The MSCs-Exo could inhibit TGF-β1-induced proliferation and activation of LX-2 cells in vitro, and needs further investigation.