Objective: To explore the effect of macrophage-derived exosomes on the activation of hepatic stellate cells and its possible mechanism. Methods: Differential ultracentrifugation was used to extract macrophage exosomes. The exosomes were co-cultured with the mouse hepatic stellate cell line JS1, and a control group was established with phosphate buffered saline (PBS). Cell immunofluorescence was used to observe the expressional conditions of F-actin. Cell counting kit-8 (CCK8) was used to detect the survival rate of JS1 cells in the two groups. The activation indices of JS1 cells [collagen type Ⅰ (Col Ⅰ) and α-smooth muscle actin (α-SMA)] and its key signal pathway activation index expression level [transforming growth factor (TGF)-β1/Smads, platelet-derived growth factor (PDGF)] in the two groups were determined using Western blot and RT-PCR. Data comparison between two groups was performed using an independent sample t-test. Results: The membrane structure of exosomes was clearly observed by transmission electron microscopy. The expression of exosome marker proteins CD63 and CD81 was positive, suggesting that exosomes were successfully extracted. Exosomes were co-cultured with JS1 cells. Compared with the PBS control group, there was no statistically significant difference in the proliferation rate of JS1 cells in the exosomes group (P＞0.05). The expression of F-actin was significantly increased in the exosome group. The mRNA and protein expression levels of α-SMA and ColⅠwere significantly increased in exosome group JS1 cells (all P＜0.05). The mRNA relative expression levels of α-SMA in PBS and exosome group were 0.25±0.07 and 1.43±0.19, respectively, while that of ColⅠ was 1.03±0.04 and 1.57±0.06, respectively. The mRNA and protein expressions of PDGF were significantly increased in exosome group JS1 cells (P＜0.05). The mRNA relative expression levels of PDGF in the PBS group and exosome group were 0.27±0.04 and 1.65±0.12, respectively. There were no statistically significant differences in the mRNA and protein expressions of TGF-β1, Smad2 and Smad3 between the two groups (P＞0.05). Conclusion: Macrophage-derived exosomes significantly promote the activation of hepatic stellate cells. JS1 cells may be the underlying mechanism for the up-regulation of PDGF expression.
目的： 探究巨噬细胞来源的外泌体对肝星状细胞活化的作用及其可能的机制。 方法： 采用差异超速离心法提取巨噬细胞外泌体，将外泌体与小鼠肝星状细胞株JS1细胞共培养，设立磷酸盐缓冲液（PBS）对照组。细胞免疫荧光观察F-actin表达情况，细胞活力检测（CCK8）法检测两组JS1细胞的存活率，western blot和RT-PCR法检测两组JS1细胞的活化指标［Ⅰ胶型原蛋白（ColⅠ）、α-平滑肌肌动蛋白（α-SMA）］及其关键信号通路激活指标［转化生长因子（TGF）-β1/Smads、血小板衍生生长因子（PDGF）］的表达水平。两组间数据比较采用独立样本t检验。 结果： 透射电镜观察外泌体膜结构清晰，外泌体标志蛋白CD63、CD81表达阳性，提示外泌体提取成功。外泌体与JS1细胞共培养，与PBS对照组比较，外泌体组JS1细胞的增殖率差异无统计学意义（P＞0.05）；外泌体组细胞F-actin表达明显增多；外泌体组JS1细胞α-SMA和ColⅠ的mRNA和蛋白表达水平显著升高（P值均＜0.05），PBS组和外泌体组α-SMA的mRNA相对表达量分别为0.25±0.07、1.43±0.19，ColⅠ的mRNA相对表达量分别为1.03±0.04、1.57±0.06；外泌体组JS1细胞PDGF的mRNA和蛋白表达水平显著升高（P＜0.05），PBS组和外泌体组PDGF的mRNA相对表达量分别为0.27±0.04、1.65±0.12；两组细胞的TGF-β1、Smad2、Smad3的mRNA和蛋白表达水平差异无统计学意义（P＞0.05）。 结论： 巨噬细胞来源的外泌体显著促进肝星状细胞活化，其机制可能与上调JS1细胞内PDGF的表达有关。.