原代肝细胞外泌体对肝星状细胞活化的影响

Effects of primary hepatocyte exosomes on hepatic stellate cell activation

目的探讨原代肝细胞外泌体对肝星状细胞(HSC)活化的影响。方法在原位两步胶原酶灌流法的基础上,分别分离培养小鼠原代肝细胞及HSC,采用肝细胞糖原染色法和细胞角蛋白18(CK-18)细胞化学染色法鉴定原代肝细胞,采用α-平滑肌肌动蛋白(α-SMA)细胞免疫荧光染色法鉴定原代HSC;采用exoEasy Maxi Kit试剂盒提取肝细胞外泌体,用电镜法、纳米颗粒跟踪分析(NTA)及蛋白免疫印迹法(Western blot)鉴定肝细胞外泌体;将肝细胞外泌体与活化HSC共培养24 h,采用逆转录-实时定量PCR(qRT-PCR)法检测HSC中α-SMA及Ⅰ型胶原(COL 1 A 1)mRNA表达。结果原位两步胶原酶灌流法成功分离培养获得小鼠原代肝细胞及HSC,肝细胞糖原染色法及CK-18细胞化学染色法证实原代肝细胞纯度>95%,免疫荧光染色显示HSC的α-SMA阳性率>90%;exoEasy Maxi Kit试剂盒提取的肝细胞外泌体在电镜下呈圆形或椭圆形,NTA结果显示其直径约140 nm,Western blot实验可见外泌体标志物分化抗原9(CD9)和肿瘤易感基因101(TSG101)表达;与空白对照组比较,HSCs与肝细胞外泌体共培养后HSCs的α-SMA及COL 1 A 1 mRNA表达水平降低(P<0.05)。结论成功提取原代肝细胞外泌体,该外泌体可抑制HSC活化。

Objective To investigate the effect of primary hepatocyte exosomes on the activation of hepatic stellate cell(HSC).Methods On the basis of in situ two-step collagenase perfusion method,primary mouse hepatocytes and HSCs were isolated and cultured respectively.Hepatocyte glycogen staining and cytokeratin 18(CK-18)cytochemical staining were used to identify primary hepatocytes,α-smooth muscle actin(α-SMA)immunofluorescence staining was used to identify the primary HSC.Hepatocyte exosomes were extracted by exoEasy Maxi Kit,and identified by electron microscopy,nanoparticle tracking analysis(NTA)and Western blot.Hepatocyte exosomes were co-cultured with activated HSC for 24 hours,and the mRNA expressions ofα-SMA and typeⅠcollagen(COL 1 A 1)in HSC were detected by real-time quantitative reverse transcription PCR(qRT-PCR).Results Mouse primary hepatocytes and HSCs were successfully isolated and cultured by in situ two-step collagenase perfusion method.Hepatocyte glycogen staining and CK-18 cytochemical staining confirmed that the purity of primary hepatocytes was more than 95%.HSC immunofluorescence staining showed that the positive rate ofα-SMA was more than 90%.The hepatocyte exosomes extracted by exoEasy Maxi Kit were round or oval under electron microscope,and the diameter tracked by NTA was about 140 nm,the expression of exosome markers such as differentiation antigen 9(CD9)and tumor susceptibility gene 101(TSG101)was detected by Western-blot.Compared with the blank control group,the expression levels ofα-SMA and COL 1 A 1 mRNA in HSCs were decreased after co-culture with hepatocyte exosomes(P<0.05).Conclusion Primary hepatocyte exosomes are successfully extracted,which can inhibit HSC activation.