体外诱导人脂肪干细胞向Leydig细胞定向分化的研究

Induced differentiation of human adipose-derived stem cells into Leydig cells in vitro

目的:探讨体外诱导脂肪干细胞(ADSCs)向Leydig细胞分化的可能性和条件。方法:应用I型胶原酶消化法分离人皮下脂肪组织中ADSCs,原代培养,适时传代。免疫组化方法检测波形蛋白的表达。以人绒毛膜促性腺激素(hCG)不同浓度及不同作用时间进行诱导后,实时荧光PCR检测类固醇激素合成急性调节蛋白(StAR)基因的表达。MTT法测定hCG诱导下的细胞增殖情况。在hCG、DMSO诱导1周后行3β-HSD免疫组化染色,并采用放射免疫法检测其培养上清及细胞裂解液的睾酮水平。结果:ADSCs增殖迅速,ADSCs波形蛋白呈黄褐色阳性表达;ADSCs中StAR基因的表达在一定范围内与hCG诱导浓度的增加成正相关,hCG 10 U/ml达到高峰,该浓度诱导1周内StAR表达随时间延长而增加;hCG诱导增加ADSCs细胞增殖;hCG 10 U/ml、DMSO 3.2×10-6mol/L条件下诱导1周后细胞中3β-HSD免疫组化染色可见胞质呈浅褐色弱阳性,且该条件下细胞裂解液睾酮测定值明显高于其它组。结论:①hCG在一定范围内促进ADSCs的StAR基因表达;②hCG可以促进ADSCs增殖;③在hCG 10 U/ml、DMSO 3.2×10-6mol/L诱导下,人ADSCs有向Leydig细胞分化的可能。

Objective： To explore the feasibility of inducing the differentiation of human adipose tissue-derived stem cells（ADSCs） into Leydig cells in vitro.Methods： We isolated ADSCs by digestion with CollagenaseⅠ from the subcutaneous adipose tissue,cultured them in the DMEM/F12 medium with 10% fetal bovine serum,and detected the expression of vimentin by immunohistochemistry.We exposed the ADSCs to different concentrations of human chorionic gonadotropin（HCG） for different times,determined the expression of StAR mRNA by real-time PCR,and measured the HCG-induced proliferation of ADSCs by MTT.After a week of induction by HCG and DMSO,we conducted 3β-HSD immunohistochemistry,and detected the testosterone level in the supernatant and lysis of the cells by radioimmunoassay.Results： The ADSCs grew well with a positive expression of vimentin.The expression of the StAR gene was positively correlated with the increased concentration of HCG,reaching the peak at HCG 10 U/ml in 1 week culture.The proliferation of ADSCs was significantly increased by HCG induction.A positive expression of 3β-HSD was observed after 1 week induction with HCG 10U/ml and DMSO 3.2×10-6mol/L.Conclusion： HCG enhances the expression of the StAR gene and the proliferation of ADSCs.Induced by HCG 10U/ml and DMSO 3.2×10-6mol/L,ADSCs tend to differentiate into Leydig cells.