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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 investigate the possibility and conditions of differentiation of adipose-derived stem cells (ADSCs) into Leydig cells in vitro. Methods: ADSCs were isolated from human subcutaneous adipose tissue by collagenase I digestion method, primary cultured and passaged in time. Immunohistochemistry was used to detect the expression of vimentin. After induced by human chorionic gonadotropin (hCG) at different concentrations and different time, real-time fluorescence PCR was used to detect the expression of steroid-associated acute regulatory protein (StAR) gene. MTT assay hCG induced cell proliferation. After hCG and DMSO were induced for 1 week, 3β-HSD immunohistochemical staining was performed. The levels of testosterone in the culture supernatant and cell lysate were detected by radioimmunoassay. Results: The proliferation of ADSCs was rapid and the vimentin of ADSCs was positively expressed in brown. The expression of StAR gene in ADSCs was positively correlated with the increase of hCG concentration in a certain range. The peak of hCG 10 U / ml reached the peak, which induced the expression of StAR in 1 week HCG increased the proliferation of ADSCs; the expression of 3β-HSD in the cells after being induced by hCG 10 U / ml and DMSO 3.2 × 10-6 mol / L for 1 week showed weak brownish positive cytoplasm , And the measured values of testosterone in cell lysate under this condition were significantly higher than other groups. Conclusion: hCG can promote the expression of StAR gene in ADSCs within a certain range; hCG can promote the proliferation of ADSCs; ③the differentiation of human ADSCs into Leydig cells may be induced by hCG 10 U / ml and DMSO 3.2 × 10-6 mol / L.