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目的:探讨睾丸Leydig细胞(LC)来源的条件培养液诱导人脐带间充质干细胞(Hu MSCs)分化为LC的可行性。方法:用贴壁法和酶消化法分别分离培养Hu MSCs和LC。用LC来源的条件培养液诱导Hu MSCs,诱导前的Hu MSCs作为对照。培养3、7、10 d后,对干细胞进行RT-PCR检测LHR、St AR和3β-HSD的mRNA表达;培养2周后,对诱导后的干细胞进行CYP11A1、CYP17A1和3β-HSD免疫荧光检测;培养4周后,Western印迹检测3β-HSD。结果:干细胞诱导3、7、10 d后,RT-PCR发现LHR、St AR及3β-HSD表达阳性;诱导2周后,免疫荧光检测发现CYP11A1、CYP17A1、3β-HSD表达阳性;诱导4周后,Western印迹检测发现3β-HSD表达阳性。对照组各项检测均为阴性。结论:在LC来源的条件培养液诱导下,Hu MSCs具有向合成类固醇激素的细胞分化的可能,并最终可能分化为LC。
Objective: To investigate the feasibility of inducing human umbilical cord mesenchymal stem cells (Hu MSCs) to differentiate into LC by conditioned medium derived from testicular Leydig cells (LC). Methods: Hu MSCs and LC were isolated and cultured respectively by adherent method and enzyme digestion method. Hu MSCs were induced by LC-derived conditioned medium and pre-induction Hu MSCs served as controls. After cultured for 3, 7 and 10 days, the mRNA expressions of LHR, St AR and 3β-HSD were detected by RT-PCR. After 2 weeks of culture, the induced stem cells were immunofluorescently detected by CYP11A1, CYP17A1 and 3β-HSD. After 4 weeks of culture, 3β-HSD was detected by Western blotting. Results: The expression of LHR, St AR and 3β-HSD were detected by RT-PCR at 3, 7 and 10 days after induction. After 2 weeks of induction, the positive expression of CYP11A1, CYP17A1 and 3β-HSD was detected by immunofluorescence. After 4 weeks of induction Western blotting revealed that 3β-HSD expression was positive. All the tests in the control group were negative. CONCLUSION: Hu MSCs, under the induction of LC-derived conditioned medium, have the potential to differentiate into synthetic steroid hormones and may eventually differentiate into LCs.