人骨髓间充质干细胞向Leydig细胞或产类固醇激素细胞体外诱导分化的研究

目的探讨体外诱导人骨髓间充质干细胞(MSCs)向睾丸Leydig细胞分化,并确定诱导条件。方法将分离培养所获得的第3代MSCs细胞经含黄体生成素(LH)、人绒毛膜促性腺激素(hCG)、血小板衍化生长因子(PDGF)及白介素-1α(IL-1α)的诱导液诱导,按诱导液中各因子浓度不同分为A、B、C组和对照组,分别于7d、14d、21d时观察细胞形态变化,并行3β-羟类固醇脱氢酶(3β-HSD)免疫细胞化学染色和免疫荧光染色,检测各组细胞3β-HSD的表达。以人睾酮ELISA试剂盒检测诱导细胞分泌类固醇的功能情况。结果诱导10～14d后,细胞具有Leydig细胞的形态特点;免疫细胞化学染色及免疫荧光染色显示,本研究条件下,随着诱导液中各因子浓度加大及诱导时间延长,诱导细胞3β-HSD表达阳性数和阳性率逐渐增多,以A组浓度及21d为最佳诱导浓度及时间(P<0.05),并且A组诱导21d分泌睾酮量最高(P<0.05)。结论人骨髓MSCs经体外诱导后可以分化为Leydig细胞或产类固醇细胞。

Differentiation of Human Bone Marrow Mesenchymal Stem Cells into Leydig or Steroidogenic Cells in vitro

Objective To investigate the possibility of differentiating mesenchymal stem cells(MSCs) into steroidogenic or testicular Leydig cells in vitro.Methods The 3rd-passage cells of MSCs were divided into 4 groups to be induced and cultured.The experimental groups were cultured with conditional medium which consisted of luteinizing hormone(LH),human chorionic gonadotrophin(hCG),platelet-derived growth factor(PDGF) and interlukin-1α(IL-1α) as follows.Group A: LH 0.75 U/mL,hCG 40 U/mL,PDGF 10 ng/mL,IL-1α 0.0005 ng/mL;Group B: LH 0.375 U/mL,hCG 20 U/mL,PDGF 10 ng/mL,IL-1α 0.0005 ng/mL;Group C: LH 0.1875 U/mL,hCG 10 U/mL,PDGF 10 ng/mL,IL-1α 0.0005 ng/mL.Meanwhile,the control group was cultured in basal medium with normal sodium.The results were analysed by microscopic observations,3β-hydroxysteroid dehydrogenase(3β-HSD) immunocytochemistry and immunofluorescence on the 7th,14th and 21st day of induction respectively.The function of the induced cells was characterized by testosterone ELISA.Results 10-14 days after induction,the induced cells with a typical reticular intercellular connection possessed the morphologic characteristic of Leydig cells.3β-HSD immunocytochemistry and immunofluorescence showed that Group A at 21th day had the most positive cells(P<0.05).21 days of induction revealed a higher testosterone production than others(P<0.05).Conclusion MSCs from human bone marrow are able to differentiate into steroidogenic or testicular Leydig cells in vitro.Human bone marrow-derived MSC may become important cells for studies of steroidogenic differentiation and offer a potential clinical stem cell source for diseases of steroidogenic organs.