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

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

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。

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.