人脐带间充质干细胞在大鼠睾丸间质内向Leydig细胞分化的实验研究

Differentiation of human umblical cord mesenchymal stem cells into Leydig cells in the rat testis interstitium: An experimental study

目的:研究人脐带间充质干细胞(HUMSCs)在大鼠睾丸间质内向Leydig细胞分化的可行性。方法:贴壁法获得HUMSCs,分别通过流式细胞表面抗原染色与三系分化验证其纯度与多向分化能力,用CM-Dil标记HUMSCs后将其移植入大鼠睾丸间质内,并在移植后4周与8周对大鼠睾丸进行免疫荧光染色观察HUMSCs的存活与分化情况,移植后8周获得大鼠睾丸细胞悬液,通过流式细胞染色检测HUMSCs表达Leydig细胞标志物3β-HSD判断细胞分化的效率,通过流式细胞分选获得悬液内CM-Dil标记的在睾丸内分化后的HUMSCs,并对其进行培养,培养3 d后收集培养液,检测其睾酮水平。结果:Leydig细胞标志物CYP11a1在HUMSCs移植8周后有表达,而在移植4周后未见表达。3β-HSD流式染色显示其分化效率约为14.5%,流式细胞分选后细胞可存活,且其培养液内能检测出睾酮。结论:HUMSCs在大鼠睾丸间质内可向Leydig细胞分化。

Objective： To explore the feasibility of inducing human umbilical cord mesenchymal stem cells（HUMSCs） to differentiate into Leydig cells in the interstitial tissue of the rat testis. Methods： HUMSCs were obtained by tissue blocks culture attachment and their purity and multi-lineage differentiation ability were verified by flow cytometry and chondrogenic / adipogenic / osteogenic differentiation. Then the HUMSCs were marked by CM-Dil and transplanted into the interstitial tissue of the rat testis. At 4 and 8weeks after transplantation,the survival and differentiation status of the HUMSCs were observed by immunofluorescence staining and flow cytometry. The suspension of the rat Leydig cells was obtained at 8 weeks for determining the expression of the Leydig cell marker3β-HSD in the HUMSCs,the cells labeled with CM-Dil were sorted and cultured,and the medium collected after 3 days of culture for measurement of the testosterone level. Results： The expression of the Leydig cell marker CYPllal was not observed in the HUMSCs at4 weeks but found at 8 weeks after transplantation and the differentiation rate of 3β-HSD was about 14. 5% at 8 weeks. CM-Dil labeled cells survived after sorting and testosterone was detected in the medium. Conclusion： HUMSCs are likely to differentiate into Leydig cells in the interstitium of the rat testis.