人BMSCs移植小鼠睾丸后向Leydig细胞或产类固醇激素细胞诱导分化的实验研究

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

目的研究人体骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)在体内条件下能否向Leydig细胞或产类固醇激素细胞定向诱导分化,及是否发生免疫排斥反应。方法将分离培养获得的第3代BMSCs经核荧光标记物标记后,制备成细胞悬液,注射至经二甲磺基乙烷(EDS)处理后的20只BALB/c小鼠睾丸内。从移植BMSCs前1d开始,每隔2d处死1只小鼠,测尾静脉游离睾酮浓度,对照组为同期未作任何处理BALB/c小鼠20只。同时睾丸组织经荧光显微镜摄片,追踪观察BMSCs,行3β-羟类固醇脱氢酶(3β-hydroxysteroid dehydrogenase,3β-HSD)单克隆抗体和鼠抗人细胞核单克隆抗体双重免疫荧光检测,寻找3β-HSD和鼠抗人细胞核单克隆抗体荧光染色均阳性的细胞。结果 EDS对小鼠Leydig细胞有一定杀伤作用;采用睾丸网注射法行人BMSCs移植小鼠睾丸获得了成功,未见免疫排斥反应;移植BMSCs后的睾丸组织未检测到3β-HSD和鼠抗人细胞核单克隆抗体免疫荧光染色均阳性的细胞。结论采用人BMSCs移植小鼠睾丸的方法获得成功,未见免疫排斥反应,移植后的BMSCs未向Leydig细胞或产类固醇细胞定向分化。

Objective To study the differentiation of human bone marrow mesenchymal stem cells （BMSCs） into Leydig or steroidogenie cells in vivo and the immunoreaction related to transplantation into mouse testis. Methods After differentiation and cultivation, the 3rd-passage BMSCs were collected and labeled with Hoechest 33342, and joined the saline fluorouracil to form cell-suspending fluid. After injection of the etgane dimethane sulphonate （EDS）, the mice received the transplanted cell-suspending fluid by testis net injection with a dose of each side testicular 0.05 mL. Since the first day prior to transplantation, mice were executed every 2 d （one mouse each time） and the testosterone concentrations were analyzed. The control group included 20 BALB/c mice without any treatment during the same period. The results were analyzed by microscopic observation, using 3β-hydroxysteroid dehydrogenase （3β-HSD） monoclonal antibody and mouse anti-human cell nucleus monoclonal antibody for immunofluorescence assay on the testis. For tracking the BMSCs, the cells which were positively stained with both 3β-HSD and mouse anti-human cell nucleus monoelonal antibodies were retrieved. Results Certain killing effect of EDS to the mouse Leydig cells was observed. Transplantation of human BMSCs into the mouse testis by testis net injection was effective and feasible, no immunoreactions were detected. After transplantation, no positive cells of 3β-HSD and mouse anti-human cell nucleus monoclonal antibody were found. Conclusion Transplantation of human BMSCs into the mouse testis by testis net injection was effective and feasible, no immunoreactions were detected. After transplantation, the human BMSCs failed to differentiate into Leydig cells or steroidogenic cells.