精原干细胞移植后的体内迁移、增殖和分化

Migration,proliferation and differentiation of spermatogonial stem cells in vivo after transplantation

背景:精原干细胞移植对男性不育的治疗具有潜在的临床应用价值,但移植后干细胞体内迁移、增殖、分化的过程目前尚不完全清楚。目的:观测精原干细胞移植后的体内迁移、增殖和分化过程。方法:以出生后6～10d的雄性C57BL/6小鼠为供体,通过复合酶消化、差速贴壁结合非连续性Percoll密度梯度离心的方法获取精原干细胞;以出生后6周的雄性C57BL/6小鼠为受体,腹腔注射白消安,破坏其内源性生精功能。实验组采用曲细精管微注射法将供体精原干细胞移植入受体睾丸内,对移植后细胞进行PKH26-GL荧光追踪分析,观察其体内迁移过程,以Western Blot和RFQ-PCR法检测睾丸组织α6-Integrin,c-kit,SCF蛋白及mRNA的变化。以未接受化疗和细胞移植的正常同系生小鼠作为阳性对照组,以单侧睾丸曲细精管微注射移植细胞递质作为阴性对照组。结果与结论:PKH26-GL荧光追踪移植细胞,移植后1周部分精原干细胞已向曲细精管基底膜迁移,移植后1个月精原干细胞已从曲细精管管腔迁移至曲细精管基底膜,并分裂增殖,移植后3个月曲细精管管腔内可见大量精子细胞形成。移植后1,2,3个月,各组α6-Integrin,c-kit蛋白表达均呈增加趋势(P<0.01),阴性对照组、实验组SCF蛋白表达有增加趋势(P<0.05);各组α6-Integrin,c-kit,SCF mRNA的表达均有增加趋势(P<0.05)。提示大剂量化疗后,生精上皮内仍存在一定数量的Sertoli细胞,这种精子发生的微环境并未完全破坏,外源性精原干细胞移植后能在受体Sertoli细胞所提供的微环境中增殖和分化。

BACKGROUND：Spermatogonial stem cell transplantation has potential clinical value for the treatment of male infertility. However,the process of stem cell migration,proliferation and differentiation in vivo is not yet entirely clear. OBJECTIVE：To observe the migration,proliferation and differentiation of spermatogonial stem cells in vivo after transplantation. METHODS：Using C57BL/6 mice of postnatal 6-10 days as the germ cell donors,and male germ cells were obtained by combination with compound enzymatic digestions,differential velocity adherent technique and discontinuous Percoll density gradient centrifugation. Male C57BL/6 mice of postnatal 6 weeks were used as the recipients. They had been injected busulfan intraperitoneally,which could destroy endogenous spermatogenic function. In the experimental group,spermatogonial stem cells from donors were infused into the receptor testis using seminiferous tubule microinjection. We traced the PKH26-GL fluorescent-labeled cells in the recipient testes after transplantation. The migration process was observed in vivo. Western blot and RFQ-PCR were utilized to measure α6-Integrin,c-kit,SCF protein and mRNA changes in testis tissues. Normal mice without receiving busulfan treatment and cell transplantation were selected as the parallel positive control group. Mice which were microinjected media into seminiferous tubules in unilateral testes served as the parallel negative control group. RESULTS AND CONCLUSION：Tracing the PKH26-GL fluorescent-labeled cells,partial transplanted cells immigrated into basal membrane of the seminiferous tubules at one week after transplantation. At one month after transplantation,these cells had immigrated into the basal membrane and had division growth. At 3 months,a large number of spermatid formed in the seminiferous tubule. At 1,2 and 3 months,the expression level of α6-Integrin and c-kit protein was increased gradually in each group （P 0.01）. In negative control and experimental groups,SCF protein expression levels were increased gradually （P 0.05）. α6-Integrin,c-kit and SCF mRNA expression had increased tendency in each group （P 0.05）. Results have suggested that after high-dose chemotherapy,there are a certain number of Sertoli cells in the seminiferous epithelium. This microenvironment of spermatogenesis is not been destroyed completely. Following transplantation,spermatogonial stem cells can proliferate and differentiate in microenvironment provided by recipient Sertoli cells.