摘要
目的:探讨小鼠精原干细胞移植的关键技术,寻求有效可行的精原干细胞移植方法。方法:选用4-5周龄雄性BALB/c小鼠45只腹腔内注射40 mg/kg的白消安建立受体模型,消化分离新生绿色荧光小鼠睾丸精原细胞作供体细胞。在三维显微注射系统下分别采用睾丸网注射法(n=15),输出小管注射法(n=30),经输出小管注射失败后的补救方式为经输出小管-睾丸网注射,将供体细胞注射到受体小鼠左侧睾丸精曲小管内,右侧睾丸作为对照。观察注射后精曲小管的充盈度和外漏的情况;移植后2个月观察受体小鼠的生精功能恢复情况。结果:经睾丸网注射成功率仅为35.7%(5/14),其中9只因睾丸网穿破、细胞悬液外漏到间质而失败。经输出小管注射成功率为72.4%(21/29),失败的主要原因为穿刺过程中穿破输出小管,进一步采用经输出小管-睾丸网注射补救,总成功率提高为93.1%(27/29),注射成功后可见70%-80%的精曲小管被蓝染的细胞悬液充盈,均无外漏现象。移植后2个月,体视荧光显微镜下可见移植侧睾丸包含多段绿色阳性克隆;组织学染色示移植侧睾丸大部分精曲小管恢复正常生精。结论:经输出小管和经输出小管-睾丸网显微注射技术是小鼠精原干细胞移植的有效方法。
Objective： To search for an effective method for transplanting mouse spermatogonial stem cells （SSCs）. Methods： Forty-five adult male BALB/c mice, aged 4-5 weeks, were injected intra- peritoneally with of 40 mg/kg busulfan as recipient mice. Donor cells were collected from newborn transgenic mice that expressed EGFP by modified two-step enzymatic digestion method. Fifteen recipient mice underwent SSCs transplantation using rete injection with micromanipulator. Another 30 recipient mice underwent efferent duct injection. Efferent duct-rete injection was used as a remedy when efferent duct injection failed. The left testis of all recipient mice received injection and right testis were set as self-control. Transplantation efficiency was evaluated by observ-ing seminiferous tubules two months after transplantation. Results： For rete injection, the success rate was only 35.7%（5/14）, the rete was easily be penetrated which led to the donor cell suspension leaking into intertubular tissue. Efferent duct injection was fast and reliable, and the success transplantation rate was 72. 4%, and when the efferent duct-rete injection was taken as its remedy, the success rate was further increased to 93.1%. Two months after transplantation,green positive colonization was confirmed by fluorescence stereomicroscope, and histological ex- amination showed most of seminiferous tubules from left testis had apparently normal spermatogenesis. Conclusion： Rather than the rete injection, efferent duct-rete injection can significantly improve the transplantation efficiency for SSCs transplantation.
出处
《武汉大学学报（医学版）》
CAS
北大核心
2009年第4期518-521,I0004,共5页
Medical Journal of Wuhan University
基金
国家自然科学基金资助项目(编号:30400160)
关键词
精原干细胞
移植
小鼠
Spermatogonial Stem Cell
Transplantation
Mouse
