亮丙瑞林可促进供体精原干细胞(SSCs)在受体小鼠睾丸的归巢效率

Enhancement of Donor Cell Colonizations in Seminiferous Tubules of Recipient by Leuprolide after Spermatogonial Stem Cells Transplantation is Probably through Promoting the Homing Process in Mice

目的:研究亮丙瑞林(GnRH-激动剂)处理受体小鼠促进精原干细胞(SSCs)移植后克隆率的作用时间、方式。方法:使用亮丙瑞林(7.6 mg/kg)或其载体剂(生理盐水)皮下注射处理受体小鼠,然后将供体SSCs(具有GFP标记)通过睾丸输出管注射法移植到经白消胺处理过的受体睾丸曲细精管内,分别于移植后1周和4周采样,在荧光显微镜下观察供体细胞在受体睾丸曲细精管中的归巢数量,供体细胞来源的睾丸数量及曲细精管数量;反转录PCR检测移植后1周和4周睾丸组织中β1-整合素(β1-integrin)mRNA表达水平。结果:在SSCs移植1周后,显微镜下可见亮丙瑞林处理组(实验组)曲细精管中SSCs的归巢数量为20.4±4.3个/曲细精管,显著高于对照组(11.5±3.8个/曲细精管)(P<0.05);在SSCs移植后4周,处理组显示绿色荧光的曲细精管比率(24.1±4.5%)也显著高于对照组(13.4±5.3%,P<0.05)。用反转录PCR检测mRNA表达量,在移植后1周时实验组小鼠睾丸内β1-整合素明显增加(P<0.05),而在移植后4周时β1-整合素与对照组相比无显著差异(P>0.05)。结论:GnRH-激动剂对SSCs的归巢具有重要促进作用,并且提示其归巢作用的发生可能是通过β1-整合素来完成的,从而使SSCs在受体小鼠曲细精管的数量一直(到移植后4周)保持着高于对照组的数量优势。

Objective： To study effects of the GnRH-agonist leuprolide on homing of donor derived spermatogonial stem cells（SSCs） in recipient testes after SSCs transplantatin in mice.Methods： Recipient mice were injected with GnRH-agonist leuprolide（7.6 mg/kg,i.h.） or physiological saline subcutaneously.The donor GFP cell suspensions were introduced by efferent duct injection into seminiferous tubules of recipient mice pretreated with busulfan for eliminating the endogenous SSCs.At 1 week and 4 weeks after transplantation,numbers of donor cell-derived（GFP） and clones in seminiferous tubules in recipients were observed by fluorescence stereomicroscope.The number of homing SSCs was evaluated by counting the number of seminiferous tubules with GFP under fluorescence microscope.Meanwhile,mRNA expressions of b1-integrin in recipient testes at above time points were examined using RT-PCR.Results： At 1 week after the transplantation,the number of homing SSCs（GFP） in seminiferous tubules of recipient mice treated with leuprolide was significantly（P0.05） higher（20.4±4.3） than that of sham treatment（11.5±3.8）.At the same time,mRNA expressions of b1-integrin in recipient testes with leuprolide treatment group were also significantly increased compared with that in sham group（P0.05）.At 4 weeks after transplantation,percentage of seminiferous tubules with GFP cells in recipient mice pretreated with leuprolide was significantly elevated（24.1±4.5% vs 13.4±5.3%）（P0.05）.However,in this time there was no significant difference（P0.05） in mRNA expression of b1-integrin between leuprolide treated and nontreated groups.Conclusion： Enhancement of SSC in recipient testes by leuprolide administration after transplantation was through the promoting SSC homing process,at least,probably with increasing b1-integrin expression in recipient testes.