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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 the effect of leuprolide (GnRH-agonist) on the cloning efficiency of spermatogonial stem cells (SSCs) in recipient mice after transplantation. METHODS: Recipient mice were treated subcutaneously with leuprorelin (7.6 mg / kg) or its vehicle (saline) and then transplanted with donor SSCs (labeled with GFP) into the testis via the testis Amine-treated recipient testicular seminiferous tubules, respectively, at 1 week and 4 weeks after transplantation samples were observed under a fluorescence microscope donor cells in the testis seminiferous tubules of the amount of homing, donor cell-derived The number of testes and the number of seminiferous tubules. The expression of β1-integrin mRNA in testis tissue was detected by RT-PCR at 1 and 4 weeks after transplantation. Results: After 1 week of SSCs transplantation, the numbers of homing SSCs in seminiferous tubules of leuprolide-treated group (experimental group) were 20.4 ± 4.3 / seminiferous tubules, which was significantly higher than that of control group (11.5 ± 3.8 / seminiferous tubules) (P <0.05). At 4 weeks after transplantation, the green fluorescent seminiferous tubules (24.1 ± 4.5%) in the treated group were also significantly higher than those in the control group (13.4 ± 5.3% , P <0.05). Reverse transcriptase-PCR was used to detect the mRNA expression of β1-integrin in testis of test group (P <0.05), and the level of β1-integrin in test group was significantly increased at 1 week after transplantation No significant difference (P> 0.05). CONCLUSIONS: GnRH-agonists play an important role in homing of SSCs and suggest that the homing effect may be mediated by β1-integrin so that the number of SSCs in the recipient mouse seminiferous tubules remains constant ( 4 weeks after transplantation) maintained a quantitative advantage over the control group.