白消安处理消融猪内源精原干细胞的效果及外源精原干细胞移植

Ablation effect of busulfan on pig endogenous spermatogonial stem cells and transplantation of exogenous spermatogonial stem cells

【目的】研究猪睾丸组织注射白消安消融猪内源精原干细胞(Spermatogonial stem cell,SSC)的效果,以及猪SSC同种异体移植后对内源性SSC消融受体生殖能力恢复的影响。【方法】采用3 mg/kg剂量的白消安对9头6周龄大白公猪进行睾丸注射,另外3头注射2 mL二甲基亚砜作为对照。3周后,对试验组公猪以相同剂量进行第2次睾丸注射。第2次注射3周后,采集试验组和对照组公猪睾丸进行相关检测,评估内源性SSC消融情况。第2次白消安注射1个月后,以两步酶消化法处理5~7日龄大白仔猪睾丸分离得到睾丸单细胞悬液,并用明胶差速贴壁法进行纯化,纯化后以免疫荧光和流式细胞术分析SSC的纯度。异体移植SSC 4个月后,用微卫星标记检测受体公猪精液以及睾丸组织中供体来源SSC的存在。【结果】以3 mg/kg剂量的白消安处理大白公猪2次后,睾丸组织苏木精−伊红染色以及免疫组织化学染色结果显示,试验组睾丸曲细精管中各级生精细胞消融但其支持细胞结构完好,可以支持外源性SSC的定植及发育。免疫荧光以及流式细胞术结果表明,分离得到的睾丸单细胞悬液经纯化后UCHL-1阳性细胞占比由差速贴壁前的16.3%提高到了50.8%。苏木精−伊红染色以及免疫组织化学染色结果显示,猪SSC移植4个月后,移植组睾丸组织生精细胞恢复,在受体睾丸曲细精管基底膜上可检测到UCHL-1阳性SSC。受体睾丸组织的微卫星标记分析显示了供体SSC的存在,表明移植进入受体睾丸中的供体SSC可以在受体睾丸中定植存活超过4个月;精液微卫星标记未检测到供体来源的精子。【结论】以3 mg/kg剂量的白消安注射公猪睾丸能有效消融内源性SSC,可以用来制备SSC移植的受体猪。两步酶消化及明胶差速贴壁法可成功分离纯化猪SSC。猪SSC经同种异体移植后可以在受体睾丸中定植存活超过4个月。

【Objective】To study the effect of intratubular injection of busulfan on ablation of pig endogenous spermatogonial stem cells(SSCs)and allotransplantation of SSCs on the recovery of the reproductive ability of the recipient after ablation of endogenous SSCs.【Method】Nine 6-week-old Large-white boars were injected through seminiferous tubules with busulfan at a dosage of 3 mg/kg,and other three pigs were injected with 2 mL DMSO as controls.After three weeks,each boar in the test group was given a second injection at the same dosage.At three weeks after the second injection,testes from test and control groups were collected to evaluate the endogenous SSC ablation conditions.At one month after the second busulfan injection,testicular single-cell suspension was isolated from the testes of 5−7-day-old Large-white piglets by two-step enzyme digestion method and purified by gelatin differential adherence method.After purification,the purity of SSCs was analyzed by immunofluorescence and flow cytometry.At four months after allotransplantation of SSCs,microsatellite marker analysis was used to detect the presence of donor-derived SSCs in recipient boar semen and testicular tissue.【Result】After treating the Large-white boar twice with busulfan at a dosage of 3 mg/kg,the results of HE staining and immunohistochemistry staining of testis tissue showed that germ cells at all levels in the seminiferous tubules of the testes in test group were ablated but the structure of sertoli cell was intact,which could support the colonization and development of transplanted exogenous SSC.The results of immunofluorescence and flow cytometry showed that the rate of UCHL-1 positive cells in the isolated testicular single-cell suspension increased from 16.3%before differential adhesion to 50.8%after purification.At four months after allotransplantation of porcine SSC,HE staining and immunohistochemical staining of testicular tissues in the transplanted group showed recovery of germ cell layers compared with the group injected with busulfan but not transplanted with donor cells.UCHL-1 positive SSCs were detected on the basement membrane of seminiferous tubules in recipient testes.Microsatellite marker analysis of recipient testis tissue showed the presence of donor SSCs,suggesting that donor SSCs transplanted into recipient testes could colonize and survive in recipient testes for more than four months.However,microsatellite marker analysis of semen did not detect donorderived sperms.【Conclusion】Intratubular injection of busulfan into boar testes at a dosage of 3 mg/kg can effectively ablate endogenous SSCs and can be used to prepare recipient pigs for SSC transplantation.Two-step enzyme digestion and gelatin differential adhesion method can be used to successfully isolate and purify porcine SSCs.Porcine SSCs can colonize in recipient testes and survive for more than four months after allotransplantation.