睾丸生精小管干细胞移植受体模型建立与移植技术改进  

Establishment of Recipient Mouse Model of Stem Cell Transplantation into Testicular Seminiferous Tubules and Improvement of Transplantation Techniques

作　　者：张伟[1] 夏小雨[2] 丁慧[1] 刘平[1] 蔡恒[2] 李朋[1] 徐晨[2] 李铮[1] 

机构地区：[1]上海交通大学医学院附属仁济医院泌尿外科,上海交通大学Med-X/仁济医院干细胞临床研究中心,上海200001 [2]上海交通大学医学院生殖医学重点实验室,上海200025

出　　处：《中华男科学杂志》2009年第8期703-707,共5页National Journal of Andrology

摘　　要：目的:探讨建立稳定的、适合干细胞睾丸生精小管移植的受体小鼠模型和移植技术改进。方法:将雄性ICR小鼠60只随机分为A、B、C、D4组,每组15只。A、B、C组注射白消安的剂量分别为15、30、40mg/kg体重,D组注射50%二甲亚砜作为对照。注射后常规饲养,观察小鼠死亡情况。于注射后4、8、12周称量各组小鼠的睾丸重量,制作睾丸石蜡切片,观察生精小管的中空率和生精上皮结构变化。自主设计干细胞移植装置,改进干细胞移植技术。该装置通过三通连接装置将口含端、注射器端及穿刺端连接在一起,口含端负责试探性的注射,注射器端负责抽吸细胞悬液和注射。结果:注射后只有C组出现1只小鼠死亡。4周时A、B、C组睾丸重量的变化,与作为对照的D组相比有显著性差异(P<0.05);8周时A组与D组无差异(P>0.05),而B、C组与D组差异仍有显著性(P<0.05);12周时各组与D组均无显著性差异(P>0.05)。4、8周时A组的生精小管中空率<50%,B、C组中空率都在50%以上,而D组无明显改变;12周时A、B、C组无显著性差异(P>0.05),恢复近正常状态。自主设计的三通式注射装置注射成功率高,可达90%以上;细胞浪费少,注射双侧睾丸所需细胞悬液量<50μl;所需时间短,注射一侧睾丸<10min。结论:雄性ICR小鼠腹腔内单次注射30mg/kg体重白消安,无小鼠死亡,且4周后生精小管的中空率高(均>50%),适合作为干细胞移植的模型。改进后的移植装置和方法,提高了移植的效率和成功率。为探讨干细胞向雄性配子发育研究提供了简单易学、适于推广的新技术。Objective： To establish a stable recipient mouse model for stem cell transplantation into seminiferous tubules and improve the traditional techniques for transplantation. Methods： Sixty male ICR mice were equally divided into Groups A, B, C and D, and injected with Busulfan at 15 mg/kg, 30 mg/kg, 40 mg/kg and 0 mg/kg, respectively. The survival rate was recorded every day, and the testis weight and spermatogenesis of testicular tubules were determined at 4, 8 and 12 weeks after the injection. We improved the stem cell transplantation technique and designed a new transplantation device, which connected the nozzle end, syringe and puncture needle by a three-way joint. The nozzle end was used for tentative injection, and the syringe for drawing and then injecting the cell suspension. Results： Only one mouse in Group C died after the injection. At 4 weeks after Busulfan treatment, the testis weight decreased apparently in Groups A, B and C, with significant differences from D （ P 〈 0.05 ）. The differences remained significant at 8 weeks （P 〈 0.05 ）, except between Groups A and D （P 〉 0.05 ）, but at 12 weeks none of the first three groups showed any significant difference from Group D （ P 〉 0.05 ）. At 4 and 8 weeks, the rate of hollow seminiferous tubules was 〈 50% in Group A and 〉 50% in Groups B and C, and almost returned to normal at 12 weeks, with no significant differences among the three groups （P 〉 0.05 ）, but it remained unchanged in Group D. The improved transplantation device increased the success rate （ 〉 90% ） , lowered the donor cell loss （ 〈50 μl cell suspension needed for both testes） and shortened the process （ 〈 10 rain for one testis）. Conclusion： Intraperitoneal injection of Busulfan at 30 mg/kg is suitable for the establishment of the recipient mouse model of stem cell transplantation. The improved transplantation device and methods help promote the efficiency and success rate of the transplantation operation.

关 键 词：干细胞移植 白消安 小鼠 精子发生障碍模型 

分 类 号：R-332[医药卫生] R699