体外培养方法诱导大鼠精母细胞减数分裂

Rat Spermatocytes to Meiosis Induced by Culture in vitro

目的:探讨大鼠睾丸精母细胞在体外减数分裂、分化为精子细胞或精子的可能性。方法:取出生后20-22dWistar大鼠睾丸组织,经胶原酶消化分离细胞,分别用含2%胎牛血清(FBS)的DMEM/F12培养基(对照组)和含2%FBS的DMEM/F12培养基+多种性激素+多种细胞生长因子等(实验组)作体外培养,于培养后d3、d7、d14收集细胞,采用流式细胞技术,检测培养前后生精细胞染色体倍体的变化;RT-PCR法检测精子细胞特异性基因过渡蛋白1(TP1)mRNA的表达。结果:培养d2,可见支持细胞贴壁生长,生精细胞粘附于支持细胞表面,实验组生精细胞存活率>90%,对照组<5%;培养14d,实验组可见长有鞭毛的长形精子细胞。流式细胞检测结果显示,培养前生精细胞为二倍体和四倍体细胞群,培养d14实验组单倍体细胞占总细胞数的6.2%,对照组未见单倍体峰。RT-PCR结果显示与流式细胞技术检测结果一致:培养前生精细胞未检测到TP1mRNA的表达,培养7d后实验组可检测到该基因的表达,培养14d,其表达量显著增加。结论:采用生精细胞与支持细胞混合培养并在培养液中添加性激素、细胞生长因子等的方法,大鼠精母细胞可以在体外进行减数分裂,分化成长形精子细胞。

Objective： To explore the possibility of in vitro differentiation from sperrnatocytes to sperrnatids in rats. Methods： Testicular tissues from Wistar rats （20-22 d-old） were sheared and digested by collagenase at 34℃ for 15-30 min. Spermatogenic and Sertoli cells were co-cultured in 6-well dishes. The cells were divided into control （DMEM/F12 ＋ 2% FBS） and experimental group （DMEM/F12 ＋ 2%FBS, supplemented with sex homrmone＋cell factors, etc）. The cells were harvested on d 3, d 7, d 14 and used for the following experiments. Chromosome ploidy of the spermatogenic cells was analyzed by flow cytometry （FCM） and spermatid specific gene transition protein 1 （TP1） was detected in the cultured cells by RT-PCR. Results： On d 3 of culture, Sertoli cells grew to form a single layer on the plate, and spermatogenetic cells loosely placed on the surface of Sertoli cells. Trypan blue exclusion showed that the survival rate of the spermatogenetic cells maintained to exceed 90% but less than 5% in experimental group and control group, respectively. FCM analysis indicated that only could 4N cells （sperrnatogonia and spermatocytes） and 2N cells （spermatogonia） were detected before the culture. On d 14, 1N cell population reached about 6.2% of total cells in experimental group. Expression of TP1 mRNAs could not be detected by RT-PCR before the culture. The gene was detected on d 7 and significantly increased on d 14. Conclusion： With the co-culture system, rat spermatocytes could complete the meiotic process in vitro and differentiate into enlonged spermatids.