亮丙瑞林联合视黄酸辅助小鼠骨髓间充质干细胞向雄性生殖细胞分化

Combination of leuprorelin acetate and retinoic acid induces bone marrow mesenchymal stem cells differentiate into male germ cells

目的研究亮丙瑞林联合视黄酸诱导小鼠骨髓间充质干细胞向雄性生殖细胞方向分化的可行性。方法 66只C-kit W/Wv成年小鼠随机分成两组,实验组小鼠给予亮丙瑞林（7.8 mg.kg-1）肌肉注射1周,对照组给予同等体积生理盐水注射1周。小鼠骨髓间充质干细胞体外分别给予2μmol＂L-1视黄酸（视黄酸组）或0.1%二甲基亚枫（二甲基亚枫组）,诱导定向分化为生殖细胞。使用流式细胞仪分选处理后的细胞,筛出阶段特异性抗原1阳性的细胞即为原始生殖细胞,通过睾丸网显微注射技术将其注入实验组和对照组小鼠生精小管中。在注射后7~70 d收集睾丸组织,采用RT-PCR方法检测生殖细胞特异性标志物,如视磺酸诱导基因、联会复合体蛋白3及过渡蛋白1基因的表达。通过对移植后120d小鼠睾丸组织进行HE染色,观察生精小管中生精细胞分化。结果视黄酸作用后的小鼠骨髓间充质干细胞,阶段特异性抗原1（SSEA1）阳性细胞分选率为（1.50±0.29）%,二甲基亚枫作用后分选率为（0.01±0.01）%,两者差异有统计学意义（P〈0.05）。实验组小鼠睾丸组织移植后7d开始表达视磺酸诱导基因,移植后70d表达联会复合体蛋白3及过渡蛋白1基因,对照组小鼠睾丸组织中未见明显的生殖细胞基因表达。移植后120d实验组小鼠睾丸组织HE染色显示部分生精小管中管腔中可见精子发生过程,并可见少量单倍体精子细胞。结论亮丙瑞林可促进视黄酸诱导小鼠骨髓间充质干细胞通过减数分裂,并形成单倍体精子细胞。

Objective To explore the feasibility of Leuprorelin Acetate combined with retinoic acid （ RA ） can inducing bone mar-row mesenchymal stem ceils （BMMSCs） differentiate into male germ cells. Methods 66 adult C-kit W/Wv mice were treated with 7.8 mg·kg^-1 leuprorelin Acetate or saline for 1 week. Embryoid Bodys（EB） derived from BMMSCs were treated with culture medium containing 2μmol·L^-1RA or 0.1% DMSO. SSEA1 （ stage specific embryonic antigen 1 ） positive ceils were isolated by fluorescence activated cell sorting （FACS）. The SSEAI positive cells were injected via the rete testis into the mice seminiferous tubules after treated by leuprorelin or saline. RT-PCR was used to detect the expression of germ cell special markers. HE staining was used to investigated the expression of male germ cells. Results Significantly differences were observed in the percentage of PGC （SSEA1 positive cells） between the RA group and DMSO group （RA group （1.5±0.29） %, DMSO group （0.01±0.01）%. Stra8 expressed in 7 days after transplantation; Scp3 and Tpl expressed in 70 days＇ after transplantation. Moreover, 120s after transplantation, the haploid cells can be found in the center of seminiferous tubules. Conclusion Combination of leuprorelin acetate and retinoic acid induces BMMSCs differentiate into male germ ceils.