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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 study the feasibility of leuprolide combined with retinoic acid in inducing mouse bone marrow mesenchymal stem cells to differentiate into male germ cells. Methods Sixty-six adult C-kit W / Wv mice were randomly divided into two groups. The mice in the experimental group were given intramuscular injection of leuprolide (7.8 mg.kg-1) for 1 week, while the control group were given the same volume of normal saline for 1 week. Mice bone marrow mesenchymal stem cells were treated with 2μmol ”L-1 retinoic acid (retinoic acid group) or 0.1% dimethyl sulfoxide (dimethyl sulfoxide group) in vitro to induce directional differentiation into germ cells. Cells sorted by flow cytometry, the stage of specific antigen-1 positive cells that are primordial germ cells, injected into the seminiferous tubules of experimental mice and control mice by testicular microsurgical injection. Testicular tissues were harvested from 7 to 70 days after injection, and germ cell specific markers such as visual acyl sulfonate induced gene, synaptophysin 3 and transitional 1 gene expression were detected by RT-PCR.After 120 days of transplantation The testicular tissue was stained with hematoxylin and eosin (HE) to observe the differentiation of spermatogenic cells in the seminiferous tubules.Results The percentage of stage-specific antigen 1 (SSEA1) -positive cells was (1.50 ± 0.29) in mouse bone marrow mesenchymal stem cells ), Respectively, the sorting rate was (0.01 ± 0.01)% after the dimethyl sulfoxide treatment, the difference was statistically significant (P <0.05) .After 7 days of testicular tissue transplantation, , 70 days after transplantation, the expression of Synaptophysin 3 and transitional 1 gene was small in the control group There was no obvious germ cell gene expression in the testis tissue of mice.Hematoxylin and eosin staining of the testes in the test group 120 days after transplantation showed that spermatogenesis occurred in the lumen of some seminiferous tubules and a small amount of haploid spermatid was observed.Conclusion: Raylin can promote the retinoic acid-induced mouse mesenchymal stem cells to undergo meiosis and to form haploid sperm cells.