摘要
目的本研究拟通过miR⁃let⁃7c抑制剂转染精原干细胞(SSCs)观察其对SSCs分化和小鼠精子质量的影响。方法SSCs从新生小鼠(3~6 d龄)的睾丸中分离出来,纯度通过流式细胞术对SSCs的标志物ID4和Thy1进行测定。细胞用miR⁃let⁃7c抑制剂转染并通过qRT⁃PCR检测转染效率,转染后48 h通过免疫组化和Western blot评估SSCs的增殖(GFRa1、PLZF和ID4)和分化(C⁃Kit)标志物的表达水平。体内实验中,60 mg/(kg·d)腹腔注射环磷酰胺被用于制备小鼠少弱精子症模型,附睾内注射转染miR⁃let⁃7c抑制剂的SSCs,以观察生精细胞、间质细胞等变化和分析精子浓度、活力及形态。结果miR⁃let⁃7c抑制剂组转染后SSCs分化标志物的表达明显升高,GFRa1、PLZF和ID4蛋白降低,并显著提高少弱精子症小鼠的精子浓度、精子活力和正常形态率。结论miR⁃let⁃7c抑制剂促进精原干细胞分化并改善小鼠精子质量。
Objective To observe the effect of miR⁃let⁃7c inhibitor on spermatogonial stem cells(SSCs)by transfecting it on the differentiation of SSCs and sperm quality in mice.Methods SSCs were isolated from testes of neonate mice(3~6 days old),and their purities were performed by flow cytometry with ID4 and Thy1 markers.Cultured cells were transfected with miR⁃let⁃7c inhibitor.Evaluation of the proliferation(GFRa1,PLZF,and ID4)and differentiation(C⁃Kit)markers of SSCs were accomplished by immunocytochemistry and western blot 48h after transfection.In vivo experiments,60 mg/(kg·d)intraperitoneal injection of cyclophosphamide was used to prepare a mouse model of oligoasthenospermia,and SSCs transfected with miR⁃let⁃7c inhibitor were injected into the epidid⁃ymis to observe the changes of spermatogenic cells and interstitial cells and analyze sperm concentration,vitality and morphology.Results The expression of differentiation markers of SSCs after transfection in the miR⁃let⁃7c inhibitor group was significantly increased;the protein of GFRa1,PLZF,and ID4 was decreased,and the sperm concentration,sperm motility and regular morphological rate of oligoasthenospermia mice were significantly in⁃creased.Conclusion miR⁃let⁃7c inhibitor promotes the differentiation of spermatogonial stem cells and improves sperm quality in mice.
作者
王明
萨如拉
王超奇
燕贞
WANG Ming;SA Rula;WANG Chaoqi;YAN Zhen(Reproductive Medicine Center,Affiliated Hospital of Inner Mongolia University for Nationalities,Tongliao 028000,China;不详)
出处
《实用医学杂志》
CAS
北大核心
2022年第20期2524-2530,共7页
The Journal of Practical Medicine
基金
国家自然科学基金(编号:81860583)。
