人脐带间充质干细胞及其外泌因子对白消安致雄性小鼠生殖损伤的保护作用

Protective effects of human umbilical cord mesenchymal stem cells and its exocrine factor on reproductive damage induced by busulfan in male mice

目的探讨人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,HUMSCs)及其外泌因子对白消安(busulfan,BSF)致雄性小鼠生殖损伤的保护作用。方法将HUMSCs无血清培养48 h获得条件培养基(conditioned medium,CM),超滤浓缩为原培养基25倍。将BSF(40 mg/kg)通过腹腔注射小鼠,处理后第2天,将浓缩后的条件培养基注射至小鼠尾静脉,同时设对照组(生理盐水)、BSF组和HUMSCs组,治疗频次为每周2次,连续4周。取小鼠一侧睾丸,苏木素-伊红染色观察切片组织生精小管基底膜细胞分离情况和基底腔空泡形成情况;实时荧光定量PCR(qPCR)检测小鼠生精细胞减数分裂相关基因mRNA的表达水平;Western blot分析小鼠睾丸组织中细胞黏附相关蛋白的表达水平。结果与其他组相比,HUMSCs-CM组小鼠睾丸损伤延迟,包括生精上皮基底膜细胞形成空泡和基底膜细胞脱离;HUMSCs-CM组小鼠生精细胞减数分裂基因STRA8、TNP2、SCP3 mRNA表达水平显著增加(P<0.05),小鼠睾丸组织中N-钙黏蛋白(N-cadherin)、细胞间黏附分子-1(intercellular adhesion molecule-1,ICAM-1)和P-钙黏蛋白(P-cadherin)表达水平显著增加(P<0.05)。结论HUMSCs外泌因子通过减少生精细胞凋亡和增强细胞间的黏附作用保护BSF致精子发生的损害,上调黏附分子的表达。

Objective To investigate the protective effects of human umbilical cord mesenchymal stem cells(HUMSCs)and its exocrine factor on reproductive damage induced by busulfan(BSF)in male mice.Methods Umbilical cord mesenchymal stem cells were cultured for 48 h without serum to obtain conditioned medium(CM),which was concentrated by ultrafiltration to 25 times of the original medium.Mice were injected i.p.with BSF at a dosage of 40 mg/kg,using normal saline as normal control.On the second day after injection with BSF,the mice were injected i.v.with the concentrated CM(HUMSCs-CM group),HUMSCs(HUMSCs group)and normal saline(BSF group)respectively,twice a week for 4 weeks.Unilateral testicle of mice was prepared into sections and observed for the isolation of seminiferous tubule basement membrane cells and formation of basal cavity vacuoles by hematoxylin-eosin staining.The expression level of mRNA of genes associated with meiosis of spermatogenic cells were determined by real-time fluorescent quantitative PCR(qPCR),while those of cell adhesion-associated proteins in testicle tissue were analyzed by Western blot.Results Compared with those in other groups,the testicular damage in HUMSCs-CM group was delayed,including vacuoles formed by basement membrane cells of seminiferous epithelium and detachment of basement membrane cells.The expression levels of mRNAs of meiosis genes STRA8,TNP2 and SCP3 in spermatogenic cells as well as those of Ncadherin,intercellular adhesion molecule-1(ICAM-1)and P-cadherin in testicle tissue in HUMSCs-CM group increased significantly(each P<0.05).Conclusion The exocrine factor of HUMSCs protected the BSF-induced damage of spermatogenesis and the expression of adhesion molecules by reducing the apoptosis of spermatogenic cells and enhancing the adhesion between cells.