月经血干细胞对薄型子宫内膜大鼠的治疗修复作用

Therapeutic effect of menstrual blood stem cells in rats with thin endometrium

目的:探究月经血干细胞(MenSC)移植对薄型子宫内膜的治疗修复作用及机制。方法:选取8~10周龄无特定病原体级雌性SD大鼠30只,随机分为模型对照组和MenSC组,每组各15只。两组通过化学损伤法在大鼠一侧子宫内膜建立薄型子宫内膜损伤模型,建模7 d后分别将等渗氯化钠溶液及MenSC多点注入大鼠损伤侧子宫内,另一侧子宫作为手术对照组不做任何处理。第三代传代MenSC治疗薄型子宫内膜模型大鼠。苏木精-伊红染色观察子宫内膜组织结构;免疫组织化学染色观察子宫内膜组织细胞角蛋白(CK)18、波形蛋白表达;5-乙炔基-2’-脱氧尿嘧啶核苷(EdU)法检测子宫内膜组织中细胞增殖情况;免疫荧光染色检测子宫内膜组织血管内皮标志物CD34和血管内皮生长因子(VEGF)的表达;实时逆转录PCR检测子宫内膜组织白血病抑制因子(LIF)、整合素β3(ITGβ3)及同源异型框A10(HOXA10)的基因表达。两组分别按雌鼠和雄鼠2∶1比例进行合笼实验,观察MenSC对模型大鼠生殖功能的影响。结果:与手术对照组比较,模型对照组子宫内膜薄,腺体及血管数少(均P<0.01),MenSC移植后大鼠子宫内膜厚度、血管数、腺体数明显增加(均P<0.01)。MenSC组子宫内膜基底层增殖细胞较模型对照组多(P<0.05),且MenSC组子宫波形蛋白、CK18、CD34及VEGF的表达均明显高于模型对照组(均P<0.05),LIF、ITGβ3及HOXA10基因表达水平也较模型对照组明显升高(均P<0.05)。妊娠实验结果显示,MenSC组较模型对照组胚胎种植数增加(P<0.05)。结论:MenSC移植通过促进子宫内膜细胞增殖和子宫内膜波形蛋白、CK18、CD34及VEGF表达以利于模型大鼠子宫宫腔形态和功能恢复,从而提高模型大鼠子宫内膜容受性,改善模型大鼠的生育能力。

Objective:To explore the therapeutic effect of transplantation of menstrual blood stem cells(MenSCs) in rats with thin endometrium.Methods:Thirty SPF grade female SD rats aged 8-10 weeks were randomly divided into model control group and MenSC group,with 15 rats in each group.The thin endometrium injury model was prepared by chemical method in one side of the uterus of both groups.On the 7th day of modeling,normal saline or the third generation of MenSCs were injected into the model uterus at multiple points,and the other side of the uterus was used as an internal control without treatment.HE staining was used to observe the histological structure of endometrium;immunohistochemical staining was used to observe the expression of cytokeratin(CK) 18 and vimentin in endometrial tissue;5-ethynyl-2'-deoxyuridine(EdU) cell proliferation assay was used to evaluate the cell proliferation in endometrial tissue;immunofluorescence staining was used to detect the expression of vascular endothelial marker CD34 and vascular endothelial growth factor(VEGF) in endometrial tissue;the expression of leukemia inhibitory factor(LIF),integrin β3(ITGβ3) and homeobox A10(HOXA10) in endometrial tissue was determined by realtime RT-PCR.After treatments,the female and male rats were caged in a ratio of 2∶1 to observe the effect of MenSC on the reproductive function of thin endometrium model rats.Results:Compared with the surgical control group,the endometrium in the model control group was thinner,and the numbers of glands and blood vessels were less(all P<0.01).After MenSC transplantation,the thickness of endometrium,the numbers of blood vessels and glands were significantly increased(all P<0.01).The proliferative cells in the basal layer of endometrium in MenSC group were more than those in the model control group(P<0.05),and the expression of vimentin,CK18,CD34 and VEGF in the uterus of rats in MenSC group were significantly higher than those in the model control group(P<0.05).LIF,ITGβ3 and HOXA10 gene expression levels were also significantly higher than those in the model control group(all P<0.05).The results of pregnancy experiment showed that the number of embryo implantation in MenSC group was higher than that in model control group(P<0.05).Conclusion:MenSC transplantation can promote the proliferation of endometrial cells,upregulate vimentin,CK18,CD34 and VEGF levels,and promote the recovery of endometrial morphology and function,thus improving the endometrial receptivity and fertility of the rats with thin endometrium.