人羊膜间充质干细胞改善小鼠自身免疫性卵巢功能不全及子宫内膜容受性

Human amniotic mesenchymal stem cells improve ovarian function and endometrial receptivity in mice with autoimmune premature ovarian insufficiency

目的探索人羊膜间充质干细胞(human amniotic mesenchymal stem cells,hAMSCs)对自身免疫性早发性卵巢功能不全(premature ovarian insufficiency,POI)小鼠卵巢功能和子宫内膜容受性的影响。方法使用透明带3多肽-弗氏免疫佐剂诱导小鼠自身免疫性POI,一次性尾静脉注射移植hAMSCs 1×106细胞/只,将动物分为正常组(n=10)、模型组(n=15)、hAMSCs组(n=15),阴道涂片监测动情周期,酶联免疫法检测血清卵泡刺激素(follicle-stimulating hornome,FSH)、雌二醇与抗苗勒管激素(anti-Müllerian hormone,AMH)含量,HE染色观察卵巢和子宫病理组织学变化,免疫组织化学检测子宫同源框A10(homeobox A10,HOXA10)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和FSH受体(FSH receptor,FSHR)蛋白表达,综合评价子宫内膜容受性。结果hAMSCs移植6周,hAMSCs组动情周期异常率[40.00%(6/15)]低于模型组[86.67%(13/15),P=0.021]。与模型组血清FSH[(10.239±1.091)μg/L]、雌二醇[(103.325±4.952)ng/L]和AMH[(1.133±0.494)μg/L]水平相比,hAMSCs组FSH水平[(7.664±0.735)μg/L]明显降低(P<0.001),雌二醇[(126.883±23.370)ng/L]和AMH[(2.204±0.453)μg/L]水平均明显升高(P=0.015,P<0.001)。与模型组不同,hAMSCs组卵巢指数、子宫指数增高;卵巢组织不同发育阶段的卵泡数增加,间质纤维化和闭锁卵泡少见,子宫壁与子宫内膜增厚,腺体数量和体积增加。HOXA10吸光度(absorbance,A)值hAMSCs组(5.90±1.94)高于模型组(2.79±1.27,P=0.029),TNF-αA值hAMSCs组(3.83±1.23)低于模型组(6.26±0.96,P=0.002);FSHR A值hAMSCs组(3.61±1.66)与模型组(2.74±0.22)差异无统计学意义(P>0.05),但模型组低于正常组(4.13±0.54,P=0.006)。结论移植hAMSCs在恢复自身免疫性POI小鼠卵巢功能的同时,可明显改善子宫内膜容受性,有助于提高生育能力。

Objective To investigate the effect of human amniotic mesenchymal stem cells(hAMSCs)on ovarian function and endometrial receptivity in mice with autoimmune premature ovarian insufficiency(POI).Methods POI mouse was induced by treatment with zona pellucida 3 polypeptide fragment-Freund immune adjuvant.The animals were divided into normal group(n=10),model group(n=15)and hAMSCs group(n=15).hAMSCs(1×106 cells/mouse)were transplanted by tail vein single injection.The oestrus cycles were evaluated by vaginal smears.The levels of follicle-stimulating hormone(FSH),estrogen and anti-Müllerian hormone(AMH)in serum were detected by enzyme-linked immunosorbent assay methods.Morphological changes of ovarian and uterus tissues were observed after HE staining.The expressions of homeobox A10(HOXA10),tumor necrosis factor-α(TNF-α)and FSH receptor(FSHR)proteins in uterine were measured by immunohistochemistry.The endometrial receptivity was comprehensively assessed.Results After hAMSCs transplanting 6 weeks,the rate of abnormal oestrus cycles in hAMSCs group[40.0%(6/15)]was lower than that in model group[86.7%(13/15),P=0.021].Compared with the levels of serum FSH[(10.239±1.091)μg/L],estradiol[(103.325±4.952)ng/L]and AMH[(1.133±0.494)μg/L]in model group,the level of FSH in hAMSCs group[(7.664±0.735)μg/L]was significantly decreased(P<0.001),the levels of estradiol[(126.883±23.370)ng/L]and AMH[(2.204±0.453)μg/L]were significantly increased in hAMSCs group(P=0.015,P<0.001).Different from model group,the ovarian and uterine index were increased.A large number of healthy follicles at all stages were highly increased,but it was rare to find interstitial fibrosis and atresia follicles.The uterine wall and endometrium were thickened,and the number and volume of the glands were increased.The absorbance(A)of HOXA10 in hAMSCs group(5.90±1.94)was higher than that in model group(2.79±1.27,P=0.029).The TNF-αA value of hAMSCs(3.83±1.23)group was significantly lower than that of model group(6.26±0.96,P=0.002).Although there was no significant difference on FSHR A value between hAMSCs group(3.61±1.66)and model group(2.74±0.22,P>0.05),the FSHR A value of model group was lower than that of normal group(4.13±0.54,P=0.006).Conclusion hAMSCs transplantation could restore ovarian function of autoimmune POI mice meanwhile significantly improve uterine receptivity and fertility.