HAMSCs移植联合G-CSF有效修复IUA大鼠子宫内膜损伤并抑制其纤维化

HAMSCs transplantation combined with G-CSF effectively repairs endometrial damage and inhibits fibrosis in IUA rats

目的探讨人羊膜间充质干细胞(HAMSCs)联合粒细胞集落刺激因子(G-CSF)对宫腔粘连(IUA)大鼠子宫内膜损伤后的修复及纤维化抑制的影响。方法酶消化法提取HAMSCs体外培养,取P3代细胞通过生长特征、免疫荧光鉴定表面标记分子和三系分化能力进行鉴定。32只动情期SD大鼠随机均分为IUA组、HAMSCs组、G-CSF和H+G组。乙醚麻醉后行背部开口,IUA组从近卵巢端向左侧子宫腔内注射1 mL无水乙醇作用90 s,1 mL生理盐水冲刷子宫2次,右侧子宫注射等体积生理盐水作为假手术自身对照(Sham对照)。在造模后第7天和第10天进行2次治疗,每次注射体积均为0.2 mL。其中IUA组注射PBS,HAMSCs组宫腔灌注含1×106个细胞的PBS;G-CSF组灌注含20μg G-CSF的PBS;H+G组则在G-CSF灌注后再行HAMSCs灌注。各组大鼠在第2次治疗结束7 d后处死取子宫角备用。HE、Masson染色法检测各组大鼠子宫内膜的修复情况;qRT-PCR和免疫组化法检测各组大鼠子宫中TGF-β1、SMAD3、SMAD7、β-catenin、孕酮受体(PR)及雌激素受体(ER)的表达情况;另取32只大鼠同样分组造模后与正常雄鼠交配,计算其怀孕率和胚胎数。结果所获细胞经生长特性、细胞表面分子和分化能力的鉴定符合MSCs特征。IUA组大鼠子宫出现水肿、充血等现象,其余各组大鼠子宫形态均在一定程度上得以恢复,子宫内膜厚度和腺体数目从高到低依次为Sham对照、H+G组、HAMSCs组、G-CSF组、IUA组。Masson染色结果显示,在纤维化面积占整个子宫内膜面积的比例上,从高到低依次为IUA组、G-CSF组、HAMSCs组、H+G组、Sham对照。qRT-PCR结果显示,Sham对照TGF-β1、SMAD3和β-catenin mRNA最低,在IUA组中表达最高(P<0.05)。与IUA组相比,HAMSCs、G-CSF和H+G处理不同程度降低TGF-β1、SMAD3和β-catenin mRNA表达,尤其H+G组的表达水平接近于Sham对照水平(P<0.05)。SMAD7、PR和ER mRNA的表达趋势则与TGF-β1、SMAD3、β-catenin相反(P<0.05)。免疫组化结果显示,与IUA组相比,HAMSCs、G-CSF和H+G处理可以降低TGF-β1、SMAD3和β-catenin表达而升高SMAD7、PR和ER表达(P<0.05)。怀孕率和胚胎数计数结果显示,IUA组怀孕率为0,HAMSCs、G-CSF、H+G可以提高怀孕率和胚胎数(P<0.05)。结论HAMSCs移植及G-CSF宫腔灌注可能通过TGF-β1/SMAD3/β-catenin信号促进IUA大鼠子宫内膜损伤后的修复并抑制其纤维化,并且H+G处理的治疗效果优于单一的HAMSCs和单一G-CSF处理。

Objective To investigate the effects of human amniotic mesenchymal stem cells(HAMSCs)combined with granulocyte colony stimulating factor(G-CSF)on the repair and fibrosis inhibition of endometrial injury in intrauterine adhesions(IUA)rats.Methods HAMSCs were extracted using enzymatic digestion and cultured in vitro.The identification of the characteristics of P3 generation HAMSCs was carried out through their growth characteristics,surface marker molecules identification using immunofluorescence,and ability to induce differentiation into three directions:bone,fat,and cartilage.A total of 32 SD rats during estrus were randomly divided into IUA,HAMSCs,G-CSF and H+G group,with 8 rats in each group.After anesthetizing rats with ether,a surgical incision was made on their back to expose the uterus.The IUA group injected 1 mL of anhydrous ethanol into the left uterine cavity of rats near the ovary for 90 seconds,then washed the uterus twice with 1 mL of physiological saline.At the same time,an equal volume of physiological saline was injected into the right uterine cavity as a sham surgery control(Sham control).On the 7th and 10th day after IUA modeling,two treatments were performed with a liquid volume of 0.2 mL each time.The IUA group injected only PBS,the HAMSCs group perfused PBS containing 1×106 cells,the G-CSF group perfused PBS containing 20μg G-CSF,and the H+G group received HAMSCs transplantation after G-CSF perfusion.After 7 days of the second treatment,rats in each group were euthanized and uterine horns were collected.HE and Masson staining methods were used to detect the repair of rat endometrium in each group.qRT-PCR and immunohistochemistry were used to detect the mRNA and protein expression of TGF-β1,SMAD3,SMAD7,β-catenin,progesterone receptor(PR),and estrogen receptor(ER)in the uterus of rats in each group.Another 32 rats were also divided into 4 groups as mentioned earlier and mated with normal male rats after modeling.On the 18th day after pregnancy was determined,the rats were euthanized and their pregnancy rate and number of embryos were counted.Results The cells obtained in this study were identified to conform to MSCs characteristics in terms of growth characteristics,cell surface molecular expression,and differentiation ability.The uterus of the IUA group rats showed edema and congestion,while the morphology of the rat uterus was partially restored in the other groups.The thickness and number of glands in the rat endometrium in descending order are Sham control,H+G group,HAMSCs group,G-CSF group,and IUA group.In terms of the proportion of fibrotic area to the total endometrial area of rats,from high to low,it is ranked as IUA,G-CSF,HAMSCs,H+G group and Sham control.qRT-PCR results indicated that TGF-β1,SMAD3 andβ-catenin mRNA expression was lowest in the Sham control and highest in the IUA group(P<0.05).Compared with the IUA group,the mRNA expression of these molecules was reduced to varying degrees in the HAMSCs,G-CSF,and H+G groups,especially in the H+G group,where their expression levels were reduced to near the Sham control level(P<0.05).However,the expression trends of SMAD7,PR,and ER mRNA in the rat uterus of each group were opposite to those of TGF-β1,SMAD3 andβ-catenin(P<0.05).The immunohistochemical results showed that compared with the IUA group,HAMSCs,G-CSF,and H+G treatments could reduce the expression of TGF-β1,SMAD3 andβ-catenin while the expression of SMAD7,PR,and ER was reduced(P<0.05).IUA group rats showed no pregnancy,while HAMSCs,G-CSF,and H+G treatment could increase the pregnancy rate and embryo number of rats(P<0.05).Conclusion HAMSCs transplantation and G-CSF intrauterine perfusion may promote the repair of IUA induced endometrial injury and inhibit its fibrosis by affecting the expression of TGF-β1/SMAD3/β-catenin signaling pathway and the therapeutic effect on IUA of H+G treatment is superior to that of HAMSCs alone and G-CSF alone.