HAMSCs transplantation combined with G-CSF effectively repairs endometrial damage and inhibits fibrosis in IUA rats
Objective To investigate the effects of human amniotic mesenchymal stem cells(HAMSCs)com-bined with granulocyte colony stimulating factor(G-CSF)on the repair and fibrosis inhibition of endometrial in-jury in intrauterine adhesions(IUA)rats.Methods HAMSCs were extracted using enzymatic digestion and cul-tured 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 e-ther,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 uter-ine cavity as a sham surgery control(Sham control).On the 7th and 10th day after IUA modeling,two treat-ments were performed with a liquid volume of 0.2 mL each time.The IUA group injected only PBS,the HAM-SCs group perfused PBS containing 1 × 106 cells,the G-CSF group perfused PBS containing 20 μg 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 deter-mined,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 indi-cated that TGF-β1,SMAD3 and β-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 expres-sion 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 and β-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 and β-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 in-jury and inhibit its fibrosis by affecting the expression of TGF-β 1/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.
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