A study of PPCNg-mediated effect of human amniotic mesenchymal stem cells in the treatment of intrauterine adhesion in rats

Abstract:

Objective：To investigate polyethylene glycol citrate-co-N -isopropylacrylamide gelatin（PPCNg）-mediated feasibility and effectiveness of human amniotic mesenchymal stem cells（hAMSCs） in the treatment of intrauterine adhesion（IUA）. Methods：The PKH26-labeled hAMSCs were cultured in different concentrations of PPCNg and were observed for cell status with a fluorescence microscope after 48 hours. CCK-8 assay was used to evaluate the cytotoxicity and material toxicity of PPCNg. The female Sprague-Dawley rats were randomly divided into sham operation group，IUA group（model group），PPCNg-treated IUA group（PPCNg group），hAMSCs-treated IUA group（hAMSCs group），and PPCNg+hAMSCs-treated IUA group（PPCNg+hAMSCs group）；two weeks after IUA modeling，the above five groups except the sham operation group（left untreated） were injected with PBS，PBS-suspended PPCNg，PBS-suspended hAMSCs，and PBS-suspended hAMSCs and PPCNg solution，respectively，in the uterine cavity. Two weeks after the intrauterine injection，paraffin sections of the uterine tissue were treated with HE staining and Masson staining to measure the number of endometrial glands and fibrosis status. Immunohisto－chemistry was used to determine the expression of transforming growth factor-β1（TGF-β1），vascular endothelial growth factor （VEGF），vimentin（VIM），α-smooth muscle actin（α-SMA），cytokeratin 19（CK19），and E-cadherin（E-Ca） in the endometrium. The distribution of hAMSCs in the endometrium was evaluated with a fluorescence microscope using frozen sections. Results：In different concentrations of PPCNg，hAMSCs adherently grew well，and PPCNg was not cytotoxic with its material toxicity classified as grade 1（qualified）. Compared with the sham operation group，the model group had a significantly reduced number of glands and a significantly increased fibrosis area ratio（both P=0.000）. Compared with the model group，the PPCNg group，the hAMSCs group，and the PPCNg+hAMSCs group had different degrees of increase in the number of glands（P=0.000） and different degrees of decrease in the fibrosis area ratio；meanwhile，the three groups had different degrees of decrease in the expression of TGF-β1，VEGF，VIM，and α-SMA（with a significant decrease observed in the PPCNg+hAMSCs group[P=0.000]） and different degrees of increase in the expression of CK19 and E-Ca（with a significant increase observed in the PPCNg+hAMSCs group[P=0.000]）. The PPCNg+hAMSCs group had more PKH26-labeled hAMSCs than the hAMSCs group. Conclusion：PPCNg has no cytotoxicity and can significantly improve the effectiveness of hAMSCs in the treatment of IUA.