In Situ Repair Abilities Of Human Umbilical Cord–derived Mesenchymal Stem Cells And Autocrosslinked Hyaluronic Acid Gel Complex In Rhesus Monkeys With Intrauterine Adhesion

Part 1 Construction of huMSCs/HA-GEL complex[Purpose] Obtain huMSCs with high purity and good condition,evaluate the safety of HA-GEL to huMSCs,and construct huMSCs/HA-GEL complex.[Methods] The huMSCs were extracted and purified by tissue adherence method,passaged and cryopreserved to obtain P3-P6 generation cells.The P3-P6 generation huMSCs obtained were sorted and identified by flow cytometry based on the specific molecules of huMSCs surface,and the differentiation potential in vitro Induction experiments;after huMSCs and HA-GEL co-cultured,flow cytometry and DEADLIVE kit detection methods will be used to detect the apoptosis rate and the proportion of dead and living cells.[Results] We successfully obtained P3-P6 generation huMSCs with strong proliferation ability,high purification and good differentiation potential in vitro;we successfully constructed huMSCs/HA-GEL complex.[Conclusions] huMSCs can be obtained by tissue adhesion method,and HA-GEL as a carrier has no harmful effect on huMSCs.Part 2 Observation on the effect of huMSCs/HA-GEL complex in rhesus monkeys with intrauterine adhesion[Purpose] Establish a non-human primate intrauterine adhesion model and huMSCs/HA-GEL complex transplantation to determine the therapeutic effect of huMSCs/HA-GEL complex on rhesus monkey intrauterine adhesions.[Methods] We did the dilatation and curettage of uterus to establish a non-human primate intrauterine adhesion model.After 2 months,we performed the huMSCs/HA-GEL transplantation on rhesus monkeys.We closely monitored and evaluated intrauterine physiology,and observed pathological changes of endometrium after transplantation.[Results] The rhesus monkey uterine adhesion model was successfully established.2months after the huMSCs/HA-GEL transplantation,the rhesus monkey resumed the menstrual cycle,the uterine cavity was restored.Furthermore,the endometrial collagen fiber area and inflammatory cells were both significantly reduced.A typical secretory villi structure appears in the endometrium.[Conclusions] The huMSCs/HA-GEL complex could effectively repair endometrial injury and adhesions.Part 3 Study on the mechanism of the repair abilities in situ of huMSCs/HA-GEL in rhesus monkeys with intrauterine adhesion[Purpose] Preliminarily explore the possible mechanism of the mechanism of the repair abilities in situ of huMSCs/HA-GEL in rhesus monkeys with intrauterine adhesion[Methods] Firstly,we designed primers for the non-homologous sequences of both DXZ1 and SRY genes between human and rhesus monkey,and performed non-homology verification;We used FISH probe(Vysis SRY Probe LSI SRY Spectrum Orange/Vysis CEP X Spectrum Green)to trace the DXZ1 and SRY genes in the multi-point samplles of rhesus monkey endometrium 2 months after transplantation.We detected the cytokines in the endometrium by Immunofluorescence and RT-qPCR.[Results] SRY and DXZ1 primers were specific and stable,the product was single,and there was no homology with the rhesus monkeys.The designed primers could be used for the follow-up tracer detection of huMSCs in this subject;the FISH showed that,2months after transplantation,there was no huMSCs colonization in the endometrium of rhesus monkeys.At the same time,through the detection of cytokines in the microenvironment of the endometrium,the expression of inflammatory factors in the endometrium decreased while the expression level of cell growth factors that promote intimal regeneration increased after huMSCs/HA-GEL transplantation.[Conclusions] The huMSCs were not stably implanted in the endometrial tissue 2months after transplantation.huMSCs may secrete a series of paracrine factors such as antiinflammatory factors,growth factors and cytokines.These cytokines jointly constructed microenvironmental conditions with anti-inflammation,promoting repair,maintaining cell function and angiogenesis.