| Objective To study the optimized schemes of rat bone marrow mesenchymal stem cells(BMMSCs),that is,to purify the multilineage differentiating stress enduring(Muse)cells and heme oxygenase-1(HO-1)gene modification.And then to determine the protective effect of two kinds of optimized BMMSCs on damaged intestinal epithelial cell(IECs),and explain its molecular mechanism in detail.Methods 1.Rat BMMSCs were extracted and identified by flow cytometer(FCM)and other methods.2.Rat Muse cells were purified from BMMSCs by trypsin long-term incubation,and identified by pluripotent differentiation and directional differentiation.3.The inflammatory injury model of IECs was established by tumor necrosis factor-?(TNF-?)in vitro.Muse cells were co-cultured with inflammatory injury IECs in Transwell isolation state.The protective effect of Muse cells on inflammatory injury IECs was detected,and the expression of inflammatory-related cytokines in microenvironment was detected.4.BMMSCs modified by HO-1 gene(HO-1/BMMSCs)was obtained by adenovirus transfection.5.HO-1/BMMSCs was cocultured with inflammatory injury IECs.Apoptosis of IECs was detected by Annexin V-FITC/PI and in situ Td T-mediated d UTP Nick end labeling(Tunel)assay.Hematoxylin-eosin staining,immune injury score and Tunel were used to evaluate the effect of HO-1/BMMSCs on damaged transplanted small bowel tissues in rat small bowel transplantation(SBTx)model.6.The exosomes from HO-1/BMMSCs coculture system(HBM-exo)were isolated and identified.The protective effect of IECs,on inflammatory injury was detected by stimulating IECs,and the changes of protein expression profile in IECs were detected by mass spectrometry to screen key proteins.7.Molecular biology experiments,such as real-time fluorescence quantitative PCR(q RT-PCR)and double luciferase reporting,confirmed the mechanism of key Micro RNA in HBM-exo and its target molecules mechanism in protecting IECs from inflammatory injury by HO-1/BMMSCs,and preliminarily verified them in SBTx model.Results 1.The extraction,culture and verification of the cells related to the experiment:(1)the extracted rat BMMSCs conformed to the phenotypic and purity requirements;(2)the isolated cells were isolated and purified by trypsin long-tern incubation,and could be induced to differentiate into cells with three germ layers,which met the requirements of Muse cells;(3)HO-1/BMMSCs was successfully constructed;(4)the exosomes was extracted and identified.2.The rat Muse cells isolated and purified from BMMSCs have a better protective effect on inflammatory injury of IECs.The mechanism is related to the stronger ability of Muse cells to tolerate inflammatory stimulation and reduce the inflammatory microenvironment.3.Optimized BMMSCs,modified by HO-1 gene can significantly reduce the inflammatory injury of IECs and intestinal injury of SBTx rats,and its paracrine function plays an important role.4.HBM-exo has an ideal effect on reducing inflammatory injury of IECs.5.HBM-exo significantly down-regulated the expression of high mobility group protein B3(HMGB3)and c-Jun N-terminal kinase(JNK)pathway in inflammatory injury IECs.And HMGB3/JNK was involved in the inflammatory injury process of IECs.6.Micro RNA(Mi R)-200 b in HBM-exo and its stimulated IECs was significantly increased,and targeted to bind to the 3' untranslated region of Hmgb3,thus regulating the expression of HMGB3;7.Mi R-200 b can reduce the inflammatory damage of IECs and interfere with Mi R-200 b to block the protective effect of HBM-exo on inflammatory injury IECs.Conclusion The purification of Muse cells from BMMSCs and the overexpression modification of HO-1 gene in BMMSCs are effective optimization schemes,which have a more ideal protective effect on IECs caused by inflammatory injury.The mechanism is related to the optimization of paracrine cytokines and exocrine of BMMSCs.Mi R-200 b targets Hmgb3 regulating JNK pathway was important to regulate signal molecules. |
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