Mechanism Of HucMSCs Derived Exosomes Promoting Liver Regeneration After Hepatectomy In Rats

Objective:The liver has a strong ability of regeneration.Hepatocytes will start the process of liver regeneration after undergoing various injuries or liver tissue loss.Due to the influence of age,gender,hepatic steatosis,viral hepatitis,hepatic fibrosis,cirrhosis and intraoperative ischemia-reperfusion injury,the perioperative liver regeneration ability after hepatectomy is greatly challenged,which seriously restricts the safety of liver surgery.With the development of basic and clinical transformation research,stem cells,stem cell-derived Exosomes and various non-coding RNAs have shown amazing potential in the field of regenerative medicine,and gradually attracted the attention of scholars.Based on previous studies,isolated and cultured human umbilical cord blood mesenchymal stem cells(hucMSCs),and extracted the Exosomes from hucMSCs.Upon successful identification,70% liver resection model and liver ischemia-reperfusion injury model were SD by Exocrine intervention,which proved that hucMSCs Exocrine can promote liver regeneration after partial liver resection and alleviate liver ischemia-reperfusion injury.Finally,the rat brl-3a cell line combined with model animals was used to explain the mechanism in vivo and in vitro through a variety of cellular and molecular biological techniques The molecular mechanism of hucMSCs derived Exosome miR-124 promoting hepatocyte regeneration.This study intends to explore the relevant regulatory miRNA expression profile by choosing microarray technology,verify,predicting and screening the downstream target proteins,count the effect of micro RNA expression regulation on liver regeneration,and carry out mechanism research to provide scientific basis for the prevention and treatment of clinical hepatectomy,try to find out the key factors of liver regeneration,and provide guidance for clinical treatment.Methods:hucMSCs was selected as the research object in this study.Firstly,hucMSCs was isolated and cultured from fresh umbilical cord tissue by tissue block culture method.After successful identification by flow cytometry and other techniques,the cell culture medium was extracted by LG-DMEM medium with low blood glucose,Exocrine in hucMSCs was extracted by ultracentrifugation method.The morphology and particle size of Exocrine were analyzed by transmission electron microscope and NAT system,and the CD63,CD9 of surface markers was detected.The effects of hucMSCs derived Exosomes on promoting liver regeneration and alleviating liver ischemia-reperfusion injury after partial hepatectomy were verified by 70%hepatectomy model and ischemia-reperfusion injury model in rats.Online tools such as miRDB,bioinformatics analysis using DIANA and Target Scan to predict potential targets of miR-124.Through repeated comparison and exploration,the internal causes of the experimental targets were speculated based on the prediction results.Finally,q PCR,Western Blot and other methods were used to judge whether the prediction was correct.The purpose of this study is to explore the mechanism of hucMSCs derived Exosome derived miRNA in regulating liver regeneration,and to analyze the downstream signal analysis and potential interaction network of target miRNA.Results:Under the electron microscope,the cells were spindle shaped and uniform.Flow cytometry showed that CD73-fitc and CD105-pe were positive,the expression rates were 99.49% and 99.88%,respectively;CD45-FITC and HLA-DR-PE were negative,the expression rates were 0.38% and 0.26%,respectively.The Exosome extracted from hucMSCs by ultracentrifugation were round or oval in size,with complete membrane structure and low-density substances under electron microscope;the particle size distribution was 48-150 nm according to the nanoparticle tracking analysis system;the Exosomes from hucMSCs were confirmed by screening the related marker proteins CD9 and CD63.hucMSCs derived Exosomes could significantly increase the ratio of liver to body on the 2nd and 3rd day after 70% hepatectomy in SD rats,and the difference was significant between the experimental group and the control group(P<0.05);hucMSCs derived Exosomes could significantly reduce the levels of SOD,MDA,IL-6 and other ischemia-reperfusion related liver injury indexes in the 2nd and 3rd day of ischemia-reperfusion injury group.There was significant difference between the two groups(P<0.05).Micro RNA microarray analysis showed that miR-193 a,miR-124 and miR-93 were significantly up-regulated,while miR-204 and miR-10a-5p were down regulated in the experimental group.QPCR method was used to analyze the relative expression of miR-193 a,miR-204,miR-124,miR-365,miR-93,miR-16,miR-10a-5p and miR-32 in Exosome injection group after 70% hepatectomy and non Exosome injection group after 70% hepatectomy.It was further found that there were differences in the expression of miR-193 a,miR-204,miR-124,miR-93 and miR-10a-5p between the two groups Significant difference(P<0.01).The relative expression of miR-193 a,miR-204,miR-124,miR-93 and miR-10a-5p in the Exosomes derived from hucMSCs was analyzed by q PCR.It has found that miR-193 a,miR-124 and miR-93 were highly expressed in the Exosomes.Using miRDB,DIANA and Target Scan online tools for bioinformatics analysis,the potential targets of miR-124 were predicted,and six common targets(chtf8,PASP,chsys,ADAM9,Strbp and Foxg1)were found The interaction between miR-124 and the 3'-UTR of Foxg1 was confirmed by the results of blot and dual luciferase analysis system.Foxg1 is the direct target of miR-124.The expression of Foxg1 protein expression in liver(p H,Ph+Exo,Ph+antigomir-124,Ph+Exo+antigomir-124,etc.).After repeated verification,it has found that regulating the expression level of miR-124 could negatively regulate the expression of Foxg1 and promote the proliferation of rat hepatocytes.Conclusions:1.hucMSCs is successfully isolated,and the results are reliable.hucMSCs derived Exosomes are successfully isolated.2.hucMSCs derived Exosomes significantly promote liver regeneration after hepatectomy in rats,and reduced SOD,MDA,IL-6 and other indicators of ischemia-reperfusion relate liver injury.3.Micro RNA microarray technology are used to identify miR-124 which was differentially expressed in hucMSCs derived Exosomes and related to hepatogenesis.It further prove that hucMSCs derived Exosomes miR-124 could greatly promote liver regeneration after partial hepatectomy in rats and help to reduce hepatocyte injury.4.It has found that the transcription factor Foxg1 is the direct target of miR-124,and miR-124 promotes the proliferation of rat hepatocytes by inhibiting the expression of Foxg1.