| Objective:This study focoused on the effect of mi R-122 modified human umbilical cord blood mesenchymal stem cells on the proliferation ability and induced differentiation into hepatocyte.And the effects of mi R-122-modified human umbilical cord blood mesenchymal stem cells(h UCB-MSCs)on the pathophysiology and apoptosis of liver tissue in rats with acute-on-chronic liver failure were further explored.Methods:1.Human umbilical cord blood mesenchymal stem cells collection,culture and mi R-122 transfection:The collected umbilical cord blood was separated from human umbilical cord blood mesenchymal stem cells by magnetic activated cell sorting,and the mesenchymal stem cell surface antigens(CD29,CD90,CD34 and CD45)were detected by flow cytometry.The h UCB-MSCs were divided into three groups:blank group without any intervention(Control),control group transfected with empty vector(NC-vector),and transfected group with overexpressing mi R-122 vector(mi R-122-vector).The cell morphology was observed under an inverted biologic microscope.Then using q RT-PCR to detect the transfection efficiency of mi R-122.2.Cell proliferation detection:The proliferation activity of h UCB-MSCs after mi R-122 transfection was detected by MTT assay,and the expression of PCNA was observed by immunofluorescence assay.3.Hepatocyte directed differentiation ability test:The above three groups were given or not given inducer(Inducible factor)intervention,and the h UCB-MSCs cells were cultured in hepatocyte-directed differentiation medium for 7 days.Using q RT-PCR and Western blot to detect the expression of hepatocyte differentiation marker m RNA and proteins,including AFP,CYP3A4,ALB,CK-18 and CK-19.4.Establishment of acute-on-chronic liver failure model,and analysis the efficacy of mi R-122-h UCB-MSCs transplantation:Using human serum albumin(HSA)to establish a chronic liver injury rat model,and then combined with D-galactosamine(D-gal)and lipopolysaccharide(LPS)to establish an acute-on-chronic liver failure(ACLF)rat model,and the control group were given normal saline.The established acute-on-chronic liver failure rat models were randomly divided into 3 groups:ACLF group,ACLF+NC-MSCs group,and ACLF+mi R-122-MSCs group.The three groups were injected with normal saline,NC-h UCB-MSCs suspension and mi R-122-h UCB-MSCs suspension through tail vein respectively,and transplanted 200μL(about 1.4×10~7cells).After transplantation,at24h,72h,and 120h,several rats in each group were sacrificed,and liver samples were collected for hematoxylin-eosin(HE)staining,followed by TUNEL assay to analyze cell apoptosis,and the m RNA expression of Caspase-1,IL-18 and VEGF were detected by q RT-PCR.Results:1.Under the inverted biologic microscope,the h UCB-MSCs cells showed elliptical or long-spindle adherent growth,and the cells were closely arranged and uniform in shape.The expression of CD29 and CD90 were high,and the positive rates were all>94%,CD34 and CD45 was weakly expressed,and the positive rate was<5.5%.Compared with the Control and NC-vector group,the expression of mi R-122was significantly increased in the mi R-122-vector group,indicating that the mi R-122-overexpressing h UCB-MSCs was successfully constructed.2.After transfection,the optical density values measured at 12h,24h,and 48h showed that the mi R-122-vector group was higher than the control group and the NC-vector group(P<0.05).The expression of PCNA in mi R-122-vector group was the highest among the three groups(P<0.05).It was suggested that overexpression of mi R-122 promoted the proliferation of h UCB-MSCs.3.After induction into hepatocytes,the m RNA and protein levels of AFP,ALB,CK-18,CK-19 and CYP3A4 were increased in the h UCB-MSCs that transfected with mi R-122 compared with the corresponding control group(P<0.05).Among them,compared with the Control group and NC-vector group without differentiation inducer,the m RNA and protein expression levels of five hepatic markers in the mi R-122-vector group were increased(P<0.05).Compared with the mi R-122-vector group,the mi R-122-vector+Inducible factor group had the highest expression levels of the five hepatic markers(P<0.05).4.After D-gal and LPS combined to attack the chronic liver injury rat model,the HE staining of the rat liver tissue showed that hepatocytes appeared flaky or massive necrosis,and inflammatory cell infiltration and fibrous tissue hyperplasia were seen in the central venous portal area.Pathological symptoms aggravated with time.It indicated that the acute-on-chronic liver failure rat model was successfully constructed.The livers’HE staining of the ACLF and ACLF+NC-MSCs groups at 24h,72h,and 120h showed that the liver tissue was damaged to varying degrees,manifested as partial necrosis of hepatocytes,cell edema,and inflammatory cell infiltration.Compared with the ACLF group and ACLF+NC-MSCs group,the HE staining in the ACLF+mi R-122-MSCs group showed that the necrosis of liver cells was significantly reduced,the structure of the hepatic lobules gradually recovered,the density of blood vessels in the liver tissue increased,formed new capillaries,alleviated the liver fibrotic state.Liver pathological status gradually improved with time.TUNEL staining showed that at 24h,72h,and 120h after transplantation,the apoptosis rate of hepatocytes in the ACLF+mi R-122-MSCs group was the lowest,the apoptosis rate of the hepatocytes in the ACLF group was the highest,and the apoptosis rate in the ACLF+NC-MSCs group was between the other groups.The difference between each group was statistically significant(P<0.05).5.At 24h,72h and 120h after transplantation,the m RNA expressions of Caspase-1 and IL-18 in ACLF group were the highest,and the m RNA expression of VEGF was the lowest in the three groups.The m RNA expressions of Caspase-1 and IL-18 were the lowest in the ACLF+mi R-122-MSCs group,and the m RNA expression of VEGF was the highest.between the other two groups,and the difference between each group was statistically significant(P<0.05).Conclusions:1.Overexpression of mi R-122 can improve the proliferation ability of h UCB-MSCs,and promote h UCB-MSCs differentiate into hepatic cells;2.mi R-122-modified h UCB-MSCs transplantation can reduce the degree of liver pathological damage in ACLF rats,promote the recovery of liver tissue,and play a certain protective role in the ACLF rat model. |
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