| Objective: To investigate the paracrine secretion of human umbilical cord mesenchymal stem cells(UC-MSCs)and the immune regulation of T cells by detecting the changes of cytokine secretion induced by UC-MSCs and cord blood T cells.By establishing bone marrow mesenchymal stem cells(BM-MSCs)transplantation combined with associating liver partition and portal vein ligation for staged hepatectomy(ALPPS)model,whether BM-MSCs cell transplantation can promote residual liver regeneration,improve liver function and pathological indicators,and prevent Postoperative liver failure.Methods:(1)Umbilical cord tissue was standardized by umbilical cord dissociator and supporting kit to obtain UC-MSCs;BM-MSCs were obtained by density gradient centrifugation.Surface markers of UC-MSCs and BM-MSCs were identified by flow cytometry.(2)The first part of the experiment was divided into three groups,namely UC-MSCs group,cord blood T cell group and co-culture group.The amount of cytokine secretion in the three cell culture supernatants was detected by liquid phase chip technique.The secretion of 14 cytokines in P1-P8UC-MSCs was detected,and the UC-MSCs cell passage with strong secretion ability was searched.The passage cells were used to establish a co-culture system of UC-MSCs and cord blood T cells in different proportions to detect five kinds of cells.Factor expression amount.(3)The second part of the experiment was divided into three groups,namely portal vein ligation and hepatic lobe separation group(ALPPS group),portal vein injection of saline combined with ALPPS group(hereinafter referred to as saline group)and bone marrow mesenchymal stem cell cell transplantation combined with ALPPS group(post Referred to as the cell transplantation group).Construct ALPPS surgical model and transplantation model.ALPPS surgical model and cell transplantation combined with ALPPS operation model were constructed;changes in body weight and liver regeneration rate were calculated at different time points(1d,5d,7d,10 d and 14d);Venous blood,and the levels of AST,ALT,Alb,and Tbil in serum samples were measured.The necrosis and regeneration of rat liver were detected by HE staining,and the expression of PCNA index was detected by immunohistochemical staining.The expression levels of TNF?,IL-6 and HGF in the liver of rats were detected by ELISA.Results:(1)UC-MSCs with higher viability,high expression of CD29,CD49,CD90 and CD105,low expression of hematopoietic phenotype CD34,leukocyte marker CD45 and endothelial cell phenotype CD31,low expression of monocyte surface MHC class II molecule HLA-DR.The BM-MSCs with higher viability obtained higher expression of CD29 and CD44,and did not express the white blood cell marker CD45.(2)P6UC-MSCs have higher cytokine secretion than other passage cells.The amount of GM-CSF secreted in the co-culture group was not affected by the presence of UC-MSCs.However,with the addition of UC-MSCs,the secretion of IL-4 and IL-10 increased,the secretion of TNF-? and IFN-? decreased,and the ratio of cord blood T cells to UC-MSCs was 10:1 or higher.The change in secretion was more significant(P<0.05).(3)In the second part of the experiment,the body weight of the three groups of rats was rapidly negatively increased in the 1d,and the body weight gradually increased after 5d,and gradually returned to the normal level on the10 d.On the 5d after operation,the right middle lobe of the three groups began to regenerate the hepatic lobe,and the cell transplantation group regenerated faster.On the 5d,7d,10 d and 14 d,the right middle lobe regeneration rate of the cell transplantation group was significantly greater than that of the ALPPS group and the saline group.(4)The concentration of ALT and AST in the cell transplantation group was significantly lower than that in the ALPPS group and the saline group from the5 d after operation,and the difference was statistically significant(P < 0.05),and between the ALPPS group and the saline group.The difference was not statistically significant(P > 0.05).Up to 10 d,the ALT and AST concentrations in the cell transplantation group returned to the normal level,and the ALPPS group and the saline group returned to the normal level on the 14 th.From 5d after operation,the concentration of Alb in the cell transplantation group was significantly higher than that in the ALPPS group and the saline group,and the difference was statistically significant(P<0.05).The difference between the ALPPS group and the saline group was not statistically significant.(P>0.05).On the 10 d,the Alb concentration in the cell transplantation group returned to the normal level,and the ALPPS group and thesaline group returned to the normal level on the 14 d.There was no significant difference in serum Tbil concentration between the three groups(P>0.05).(5)On the 1d after operation,the three groups showed large area edema,cytoplasmic vacuoles,and a small amount of focal necrosis.On the 5d after operation,edema increased,but the cell morphology,hepatocyte line arrangement and congestion were all reduced compared with the 1d,and the mitotic figures increased,showing new cells.On the 7d after surgery,the three groups of tissue structure were close to normal.(6)The positive expression of PCNA was the highest on the 5d after operation,and the positive expression of PCNA on the 7d,10 d and 14 d decreased,and the difference between the three groups was not statistically significant.(7)The expression of TNF?and IL-6 in the right middle lobe of the three groups was different but not statistically significant(P > 0.05).The expression level of HGF was significantly increased,peaked on the 5d,and the difference between the cell transplantation group and the other two groups was statistically significant(P<0.05).On the 10 d after surgery,the cell transplantation group returned to the normal range,and the ALPPS group and the saline group returned to the normal level on the 14 d after surgery.Conclusions: UC-MSCs were obtained by umbilical cord tissue dissociation and matching kits,and BM-MSCs were obtained by density gradient centrifugation.The obtained cells have strong cell proliferation ability,and their morphology and immunological table are in line with the biological characteristics of MSCs.After co-culture of UC-MSCs with T cells,the secretion of anti-inflammatory cytokines increased,the secretion of pro-inflammatory cytokines decreased,and the function of inflammatory reaction was reduced.Transplantation of BM-MSCs combined with ALPPS can promote residual liver regeneration,improve liver function and pathological parameters.To prevent and treat postoperative liver failure and provide a theoretical basis for the clinical application of MSCs transplantation for liver disease. |
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