| Objective:Through the establishment of animal models of liver cirrhosis, and preparation of human umbilical cord blood-derived mesenchymal stem cells(MSCs),to study the therapeutic effect of umbilical cord blood MSCs transplantation for cirrhotic rat, discussion on whether the transplanted cord blood MSCs are colonized and differentiated in cirrhotic rat, expression of the human hepatocyte marker genes.Method:1.Composite factor method (subcutaneous injection of40%of CCL4oil+20%of alcohol drinking water+high-fat diet) induce cirrhosis model, whether HE and Masson staining detection model is successful.2.Collecting umbilical cord blood of newborn under aseptic conditions, to obtain mononuclear cells of human umbilical cord blood by density gradient centrifugation separation, through the cultivation, purification, passage of amplification to get the umbilical cord blood MSCs, to detect cell surface antigens by flow cytometry (CD29, CD105, CD34, CD45), using5-bromo-2-deoxyuridine Brdu in vitro cell markers, to detect whether the mark is success by immunofluorescence assay.3.Modeling successful cirrhotic rats were randomly divided into:group A, portal vein transplantation of experimental group (n=20): transplantation of cord blood MSCs lml via the portal vein; group B, the portal vein transplantation of control group (n=20):injection of equal volume PBS via the portal vein. Normal rats were also randomly divided into two groups:group C, the portal vein transplantation of normal group (n=10):transplantation of cord blood MSCs lml via the portal vein; Group D, the normal control group (n=10):ordinary food and water feeding. Kill groups of rats after transplantation for four weeks, collecting the inferior vena cava(IVC) blood to detect the liver function and immune function; HE and Masson staining to understand the pathological changes of groups of rats after umbilical cord blood MSCs transplantation; to detect the colonization situation of Brdu marked cord blood MSCs by immunohistochemistry and immuno-fluorescene; and to detect whether there is the expression of human hepatic cell markers genes in the liver tissues of cirrhotic rats by RT-PCR technology.4.Data are expressed as mean±standard deviation, using SPSS18.0statistical software, such as t test and single-factor ANOVA for statistical analysis。 Results:1.Composite factors induced rat liver cirrhosis model successfully established after eight weeks, HE staining shows:normal rat liver lobule structure was damaged, being replaced by pseudolobule in which liver cells have varying degree of degeneration and necrosis, infiltration of inflammatory cells visible. Masson staining shows:rat model group can be seen clearly the typical pseudolobule structure, collagen fibers are blue.2.Successfully isolated and cultured human umbilical cord blood MSCs and the exchange of medium passage P3-generation cell morphology was relatively homogeneous shuttle line and parallel arranged or swirling growth. Flow cytometry prompted cell phenotype CD29and CD105has a high expression rate, respectively93.37%and89.08%.By immunofluorescence detection, Brdu can successfully mark the cord blood MSCs in vitro.3.Umbilical cord blood MSCs transplanted via the portal vein after4weeks, group A improved in general condition, body weight compared with pre-transplant and group B was obviously increased (P <0.05); liver function compared with group B,ALT,AST,TBIL and ALB had significant improvement (P<0.05). The pathological findings shows:group A compared with group B, the severity of liver cell necrosis, fatty degeneration and hepatic fibrosis had improved, semi-quantitative grading score shows significant differences (P <0.05).4.After four weeks of the umbilical cord blood MSCs transplantation, taking the liver tissue of group A for immunohistochemical examination, it can be seen that the Brdu labeled positive cells, its nuclear is brownish yellow colonized in the liver; to see Brdu labeled positive cells by immunofluorescence detection, the cell nuclear is red fluorescence colonized in the liver; detected by RT-PCR, as shown in cirrhotic rats which implanted the human umbilical cord blood MSCs,there is the expression of CK18mRNA and ALB mRNA.5.After four weeks of the umbilical cord blood MSCs transplantation, group C compared with group D, the body weight change had no obvious difference (P>0.05); ALT,AST,TBIL and ALB were no significant difference (P>0.05); Immune-related indicators of IgA, IgG, IgM, C3, C4, change there were no significant difference (P>0.05); each organ in group C such as liver tissue, spleen tissue, lung tissue and kidney tissue can be seen that a small amount of positive cells Brdu labeled colonization, but each organs found no tumor-like growth through pathology testing, and no pathological changes.Conclusion:1.Human umbilical cord blood MSCs transplantation for the treatment of cirrhotic rats, it can improve the rat liver function and histological structure to a certain extent;2.The transplanted human umbilical cord blood MSCs via the portal vein can be colonized in the cirrhotic rat liver and differentiated into hepatocytes. |
PDF Full Text Request