| PurposeTo look for the methods of isolate, amplify and identify the human umbilical cord mesenchymal stem cells(hUCMSCs),and to explore the effects of the liver injury rat’s serum on transdifferentiation of hUCMSCs into hepatocyte-like cells in vitro, provide experimental evidence for using hUCMSCs to treat patients with end-stage liver disease clinically.Methods1. Mesenchymal stem cells isolated from human umbilical cord with enzymatic digestion are cultured in the dish. Observe the morphological changes of the extracted umbilical cord-derived mesenchymal stem cells under the microscope.2. We use flow cytometry to detect the expression of mesenchymal stem cells markers as CD73,CD105and hematopoietic stem cells markers as CD34,CD45.3. HUCMSCs are induced with adipogenic differentiation medium in21days and then stained by the oil red O solution. At the same time, hUCMSCs are cultured with osteogenic differentiation medium in23days and then stained by alizarin red working solution. 4.Establish the model of acute liver failure in rats:rats are intraperitoneally injected carbon tetrachloride-soybean oil solution as the model group, meanwhile rats are intraperitoneally injected isodose soybean oil as the control group. Obtain abdominal aortic blood of the rats after48hours, then detect the level of ALT and AST of the serum. Take HE staining of rats liver tissue, and observe the two groups rats’ liver pathological changes under inverted microscope.5.Use the serum of model group rats and FBS to culture respectively. There are two groups in this experiment, experimental group and control group. The experimental group:use the medium with20%serum of liver failure rats to culture hUCMSCs; The control group:use the medium with20%FBS to culture hUCMSCs. Observe the cell morphology before induction,and the cell morphology changes of the experimental group and control group.Detect the AFP, ALB value before induction and the value changes of the two groups.Results1. Primary umbilical cord mesenchymal stem cells could be observed as adherent cells with fibroblast morphology under inverted microscope. The cells proliferate faster after six or seven days and then can be seen as braided or spiral shape.2.Adherent cells are positive for mesenchymal stem cells related antigens, such as CD73and CD105, but negative for hematopoietic related antigens, such as CD34and CD45.3.The P3-generation sample obtain cells with an adipogenic phenotype when stained by oil red O solution after cultured with adipogenic differentiation medium after23days. There are red vacuoles in the differentiated cells. The P3-generation sample form mineralized matrix when identified by alizarin red solution with osteogenic differentiation medium after21days. The size and shape of mineralized matrix is very different. The two control samples are negative.4.Compared with the control group,the level of serum transaminase in the model rats injected with carbon tetrachloride is significantly increased,and the difference is statistically significant(P<0.05).The hepatic sinusoid of the model group is disappeared under the microscope.There are hepatic steatosis and focal necrosis in the liver tissue. The cell nucleus is condensated,fragmentated or dissoluted. A little of inflammtory cell can be seen between the liver cells and portal area. The hepatocyte morphology of the control group rats is normal.5.The cell morphology of the hUCMSCs cultured with the serum of liver injury rat gradually become different. In the first day the cells can be seen as braided or spiral shape, and become short spindle in the second day. Then the cells are roud in the next two days and there are few cells floated. The control cells are fibrous.ALB is increased in the supernant of experimental group and the differences are notably significant compared with the pre-induced and FBS treated hUCMSCs. AFP can not be detected.Conclusions1.Umbilical cord mesenchymal stem cells can be successfully extracted from umbilical cord tissue.2.Stem cells isolated from umbilical cord are plastic adherent, and have high expression levels of mesenchymal stem cells CD73,CD105and low expression for hematopoietic CD34,CD45.Then stem cells also could differentiate into several lineages such as adipose cells and bone cells.3.Acute liver injury of rat could be induced by10%carbon tetrachloride-soybean oil solution intrapertoneally injection. There is high survival rate in the model group.4.The serum of liver injury rats could make umbilical cord mesenchymal stem cells transdifferentiate into hepatocyte-like cells. |
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