Experimental Study On Bone Marrow Mesenchymal Stem Cells Transdifferentiate Into Hepatocytelike Cells Induced By Acute Hepatic Failure Rat's Serum

Objective:To explore the effects of the acute hepatic failure rat's serum on the transdifferentiation of bone marrow-derived mesenchymal stem cells (BMSCs) into hepatocyte-like cell in vitro, provide experimental evidence for using BMSCs to treat patients with end-stage liver disease .Methods:BMSCs isolated from the rat are cultured by hol-bone marrow culture method. Observe the cell morphology under inverted microscope.The expression of CD29 and CD34 are detected by immunofluorescence staining .Establish model of acute hepatic injury, partially hepatectomize the rat after intraperitoneal inject carbon tetrachloride as the model group; intraperitoneal inject isodose of normol sodium then close the abdomen as the sham operation group. Detect the level of ALT,AST and TBIL of the serum of rats ,obtain rats'liver for HE staining. Observe the two groups rats'liver pathological changes under inverted microscope. Use the serum of the model group rats , the sham operation group rats and FBS to culture BMSCs respectively. There are three groups in the experiment, experimental group and control group and blank control group. The experimental group: use the serum of the model group rats and culture medium to culture BMSCs; The control group: use the serum of the sham operation group rats and culture medium to culture BMSCs;The blank control group:use FBS and culture medium to culture BMSCs. Observe the cell morphology under inverted microscope respectively. The expression of hepatocyte surface specificity marker, such as alpha-fetoprotein(AFP),albumin(ALB)and cytokeratin-18(CK18)are detected by RT-PCR at different time points after induction, on the 7th,14th and 21st day.Results:(1) BMSCs are spindle observed under inverted microscope, the primary cell under fluorescent microscope, we can see about 90% of cells express CD29, not express CD34.(2) The level of serum bilirubin and transaminase in the model rats was significantly increased then the sham group. Compare the rats'serum between the model group and sham operation group, the difference was notable significant(P<0.001). Compare the liver histological score , the hepatocyte morphology of sham operation group rats are normal, There is vacuoles in the cytoplasm and pyknosis of hepatocyte from the model group rats liver, the level of Pathological score of the model group is significantly higher than the sham operation group , the difference was notable significant(P<0.001).(3)Observe the BMSCs under inverted microscope, the cell morphology was induced gradually became triangular; the cell morphology of control group and blank control group are still spindle. The level of hepatocyte surface specificity marker, such as alpha-fetoprotein(AFP),albumin,(ALB)and cytokeratin-18(CK18)are detected by RT-PCR after BMSCs was induced on the 7th,14th and 21st day.AFP mRNA: On the 7th day after the induce of acute liver injury serum, the AFP mRNA expression appeared ; on the days of 14,the AFP mRNA expression is weakend, between the group of the 7thday and 14thday, the difference was notable significant(P<0.001).On the 21st day the AFP mRNA expression disappear. At any point , there is no AFP mRNA expression of the control group and the blank control group.ALB mRNA: on the 7th day after the induce of liver injury serum, there is no ALB mRNA expression; On the 14th day and 21st day ,the ALB mRNA expression is increased after induction. Between 7d and 14d, the difference is significant(P<0.05).Between the 14th d and 21st d, the difference was notable significant(P<0.001).CK-18mRNA: on the 7th,14th and 21st day, after the induce of liver injury serum, the CK-18mRNA expression is increased. The control group and blank control group CK-18mRNA expression on each time, but there is no significant(P>0.05),Compared with the experimental group on the time of 7d after cultured, the difference is no significance (P>0.05),compared with the experimental group on the time of 14d after cultured, the difference is significant (P<0.05), compared with the experimental group on the time of 21d after cultured, there is notable significant(P<0.001). Conclusion:(1)Acute liver injury can be induced by 25% (volume ratio)CCL4-soybean oil solution intrapertoneal injection plus 10% hepatectomy (the resected lobe weight / total weight of the liver) ,there are several advantages, for example, easily into the mold and high survival rate.(2)The serum of the model rats ,induced by partially hepatectomized after intraperitoneal injection carbon tetrachloride, can induce BMSCs differentiate into hepato-cytelike cells, provide a theoretical basis for future treatment, we can use the serum of the liver patients after liver resection to induce the BMSCs differentiated into heptocytelike cells.