Differentiation Of Induced MSCs In Vivo And Vitro With Rats Hepatic Fibrogenesis Environment And The Effects Of RGJ

The regeneration medicine is one of the key jobs this century on the biomedical science.Liver regeneration and the function of the stem cells have been hot point for research. The current research focus in the embryo stem cells and marrow stem cells transplants etc. Autologous marrow mesenchymal stem cells have much greater advantage than others because that they are easy to obtain, that they have no immunosuppression, have multi differentiating potential, have moral support and have no tumor inducing possibilities. In recent years, specialists have interested in some research work on the plasticity and its application in liver regenerative medicine, including differentiation of MSCs into hepatocytes in vitro and in vivo. To investigate mechanism that MSCs differentiate into hepatocytes . To observe differentiation of inducing of MSCs in vivo and in vitro, transplantation in rat hepatic fibrogenesis environment. MSCs and induced hepatocytes were injected into rat hepatic fibrogenesis environment through the portal vein. The distribution of cells and differentiation were observed. 1. we extract the MSCs from femur in the SD rat . Using Percoll, method realizes MSCs separation, purification and culture in vitro. Selecting the augmented MSCs and washing, making into MSCs single cell suspension, adding respectively into IgG-PE and IgG-FITC; CD44-FITC; CD34-PE; CD45-FITC; CD31-PE; CD90-FITC, incubating for 20 min in room temperature, sending for flow cytometry checking. Flow cvtometry observed negative CD34, CD45, CD31, and positive CD44, CD90 . Cells had fairly good uniformity. Meaning different from non -hematopoietic marrow cells in hematopoietic stem cells .Selecting the MSCs in the third passage, adding 20ng/ml, HGF and EGF1.5μg /ml into complete media, inducing for 14days checking on cells with optical Microscopes in different time of inducement. The expressions of ALB, ck8/ck18 were detected with immunofluorescence, counting the number of ALB positive cells and calculating the rate of differentiation; Albumin mRNA were examined by reverse transcriptase polymerase chain reaction(RT-PCR). 2.We use Bromodeoxyuridine(BrdU) labeling for S-Phase MSCs .Cells suspension was divided into four bottles; three bottles contained BrdU at 0.5μg/ml ,5μg/ml and 50μg/ml labeling for S-Phase cells and another did not contained BrdU. The cells were incubated at 37℃,containing 5%CO2 in a humidified atmosphere for up to 72h.After 12,24,48 and 72h of incubation, cell suspension was taken from each of the four bottles ,the labelling index and the cell viability was measured. We found that optimal labelling in our study was achieved after incubating the cells with BrdU at 5μg/ml for 24h.The labelling index of 58.6±1.6%(n=3)under these conditions was nearly maximum. Cell viability, which was 91.8±1.2%(mean±s.d.,n=3). SD rat hepatic fibrogenesis models was induced useing 40% carbon tetrachloride. 3. On the basis of in vitro experimental data, Incubating BMSCs and induced hepatocytes with BrdU at 5μg/ml for 24h. The rats with hepatic fibrogenesis were randomly separated into two groups (n= 10),labeled MSCs with Brdu and induced hepatocytes were injected into rat hepatic fibrogenesis environment through the portal vein. The distribution of the labelled cells and albumin expression were observed immunohistochemically after 2 weeks. The number of BrdU-positive cells In two groups , there were no statistically significant differences. (p>0.05). The number of BrdU-labeled with ALB positive cells. there were statistically significant differences. (p<0.05). Induced BMSCs in vitro can differentiate into hepatocytes .The albumin express more obvious in induced hepatocytes group. Induced MSCs in vivo and in vitro with hepatic fibrogenesis environment can differentiate into hepatocytes .The albumin express more obvious in induced hepatocytes group. 4.The Rats with hepatic fibrogenesis were randomly separated into four groups (n= 10),A group was Rats with hepatic fibrogenesis as control , B group was injected by induced hepatocytes thr...