Primary Research On The Differentiation Of Rat Bone Marrow Mesenchymal Stem Cells Into Hepatocyte-like Cells In Vitro

ObjectiveIn recent years, the incidence and mortality of cirrhosis increased year by year. It has become another major disease who seriously threat to human life after cardiovascularand tumor diseases. In China, the liver cirrhosis dued by hepatitis caused by virus is the most common. So far, this disease can not be treated. There is a symptomatic treatment, but is poor. Liver transplantation is the good treatment of end-stage liver disease, but due to limited donor sources, thus limiting the clinical progress of liver transplantation. Recent domestic and international study found that bone marrow mesen-chymal stem cells (BM-MSCs) have more potential to differentiation. Under certain conditions, in vivo and in vitro they can be differentiated into osteoblast, fat cells and neuron-like cells, heart muscle cells, and so on. BM-MSCs may become new seed cells that treat end-stage liver disease. However, the difference of differentiation conditions (basic training, cell density, mechanical power, growth factors, etc.) have a great impact on the differentiation of BM-MSCs. The best-inducible factor and its usage about the differentiation of BM-MSCs into liver cells in vitro are also worthy of further study.Owing to the purpose of this experiment, we are on the basis of separation culturingBM-MSCs in vitro, and separately and jointly induce BM-MSCs in different doses of hepatocyte growth factor (HGF) and fibroblast growth factor-2 (FGF-2), at the same time, observe the relation between the growth factor and the effects of the differentiation, so provide the experimental basis on the feasibility of BM-MSCs treatingcirrhosis. Methods一,BM-MSCs PurificationWe selected Sprague-Dawley rats (90-100g, health, male), obtained BM-MSCs, and cultured them in L-DMEM containing 10% fetal bovine serum and in 37℃and 5%CO₂ for adherent culture. Medium was changed every 3 days. When the cell layer was full, in the ratio of 1:2 for mass culture, after the three generation, we obtained more purified BM-MSCs.二,In vitro the differentiation of BM-MSCs into hepatocytesWe selected the third generation of BM-MSCs, and divided them into six groups. Adjusted the density of BM-MSCs to 3×10⁵/ml, and separately inoculated in 24 cell culture orifices that were covered placed by disinfection slides. Mediums contained different growth factor and 10% fetal calf serum L-DMEM culture. Table 1, groups of different growth factorMedium was changed every 3 days, and regularly collected culture mediums and slides.三,Identification1. Morphological identificationAfter joining various growth factors, we observated morphology changes of cells of each group by inverted microscope everyday.2. Detection characteristic markers of liver cells(1) Periodic acid-Schiff treatment for glycogen We took out the cells of each groups, on days 12, 24, and treated the cells by reagent of Periodic acid-Schiff.(2)ELISAWe took out the culture mediums of each groups, on days 3, 6, 9,12,15,18,21, 24, and detected the secretion of AFP of cells.(3) ImmunocytochemistryWe took out the cells of each groups, on days 12, 24, and detected the secretion of ALB and CK19 of cells by immunocytochemistry.Results1. We successfully cultured BM-MSCs. In optical microscopy, they were fibro-blast-like cells, high uniformity, arranged in parallel pipelines. About 10 days they can be full of cell layer. After three generations, BM-MSCs were more purified, and backgroundhybrid cells were significantly decreased.2. From the first group to the fifth group, there was a marked change at morphologyafter induced, cell shape increased gradually from the polygon to the spindle, circular or irregular polygon. About 10 days, there was oval change, and like liver cells. The sixth group was no obvious morphological changes.3. From the first group to the fifth group after induced by growth factors, the secretion of ALB, CK19 and glycogen,especially the first, second and fourth group, were significantly higher after the 12th day. On the contrary, AFP was the highest expression on the 12th day, hereafter gradually reduced. The sixth group was no positive results.Conclusions1.FGF-2(20ng/ml),FGF-2 (10ng/ml),HGF(20ng/ml),HGF(20ng/ml)+FGF-2 (10ng/ml) can respectively efficiently induce BM-MSCs into hepatocyte-like cells.2. FGF-2(20ng/ml)have stronger role in the induction of BM-MSCs into hepatocyte-like cells than HGF(20ng/ml).3. FGF-2( > 10ng/ml) can efficiently induced BM-MSCs into hepatocyte-like cells, The induced efficience of FGF-2( <10ng/ml) is weaker and weaker. 4. FGF-2 have stronger impact in cell morphology than HGF.