Experimental Research On The Therapy Of Hepatic Cirrhosis By Transplantation Of BMSCs

ObjectiveHepatic cirrhosis is a chronic,progressive and irreversible changes of the liver. On the pathology, it follows by liver fibrosis and formation of false leafit and reactivated nodule. The clinical manifestation is disorder of liver function and portal hypertension. It is one of the most common diseases and the main cause of death in our country. Current treatments for cirrhosis are limited to removing the underlying injurious stimulus. As a result, a safe and effective way is needed urgently. In resent years, bone marrow mesenchymal stem cells (BMSCs), a kind of seed cell with the ability of multipotent differentiation, has attracted the attention of many researchers. In view of it, the study cultures BMSCs in vitro and establishes hepatic cirrhosis models, detects the improvement of liver function, they may offer possible certification for clinical therapy of hepatic cirrhosis.Methods一,Isolation and purification of BMSCs and detection of surface markers1,Isolation and purification of BMSCsA male SD rat,4-weeks old, weighting 100-120g, obtained BMSCs and cultured in L-DMEM containing 10% FBS and incubated at 37℃in 5% CO₂.As 80%-90% of cells are fused, the cells were dissociated with 0.25% trypsin and 0.01% EDTA ,then subculturing them at the ratio of 1:2 .After 3 generations, purified BMSCs are obtained.2,Drawing growth curves of BMSCsDissociating first and third passage cells and replating at 1×10⁴ in 24-well plastic culture dishes. Counting the number of adherent cells of 3 wells every day. After 9 days, growth curves were assessed.3,Phenotype analysis surface markers of BMSCsTaking the third passage cells, detected phenotype CD29 and CD44 immunofluorescence and calculated positive expression rate.二,Establishment of hepatic cirrhosis in rat models80 female SD rats,6-weeks old, weighing 150g-180g, they were divided into three groups in random after raised one week in separated cages: normal group(20 rats) received normal diet; model control group(30 rats) and experimental group(30 rats), injected the mixtures of 40% of CCL4 and 60% of vegetable oil in hypo(0.3ml/kg, twice every week). Rats were fed with normal chow and water. After eight weeks , making pathological sections of livers from the three groups respectively in order to gain the results.三,Examination of liver function after infusion of BMSCsInjecting purified BMSCs into model rats, killing them on weeks1, 2, 3, 4 respectively, collected their serums and detected liver functions, making pathological sections to observe the recovery of model rats.Results1,The BMSCs have been cultured and purified successfully; Depicted growth curves; Detected the surface markers CD29n CD44 of BMSCs, the number of positive CD29 and CD44 cells are 9.75×10⁴/ml and 9.5×10⁴/ml and the positive expression rates are 97.5% and 95% .2,Liver cirrhosis rat models have been established.3,Detected liver function of rat models with injected BMSCs by serum-detecting.Discussion1,We gained purified BMSCs by adherence to the culture plastic; detected positive expression of surface markers of CD29 and CD44;2,Verified feasible method of rat liver cirrhosis model-build;3,Injected BMSCs to liver directly can improve liver functions of rats with hepatic cirrhosis in a certain degree.