Combination Interleukin-1 Beta Plus Hepatocyte Growth Factor On Hepatocytes Induced Differentiation From Bone Marrow Mesenchymal Stem Cells In Vitro

Objective:To compare the induced effency between combination Interleukin-1 beta(IL-1?)plus hepatocyte growth factor(HGF)with HGF on hepatocytes induced differentiation from Bone Marrow Mesenchymal Stem Cells(BMSCs)in vitro.And to find suitable methods to induced BMSCs differentiation into hepatocytes.Methods: 1.The separation of BMSCs 4 weeks old healthy male SD rats were killed off,with femur and tibia obtained under aseptic conditions,the removal of the epiphyses.Rinse the marrow cavity until it turns white and collect the cell suspension.2.The purification and passage of BMSCs The cell suspension was centrifugalized and resupended,and then the cells were inoculated in culture bottle and placed in the incubator.The cell morphological changes and attached were observed under inverted phase contrast microscope.Generation passage was be done when 90% the bottom of bottle filled with cells.3.The Identification of BMSCs The third passage cells with good growth and high activity was detected by flow cytometry through CD45,CD90 and CD29 antibodies.The expression of CD90 and CD29 in BMSCs was positive,and the expression of CD45 was negative.4.Induced differentiation of BMSCs Bone marrow mesenchymal stem cells at the concentration of 1x106/ml was inoculated into cell sheets in 6-well plates.After the cells adhered to the wall,24 hours later,the cells were divided into different cytokines and basic medium to induce differentiation.The BMSCs of the third generation Were collected and divided into five groups:Group A(blank control grou p): basal medium;Group B(hepatocyte growth factor):HGF 20ng/m L+basal medium;Group C(HGF+ IL-1? low concentration group):HGF 20ng/m L + IL-1? 5ng/m L+basal medium;Group D(HGF+IL-1? medium concentrati on group):HGF 20ng/m L+IL-1? 10ng/m L+ basal medium;Group E(HGF+I L-1? high concentration group):HGF 20ng/m L+IL-1? 20ng/m L+basal medium.Different concentration of cytokines according to groups were added to the basal medium continuously when the cells attached after 24 hours.Cells were removed for 7 days,14 days and 21 days,respectively.The changes of cell morphology were observed under inverted phase contrast microscope.The expression of albumin(Albumin,ALB),alpha fetoprotein(Alph Fetal,AFP)and cytokeratin 18(Cytokeratin 18,CK18)were detected by immunocytochemistry.The expression of albumin in the cells could be used to indicate that the cells were hepatocytes.Each group took 6 cell climbing slices,each slice randomly selected 5 vision of non repetitive high power field(X200),counted the 100 cells,counted the percentage of positive staining cells,and took the average value.The cytoplasm was positive for brown granules.Use SPSS20.0 for statisticald escription and analysis,Measurement data using mean±standard deviation.Comparison between groups using single factor analysis of variance,comparison among groups analysis with the least significant difference method,difference had statistical significance when P<0.05.Results:1.Identification of bone marrow mesenchymal stem cells flow cytometry was used to detect the expression of CD29 and CD90 were high,and the expression of CD45 was low,which was consistent with th ephenotype of BMSCs.2.Induction of cell morphology under light microscope,there was no morphological change in A group.The B group to the E group cell morphology changed obviously,Cells from the spindle,prismatic gradually to the polygon and class round development,7 days later with oval change,similar to liver cells,and induced cell mo rphologywere roughly the same.3.Albumin expression and Liver cell transformation rate A group had no ALB expression.The positive expression of albumin in group B and group E showed that the experiment could induce differentiation into hepatocytes.In the 7 days And 14 days,B group,C group,D group,E group,albumin expression gradually increased,P<0.05,there were statistically significant differences;21 days,B group(69.45±2.27),C group(77.81±0.90),D group(87.68±2.10),E group(98.71±0.71)increased gradually,P<0.05,the differences were statistically significant;Liver cell transformation rate in the 21 day,B group(69.45±2.27),C group(77.81±0.90),D group(87.68±2.10),E group(98.71±0.71)increased gradually,P <0.05,there were statistically significant differences;Albumin expression in B group to E group from 7 days to 21 days were increased with the number of days increasing,P<0.05,the differences were statistically significant.4.AFP expression the expression of AFP was not observed in A group.On the 7 day,the 14 day and the 21 day,the expression of alpha fetoprotein in B group,group C,group D and group E increased gradually,P<0.05,the difference was statistically significant;From group B to group E,the expression of AFPi n each group decreased gradually with the increase of the number of days from 7 days to 21 days,P<0.05,the difference was statistically significant;5.Expression of CK18 there was no CK18 expression in Group A.7 days,14 days and 21 days,CK18 expression gradually increased ingroup B to group E,P<0.05,there were statistically significant differences;B group to E group,each group from 7 days to 21 days,the expression rate of CK18 increased with the number of days increasing,P <0.05,the differences were statistically significant.Conclusion:Hepatocytegrowth factor and hepatocyte growth factor plus I L-1? can induce the differentiation of BMSCs into hepatocytes.Moreover,the induction effect of IL-1? combined with hepatocyte growth factor was stronger than that of hepatocyte growth factor.With the increase of IL-1? concentration,the induction ability was strengthened.