Effect Of RhoA/ROCK1 Signaling Pathway On Differentiation Of Bone Marror Stem Cells Into Hepatocytes Induced By HGF

Objective:The present work aims to certify the effect of RhoA/ROCK1 signaling pathway on hepatocyte differentiation by adult bone marrow stem cell.It provides a new target for the regulation mechanism of BMCSs hepatocyte differentiation,and offer a theoretical foundation for better clinical application of BMSCs transplantation in the treatment of cirrhosis.Methods:1.Build pcDNA3.1(+)-RhoA and plvx-shrna1-rhoa vector,then transfect them into BMSCs via Lipofectamine 2000.BMSCs are cultured and grouped into BMSCs cell group(normal control group),pcDNA3.1(+)-HK group(RhoA empty expression vector group),pcDNA3.1(+)-Rhoa group(overexpression of RhoA group),pLVX shRNA1-RhoA group(silence RhoA group),and pLVX shRNA1-HK group(group silence RhoA empty carrier).2.After cell transfection,each group was incubated with HGF at a concentration of 20 ng/ml for 7 days in vitro firstly.Then,the proliferation of the cells was detected by MTT assay and the cell growth curve was plotted.Next,The expression of hepatocyte-specific protein alpha-fetoprotein(AFP)in each group of BMSCs was detected by immunofluorescence.At last,Western blot technique was used to detect the expression of RhoA,ROCK1 and p-MLC proteins in BMSCs after differentiation.Results:1.Cell morphology changesFlat or triangular,Epithelioid cells are observed in the plvx-shrna1-rhoa at the 7th day after transfection.A small number of cells showed irregular shape in in BMSCs group,pcDNA3.1(+)-HK group and plvx-shrna1-HK group.Their plasma is larger than cells in pLVX-shRNA1-RhoA group.However,The cells are arranged in a spindle and the morphological changes are not obvious in pcDNA3.1(+)-RhoA group.2.BMSCs proliferation results in each group.After 3 days cell culture,the OD value of each group of BMSCs increased,among which,the OD value of pLVX-shRNA1-RhoA group increased significantly and higher than other groups.However,the OD value of pcDNA3.1(+)-RhoA group represented a minimum value and was statistically significant(P<0.05),compared with BMSCs group,pcDNA3.1(+)-HK group,pLVX-shRNA1-HK group and pLVX-shRNA1-RhoA group after transfection in 3d,5d,and 7d.Besides,the OD value of pLVX-shRNA1-RhoA group increased and was statistically significant(P<0.05),compared with BMSCs group,pcDNA3.1(+)-HK group,pLVX-shRNA1-RhoA group and pLVX-shRNA1-HK group after transfection in 3d,5d and 7d.3.Related protein expression of RhoA/ROCK1 in each group.3 days after transfection,the expression difference of RhoA,ROCK1 and p-mlc protein between BMSCs group,pcDNA3.1(+)-HK group,pcDNA3.1(+)-RhoA group,pLVX-shRNA1-HK group,and pLVX-shRNA1-RhoA group was no statistically significant(p>0.05).The RhoA,ROCK1 and p-mlc protein expression of pLVX-shRNA1-RhoA increased and was statistically significant(P<0.05),compared with BMSCs group,pcDNA3.1(+)-HK group,pLVX-shRNA1-RhoA group and pLVX-shRNA1-HK group after 7d.whereas,the RhoA,ROCK1 and p-mlc protein expression of pcDNA3.1(+)-RhoA decreased and was statistically significant(P<0.05),compared with other groups.4.BMSCs immunofluorescence results in each group.No obvious fluorescence signal was found in each group of cells after 3 days induction.At the same time,the green fluorescent AFP gene expression was observed in the cytoplasm after 7 days induction under fluorescence microscope.Compared with the other four groups,the plvx-shrna1-rhoa group had a higher AFP fluorescence intensity,while pcDNA3.1(+)-RhoA group had a weak AFP fluorescence intensity.Conclusions:1.Inhibiting RhoA/ROCK1 Signaling Pathway can promote cell proliferation and liver differentiation of BMSCs.2.Over-expressing RhoA/ROCK1 Signaling Pathway can inhibit cell proliferation and liver differentiation of BMSCs.