Effect of Rats Bone Marrow Mesenchymal Stem Cells on Hepatic Stellate Cell Proliferation
|School||Guangxi Medical University|
|Course||Digestion within the science|
|Keywords||Bone marrow mesenchymal stem cells Hepatic stellate cells Co-culture Cell cycle Cyclin D1 P27|
Objective: To observe the bone marrow mesenchymal stem cells (MSC) on hepatic stellate cells (HSC) cell cycle and regulation of protein expression, and to further explore the mechanism of MSC inhibit the proliferation of HSC. Methods: MSC separation: SD male rat femur bone marrow isolated under sterile conditions using adherent culture MSC culture, purified and amplified, the passage to the 4th generation; rat hepatic stellate cells (HSC-T6 ) and fibroblasts passaged cell lines after freezing and thawing use. Application 6 holes plastic cell culture box, was inoculated on a semipermeable membrane (transwell insert) MSC (2 × 105cells / well), inoculated in 6-well plastic culture plate HSC-T6 cells (2 x 105cells / well), to establish down double layer cell co-culture system, conventional cultivation; were divided into 3 groups: ① experimental group in the semi-permeable membrane inoculated with rat bone marrow mesenchymal stem cells in 6-well plastic culture board inoculation hepatic stellate cells, establish upper and lower bunk cell total culture system; ② control group rat bone marrow mesenchymal stem cells to replace the fibroblasts; (3) control group only line of hepatic stellate cells cultured alone. The above system culture observed for 72 h. Inverted phase contrast microscope every day in the dynamic observation of living cells morphological changes; WST-8 assay hepatic stellate cell growth inhibition rate, flow cytometric analysis of cell cycle, RT-PCR detection of hepatic stellate cells CyclinD1 and P27 mRNA expression , Western blot detection of hepatic stellate cells CyclinD1 and P27 protein expression. SPSS13.0 statistical software for data analysis. Results: Compared with the control group, the experimental group was co-cultured for 24 h, 48 h, 72 h hepatic stellate cell growth inhibition rate was significantly higher (P lt; 0.01); 72h G0/G1 phase cells co-cultured significant increase (P lt; 0.01), S-phase cells was significantly reduced (P lt; 0.01), bone marrow mesenchymal stem cells can block hepatic stellate cells from G0/G1 to S phase transition. Co-cultured for 24 h CyclinD1 mRNA and protein expression of the experimental group starts to rise again, to 72 h, expression was significantly lower than that of the control group, blank group (P lt; 0.01); culture of each group in the whole process of P27 mRNA expression was no significant difference (P gt; 0.05), when co-cultured for 24 h experimental group P27 protein expression compared with the control group, blank group were significantly raised (P lt; 0.01). Result confirmed rat bone marrow mesenchymal stem cells are able to inhibit the proliferation of hepatic stellate cells. Conclusions: (1) bone marrow mesenchymal stem cells in vitro inhibition of hepatic stellate cell proliferation. (2) bone marrow mesenchymal stem cells inhibit hepatic stellate cell proliferation mechanism may be by inhibiting CyclinD1, raised P27 protein expression and cell cycle arrest in G0 / G1 phase play thus inhibit the proliferation of rat hepatic stellate cells .