The Study Of MicroRNA-148a Inducing The Autophagy In Primary Hepatic Stellate Cells Of Rats

Objective Improving the extraction method of primary hepatic stellate cells(HSCs),and observing the autophagy of HSCs induced by miR-148a in rats.Methods The primary HSCs were isolated from adult male SD rats,and were cultured and identified,including morphology,yield,vitality and purity determination.The cell yield was determined by cytometry,and the cell viability was determined by trypan blue assay.Confocal laser microscopy was used to detect the spontaneous fluorescence.Desmin and?-SMA immunocytochemical staining were used to determine the cell purity.The primary HSCs were transfected with miR-148a mimic and inhibitor,and Western Blot was applied to detect the autophagy marker protein LC3B and the degradation of SQSTM1/p62.Primary HSCs were co-transfected with GFP-LC3 and miR-148a,and the green fluorescence spots were detected by confocal laser microscopy to determine autophagy.Results 1.Separation,culture and identification of primary HSCs:the newly separated cells were round under the microscope,containing a large number of highly refractive particles.After24-48h of the culture,they adhered to the wall,showing a round,spindle shape and a polygon.About 7 days after culture,focal growth appeared.After 14 days,the cells began to take on the appearance of fibroblasts.The cell yield of rat was about 4.2X10~7 per rat.The cell activity was above 90%.Initially separated HSCs can emit blue-green fluorescence under excitation light of 328nm.The cell purity was above 90%.2.Western blot assays revealed that miR-148a mimic could increase LC3B-II expression,promote LC3B class conversion and SQSTM1/p62 degradation,then enhance the autophagy.While miR-148a inhibitor could reduce LC3B-II expression,inhibit LC3B class conversion and SQSTM1/p62 degradation,and then reduce the autophagy.3.Fluorescence protein detection with confocal laser microscopy:the green fluorescence spots increased significantly in the group transfected with miR-148a mimic cells,promoting the GFP-LC3 fluorescence protein aggregation,indicating that the miR-148a mimic could enhance autophagy.While the green fluorescence spots decreased significantly in the miR-148a inhibitor cell transfection group,inhibiting the GFP-LC3 fluorescence protein aggregation,indicating that the miR-148a inhibitor inhibited autophagy.Conclusion 1.The improved liver two-step enzyme perfusion method can isolate HSCs cells easily,economically and efficiently.2.MiR-148a can induce autophagy activity of primary HSCs in rats.