Construction Of PEGFP-N1-BKβ₁ Fusion Gene Vector And Research Of Amygdalin On Smooth Muscle Cell Proliferation

The methods of obtaining bronchus smooth muscle cell (BSMC) were dicussed: digest method.after getting the cells from the wista rat,then 0.05% Trypsin for 1hr and 30mins in 37℃.one week later,cells were full of the base of the bottle.the BSMC were subcultured in DMEM-F12 containing 10% FBS with zobary and spindly smooth membrane.The Objection of the thesis is to construct green fluorescent protein (pEGFP-N1)- BKβ₁ fusion gene vector for observing the expression and localization of BKβ₁ in HEK-293 cells. the coding region in cDNA was amplified by RT-PCR from rat airway smooth muscle cells and rat bronchia smooth muscle cells.Total RNA is extracted from the 3rd - 5th generation cells,reversely translated,then followed with PCR and recombined into pEGFP-N1 plasmid expressing pEGFP-N1.After identification with restriction endonucleases and sequence analysis,the recombinant plasmid will be transfected into HEK-293 cells with the cationic liposome DOTAP as the transfection reagent.Amygdalin (D-mandelonitrile-β-D-gentiobioside) is a natural compound with the anti-tussive and anticancer activities.It is decomposed by the action ofβ-D-glucosidase to yield hydrocyanic acid which stimulates the respiratory center reflexively and produces a kind of antitussive and antiasthmatic effects.amygdalin is important in respiratory disease and cardiovascular disease.In this thesis,Amygdalin inhibited BSMC proliferation by 50% after 24hrs.