摘要：

Objective:To construct and identify eukaryotic expression vector containing short hairpin RNA(shRNA) of maspin and investigate its effect on apoptosis of gastric cancer cell line MKN-28.Methods:ShRNA targeting maspin gene was designed,synthesized,conjugated with eukaryotic expression vector pGenesil 1.1 to construct recombinant plasmid pGenesil-maspin.RT-PCR and Western blotting were used to detect the mRNA and protein expressions of maspin,bcl-2,and bax after transfection,respectively.Flow cytometry was used to determine the effects of transfection of pGenesil-maspin on cell cycle distribution and apoptosis.Results:We successfully constructed recombinant plasmids containing maspin specific shRNA(pGenesil-maspin1 and pGenesil-maspin2) and pGenesil-HK(as the blank control),which were validated by gel electrophoresis and DNA sequence analysis.RT-PCR and Western blotting analysis indicated that maspin and bax mRNA and protein expression was dramatically down-regulated(P0.01) and bcl-2 mRNA and protein expression was up-regulated(P0.01) in gastric cancer cell line MKN-28 after transfection with pGenesil-maspin positive plasmids.Flow cytometric profiles indicated that positive plasmid transfection led to a decrease in the percentage of cells in G0/G1phase(P0.01) and a decrease in apoptotic rate(P0.01).Conclusion:Transfection of exogenous maspin shRNA effectively down-regulated the expression of maspin,inhibited apoptosis,altered cell cycle distribution of human gastric cancer cell line MKN-28.The molecular mechanism might be associated with altering cell cycle,up-regulating bcl-2 expression and down-regulating bax expression.

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