| Objective:1. To construct the recombinant eukaryotic vector expressing MASPIN cDNA and detect its effect on MASPIN expression of SGC7901.2. To detect the influence of cell proliferation, cell cycle and apoptosis of SGC7901 before and after transfection.3. To detect the expression changes of Bax/Bcl-2 gene and investgate the molecular mechanism of apoptosis of SGC7901induced by MASPIN overexpresing.Methods:1. The fragment of MASPIN gene was amplified by polymerase chain reaction(RT-PCR).A eukaryotic expression vector MASIPIN/PCR2.1 was constructed with PCR2.1 and transfected into the SGC7901.2. The characteristic DNA ladders of apoptosis were determined by agarose gel electrophoresis (AGE).3.TNF-related apoptosis inducing ligand (TRAIL) was used to induce apoptosis during the different time (50ng/ml). Cell apoptosis rate was measured by FCM.4. RT-PCR and Western blot methods were used to detect the expression changes of MASPIN,Bax/Bcl-2 gene.5. The effects of MASPIN overexpressing on the cell proliferation, cell cycle of SGC7901 were detected by MTT and FCM.Results:1. Recombinant plasmid MASIPIN/PCR2.1 was successfully constructed and transfected into SGC7901 cells. The mRNA and protein level of MASPIN were significantly higher in the MASPIN/PCR2.1 group (33.62±1.2, 23.36±1.6)than in the PCR2.1 group(15.01±1.5,12.32±1.5)and the untreated group(13.72±2.0,12.01±1.3, P<0.05).2. DNA ladders appeared by AGE. A time-dependent apoptosis inducing effect was demonstrated in each group: The apoptosis rate was the highest in the MASPIN/PCR2.1 group with TRAIL inducing and the rates at 12h,24h,48h were 8%,16.3%,25.8%; the MASPIN/PCR2.1 group's were 3.0%,8.2%,14.4%; the TRAIL inducing group's were 4.1%,9.8%,15.9%(P<0.05).3.The Bax mRNA and protein expressing level at 48h of MASPIN/PCR2.1 vector group with TRAIL inducing(55.32±,75.38±1.3) were significantly higher than the PCR2.1 group (34.32±1.2,40.67±1.8)and the TRAIL inducing group(43.25±1.8,36.18±1.3,P<0.05). The Bcl-2 mRNA expression were 28.33±2.5,34.31±1.2,32.84±2.1(P<0.05)and the protein expression were 17.43±1.5,45.11±2.1,42.84±1.5(P<0.05).4. The proliferation of SGC7901 cells was significantly lower(93.07%,81.97%,66.5%) in the MASPIN/PCR2.1 group than in the PCR2.1 group(95.98%,95.79%,95.59%,P < 0.05) at 24h,48h,72h. the G0/G1 phase proportion of the MASPIN/PCR2.1 group at 72h was significantly higher than in the PCR2.1 group(70.3%vs55.6%,P < 0.05).the S phase proportion was 19.8% and 34.9%(P<0.05)respectively .Conclusions:1. The expression vector MASPIN/PCR2.1 is constructed successfully and can be expressed in eukaryotic cells.2. The MASPIN overexpression can induce apoptosis and significantly enhance the sensibility to the apoptotic inducer in gastric carcinoma. The onset of apoptosis in gastric carcinoma cell lines is related to the up-regulation of Bax and down-regulation of Bcl-2 in mRNA and protein level that induced by the MASPIN overexpression.3. MASPIN overexpressing can suppress the proliferation of gastric carcinoma cells SGC7901. |
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