| Objectives: To evaluate the mechanisms of immune escape of gastric cancer cell by detecting the expression of Fas, FasL in human gastric adenocarcinoma cell line SGC-7901; To evaluate the inhibition of gastric adenocarcinoma cell by tranfecting heparanase (HPSE) antisense oligodeoxynucleotide (AS-ODN) into SGC-7901 cells before and after AS-ODN transfection in vitro. To evaluate the mechanisms of HPSE AS-ODN inhibited and induced apoptosis of gastric cancer cells by detecting expression of HPSE mRNA, Fas, FasL, Bcl-2 in human gastric adenocarcinoma cell line SGC-7901 ; To evaluate the inhibition of invasiveness of gastric adenocarcinoma cell by detecting the expression of basic fibroblast growth factor(bFGF) and CD44v6 in human gastric carcinoma cell line SGC-7901 by transfecting HPSE AS-ODN into SGC-7901 cells.Methods: The AS-ODN complementary to the start codon region of heparanase mRNA and its control, scrambled nonsense oligodeoxyuncleotide (NS-ODN) were desiged and synthesized and phosphorthioated. The experiment was divided into 4 groups: controls (no transfection) , liposome controls (liposome transfected only), nonsense oligodeoxynucleotide groups(ODNs, ODNs were embedded in cationic liposome), AS-ODN groups (AS-ODN were embedded in cationic liposome Lipofection and transfected into SGC-7901 cell) .Choosing 0.1 > 0.2, 0.4, 0.8umoL/L AS-ODN to do the experiment according to the pre- experiment. They were embedded in cationic liposome Lipofection and transfected into SGC-7901 cells and cultured in 37 °C,5%CO2 culture box for 48h. The expression of HPSE mRNA, Fas, FasL and Bcl-2 were detected by reverse transcription-polymerase chain reaction analysis (RT-PCR) before and after transfection. The morpologic test, Flow cytometery were used to measure apoptosis of gastric cancer cells. The cells proliferation were detected by MTT method.The expression of Fas, FasL> bFGF and CD44V6 were detected by immunohistochemical staining method before and after transfection. The invasiveness of transfected gastric adenocarcinoma cells were measured quantitatively by matrigel invasion assays.Results:The first part: The effect of HPSE AS-ODN on immune escape in human gastric adenocarcinoma cell line SGC-7901.The results of RT-PCR slowed that HPSE mRNA, Fas mRNA, FasL mRNA and Bcl-2 mRNA of SGC-7901 treated with AS-ODN of different final concentration were significantly changed (HPSE mRNA, FasL mRNA and Bcl-2 mRNA were decreased, Fas mRNA was increased)compared with that of the controls. AS-ODN has a significant inhibitory effect on the SGC-7901 and influence on the expression of Fas mRNA, FasL mRNA and Bcl-2 mRNA in a dose-dependent manner. But the controls, the ODNs and the liposome controls have no difference.The second part: The effect of HPSE AS-ODN on apoptosis and proliferation in human gastric adenocarcinoma cell line SGC-7901.The results of flow cytometry showed typical sub-diploid "apoptotic peak" in DNA histogram.48 hours after the transfection with AS-ODN at the concentration of 0.1, 0.2, 0.4 and 0.8p.moL/L, the apoptotic rates of SGC-7901cells were 9.13%, 16.00%, 15.63% and 13.32% respectively; In a multivariate analysis, there were significant difference in different groups with concentration (P < 0.01). There was no difference among normal controls, NS-ODN groups and liposome controls (P>0.05) .The percentage of S phase or G2M+S phase is regarded as proliferation index in flow cytometry.In our experiment, flow cytometry show that the phases of gastric adenocarcinoma cells were changed after transfected with AS-ODN of different final concentration. The percentage of S phase was decreased. They were 47.26%,, 47.72% and 46.98% in normal controls, liposome controls and 0.4 u moL/L NS-ODN groups ( P > 0.05 ) respectively. There were no difference among the groups(i>>0.05). The S phase percentages of AS-ODNs are 33.46%, 32.98%, 29.48% and 13.27% respectively. Compared with normal controls, there was significant difference (i><0.01). The results are coincided with those observed in MTT.The results by MTT showed that AS-ODN could inhibit the proliferation of SGC-7901 cells. In a multivariate analysis, the mean of proliferative rates of cells showed statistical significance in different group with time and concentration. And the inhibition enhanced gradually with the extension of time and the increasing of concentration. The inhibitory effects on SGC-7901 were in a dose-dependent and time-dependent manner(P < 0.01). The proliferation of SGC-7901 cells were 90.23, 85.35, 78.81, 65.23; 80.22, 74.64,48.50,42.57; 73.56,37.68,26.53,20.65 respectively, after transfected with AS-ODN at the concentration of 0.1, 0.2, 0.4 and 0.8^moL/L in 24, 48, 72h.The third part: The effect of HPSE AS-ODN on invasiveness in human gastric adenocarcinoma cell line SGC-7901The results by immunohistochemical staining showed that AS-ODN could inhibit the expression of bFGF and CD44v6 in human gastric adenocarcinoma cell line SGC-7901.The positive percentages of bFGF in SGC-7901 cells are 57.2%, 51.1%, 42.5% and 36.2% respectively, aftertransfected with AS-ODN at the concentration of 0.1, 0.2, 0.4 and 0.8umoL/L in 48h. The positive percentages of CD44v6 in SGC-7901 cells are 56.5%, 54.1%, 46.5% and 41.3%.The results above showed statistical significance in different groups, compared with the controls, the ODNs and the liposome controls groups. The invasive cells among the controls, the ODNs and the liposome controls groups have no difference(P>0.05). The invasive cells in AS-ODNs of different concentrations decreased and have significant difference (P<0.01). The inhibitory effects on SGC-7901 were in a dose-dependent manner (P< 0.01).There were statistical significance in different AS-ODNs groups(P< 0.01).Conclusions:1. The expression of HPSE mRNA in human gastric adenocarcinoma cell line SGC-7901 decreased after treated with AS-ODN, which indicated heparanase AS-ODN could downregulate the expression of HPSE by closing genes of HPSE.2. The expression of FasL mRNA in human gastric adenocarcinoma cell line SGC-7901 decreased after treated with AS-ODN and Fas mRNA increased, which indicated heparanase AS-ODN could inhibit immune escape of gastric cancer cell.3. The results of flow cytometry showed typical sub-diploid "apoptotic peak" in DNA histogram after treated with AS-ODN. The apoptotic rates of SGC-7901 cells increased and the proliferation of SGC-7901 cells decreased, which indicated heparanase AS-ODN could induce apoptosis to inhibit gastric cancer cells.4. AS-ODN could accelerate the expression of Fas and inhibit the expression of FasL ^ Bcl-2, which indicated heparanase AS-ODN induce apoptosis by upregulating the expression of Fas and downregulating the expression of FasL and Bcl-2 genes.5. After transfected HPSE AS-ODN, the expressions of bFGF andCD44v6 decreased obviously > which indicated heparanase AS-ODN could inhibit the invasiveness of gastric cancer cells.The results of the study provid experiment evidences for gastric cancer therapy using HPSE AS-ODN.They also show a new method for gastric cancer therapy. The results of the study should be further studied on animals, which could make them be applied in clinical therapy.
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