| Background Gastric carcinoma is a very common tumor in China, both its incidence rate and mortality occupy the first place among the gastrointestinal malignant tumors. Gastric carcinoma is a severe threat to people's health. Nowdays operation and other comprehensive measures such as chemotherapy and radiotherapy afterwards are still main therapeutic approaches, but postoperative recurrence rate and metastasis rate remain as high as 40% to 65%, the overall 5-year survival rate is only about 30% . Postoperative recurrence and metastasis are the main factors contributing to patients' death. So it is absolutely necessary to explore new modalities of treatment to inhibite postoperative metastasis, and to increase efficacy of comprehensive therapies, thus to improve postoperative prognosis.Antisense techinique, one of the gene therapy, is based on the base pair when antisense oligonucleotides(AsODN) incubated with tumor cells, sequence specific complementary to the target sequence would block transcription and expression of the relevant gene. Because of its effectiveness, safety and simplicity, antisense techinique is a promising approach in the treatment of gastric caicinoma.Ets-1 gene is a member of Ets family and was originally characterrized as the v-ets retroviral gene in the avian leukemia retrovirus E26. The protein coded by the Ets-1 gene is a transcription factor that regulates the expression of matrix proteases. Ets-1 appears to play an important role in angiogenesis, regulating the expression of matrix proteases.Ets-1 gene is transiently expressed in endothelial cells during vasculardevelopment in embryos, as well as during angiogenesis in adults in normal conditions. Researches showed that Ets-1 gene expressed highly in breast carcinoma, colon carcinoma, rectal carcinoma, esophageal carcinoma, uterine endometrial and so on, these results provided theoretical basis for gene therapy targeted to Ets-1 gene on malignant tumors.Objective (1) To study the relationship between Ets-1 gene expression and Ihe biological behavior, prognosis in patients with gastric caicinoma. ( 2 ) To investigate the effect of Ets-1-AsODN on expression of Ets-lmRNA, Ets-1 protein of gastric caicinoma cell line SGC-7901, to investigate the effect of Ets-1-AsODN on Ihe capability of heterotypic adhesion, invasion, motility induced by chemokine and vessel channel formation in vitro. ( 3 ) To study the inhibitory effect of Ets-1-AsODN on growth of human gastric caicinoma in nude mice.Methods (1) Expression of Ets-1 protein was detected immunohistochemically in paraffinembedded specimen of 86 cases of gastric caicinoma Relationship between Ets-1 expression and clinicopathological parameters , postoperative prognosis were analysed. (2) The AsODN were complementary to Ets-lmRNA sequences. Human gastric carcinoma cell line SGC-7901 was treated with AsODN. Effect of Ets-1-AsODN on expression of Ets-lmRNA, Ets-1 protein of SGC-7901 cells were detected by RT-PCR and immunocytochemistry technique respectively. Modified Boyden chamber model was used for quantitative invasion and chemotaxis motility assay. MTT assay was used for heterotypic adhesion tests. Vessel channel formation in vitro was studied by tube formation assay. Flow cytometry(FCM) was used to detecte expression of matrix metalloproteinase-7(MMP-7). (3) Human gastric caicinoma transplanted subcutaneously in nude mice was constructed. A total of 12 mice were divided into 3 groups, namely, control group, Ets-1-AsODN group and Ets-1-sODN group. Different treatments were given respectively for 12 days. The weight and volume of the subcutaneous tumors were measured. Expression of Ets-1 gene , MMP-7 and microvessel density (MVD) of the subcutaneous tumors were examined immunohistochemically.Results (1) Ets-1 protein expressed in all tumor tissues. Ets-1 labelling indices(LI) ranged from 24.00% to 80.00% with the mean of (56.00±13.26) %. Ets-1 protein expressed highly in 44 cases, lowly in 42 cases. Ets-1 LI correlated with tumor diameter,regional nodal metastasis and infiltrative depth positively(P<0.05). Ets-1 LI in tumors with diameter>4cm was significantly higher than those with diameter<4cm(61.41%±11.73%vs49.78%±9.35%, P <0.05) ; Tumors with regional nodal metastasis showed higher Bs-1 LI than those without nodal involvement (61.71%±10.68% vs 44.18%±9.90%, i><0.05); The deeper of the tumor infiltration, the higher of Bs-1 expression. Ets-1 LI in tumors of Ti ,T2 ,T3, T4 was (28.50±3.42) %, (45.53±7.83) %, ( 57.23±9.27) %, ( 67.30± 10.32) % respectively (P<0.05). No significant difference was found between Ets-1 LI and age, site of tumor, and differentiation (P >0.05) . The overall 5-year survival rate of these 86 cases was 32.56%. Patients with high Ets-1 expression had a lower survival rate than those' with low expression (42.85% vs 22.73%,P<0.01), multiple variables analysis indicated that expression of Ets-1 was an independent risk factor affecting the prognosis of gastric carcinoma. ( 2 ) The effect which Ets-1-AsODN reduced level of Ets-1 mRNA was detected at 0, 10, 15, 20, 25, 30umol/L levels, the results showed that the antisense inhibitory activity enhanced gradually from 0 to 20umoI/L after treatment for 48h. However the activity did not increase sharply at more than 20umol/L. Then the effect which Ets-1-AsODN reduced level of Ets-1 mRNA was observed at 0, 12, 24, 36, 48, 60h respectively, the antisense inhibitory activity was the most obvious after treatment for 48h afterwards the activity reduced. At last the SGC-7901 cells were treated with different ODN, the Ets-1/p-actin ratio of AsODN group was significantly lower than that of sODN group and control group (0.510±0.006 vs 0.980±0.012vsl.010±0.015, P<0.05), no significant difference was found between sODN and control group (P>0.05) . Immunocytochemistry detection showed that average gray degree of AsODN group was significantly lower than the other two groups( 1.35±0.66vsI.88±0.84vs2.01±1.02, P<0.05) . At the concentration of 20umol/L, Ets-1-AsODN had significant effect in terms of adhension to matrigel on SGC-7901 cells at the time of treatment for 120min and 180min(P<0.05) . Ets-1-AsODN could inhibite the ability of invasion and motility of SGC-7901 cells, with inhibition rate of 52.82% and 45.10% respectively. Tube formation assay also showed that vessel channel formation in vitro was inhibited by Ets-1-AsODN. FCM analysis indicated that fluorescent intensity and percent of positive cells of MMP-7 of control group , sODN group and AsODN... |
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