| Objectives: Esophageal cancer is one of the malignant tumors of digestive tracts, which makes a serious threat to human health. The mortality of esophageal cancer in the world ranks the seventh of malignant tumors. China is a country of high incidence of esophageal cancer, especially for the high incidence in the area of Taihang Mountains. It is important for theory and clinical that the the pathogenesis of esophageal cancer was explored and than effective prevention and treatment measures was come up with. Studies have shown that the incidence and development of esophageal cancer is a multi-factor, multi-stage, multi-step process, Many factor work in the progress, include environment, habits and oncogene activated and inactivation tumor suppressor genes and abnormal cell regulation mechanisms.Metallothionein (MT) is a class of low molecular weight cysteine-rich metal-binding proteins. MT could Scavenge free radicals, detoxificate heavy metals and involve trace elements metabolism in vivo and so on. MT-3 (also known as growth inhibitory factor, GIF,) has growth inhibitory activity. It has been reported that MT-1 & MT-2 was high expression condition in esophageal cancer. But there is little research about MT-3. Our previous study showed that the hypermethylation of MT-3 gene extensively exists in esophageal carcinoma, moreover hypermethylation of MT-3 can lead to reduced expression level. To continue to study the role of MT in esophageal cancer, The gene transfection and RT-PCR technique were used in our experiments. When transfected Eca-109 cells were cultured ,The state of cell growth and gen expression in Eca-109 cell were observed.Method:1 There were 3 kinds of plasmid in E.coli strains, which were EX-T3737-M03-MT-3, EX-T2598-M56-MT-1E and the EX- T2598-M56. The MT-3 gene is in the first plasmid, the MT-1E gene is in the second plasmid, the latter plasmid is an empty. Plasmid was extracted from the E.coli strains after which cultured through one night ,by the plasmid mentioned night kit (TIANGEN company) , than concentration and purity of the plasmid were determined .2 The Eca-109 cells (a kind of squamous cell carcinoma of the esophagus) were cultured in six-well plate with RPMI1640 medium. When cell confluence of 70%, preparating for cell transfection.3 The plasmid was transiently transfected into Eca-109 cells using cationic liposome. After the Eca-109 cells were transfected by plasmid for 48h, the cells of the transfection groups and control groups were collected in six-well plate, than the cell were counted.4 The mRNA was extracted from the cells cultured, than mRNA expression were measured and study. 5 All measurement data were analyzed by the SPSS13.0 statistical software. Variance analysis and LSD-t test were selected, Test standard isα=0.05 , P<0.05 was considered statistically significant.Results:1 When the Eca-109 cells were transfected by 3 kinds of plasmid 48 hours, fluorescent marker were observed in all groups by fluorescence microscope. The cells transfected by EX-T2598-M56-MT-1E and EX-T2598-M56 were red fluorescent. The cells transfected by EX-T3737-M03-MT-3 were green fluorescent. Counts of fluorescence marker was 80% of the total number of cells, suggesting that transfection was successful.2 The count of the 3 groups transfected was weaker than control group (P<0.05),there is statistical significance. The count of cells between transfected by MT-1E and by blank vector was no obviously different (P>0.05), there is no significant difference. The count of cells transfected by MT-3 was was weaker than other 3 groups ( P<0.05),. there is statistical significance.3 MT-1E gen mRNA expression of the group transfected by MT-1E was greater than the group transfected by blank vector and control group (P<0.05), there is statistical significance. MT-1E gen mRNA expression between the control group, the group transfected by blank vector and he group transfected by MT-3 were on obviously different (P>0.05), there is no significant difference.4 MT-3 gen mRNA expression of the group transfected by MT-3 was greater than the g other 3 groups (P<0.05), there is statistical significance. MT-3 gen mRNA expression between the control group, the group transfected by blank vector and he group transfected by MT-1E were on obviously different (P>0.05), there is no significant difference.Conclusion:1 MT-1E & MT-3 gen was transfected into Eca-109 cells and expressed successfully.2 When MT-1E gen exppressed in Eca-109 cells, after transfection. It was not significant effect on cell growth, indicating that the effect of MT-1E gene is not obvious in the pathogenesis of esophageal cancer.3 MT-3 gen exppressed in Eca-109 cells, after transfection. It showed inhibition of cell proliferation, indicating that MT-3 gene could inhibit esophageal cancer cell.4 When MT-1E or MT-3 gen was successfully transfected into Eca-109 cells and expressed, another expression of the gene had no significant change, indicating that the expression of different MT subtypes in esophageal cancer should have different mechanisms. . |
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