| Objective: Esophageal cancer is a common carcinoma of digestive system which severely harms the health of mankind. The global morbidity of the esophageal cancer is 2.5-5.0/100, 000 in male and 1.5-2.5/100, 000 in female. The epidemiological data show that lack of vitamin, high nitrite contents and contaminated food with mycetes may have the relationship with esophageal cancer in central-Asia and China. With the development of molecular biology, more gene alterations were involved in the esophageal cancer which is the result of oncogene activation and anti-oncogene inactivation. For this reason, it is extremely important to study the mechanism of esophageal cancer occurrence, for the prevention or treatment of the malignancy. Metallothionein (MT) is a low-molecular-weight protein, rich of cysteine, and generally distributes in living body. MT protein has many important effects such as regulating entatic state of metal ion, detoxicating heavy metals and eliminating free radicle. There are four isomerides of MT in nature, MT-1, MT-2, MT-3 and MT-4. Compared to other MT proteins, MT-3 has a special structure and biological functions and was first identified as the growth inhibitory factor in nervous system. In order to study metallothionein -3 and esophageal cancer the relationship,The MT-3 gene was cloned into the eukaryotic expression vector EX-T3737-M33, by using an empty vector and carrying MT-1 gene EX-H2598-M15 expression vector as control.Plasmids were transfected into EC109, TE13 esophageal cancer cells in two, so that overexpression of MT-3 to study the MT-3 on esophageal carcinoma cell behavior, in order to investigate if MT-3 play a role in esophageal cancer.Methods: 1 Squamous cell carcinoma cell line of the esophagus Eca-109, and TE13 were selected, and cultured in six-well plate with RPMI1640 culture medium to the cells by up to 80% in preparation for transfection.2 Get containing EX-T3737-M33, EX-T3737-M33-MT3, and EX -H2598-M15-MT1 three kinds of strains of E. coli plasmid, and then shake bacteria, overnight culture, extract high purity plasmid and store at -70℃, ready to transfection.3 The preparation of EX-T3737-M33, EX-T3737-M33-MT3, and EX-H2598-M15-MT1 three kinds of plasmid DNA / liposome complexes, which with two kinds of cell lines in six plates were cultured within 4-6 hours, Then we observe the eukaryotic expression vector in the fluorescent protein expression in cells under the inverted fluorescence microscope respectively at 24 hours, 48 hours, 72 hours, and calculate the cell transfection efficiency in order to determine the three kinds of eukaryotic expression plasmids in the cell would be best to turn dying time.4 Two kinds of cell lines with the method in 96-well plates were transiently transfected by non-professionals. We are re-established set of six holes, each hole has been re-repeat the above operation, which repeated three times, and then select the 490nm wavelength, measure by enzyme-linked absorption of luminosity detector application of MTT method, respectively at 0 hours,24 hours,48 hours , 72 hours, and record the results. And to the results of time as the horizontal axis, OD values of cell growth curve drawn for the vertical axis.5 Collection of Eca-109, and TE13 cells transfected 6-well plates for 48 hours, the application of flow cytometry cell apoptosis.6 All measurement data is expressed as mean plus or minus standard deviation, all data are SPSS13.0 package complete. P <0.05 for the difference statistically significant.Results:1 after three kinds of plasmid DNA transfection of esophageal squamous cell carcinoma Eca-109, and TE13 cells, each transfection group were observed with fluorescent labeling under fluorescence microscope 24 hours, 48 hours, 72 hours later. MT-3 and the empty lanes are green fluorescent, MT-1 Red fluorescence, fluorescent cells, the highest expression after 24 hours, about 70-80% of the total cells, suggested that transfection is successful and efficient.2 We have observed that transfection of exogenous genes EC109, TE13 esophageal cancer cells under a microscope, found that transfection EX-T3737-M33-MT3 of the EC109, TE13 two cells becoming round after 48 hours in training, showing varying degrees of atrophy , some cells dying off; while transfected EX-T3737-M33, EX-H2598-M15-MT1 of the EC109, TE13 two cells, found no obvious abnormality, the basic and normal cell growth state of basically the same.3 The OD values of three groups were 0.282±0.015 for 0 hours after transfection. After transfection for 24 hours by empty vector group, MT3 group and MT1 group, OD values were 0.580±0.023, 0.577±0.035, 0.475±0.025 (P<0.05, MT3 group vs empty vector group and MT1 group), respectively. After transfection for 48 hours vector group, MT3 group and MT1 group OD values were 0.868±0.035,0.877±0.035,0.489±0.030 (p <0.05, MT3 group vs empty vector group and MT1 group). After transfection, 72 small-space-time vector group, MT3 group and MT1 group OD values were 1.079±0.110,1.075±0.113,0.410±0.027 (p <0.05, MT3 group vs empty vector group and MT1 group). Growth curves from the cells can be seen MT3 group in 24 hours, 48 hours, respectively, inhibited, while in the 72 hours being the maximal inhibition. And empty vector group and MT1 group suppression is not obvious at all times.4 Normal esophageal cancer cell apoptosis rate was (5.10±0.65 ) %,the apoptosis rate of esophageal cancer cell transfected with empty vector after 48 hours was (5.26±0.73) %, the apoptosis rate of esophageal cancer cell transfected with MT1 Genes after 48 hours was (4.98±0.78) %, the apoptosis rate of esophageal cancer cell transfected with MT3 Genes after 48 hours was (14.10±0.80 ). (P <0.05, MT3 group vs empty vector group and MT1 group) .Conclusions:1 The first time using transient transfection studies MT3 gene on cell proliferation of esophageal cancer.2 MT1 gene on esophageal cancer cell growth without inhibition, MT3 gene on esophageal cancer cells significantly inhibited the growth of.3 MT3 can promote apoptosis in esophageal cancer.4 MT-3 in esophageal cancer cell growth inhibition and pro-apoptotic effect, suggesting that MT -3 gene in esophageal cells may be apoptotic genes.5 Transfected with MT -3 gene, inducing its overexpression is expected to become a new method of treatment of esophageal cancer. |
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