| BackgroundEsophageal cancer is one of the world within the scope of one of the most common gastrointestinal malignancy, the fatality rate of malignant tumor mortality around the world is located in the sixth. Now, incidence of esophageal cancer showed a trend of rising year by year, the high incidence are mainly distributed in Turkmenistan, Kazakhstan, South Africa, France, etc., China is a high incidence of esophageal cancer area, too, especially in some provinces of northern China, such as Henan, Hebei, Shanxi. Esophageal cancer has no early clinical symptom, always ignored by the patients. Large numbers of the patients who appeared more obvious symptoms may have been characterized by advanced esophageal tumors that lost treatment opportunities. In recent years, the treatment of esophageal cancer(including operation method, radiation and chemotherapy and targeted therapy, etc.) has made great progress, the survival and prognosis of patients with esophageal cancer have a certain degree of improvement, but the case fatality rate is still high. At present, the overall 5-year survival rate less than 30%. The esophageal cancer with different types of cytotoxic drugs appeared different degree of drug resistance, find new esophageal tumor targeting effect locus and new cancer drugs for the current research new and hot field.For new anti-cancer drugs, the use of small subclass protein material treatment of malignant tumor is a very promising research direction, this experiment, the m/z 6455.5 Da protein is a functional peptide fragments of small molecule protein. Application of small molecule protein substances as benefits include cancer drugs, can undertake artificial synthesis, and can be easily in the structure of the peptide in artificial. Based on people has a understanding of microstructure and function of protein, the researchers have the ability to design a new type of small molecule protein can be used in the tumor treatment. For small molecular protein in the treatment of tumor research focused on the function of peptides, including two aspects: 1, some peptides can serve as a link 2 targeted therapy of cancer, some peptides have the ability to penetrate the tumor cell membrane and poisonous to the cells of the tumor cells. In recent years, some studies show that, with cluster hydrophobic and with a nature characteristics of the structure of cationic antimicrobial peptides(AMPs) have the ability to kill microorganisms, and showed strong cytotoxicity to tumor cells. These peptides can be gathered in the membrane surface, destroy the cell membrane or organelles membrane, causing cell apoptosis or necrosis. Cell membrane penetration peptide(CPPs) and tumor targeting peptide(TTPS) employed for the tumor cell surface(such as cell membrane or tumor vascular endothelial cells have the ability of specific markers, can be used as a carrier of carrying anticancer drugs, cytoplasmic membrane of tumor cells or tumor vascular specific targeted killing. The present study the difficulty in the treatment of the peptides used in clinical trials, such as how to increase the peptide effects on tumor targeting ability, clear therapeutic peptides pharmacokinetic characteristics etc. objectiveTo explore the m/z6455.5 Da protein effect on esophageal TE1 and EC109 cells; Preliminary discussion on m/z6455.5 Da protein affect TE1 and EC109 esophageal cancer cells channel; For m/z6455.5 Da protein as a new drug in vitro experimental data applied to clinical. Materials and methodsMaterial: M/z 6455.5 Da protein comes from my team before the experimental research results, esophageal cancer, TE- 1 EC109 cells are purchase Shanghai cell bank, Chinese academy of sciences, recovery at 37 ? and 5% CO2 concentration environment, using 10% serum medium continues to develop, extend at least 2 times, to choose the good cells biological activity. MethodsGet in good shape and TE1 EC109 cells after the test according to the following methods:(1) Indirect cell immunofluorescence: using different concentrations of m/z 6455.5 Da protein with FITC fluorescence labeled cells, respectively deal with Te1 and Ec109 cells, cells with indirect immunofluorescence technique showed nuclei and cytoskeleton, and the m/z 6455.5 Da protein track position(2). Cell toxicity test: using different concentrations of m/z 6455.5 Da protein processing TE- 1 Ec109 cells, respectively, using Cell Counting Kit- 8(CCK 8) method to detect and describe the Cell growth curve, evaluation of m/z 6455.5 Da protein intervention effect on the esophageal cancer cells.(3) Flow cytometry techniques: EC109 cells, TE- 1 cell preparation into single cell suspension respectively, with a fluorescent dye(iodide pyridine PI) after dyeing, analysis of different doses of m/z 6455.5 Da protein to intervene early esophageal cancer cells apoptosis, the differences between the late apoptosis.(4)Western blot protein imprinting technology: the purified by different concentration of 6455.5 m/z Da protein after intervention in two kinds of esophageal cancer cells protein, quantitative analysis, through the system of glue, electrophoresis, transfer printing, color, such as imaging process, finally nitrocellulose membrane was analyzed by protein immunoblot coloring location and shading depth analysis on target protein expression in esophageal cancer cells Results1. Indirect immunofluorescence test cells: m/z 6455.5 Da proteins under different concentrations and the role of the specific time, all can with EC109 cells and TE- 1 phase, and the concentration dependent effect relationship(P < 0.001)2.Cytotoxicity experiment(CCK 8) : 6455.5 m/z Da proteins under different concentrations and the role of the specific time, and TE- 1 for EC109 cells have inhibition of proliferation, and show the time dependent effects- concentration relationship(P < 0.001)3.Flow cytometry techniques: flow's cells in cell apoptosis detection, along with the load of 6455.5 m/z Da protein concentration increased, apoptotic cells increased gradually, and with 6455.5 m/z Da protein concentration is concentration dependent effect(P < 0.05)4.Western blot experiments: after the specificity of PCNA protein, BAX enhancement tips, use different m/z 6455.5 Da protein solubility, this two kinds of specific protein expression present trend, in which the expression of PCNA protein with m/z 6455.5 Da concentration increases present a trend of gradual decline(P < 0.05), while the expression of BAX protein with m/z 6455.5 Da protein concentration increased, showing a rising trend(P < 0.05) ConclusionM/z 6455.5 Da protein showed inhibitory effect both on TE1 and EC109 cells The higher concentration of the m/z 6455.5 Da protein used to intervene on the esophageal cancer cells, the cells inhibition rate is getting higher. |
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