Establishment Of Syncytin Gene Abnormal Expression Cell Line And Establishment Of Animal Leukemia Model

Human Endogenous retroviruses (human endogenous retroviruses, genome) is ancient retrovirus infections in the process of evolution of the human genome, which was in turn by Mendel relics left by genetic offspring.HERV can be divided into at least 31 families, and the most studied is the genome-K, genome-H, genome-W three family.The Syncytin genome-W capsule membrane protein gene encoding, is composed of 538 amino acids.This research group in the study found that syncytin mRNA and protein in eight syncytin leukemia or lymphoma cell line have different degree of expression, and syncytin genes expression levels is associated with leukemia patients. By using density gradient centrifugation separation of peripheral blood mononuclear cells of the patients and normal controls, peripheral blood mononuclear cell, PBMC), and using self-made antibody detection of leukemia patients expressed on the cell membrane,we confirmed the syncytin protein expression with leukemia cell lines and some of the peripheral blood cell membrane in patients with leukemia.Leukemia can cause great threat to human life and health, is a malignant tumour of the hematopoietic stem cells cloned 10 high incidence. Incidence of male was higher than female, and incidence rates of crowd under 35 are highest.The etiology and pathogenesis of leukemia is very complex, it is not entirely clear, and virus factors, genetic factors, chromosome and immune genetic were associated with the onset of leukemia. Since the 1970 s immunodeficiency animals directly was used to study the occurrence and development of human leukemia cells and its biological characteristics, and used to carry out the experimental treatment, is the new development of leukemia experimental study to seek a new target for the diagnosis of leukemia.Mice is similar to the characteristics and human hematopoietic system, high pure degree of inbred line animal genes at the same time, genetic background of uniform, good repeatability, and easy to control.Therefore, choosing inbred line build syncytin abnormal gene expression in mice leukemia animal models in mice can approximately reflect the actual situation of the human body, for studying the role of syncytin gene in the pathogenesis of leukemia and to provide an ideal model for the study of the mechanism of syncytin gene involved in the molecular mechanism of leukemia and targeted gene therapy.This experiment adopts the lipofectamine 2000 Reagent will syncytin expression vector transfection to EL4 cells and t cells,293 transfection.After 48 h fluorescence were observed under inverted microscope, only discovered in 293 t cells empty carrier fluorescence, by Western Blot and rt-pcr detection, a set of syncytin protein expression and gene expression is significantly higher than the control group, which suggests that effective available expression vector.Use Lenti-Pac slow HIV virus packaging Syncytin expression plasmid, infection EL4 cells, with culture medium containing the puro for positive selection, stability express Syncytin cell lines.By Western Blot test, expression of syncytin syncytin/beta Actin gray ratio in the cell group is significantly higher than the control group;results of QPCR detection stable expression cell mRNA expression show only express syncytin cells have a relatively high amount of expression.The experiment reveals the stable expression of Syncytin protein EL4-building a successful Syncytin cell line.In this study, the Jurkat cell line with low expression of syncytin was used to make the mouse leukemia animal model pre experiment.Choose C57BL/6 mice for mice in male mice, the mouse for the females.Six days before a bone marrow transplant to contain gentamycin sulfate (0.004 ml/only) sterile water feeding, until 2 weeks after transplantation.7 days away to wash the treasure before fluid (0.1 g/L) medicated bath, three days before start once a day all the mice respectively through tail vein injection of cyclophosphamide (CTX), injection quantity is 100 mg/kg.Will be 2 days before (1-2) x 106 0.5 ml each cell vaccine by tail vein to C57BL/6 mice.After bone marrow transplantation was observed between groups of mice body weight changes are modest, and in good mental state.Two weeks after the mice tail blood smear observation, between the control group (EL4 cells) and experimental group (syncytin lower expression cell group) the proportion of the original cell are of no significant difference (P> 0.05).To sum up, this study successfully established the stable expression of EL4-syncytin protein in Syncytin cell line, which provides a cellular model for the analysis of syncytin and leukemia immune escape mechanism. Animal model of mice leukemia preliminary experiments confirmed the indirect syncytin established stable expression cell lines may not appear in the process of cultivating syncytin genes expression or missing, guessing syncytin in stable expression of syncytin cells expressed in cells after repeatedly represented by inhibition of other genes.The establishment of the model provides more specific experimental methods for the subsequent syncytin stable expression cell line of mice leukemia animal model, so as to be a foundation for revealing syncytin mechanism in mice leukemia research.