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Glioma is the most common malignant tumors of the nervous system, with a high incidence of high morbidity and high mortality, its clinical effect is far from satisfactory. Oncogene activation and (or) the inactivation of tumor suppressor genes glioma incidence of the development of the basic molecular events. Thus gene therapy in the treatment of glioma is very promising. Transferred to the patient's body to enhance human anti-tumor ability or promote tumor demise of gene therapy of choice for the use of tumor suppressor genes. The the people ndrg2 gene is Professor Deng Yanchun using subtractive hybridization was cloned from human brain tissue, a new gene, has landed the gene bank (accession number AF159092). ndrg2 a variety of tumor tissue and corresponding normal tissues of differentially expressed genes highly expressed in the brain, skeletal muscle, and salivary glands; moderate expression in the liver, the gastrointestinal epithelium and lung epithelial; low expression in the thymus, bone marrow, and testes. Can found ndrg2 expression in glioma tissue and cell lines, reduce. The when the same time ndrg2 overexpression in glioma, inhibit cell proliferation, while the cells appear obvious G1 arrest ndrg2 by the studies of two subtypes of ndrg2S and ndrg2L. ndrg2S full length of 2024bp, encoding 357 amino acids; ndrg2L full length of 2066bp, encoding 371 amino acids. Past our study focused on multi-ndrg2 shorter gene (ndrg2S) of the research method is the use of immunohistochemistry, RT-PCR and gene transfection. Long gene and the short ndrg2 gene coding for a difference of only 42bp of nucleotides i.e. a sequence of 14 amino acids, the nucleotide sequence of a high degree of consistency and similarity of the protein antigen, such that in the above-mentioned results can not separate ndrg2 length gene or the role of the short gene, gene transfection experiments only proved ndrg2 short gene anti-tumor effect, no one has ever proved whether ndrg2 long gene have the same anti-tumor effect. Adenoviruses are non-enveloped linear double-stranded DNA virus, is widely distributed in nature, human pathogenic small, does not induce cancer, more secure, do not integrate into the cell genome, genetic toxicity is low, wide host range, transfection efficiency high, easily prepared and purified. Adenovirus vectors used for gene expression, transfer and treatment. The high transfection efficiency, easy to obtain a high titer virus vector, full high, into the cell is not integrated into the host cell genome, only transient expression. Currently The recombinant virus already contains ndrg2S successfully constructed. In order to in-depth function of research ndrg2 gene clear ndrg2L of glioma cells and its mechanism, to explore ndrg2 anti-tumor mechanisms, as well as to further explore the glioma pathogenesis and new therapeutic strategies, our group intends to establish ndrg2 length of two subtypes of recombinant adenovirus and infection of human glioma cell lines after the detection of cell biology, further in-depth study of the gene. First, the use of an adenovirus expression system ViraPowerTM Adenoviral Expression System Construction of the adenovirus vector. First the ndrg2 two subtypes (the length of two subtypes are named ndrg2L, ndrg2S) using enzyme digestion, ligation, transformation, amplification methods such as extracting plasmid was cloned into entry vector pENTR2B to get entry-to clone pENTR2B-ndrg2L, pENTR2B- LR Clonase correct the ndrg2S, double digestion and PCR, recombinant enzyme? expression cloning pAd-CMV/V5-DEST-ndrg2L II Enzyme Mix entry Cloning and expression the carrier pAd-CMV/V5-DEST between recombination reaction to and plasmid pAd-CMV/V5-DEST-ndrg2S. Expression of cloned by PCR after linearized with restriction endonuclease PacI the transfection HEK293A packaging cell recombinant adenovirus (respectively named the Ad-ndrg2L and Ad-ndrg2S) after after repeated infections amplification, half of the organizations culture infectious dose (Tissue culture infective dose 50, TCID50) to detect viral titer, Western Blot analysis of recombinant virus carrier correctly express ndrg2 protein. Was successfully constructed a recombinant adenoviral vector ndrg2 gene length subtypes, packaging amplification of viral, provide the basis for the study of cancer gene therapy. In order the Discovery of ndrg2 new variant (ndrg2L) whether the anti-tumor effect, if any, role in anti-tumor effect of, ndrg2 gene length of two subtypes there are no differences? Experiments using flow cytometry, MTT method, and colony formation experiments with ndrg2 gene length of two subtypes of adenovirus infection glioma cells were detected. Can be seen: the cell cycle was detected by flow cytometry, and the adenovirus infection ndrg2 two subtypes of glioma cells in the G1 phase of the cell number, more than of infection EGFP virus and uninfected glioma cells in G1 phase cells . The the MTT experiment results show that: of infection ndrg2 two subtypes of adenovirus glioma cells infected with EGFP virus and uninfected glioma cells, and its ability to grow decline; but infection ndrg2L adenovirus infection ndrg2S adenovirus glioma cell growth capacity did not differ significantly. The results showed that the colony formation assay: adenovirus infection ndrg2 two subtypes of glioma cells infected with EGFP virus and uninfected glioma cells, their colony forming ability decreased; but the infection ndrg2L adenovirus and infection The glioma cells ndrg2S Adenovirus colony forming ability did not differ significantly.
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