Study On The Effect Of HARD1Over-Expression On The Proliferation Of Human Colorectal Adenocarcinoma

OBJECTIVE:This experiment use SW620、HCT-8and LS-174-T as study objects of Colorectal Cancer cell lines, and establish pc-DNA3.1(-)-hARD1Eukaryotic Expression Vector, then transfect this vector into Colorectal Adenocarcinoma cell lines cell. Through the establishment of stable ARD1over-expression Colorectal Adenocarcinoma cell lines to observe the proliferation of cell lines and to investigate the effect of hARD1over-expression in the period of occurrence and development of Colorectal Cancer.METHODS:1. Design and synthesis hARD1gene.2. Use DNA Recombinant Technology and establish Restrictive Endonuclease Construct to check PcDNA3.1/hARD1Eukaryotic Expression Vector.3. Culture SW620、HCT-8and LS-174T cells.4. Use Liposomes transfectiong and G418Filtering Methods to import into three Human Colorectal Adenocarcinoma cell lines, and establish stable ARD1over-expression Colorectal Cancer cell lines.5. Use Western blot to check the expression of hARDl in the stable ARD1over-expression Colorectal Cancer cell lines.6. Use EdU and Flow Cytometry to check the effect of hARD1over-expression on the proliferation of Colorectal Adenocarcinoma cell.RESULTS:1. Used PCR to amplificate hARDl gene; Agarose Gel Electrophoresis obtained about700bp genes, and appealed the gene amplification. 2. Through Restrictive Endonuclease Construct appraisaled about700bp hARD1genes and500bp Vector fragment, established PcDNA3.1/hARD1Recombinant Plasmid. G418filtered stable ARD1over-expression Colorectal Adenocarcinoma cell lines.3. Compared the experimental group compared with blank control group and negative control group, the rate of cell lines proliferation of experimental group is obviously elevated, and is obviously reduced of negative control group.CONCLUSION:1.Established pcDNA3.1(-)hARD1Recombinant Plasmid.2.Three Colorectal Adenocarcinoma cell lines which were transfected by pcDNA3.1(-) hARD1Recombinant Plasmid could achieve hARDl over-expression.3.Over-expressed hARDl in human Colorectal Adenocarcinoma would affect the proliferation of cells.4.Constructed hARD1Recombinant Plasmid will provide an effective tool for the further study about hARDl and Colorectal Adenocarcinoma, and it will also be a basement for studying the effect of manual-intervent hARD1over-expression to different kinds of tumos.