AIM： To construct p27mt recombinant adenovirus, transfect the colorectal cell line Lovo and observe the effects of p27mt on Lovo cell apoptosis and cell cycle inhibition. METHODS： We constructed recombinant adenovir...AIM： To construct p27mt recombinant adenovirus, transfect the colorectal cell line Lovo and observe the effects of p27mt on Lovo cell apoptosis and cell cycle inhibition. METHODS： We constructed recombinant adenovirus containing p27mt by homologous recombination in bacteria. The colorectal cancer cell line Lovo was infected with recombinant replication-defective adenovirus Ad- p27mt, and expression of p27mt was determined by Western blotting; the inhibitory effect of p27mt on Lovo cells was detected by cytometry. Cell cycle was determined by flow cytometry. DNA fragment analysis identified the occurrence of apoptosis. RESULTS： The recombinant adenovirus which already contained p27mt target gene was successfully constructed. When multiplicity of infection was ≥50, the infection efficiency was 100%. After transfection of Lovo cells with Ad-p27mt the cells had high p27 expression which was identified by immunoblotting assay. PI staining and flow cytometry showed that 77.96% of colorectal cancer cells were inhibited in phase G0/G1, while in the Ad-LacZ group and blank control group, 27.57% and 25.29% cells were inhibited in the same phase, respectively. DNA fragment analysis, flow cytometry and TUNEL assay demonstrated that p27mt is able to induce apoptosis in colorectal cancer cells. CONCLUSION： p27mt has an obvious blocking effect on colorectal cancer cell cycle, and most cells were inhibited in phase G0/G1. Therefore, p27mt can induce apoptosis in colorectal cells.展开更多
Objective： The aim of this study was to establish the foundation for studying the role of pokemon gene in tumori- genesis and development by constructing recombinant plasmids that can express small interfering RNA （...Objective： The aim of this study was to establish the foundation for studying the role of pokemon gene in tumori- genesis and development by constructing recombinant plasmids that can express small interfering RNA （siRNA） targeting human Pokemon gene. Methods： Hairpin siRNA templates targeting Pokemon gene were synthesized and cloned into plasmid vector psiRNA-Hineo. Three vectors derived siRNAs （psiRNA1, 2, 3） and one mocking psiRNAc （as control） were constructed. The recombinant Pokemon siRNA plasmids were constructed and identified using restrictive enzyme analysis and DNA sequencing, Results： Restrictive enzyme analysis and DNA sequencing revealed the successful construction of siRNA expression plasmids. Conclusion： Constructing siRNAtemplates targeting Pokemon gene may provide us with practical tools for further study the role of Pokemon gene in the development of some diseases and gene therapy of tumor.展开更多
基金Supported by The Natural Science Foundation of Hubei Province, No. 2003ABA193Bureau of Science and Technology of Shiyan City, No. 2005ZD036
文摘AIM： To construct p27mt recombinant adenovirus, transfect the colorectal cell line Lovo and observe the effects of p27mt on Lovo cell apoptosis and cell cycle inhibition. METHODS： We constructed recombinant adenovirus containing p27mt by homologous recombination in bacteria. The colorectal cancer cell line Lovo was infected with recombinant replication-defective adenovirus Ad- p27mt, and expression of p27mt was determined by Western blotting; the inhibitory effect of p27mt on Lovo cells was detected by cytometry. Cell cycle was determined by flow cytometry. DNA fragment analysis identified the occurrence of apoptosis. RESULTS： The recombinant adenovirus which already contained p27mt target gene was successfully constructed. When multiplicity of infection was ≥50, the infection efficiency was 100%. After transfection of Lovo cells with Ad-p27mt the cells had high p27 expression which was identified by immunoblotting assay. PI staining and flow cytometry showed that 77.96% of colorectal cancer cells were inhibited in phase G0/G1, while in the Ad-LacZ group and blank control group, 27.57% and 25.29% cells were inhibited in the same phase, respectively. DNA fragment analysis, flow cytometry and TUNEL assay demonstrated that p27mt is able to induce apoptosis in colorectal cancer cells. CONCLUSION： p27mt has an obvious blocking effect on colorectal cancer cell cycle, and most cells were inhibited in phase G0/G1. Therefore, p27mt can induce apoptosis in colorectal cells.
文摘Objective： The aim of this study was to establish the foundation for studying the role of pokemon gene in tumori- genesis and development by constructing recombinant plasmids that can express small interfering RNA （siRNA） targeting human Pokemon gene. Methods： Hairpin siRNA templates targeting Pokemon gene were synthesized and cloned into plasmid vector psiRNA-Hineo. Three vectors derived siRNAs （psiRNA1, 2, 3） and one mocking psiRNAc （as control） were constructed. The recombinant Pokemon siRNA plasmids were constructed and identified using restrictive enzyme analysis and DNA sequencing, Results： Restrictive enzyme analysis and DNA sequencing revealed the successful construction of siRNA expression plasmids. Conclusion： Constructing siRNAtemplates targeting Pokemon gene may provide us with practical tools for further study the role of Pokemon gene in the development of some diseases and gene therapy of tumor.
