Double-stranded oligomeric nucleotide encoding PEP-1 peptides was synthesized, pro- karyotic expression pET15b-pep-1-p27mt recombinant constructed, E. coli BL21 (DE3)pLysS trans- formed and induced with IPTG to highly...Double-stranded oligomeric nucleotide encoding PEP-1 peptides was synthesized, pro- karyotic expression pET15b-pep-1-p27mt recombinant constructed, E. coli BL21 (DE3)pLysS trans- formed and induced with IPTG to highly express fusion protein PEP-1-P27mt. Fusion protein with an N-terminal His-tag could be purified by Ni2+-resin affinity chromatography and identified by SDS-PAGE and Western blotting. Cultured EC9706 cells treated with PEP-1-P27mt revealed that PEP-1-P27mt was transduced into cells after 15 min and reached maximal intracellular concentra- tions in 2 h. PEP-1-P27mt of 1 μmol/L final concentration could most strongly suppress the growth. It was suggested that PEP-1 can carry P27mt across membrane, which provides a new biological pro- tocol for using cyclin dependent kinase inhibitors p27mt in suppressing the growth of tumor cells.展开更多
目的:探讨突变型p27基因(p27mutant type gene;p27mt)对恶性黑素瘤A375细胞凋亡的调节作用。方法:利用重组腺病毒Ad-p27mt转染培养的人恶性黑素瘤A375细胞,用3H-TdR掺入法检测细胞增殖;流式细胞术、DNA片段分析法、TUNEL法检测细胞凋亡...目的:探讨突变型p27基因(p27mutant type gene;p27mt)对恶性黑素瘤A375细胞凋亡的调节作用。方法:利用重组腺病毒Ad-p27mt转染培养的人恶性黑素瘤A375细胞,用3H-TdR掺入法检测细胞增殖;流式细胞术、DNA片段分析法、TUNEL法检测细胞凋亡。结果:重组腺病毒Ad-p27mt在MOI≥50时,可达到100%的转导效率。Ad-p27mt转染恶性黑素瘤细胞A375后,3H-TdR掺入法检测发现细胞增殖抑制;流式细胞术检测在G1期前出现亚二倍体凋亡峰;细胞DNA抽提电泳后发现凋亡特征性梯带。TUNEL法检测凋亡指数分别为48.5±3.6(Ad-p27mt组)及4.2±0.8(空白对照组),差异有显著性(P<0.01)。结论:重组腺病毒介导的p27mt基因转移可诱导恶性黑素瘤A375细胞在Gl期阻滞并导致细胞凋亡。展开更多
AIM:To investigate the inhibitory and anti-metastatic effect of mutant p27 gene(p27mt) on the growth of colorectal cancer xenografts in nude mice and its underlying mechanism.METHODS:Inhibitory effect of p27mt gene on...AIM:To investigate the inhibitory and anti-metastatic effect of mutant p27 gene(p27mt) on the growth of colorectal cancer xenografts in nude mice and its underlying mechanism.METHODS:Inhibitory effect of p27mt gene on the growth of colorectal cancer xenografts was determined by measurement of tumor size before and after direct intratumoral injection of Ad-p27mt in a preestablished transplantation model of human colorectal cancer in nude mice.Cell cycle and apoptosis were detected by flow cytometry performed on single-cell suspension from an isolated tumor.Expression of MMP-9 in tumor tissue was detected by immunohistochemistry.RESULTS:The average sizes of transplantation tumors were 1.94 ± 0.67 cm3,2.75 ± 0.83 cm3 and 3.01 ± 0.76 cm3 in the Ad-p27mt,Ad-LacZ and control groups,respectively(P < 0.05).The average proliferation rates were 37.34% ± 1.45%,53.16% ± 3.27% and 54.48% ± 2.43%,in the Ad-p27mt,Ad-LacZ and control groups,respectively(P < 0.05).The average apoptosis rates were 19.79% ± 3.32%,6.38% ± 4.91% and 7.25% ± 5.20% in the Ad-p27mt,Ad-LacZ and control groups,respectively(P < 0.01).The average MMP-9 expression rates were 20%,75% and 66.7% in the Ad-p27mt,Ad-LacZ and control groups,respectively(P < 0.01).CONCLUSION:p27mt inhibits the growth of transplanted tumor by blocking the proliferation of cancer xenografts and by promoting apoptosis of transplantated tumor cells,as well as decrease transpl-anted tumor metastasis.展开更多
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
In this study, the mechanism by which Ad-p27mt inhibits the growth, invasion and metastasis of transplanted liver tumor was studied by examining the effects of Ad-27mt gene transfer on the expression of Bax, Bcl-2, VE...In this study, the mechanism by which Ad-p27mt inhibits the growth, invasion and metastasis of transplanted liver tumor was studied by examining the effects of Ad-27mt gene transfer on the expression of Bax, Bcl-2, VEGF and MMP-9 in the transplanted liver tumors in nude mice.The model of transplanted hepatic tumor was established in nude mice.The mice were then divided into three groups, which were injected with PBS, Ad-LacZ and Ad-p27mt and the growth of the transplanted liver tumor was observed.The expressions of P27, Bax and Bcl-2 proteins were detected by Western blotting and the expressions of VEGF and MMP-9 were immunohistochemically determined.Our result showed that the tumor size, expressions of Bax, Bcl-2 proteins, VEGF and MMP-9 were all lower than those in PBS and Ad-LacZ groups and the differences were statistically significant (P【0.05).Our study suggested that Ad-p27mt could inhibit the growth, invasion and metastasis of hepatic cancer by lowering the expressions of VEGF and MMP-9.展开更多
基金a grant from Department of Education of Hubei Province, China (No. Q200524001).
文摘Double-stranded oligomeric nucleotide encoding PEP-1 peptides was synthesized, pro- karyotic expression pET15b-pep-1-p27mt recombinant constructed, E. coli BL21 (DE3)pLysS trans- formed and induced with IPTG to highly express fusion protein PEP-1-P27mt. Fusion protein with an N-terminal His-tag could be purified by Ni2+-resin affinity chromatography and identified by SDS-PAGE and Western blotting. Cultured EC9706 cells treated with PEP-1-P27mt revealed that PEP-1-P27mt was transduced into cells after 15 min and reached maximal intracellular concentra- tions in 2 h. PEP-1-P27mt of 1 μmol/L final concentration could most strongly suppress the growth. It was suggested that PEP-1 can carry P27mt across membrane, which provides a new biological pro- tocol for using cyclin dependent kinase inhibitors p27mt in suppressing the growth of tumor cells.
文摘目的:探讨突变型p27基因(p27mutant type gene;p27mt)对恶性黑素瘤A375细胞凋亡的调节作用。方法:利用重组腺病毒Ad-p27mt转染培养的人恶性黑素瘤A375细胞,用3H-TdR掺入法检测细胞增殖;流式细胞术、DNA片段分析法、TUNEL法检测细胞凋亡。结果:重组腺病毒Ad-p27mt在MOI≥50时,可达到100%的转导效率。Ad-p27mt转染恶性黑素瘤细胞A375后,3H-TdR掺入法检测发现细胞增殖抑制;流式细胞术检测在G1期前出现亚二倍体凋亡峰;细胞DNA抽提电泳后发现凋亡特征性梯带。TUNEL法检测凋亡指数分别为48.5±3.6(Ad-p27mt组)及4.2±0.8(空白对照组),差异有显著性(P<0.01)。结论:重组腺病毒介导的p27mt基因转移可诱导恶性黑素瘤A375细胞在Gl期阻滞并导致细胞凋亡。
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 investigate the inhibitory and anti-metastatic effect of mutant p27 gene(p27mt) on the growth of colorectal cancer xenografts in nude mice and its underlying mechanism.METHODS:Inhibitory effect of p27mt gene on the growth of colorectal cancer xenografts was determined by measurement of tumor size before and after direct intratumoral injection of Ad-p27mt in a preestablished transplantation model of human colorectal cancer in nude mice.Cell cycle and apoptosis were detected by flow cytometry performed on single-cell suspension from an isolated tumor.Expression of MMP-9 in tumor tissue was detected by immunohistochemistry.RESULTS:The average sizes of transplantation tumors were 1.94 ± 0.67 cm3,2.75 ± 0.83 cm3 and 3.01 ± 0.76 cm3 in the Ad-p27mt,Ad-LacZ and control groups,respectively(P < 0.05).The average proliferation rates were 37.34% ± 1.45%,53.16% ± 3.27% and 54.48% ± 2.43%,in the Ad-p27mt,Ad-LacZ and control groups,respectively(P < 0.05).The average apoptosis rates were 19.79% ± 3.32%,6.38% ± 4.91% and 7.25% ± 5.20% in the Ad-p27mt,Ad-LacZ and control groups,respectively(P < 0.01).The average MMP-9 expression rates were 20%,75% and 66.7% in the Ad-p27mt,Ad-LacZ and control groups,respectively(P < 0.01).CONCLUSION:p27mt inhibits the growth of transplanted tumor by blocking the proliferation of cancer xenografts and by promoting apoptosis of transplantated tumor cells,as well as decrease transpl-anted tumor metastasis.
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.
基金supported by a grant form the Program of Scientific Research of Bureau of Science and Technology of Hubei Province,China (No.2005ABA082)
文摘In this study, the mechanism by which Ad-p27mt inhibits the growth, invasion and metastasis of transplanted liver tumor was studied by examining the effects of Ad-27mt gene transfer on the expression of Bax, Bcl-2, VEGF and MMP-9 in the transplanted liver tumors in nude mice.The model of transplanted hepatic tumor was established in nude mice.The mice were then divided into three groups, which were injected with PBS, Ad-LacZ and Ad-p27mt and the growth of the transplanted liver tumor was observed.The expressions of P27, Bax and Bcl-2 proteins were detected by Western blotting and the expressions of VEGF and MMP-9 were immunohistochemically determined.Our result showed that the tumor size, expressions of Bax, Bcl-2 proteins, VEGF and MMP-9 were all lower than those in PBS and Ad-LacZ groups and the differences were statistically significant (P【0.05).Our study suggested that Ad-p27mt could inhibit the growth, invasion and metastasis of hepatic cancer by lowering the expressions of VEGF and MMP-9.
