To study the effect on regulation of cell cycle of osteosarcoma cell line MG63 tranceduced with exogenous p16ink4a and hRbl genes, pIRES-p16ink4a-hRb1, pIRES-p16ink4a and pIREShRbl plasmids were constructed by gene re...To study the effect on regulation of cell cycle of osteosarcoma cell line MG63 tranceduced with exogenous p16ink4a and hRbl genes, pIRES-p16ink4a-hRb1, pIRES-p16ink4a and pIREShRbl plasmids were constructed by gene recombination technology. The recombinant plasmid was transferred into osteosarcoma cell line MG63 by metafectene, and the resistant clones were selected by G418 selective medium, mRNA and protein expression of osteosarcoma cell line were assayed by RT-PCR and Western-Blot respectively. Cell cycle and apoptosis were analyzed by subG1 flow cytometric. Cell proliferation was tested by MTT. In the genome of these transfected target cells, the expression of p16ink4a and hRb1 mRNA and protein were detected respectively in vitro. It was demonstrated with subG1 flow cytometric analysis and MTT method that p16ink4a and hRbl genes cooperation more significantly inhibited cell growth and induced a more marked G1 arrest and apoptosis than p16ink4a/hRb1 alone （P〈0.01）. Coexpression of exogenous p16ink4a with hRbl broke the regulatory feedback loop of p16ink4a-cyclinD1/CDK-hRbl and played a more significant role in inhibiting cell growth as well as inducing cell apoptosis than p16ink4a or hRbl did alone in vitro.展开更多
The aim of this study is to investigate cyclin E, pl6ink4a and ki67 as possible diagnostic biomarkers for cervical preneoplasia using cervical exfoliated-cell specimens, and evaluate the significance for screening pat...The aim of this study is to investigate cyclin E, pl6ink4a and ki67 as possible diagnostic biomarkers for cervical preneoplasia using cervical exfoliated-cell specimens, and evaluate the significance for screening patients at high risk of de- veloping cervical carcinoma. The expression of cyclin E, p16ink4a and ki67 was examinated in 78 cervical exfoliated epithe- lial specimens diagnosed as atypical squamous cells of undetermined significance (ASCIIS) (12 cases), cervical intraepithe- lial neoplasia (CIN) of type 1 (17 cases), CIN2-3(38 cases) and invasive carcinoma (11 cases) using immunohistochemical analysis, and simultaneously, the DNA status of human papillomavirus (HPV) type 16/18 was detected by polymerase chain reaction (PCR) using type specific primers, cyclin E, pl6ink4a and ki67 were all overexpressed in CINs and invasive carci- noma, compared with little expression in ASCUS ( P < 0.005). Overexpression of cyclin E was observed in CIN1 (94.1 % , x2 = 21.16, P < 0.01), and pl6ink4a and ki67 were overexpressed in invasive carcinoma (100% and 90.9% respective- ly) . The degree of pl6ink4a and ki67 expression correlated well with that of epithelial lesions ( P < 0.005). HPV16/18 infec- tion was assessed in CINs and invasive carcinoma samples, and revealed a significant relationship with the degree of cervical epithelial lession. The expression level of pl6ink4a and ki67 seemed more closely associated with HPV16 infection than that of cyclin E ( r s = 1.0 vs rs = 0 . 4 ) . Only 1 case in CIN, and 4 cases in CIN2-3 of HPV18 positive samples were detected. Therefore no statistical significance was found by statistical analysis. Overexpression of cyclin E, p16ink4a and ki67 in CINs and invasive carcinoma cells demonstrates the potential use of cyclin E, p16ink4a and ki67 as diagnostic biomarkers for HPV-related cervical neoplastic lesions. In addition, this technique can be used for screening patients at high risk of devel- oping cervical carcinoma.展开更多
文摘To study the effect on regulation of cell cycle of osteosarcoma cell line MG63 tranceduced with exogenous p16ink4a and hRbl genes, pIRES-p16ink4a-hRb1, pIRES-p16ink4a and pIREShRbl plasmids were constructed by gene recombination technology. The recombinant plasmid was transferred into osteosarcoma cell line MG63 by metafectene, and the resistant clones were selected by G418 selective medium, mRNA and protein expression of osteosarcoma cell line were assayed by RT-PCR and Western-Blot respectively. Cell cycle and apoptosis were analyzed by subG1 flow cytometric. Cell proliferation was tested by MTT. In the genome of these transfected target cells, the expression of p16ink4a and hRb1 mRNA and protein were detected respectively in vitro. It was demonstrated with subG1 flow cytometric analysis and MTT method that p16ink4a and hRbl genes cooperation more significantly inhibited cell growth and induced a more marked G1 arrest and apoptosis than p16ink4a/hRb1 alone （P〈0.01）. Coexpression of exogenous p16ink4a with hRbl broke the regulatory feedback loop of p16ink4a-cyclinD1/CDK-hRbl and played a more significant role in inhibiting cell growth as well as inducing cell apoptosis than p16ink4a or hRbl did alone in vitro.
文摘The aim of this study is to investigate cyclin E, pl6ink4a and ki67 as possible diagnostic biomarkers for cervical preneoplasia using cervical exfoliated-cell specimens, and evaluate the significance for screening patients at high risk of de- veloping cervical carcinoma. The expression of cyclin E, p16ink4a and ki67 was examinated in 78 cervical exfoliated epithe- lial specimens diagnosed as atypical squamous cells of undetermined significance (ASCIIS) (12 cases), cervical intraepithe- lial neoplasia (CIN) of type 1 (17 cases), CIN2-3(38 cases) and invasive carcinoma (11 cases) using immunohistochemical analysis, and simultaneously, the DNA status of human papillomavirus (HPV) type 16/18 was detected by polymerase chain reaction (PCR) using type specific primers, cyclin E, pl6ink4a and ki67 were all overexpressed in CINs and invasive carci- noma, compared with little expression in ASCUS ( P < 0.005). Overexpression of cyclin E was observed in CIN1 (94.1 % , x2 = 21.16, P < 0.01), and pl6ink4a and ki67 were overexpressed in invasive carcinoma (100% and 90.9% respective- ly) . The degree of pl6ink4a and ki67 expression correlated well with that of epithelial lesions ( P < 0.005). HPV16/18 infec- tion was assessed in CINs and invasive carcinoma samples, and revealed a significant relationship with the degree of cervical epithelial lession. The expression level of pl6ink4a and ki67 seemed more closely associated with HPV16 infection than that of cyclin E ( r s = 1.0 vs rs = 0 . 4 ) . Only 1 case in CIN, and 4 cases in CIN2-3 of HPV18 positive samples were detected. Therefore no statistical significance was found by statistical analysis. Overexpression of cyclin E, p16ink4a and ki67 in CINs and invasive carcinoma cells demonstrates the potential use of cyclin E, p16ink4a and ki67 as diagnostic biomarkers for HPV-related cervical neoplastic lesions. In addition, this technique can be used for screening patients at high risk of devel- oping cervical carcinoma.
