Objective:To explore the inhihitive and apoptosis inductive effect of IL-24 genes on CD133^+laryngeal cancer cells in Hep-2 line.Methods:Human peripheral blood monocytes were isolated.The total RNA was extracted by us...Objective:To explore the inhihitive and apoptosis inductive effect of IL-24 genes on CD133^+laryngeal cancer cells in Hep-2 line.Methods:Human peripheral blood monocytes were isolated.The total RNA was extracted by using Trizol method and reverse transcripted into cDNA using RT-PCR method.Primers P1 and P2 was designed for the amplification of human IL-24 genes.After confirmation of agarose gel electrophoresis tests,TA was cloned into pMD19-T simple vector.Nhe Ⅰ and Xho Ⅰ double digesting human IL-24 and pIRES2-ZsGreen1 and eukaryotic expression vector were used to establish the pIRES2-ZsGreen1-hIL-24 vector,and detected by enzyme digestion and gene sequencing methods.Flow cytometry(FCM) was used to isolate CD133^+ cells from Hep-2 cells.CD133^+ cells were transfected with pIRES2-ZsGreen1-hIL-24 through liposome 2000.After detection,MTT and FCM were used to observe the effect of IL-24 gene on CD133^+ laryngeal cancer Hep-2 cells.Results:Lipotin mediated transfection of recombinant pIRES2-ZsGreen1-hIL-24 plasmid into CD133^+ Hep-2 could expressed IL-24 gene in cells stably.MTT results showed that IL-24 transfected group was significantly suppressed compared to empty vector group and control group(P<0.05);FCM results showed that the apoptosis rate of experimental group increased significantly compared to empty vector group and control group(P<0.05).Conclusions:IL-24 gene expressions can inhibit proliferation of CD133^+laryngeal cells in Hep-2 line and promote their apoptosis.展开更多
In order to investigate the effect of replication-incompetent adenovirus vector expressing MDA-7/IL-24 on tumor growth and apoptosis of human hepatocellular carcinoma （HCC） cell line SMMC-7721 and normal liver cell ...In order to investigate the effect of replication-incompetent adenovirus vector expressing MDA-7/IL-24 on tumor growth and apoptosis of human hepatocellular carcinoma （HCC） cell line SMMC-7721 and normal liver cell line L02, the recombinant replication-incompetent Ad.mda-7 virus vector was constructed and infected into the HCC cell line SMMC-7721 and normal liver cell line L02. RT-PCR was performed to examine the expression of MDA-7 mRNA. The concentrations of MDA-7/IL-4 in culture superuatants were determined by using ELISA. MTT and Hoechst staining assay were applied to observe the inhibitory and killing effects of MDA-7 on the HCC cells. By using flow cytometry, the apoptosis, cell cycle and proliferation of SMMC-7721 and L02 cells were measured. The results showed recombinant replication-incompetent virus expressing MDA-7/IL-24 was constructed successfully, and RT-PCR revealed that it could mediate the high expression of the exogenous gene MDA-7/IL-24 in SMMC-7721 and L02 cells. The expression of MDA-7/IL-24 proteins in the culture superuatant was detectable by ELISA. Ad.mda-7 infection induced apoptosis and growth suppression in SMMC-7721 cells and an increased percentage of HCC cells in the GyM phase of the cell cycle, but not in L02 cells. It was concluded that mda-7/IL-24 gene, mediated with replication-incompetent adenovirus vector, could selectively induce growth suppression and apoptosis in HCC cell line SMMC-7721 but without any toxic side-effect on normal liver line L02.展开更多
According to the amino acid sequence and codon preference of E. coli, the human interleukin-18（IL-18） gene was optimized to avoid the rare codons. The total length of the synthesized gene is 571 bp; 18 oligonucleoti...According to the amino acid sequence and codon preference of E. coli, the human interleukin-18（IL-18） gene was optimized to avoid the rare codons. The total length of the synthesized gene is 571 bp; 18 oligonucleotides, DNA fragments were designed and synthesized by the phosphoramidite four-step chemical method. The whole DNA sequence was synthesized by a one-step total gene synthesis method, and then inserted in pUC18 vector. Five positive clones identified by blue-white colony screening were sent to Shanghai Sangon Biological Engineering Technology and Service Co., Ltd. for sequencing. The sequencing result shows that one clone contained the complete correct gene in all the five positive clones.展开更多
基金supported by Jilin Province Natural Science Foundation of China.No:20130101151JCChinese Ministry of Education Projects of Doctoral New Teachers.No:20120061120092
文摘Objective:To explore the inhihitive and apoptosis inductive effect of IL-24 genes on CD133^+laryngeal cancer cells in Hep-2 line.Methods:Human peripheral blood monocytes were isolated.The total RNA was extracted by using Trizol method and reverse transcripted into cDNA using RT-PCR method.Primers P1 and P2 was designed for the amplification of human IL-24 genes.After confirmation of agarose gel electrophoresis tests,TA was cloned into pMD19-T simple vector.Nhe Ⅰ and Xho Ⅰ double digesting human IL-24 and pIRES2-ZsGreen1 and eukaryotic expression vector were used to establish the pIRES2-ZsGreen1-hIL-24 vector,and detected by enzyme digestion and gene sequencing methods.Flow cytometry(FCM) was used to isolate CD133^+ cells from Hep-2 cells.CD133^+ cells were transfected with pIRES2-ZsGreen1-hIL-24 through liposome 2000.After detection,MTT and FCM were used to observe the effect of IL-24 gene on CD133^+ laryngeal cancer Hep-2 cells.Results:Lipotin mediated transfection of recombinant pIRES2-ZsGreen1-hIL-24 plasmid into CD133^+ Hep-2 could expressed IL-24 gene in cells stably.MTT results showed that IL-24 transfected group was significantly suppressed compared to empty vector group and control group(P<0.05);FCM results showed that the apoptosis rate of experimental group increased significantly compared to empty vector group and control group(P<0.05).Conclusions:IL-24 gene expressions can inhibit proliferation of CD133^+laryngeal cells in Hep-2 line and promote their apoptosis.
文摘In order to investigate the effect of replication-incompetent adenovirus vector expressing MDA-7/IL-24 on tumor growth and apoptosis of human hepatocellular carcinoma （HCC） cell line SMMC-7721 and normal liver cell line L02, the recombinant replication-incompetent Ad.mda-7 virus vector was constructed and infected into the HCC cell line SMMC-7721 and normal liver cell line L02. RT-PCR was performed to examine the expression of MDA-7 mRNA. The concentrations of MDA-7/IL-4 in culture superuatants were determined by using ELISA. MTT and Hoechst staining assay were applied to observe the inhibitory and killing effects of MDA-7 on the HCC cells. By using flow cytometry, the apoptosis, cell cycle and proliferation of SMMC-7721 and L02 cells were measured. The results showed recombinant replication-incompetent virus expressing MDA-7/IL-24 was constructed successfully, and RT-PCR revealed that it could mediate the high expression of the exogenous gene MDA-7/IL-24 in SMMC-7721 and L02 cells. The expression of MDA-7/IL-24 proteins in the culture superuatant was detectable by ELISA. Ad.mda-7 infection induced apoptosis and growth suppression in SMMC-7721 cells and an increased percentage of HCC cells in the GyM phase of the cell cycle, but not in L02 cells. It was concluded that mda-7/IL-24 gene, mediated with replication-incompetent adenovirus vector, could selectively induce growth suppression and apoptosis in HCC cell line SMMC-7721 but without any toxic side-effect on normal liver line L02.
文摘According to the amino acid sequence and codon preference of E. coli, the human interleukin-18（IL-18） gene was optimized to avoid the rare codons. The total length of the synthesized gene is 571 bp; 18 oligonucleotides, DNA fragments were designed and synthesized by the phosphoramidite four-step chemical method. The whole DNA sequence was synthesized by a one-step total gene synthesis method, and then inserted in pUC18 vector. Five positive clones identified by blue-white colony screening were sent to Shanghai Sangon Biological Engineering Technology and Service Co., Ltd. for sequencing. The sequencing result shows that one clone contained the complete correct gene in all the five positive clones.
