Objective. To investigate the anti- tumor effects of human single chain interleukin- 12 (hscIL- 12). Method. pcDNA/ hscIL- 12 recombinant was transfected into human hepatic carcinoma cells (7721 cells) by lipofectin m...Objective. To investigate the anti- tumor effects of human single chain interleukin- 12 (hscIL- 12). Method. pcDNA/ hscIL- 12 recombinant was transfected into human hepatic carcinoma cells (7721 cells) by lipofectin method. The 7721/hscIL- 12 cells which secrete hscIL- 12 stably, were obtained via G418 selection, and in vitro the influence of hscIL- 12 gene transduction on the growth of tumor cells was evaluated by cell cycle analysis. In vivo, genetically engineered 7721 cells (7721/hscIL- 12, 7721/pcDNA) and parental cells were implanted into BALB/c nude mice,respectively. 7721/pcDNA and 7721/hscIL- 12 groups were divided into two sub- groups on day 8: one was administered with hPBL twice, 6 days at interval; the other was given equal volume of PBS. Mice were sacrificed on day 26, and spleens and tumors were taken out for histologic assay. Results. hscIL- 12 produced stably by 7721/hscIL- 12 cells had bioactivity, and it was proved by Western blot, immunocytochemistry, and in situ hybridization. In vitro, compared with 7721 and 7721/pcDNA, the 7721/hscIL- 12 grew much more slowly. FACS assay showed apparent G1 arrest of 7721/hscIL- 12 cells. In animal experiment, on day 8 after inoculation, the tumors of 7721 and 7721/pcDNA group were up to 5～ 7mm,while those of 7721/hscIL- 12 group were 2～ 4mm.When treated with hPBL, the tumor of 7721/hscIL- 12 group disappeared completely. Histologically, the tumors from 7721/hscIL- 12 without hPBL treatment had numerous lymphocyte infiltration, the tumor cells displayed depression looking, atrophy, focal necrosis and apoptosis , whereas the tumors of 7721 and 7721/pcDNA groups grew thrivingly. Conclusion. hscIL- 12 transduced 7721 cells could induced significant antitumor immune response which resulted in tumor regression totally when the hPBL was inoculated, and also hscIL- 12 has certain effects on mice immune system. These findings suggest that hscIL- 12 and hscIL- 12 gene therapy might have promising prospects in clinical application.展开更多
目的探讨过表达中期因子(MK)增强SMMC 7721细胞对5-氟尿嘧啶(5-Fu)耐药及其可能的机制。方法将SMMC 7721细胞转染pIRES2-EGFP-MK重组质粒过表达,采用实时荧光定量PCR(qPCR)和蛋白质印迹法(WB)检测MK mRNA和蛋白的表达。5-Fu处理后,采用...目的探讨过表达中期因子(MK)增强SMMC 7721细胞对5-氟尿嘧啶(5-Fu)耐药及其可能的机制。方法将SMMC 7721细胞转染pIRES2-EGFP-MK重组质粒过表达,采用实时荧光定量PCR(qPCR)和蛋白质印迹法(WB)检测MK mRNA和蛋白的表达。5-Fu处理后,采用MTT法检测SMMC 7721细胞对5-Fu的耐药。采用WB法检测磷酸化磷脂酰肌醇3-激酶(p-PI3K)、磷酸化蛋白激酶B(p-Akt)和NF-κB及Bcl-2、survivin、caspase-3的表达。结果将SMMC 7721细胞转染pIRES2-EGFP-MK重组质粒过表达后,MK mRNA和蛋白的表达增加,ConC组细胞对5-Fu的IC50显著高于Con-A组、Con-B组[(27.36±4.31)mg/L vs (4.57±0.34)mg/L,(4.96±0.46)mg/L],差异具有统计学意义(P<0.05)。此外,与Con-A组、Con-B组比较,Con-C组细胞p-PI3K(0.66±0.04 vs 0.35±0.03,0.57±0.03)、p-Akt (0.31±0.02 vs 0.17±0.02、0.25±0.02)、NF-κB (0.63±0.05 vs 0.27±0.02,0.53±0.04)、Bcl-2(0.42±0.04 vs 0.13±0.01, 0.38±0.04)、survivin(0.58±0.04 vs 0.18±0.02,0.51±0.04)呈现高表达,caspase-3(0.41±0.04vs 0.88±0.06,0.43±0.03)呈现低表达(P<0.05)。结论过表达MK可增强SMMC 7721细胞对5-Fu耐药,其机制可能与激活PI3K/AKT信号通路有关。展开更多
AIM: Cyclooxygenase-2 (COX-2) has been suggested to be associated with carcinogenesis. We sought to investigate the effect of the selective COX-2 inhibitor, Nimesulide on proliferation and apoptosis of SMMC-7721 human...AIM: Cyclooxygenase-2 (COX-2) has been suggested to be associated with carcinogenesis. We sought to investigate the effect of the selective COX-2 inhibitor, Nimesulide on proliferation and apoptosis of SMMC-7721 human hepatoma cells.METHODS: This study was carried out on the culture of hepatic carcinoma SMMC-7721 cell line. Various concentrations of Nimesulide (0, 200 micromol/L, 300 micromol/L, 400 micromol/L) were added and incubated. Cell proliferation was detected with MTT colorimetric assay, cell apoptosis by electron microscopy, flow cytometry and TUNEL.RESULTS: Nimesulide could significantly inhibit SMMC-7721 cells proliferation dose-dependent and in a dependent manner compared with that of the control group. The duration lowest inhibition rate produced by Nimesulide in SMMC-7721 cells was 19.06%, the highest inhibition rate was 58.49%. After incubation with Nimesulide for 72 h, the most highest apoptosis rate and apoptosis index of SMMC-7721 cells comparing with those of the control were 21.20%+/-1.62% vs 2.24%+/-0.26% and 21.23+/-1.78 vs 2.01+/-0.23 (P【0.05). CONCLUSION:The selective COX-2 inhibitor, Nimesulide can inhibit the proliferation of SMMC-7721 cells and increase apoptosis rate and apoptosis index of SMMC-7721 cells. The apoptosis rate and the apoptosis index are dose-dependent. Under electron microscope SMMC-7721 cells incubated with 300 micromol and 400 micromol Nimesulide show apoptotic characteristics. With the clarification of the mechanism of selective COX-2 inhibitors, These COX-2 selective inhibitors can become the choice of prevention and treatment of cancers.展开更多
文摘Objective. To investigate the anti- tumor effects of human single chain interleukin- 12 (hscIL- 12). Method. pcDNA/ hscIL- 12 recombinant was transfected into human hepatic carcinoma cells (7721 cells) by lipofectin method. The 7721/hscIL- 12 cells which secrete hscIL- 12 stably, were obtained via G418 selection, and in vitro the influence of hscIL- 12 gene transduction on the growth of tumor cells was evaluated by cell cycle analysis. In vivo, genetically engineered 7721 cells (7721/hscIL- 12, 7721/pcDNA) and parental cells were implanted into BALB/c nude mice,respectively. 7721/pcDNA and 7721/hscIL- 12 groups were divided into two sub- groups on day 8: one was administered with hPBL twice, 6 days at interval; the other was given equal volume of PBS. Mice were sacrificed on day 26, and spleens and tumors were taken out for histologic assay. Results. hscIL- 12 produced stably by 7721/hscIL- 12 cells had bioactivity, and it was proved by Western blot, immunocytochemistry, and in situ hybridization. In vitro, compared with 7721 and 7721/pcDNA, the 7721/hscIL- 12 grew much more slowly. FACS assay showed apparent G1 arrest of 7721/hscIL- 12 cells. In animal experiment, on day 8 after inoculation, the tumors of 7721 and 7721/pcDNA group were up to 5～ 7mm,while those of 7721/hscIL- 12 group were 2～ 4mm.When treated with hPBL, the tumor of 7721/hscIL- 12 group disappeared completely. Histologically, the tumors from 7721/hscIL- 12 without hPBL treatment had numerous lymphocyte infiltration, the tumor cells displayed depression looking, atrophy, focal necrosis and apoptosis , whereas the tumors of 7721 and 7721/pcDNA groups grew thrivingly. Conclusion. hscIL- 12 transduced 7721 cells could induced significant antitumor immune response which resulted in tumor regression totally when the hPBL was inoculated, and also hscIL- 12 has certain effects on mice immune system. These findings suggest that hscIL- 12 and hscIL- 12 gene therapy might have promising prospects in clinical application.
文摘目的探讨过表达中期因子(MK)增强SMMC 7721细胞对5-氟尿嘧啶(5-Fu)耐药及其可能的机制。方法将SMMC 7721细胞转染pIRES2-EGFP-MK重组质粒过表达,采用实时荧光定量PCR(qPCR)和蛋白质印迹法(WB)检测MK mRNA和蛋白的表达。5-Fu处理后,采用MTT法检测SMMC 7721细胞对5-Fu的耐药。采用WB法检测磷酸化磷脂酰肌醇3-激酶(p-PI3K)、磷酸化蛋白激酶B(p-Akt)和NF-κB及Bcl-2、survivin、caspase-3的表达。结果将SMMC 7721细胞转染pIRES2-EGFP-MK重组质粒过表达后,MK mRNA和蛋白的表达增加,ConC组细胞对5-Fu的IC50显著高于Con-A组、Con-B组[(27.36±4.31)mg/L vs (4.57±0.34)mg/L,(4.96±0.46)mg/L],差异具有统计学意义(P<0.05)。此外,与Con-A组、Con-B组比较,Con-C组细胞p-PI3K(0.66±0.04 vs 0.35±0.03,0.57±0.03)、p-Akt (0.31±0.02 vs 0.17±0.02、0.25±0.02)、NF-κB (0.63±0.05 vs 0.27±0.02,0.53±0.04)、Bcl-2(0.42±0.04 vs 0.13±0.01, 0.38±0.04)、survivin(0.58±0.04 vs 0.18±0.02,0.51±0.04)呈现高表达,caspase-3(0.41±0.04vs 0.88±0.06,0.43±0.03)呈现低表达(P<0.05)。结论过表达MK可增强SMMC 7721细胞对5-Fu耐药,其机制可能与激活PI3K/AKT信号通路有关。
文摘AIM: Cyclooxygenase-2 (COX-2) has been suggested to be associated with carcinogenesis. We sought to investigate the effect of the selective COX-2 inhibitor, Nimesulide on proliferation and apoptosis of SMMC-7721 human hepatoma cells.METHODS: This study was carried out on the culture of hepatic carcinoma SMMC-7721 cell line. Various concentrations of Nimesulide (0, 200 micromol/L, 300 micromol/L, 400 micromol/L) were added and incubated. Cell proliferation was detected with MTT colorimetric assay, cell apoptosis by electron microscopy, flow cytometry and TUNEL.RESULTS: Nimesulide could significantly inhibit SMMC-7721 cells proliferation dose-dependent and in a dependent manner compared with that of the control group. The duration lowest inhibition rate produced by Nimesulide in SMMC-7721 cells was 19.06%, the highest inhibition rate was 58.49%. After incubation with Nimesulide for 72 h, the most highest apoptosis rate and apoptosis index of SMMC-7721 cells comparing with those of the control were 21.20%+/-1.62% vs 2.24%+/-0.26% and 21.23+/-1.78 vs 2.01+/-0.23 (P【0.05). CONCLUSION:The selective COX-2 inhibitor, Nimesulide can inhibit the proliferation of SMMC-7721 cells and increase apoptosis rate and apoptosis index of SMMC-7721 cells. The apoptosis rate and the apoptosis index are dose-dependent. Under electron microscope SMMC-7721 cells incubated with 300 micromol and 400 micromol Nimesulide show apoptotic characteristics. With the clarification of the mechanism of selective COX-2 inhibitors, These COX-2 selective inhibitors can become the choice of prevention and treatment of cancers.
