目的：探讨Na+，K+-ATP1b1（ATP1b1）对肝癌细胞生长及侵袭能力的影响。方法：将ATP1b1表达质粒ATP1b1-CMV-FLAG转染肝癌细胞SMMC-7721，以空质粒转染作对照，通过RT-PCR测定ATP1b1的mRNA表达，在光镜下观察细胞形态学变化，MTT法检测细胞生长增殖，Transwell细胞侵袭实验分析肝癌细胞的侵袭能力。结果：ATP1b1-CMV-FLAG转染后，SMMC-7721的ATP1b1 mRNA表达水平明显增高（P＜0.05）,转染ATP1b1-CMV-FLAG组、pFLAG-CMV-1组和脂质体组的ATP1b1 mRNA表达水平分别为1.159、0.182和0.093;转染ATP1b1-CMV-FLAG的SMMC-7721细胞呈变性改变，其生长受到抑制，转染相同DNA浓度的ATP1b1-CMV-FLAG组与pFLAG-CMV-1组对细胞生长的抑制效应有显著性差异（P＜0.05）；Transwell细胞侵袭实验显示转染ATP1b1-CMV-FLAG组的SMMC-7721细胞的体外侵袭力明显受抑制（P＜0.01）。结论：实验结果说明了促进ATP1b1表达能抑制肝癌细胞的生长及侵袭行为。
Objective To observe the effects of extracellular-signal regulated kinase (ERK) 1/2 inhibitor U0126 on hepatoma carcinoma cell proliferation and apoptosis. Methods Hepatoma SMMC-7721 cell strain was divided into blank control group and different concentrations of U0126 groups. The proliferation inhibition was measured by MTT assay. FCM was used to analyze the cell cycle distribution and apoptosis. Results U0126 obviously inhibited cell proliferation, induced cell apoptosis and G0/G1 phase cell cycle arrest. There were significant differences between control group and different concentrations of U0126 groups on cell proliferation and apoptosis （P＜0.05， P＜0.01）. Conclusion Blocking ERK1/2 pathway may be an important treatment strategy for liver cancer.
Objective To study whether carbon dioxide used to establish pneumoperitoneum has an influence on port-site and intraperitoneal implantation and metastasis of tumor cells. Methods R15 hepatic cancer cells were injected into 30 Wistar rats’ peritoneal cavities 1 hour before operation, then the 30 Wistar rats were randomly divided into 3 groups: gasless group, helium group and carbon dioxide group. The suspension was exposed to the gas environment for 2 hours, all animals were killed after 28 days and the port-site and intraperitoneal implantation and metastasis of tumor cells were examined. Results On port-site, intestinal serous coat, mesentery, greater omentum and diaphragm, the weights of tumor cells, in carbon dioxide group were (326.7±230.3) mg， （626.2±215.9） mg，（476.2±204.8） mg，（2 536.5±906.7） mg and （384.5±149.9） mg respectively； in helium group were (235.6±107.3) mg， （414.2±148.4） mg, （261.8±92.6） mg, （1 633.4±247.3） mg and（220.0±57.9） mg； in gasless group were (145.0±42.4) mg， （221.5±108.2） mg， （212.5±109.6） mg, （797.5±335.9） mg and 113.0 mg.The weights of carbon dioxide group showed a significant increase, compared with helium group and gasless group (P＜0.05). The weights of helium group were greater than gasless group，but there was no significance in statistics (Pgt;0.05). Conclusion The insufflation of carbon dioxide promotes intraperitoneal tumor implantation and growth compared with helium and gaslessness in a rat model.
ObjectiveTo investigate the effects of somatostatin8 (SS8) on the apoptosis and the expression of cmyc protein of hepatocellular carcinoma cell SMMC7721. MethodsCultured in vitro, hepatocellular carcinoma cells SMMC7721 were incubated with SS8 (10 μg/ml). The apoptosis rate and expression of cmyc protein were detected by flow cytometry (FCM). ResultsSS8 can cause the spanonumber in S and G2/M phase and the auxonumber in G0/G1 phase of SMMC7721 cells . The apoptosis rate was 14.2% in the study group and 6.1% in the control group, and there was significant difference (P＜0.05); The level of expressions of cmyc protein was 0.833±0.035 after action by SS8 for 24 h. Compared with control group, there was no significant difference in the study group（P＞0.10）.But after the cells were incubated with SS8 for 48，72，96，120，144 h, the level of expressions of cmyc protein was 0.818±0.04，0.721±0.029，0.669±0.026，0.648±0.045，0.642±0.028 respectively in the study group, and there was significant difference as compared with the control group (P＜0.05). Conclusion The SS8 can induce the apoptosis and lower expression of cmyc protein of hepatocellular carcinoma cell SMMC7721.
Objective To investigate the effect of phosphorothioate antisense oligonucleotides(AS-ODN) on suppressing multidrug resistance-associated protein gene(MRP) in human drug-resistant hepatocellular carcinoma cell line (SMMC-7721/ADM). Methods Cell line was transfected with a synthetic S-ODN complementary to the coding region of MRP mRNA, Lipofectamine acting as carrier. The drug sensitivity was measured by MTT assay. The expression of MRP mRNA was detected by RT-PCR and the expression of P190 was detected by flow cytometry. Results AS-ODN inhibited expression of MRP mRNA and P190 and promoted sensitivity to daunorubicinum and adriamycinum. Conclusion AS-ODN can reduce the expression of MRP gene. MDR caused by MRP is an important cause of multidrug resistance of SMMC-7721/ADM.
Objective To investigate the reversal effect of antisense phosphorothioate oligonucleotide （ASOND) on human hepatoma resistant cells. Methods Human hepatoma resistant cells SMMC-7721 was transfected with synthetic antisense phosphorothioate oligonucleotide complementary to the 5′ region flanking the AUG initiation codon mediated by lipofectamine. In vitro drug sensitivity was measured by MTT assay. The expression of P-170 was determined by flow cytometry and mRNA was assessed by RT-PCR. Results ASOND inhibited the expression of mRNA and p-170 in SMMC-7721, enhanced the sensitivity of SMMC-7721 to chemotherapeutic drug. The best inhibitory effect was achived by the dose of 0.5μmol/L. Conclusion ASOND enhanced the sensitivity of SMMC-7721 to chemotherapeutic drug and reversed the multidrug resistance of SMMC-7721 partially.
Objective To observe the effect of a wide range of concentration of arsenic trioxide on hepatoma cell line BEL-7402 with variable duration. Methods The cell activity and morphologic changes were studied after treated with different concentration. The apoptosis were detected by flow cytometry assay and DNA Gel electrophoresis. Results The effect of arsenic trioxide on hepatoma cell lines were dependent on the time and concentration obviously. Hepatoma cells cultured with different concentration presented apoptosis features: i.e. intact cell membrane, chromatin condensation, nucleic fragmentation and apoptotic body formation; flow cytometry analysis showed an arrestment at G2/M phase and a subG1 cell peak, DNA gel electrophoresis showed a marked DNA ladder. Conclusion Arsenic trioxide can obviously inhibit the growth of hepatoma cell lines through inducing hepatoma cell apoptosis.
【Abstract】Objective To study the effects of arsenic trioxide (As2O3) on inhibiting the proliferation of hepatic carcinoma cell lines. Methods To detect the inhibiting rate of As2O3 and other 6 kinds of anticancer drugs (such as, NOV, ADM, MMC, 5-Fu, DDP and CTX) on hepatic carcinoma cell lines BEL-7404 and SMMC-7721 by using MTT assay. Results As compared with other 6 kinds of anticancer drugs, the inhibiting rate of As2O3 was the highest one (P＜0.01 or P＜0.05). The inhibiting rates of As2O3 in the groups with the concentration above 1.0 μg/ml were no different (P＞0.05). Conclusion As2O3 could inhibit hepatic carcinoma cell lines BEL-7404 and SMMC-7721 effectively in vitro. Drug sensitivity tests of different concentration’s As2O3 should be done in order to select the minimal and effective concentration of arsenic trioxide and reduce the side effects of arsenic trioxide.
ObjectiveTo evaluate the effect of ZnPP Ⅸ on the expressions of heme oxygenase-1 (HO-1) and glutathione-Stransferase-π （GST-π） and the chemosensitivity of drug-resistant hepatic carcinoma cell line Bel/Fu, and explore it’s possibility to reverse drug-resistance and the relevant regulating mechanism. Methods①MTT assay was adopted to detect the drug sensitivity for adriamycin, mitomycin, and 5-fluorouracil of Bel/Fu cell after ZnPP Ⅸ being induced for 24 h. ②RTPCR was carried out to detect the expressions of HO-1 and GST-π mRNA after Bel/FU cells being treated with different concentrations ZnPP Ⅸ for 24 h. ResultsAfter Bel/Fu cells being treated with ZnPP Ⅸ for 24 h, the 50% inhibiting concentration (IC50) for drugs was decreased dramatically （Plt;0.05）. Meanwhile, the expressions of HO-1 and GST-π mRNA in the treated cells also decreased dose-dependently (Plt;0.01). ConclusionsZnPP Ⅸ can increase the chemosensitivity of Bel/FU cells by down-regulation of HO-1 and GST-π expression. ZnPP Ⅸ is a potential agent to reverse multidrug resistance of hepatic carcinoma cells.
Objective To observe the effect of RNA interference (RNAi) on HepG2 hepatic cancer cell by small interfering RNA (siRNA). Methods siRNA targeting vascular endothelial growth factor (VEGF) gene was transfected into HepG2 cells by LipofectimineTM 2000. The VEGF mRNA and protein were respectively detected by real-time quantitive PCR and Western blot, and the concentration of VEGF protein in the cell culture supertant was determined by ELISA at 48 h after culture. Results The average efficiency of siRNA transfection was (90.4±2.9)% after 6 h cell culture. The expressions of VEGF mRNA and protein in HepG2 cells could be effectively suppressed by siRNA, and the concentration of VEGF protein in the cell culture supertant was also decreased. Conclusion siRNA can knock down the expression of VEGF gene and decrease the concentration of VEGF protein in HepG2 cells.