Gene therapy recently has become an important area of research as a new therapeutic method. In vivo and in vitro gene therapies require efficient delivery of genetic material into a cell and preferably high levels of expression of transferred gene. Traditionally, gene delivery systems are classified as viral vector-mediated systems and nonviral vector-mediated systems. Viral vectors, which have been demonstrated as systems with high transfection efficiency, however, are limited due to adverse effects such as immunogenicity, toxicity, limited DNA carrying capacity and mutagenesis caused by cell-infected viruses[1].

AIM To investigate the therapeutic effect of TNF gene transfected LAK cells on ascitic liver carcinoma-bearing mice.METHODS TNF gene was transfected into murine LAK cells by retrovirus. Low dose TNF gene-transfectcdLAK cells and IL-2 were i.p. injected into murine model. Cytotoxicity of gene transfected LAK cells wasstudied in vitro growth and the survival time of murine model was observed.RESULTS TNF gene-transfected LAK cells secreted higher level of TNF than that of normal LAK cells orcontrol gene-transfected LAK ceils. The in vitro growth ability and cytotoxicity of TNF gene-transfectedLAK cells were markedly inhibited by anti-TNF monoclonal antibodies. Significant therapeutic effect onascitic liver carcinoma-bearing mice was achieved.CONCLUSION TNF gene-transfected LAK cells have therapeutic effect on ascitic liver carcinoma-bearingmice.

AIM To explore the therapeutic potential of antisense oligodeoxynucleotides on hepatcellular cacinoma(HCC).METHODS Four antisense phosphorothioated oligodeoxynucleotides (asON), complementary to differentsites of HBV, were synthesized and assayed for their anti-HBV activity in HepG22. 2.15 cells with ELISA.The most effective asON was chosen for the following study: FACSCAN, TRAP and immuno-staining wereused respectively for checking apoptosis, telomerase activity and expression of oncogene p21ras and p62C-myc inHepG2.2.15 cells after treated by asON.RESULTS The oligomer directed against the initiator of pre-S2 was the most effective one with aninhibitory rate of 66% on HBsAg and 91% on HBeAg (P<0.02). Two inhibitory peaks (bimodal)appeared. Telomerase activity as well as the expression of p21fas and p62C-myc decreased drastically 3 days afterasON-HBpreS2 treatment. Meanwhile, apoptosis appeared in the experiments.CONCLUSION The inhibitory effects of as-preS2 on the HBV gene expression and the reversion of somemalignant behaviour in HepG2.2.15 cells were the significant, effective therapy against HBV infection andhepatocellular carcinoma.

AIM To investigate the effect of recombinant human tumor necrosis factor (rbTNF) on telomerase activityin hepatoma cell line HepG2 and HepG1-6.METHODS TRAP-ELISA method was used to determine the telomerase activity in HepG2 and HepG1-6cells which were treated by different concentrations of rhTNF. In addition, the TERTLuc (800) plasmid wastransiently transfected, which was inserted 800 bp of the human telomerase reverse transcriptase (hTERT)promoter, into HepG2 cells by Lipofect. Different concentrations of rhTNF were added into the culturemedia 2 hours later, and the activity of the hTERT promoter was detected 48 hours after transfection.RESULTS The telomerase activity of HepG2 was suppressed by rhTNF in a dose-dependent manner. Theresults also revealed that the activity of hTERT promoter was inhibited linearly with rhTNF at the dose of 10- 1000 IU/mL.CONCLUSION Inhibition of the hTERT promoter expression by rhTNF may contribute to its anti-tumoractivity.

Tol-like receptor 3 protein expression has been shown to be upregulated during cerebral ische-mia/reperfusion injury in rats. In this study, rat primary cortical neurons were subjected to oxy-gen-glucose deprivation to simulate cerebral ischemia/reperfusion injury. Chemical y synthesized smal interfering RNA (siRNA)-1280,-1724 and-418 specific to tol-like receptor 3 were transfected into oxygen-glucose deprived cortical neurons to suppress the upregulation of tol-like receptor 3 protein expression. Western blotting demonstrated that after transfection with siRNA, tol-like ceptor 3 protein expression reduced, especial y in the tol-like receptor 3-1724 group. These results suggested that siRNA-1724 is an optimal sequence for inhibiting tol-like receptor 3 expression in cortical neurons fol owing oxygen-glucose deprivation.

Objective To investigate the effect of overexpression of bcl-2 on ethanol-induced apoptosis of primary hepatocellular carcinoma (HCC) cells.Methods The retrovirus expression vector pDOR-SB containing human bcl-2 cDNA was introduced into a human HCC cell line HCC-9204 by liposome-mediated transfection. pDOR-transfected and non-transfected HCC-9204 cells were used as controls. The expression of Bcl-2 protein by transfected HCC-9204 cells was detected by the immunohistochemical method. Then the cells were cloned with the limited dilution method continually until a monoclonal cell strain whose positive rate of Bcl-2 protein was 100% detected by flow cytometry was obtained. The killing rates of cells were detected by Methabenzthiazuron assay after the treatment of 6% ethanol for 6?h. The extent of apoptosis was analyzed by transferase-mediated dUTP nick end labeling (TUNEL) staining and flow cytometry.Results Most of the pDOR-SB-transfected cells demonstrated Bcl-2 positive signals, while no signal was found in the controls. The positive rate of Bcl-2 protein detected by flow cytometry in the obtained monoclonal cell strain, which was named HCC-bcl2, was 100% after the cells had been cloned 3 times continually. The killing rate, TUNEL index and the scale of sub-G1 apoptotic peak in HCC-bcl2 cells were all significantly lower than those in the control cells.Conclusion Overexpression of Bcl-2 protein suppresses ethanol-induced apoptosis of the HCC cell line HCC-9204.

siRNA SOCS1 transfection in cancerogenesis of experimental model with altered thyroid function

野生型p53增强药物耐药的人肝癌细胞Bel7402/5-FU的化疗敏感性

AIM: To study the effect of wild type (wt) p53 gene transfection on drug resistant human hepatocellular carcinoma (HCC) cells induced by 5-Fluorouracil (5-FU). METHODS: The cytotoxicity of anticancer drugs on Be17402 and Be17402/5-FU cells was assessed using SRB assay. p53 expression was detected at its mRNA level by RT-PCR assay and at its protein level Western blot or immunocytochemistry assay in Be17402/5-FU cells transfected with either control vector or wt p53. AnnexinV-FITC/PI double labeled assay was performed to detect apoptosis. The chemosensitivity of Be17402/5-FU cells transfected with wt p53 was assessed using SRB assay. RESULTS: Be17402/5-FU cells exhibited cross-resistance to vincristine, doxorubicin, paclitaxel, and so on. wt p53 gene transfection upregulated the expression of p53 in Be17402/5-FU cells. wt p53 was able to greatly inhibit cell proliferation and significantly induce apoptosis in Bel7402/5-FU cells. Moreover, wt p53 gene transfection increased the chemosensitivity of Be17402/5-FU cells to some anticancer drugs. CONCLUSION: These results indicated that the wt p53 gene transfection not only induced suppression of cell growth, but also increased the sensitivity of Be17402/5-FU cells to 5-FU, vincristine, and doxorubicin.

Objective:To study the miRNA-21 content in cervical cancer tissue and analyze its downstream target molecules.Methods:Patients with different FIGO stages of cervical cancer and healthy subjects were selected, cervical cancer tissue and normal cervical tissue were collected, and contents of miRNA-21 and apoptotic genes were detected; cervical cancer SiHa cells were cultured, miRNA-21 mimics and inhibitors were transfected, and then apoptotic gene contents were detected.Results:miRNA-21 contents in different stages of cervical cancer tissue were all higher than those in normal cervical tissue, mRNA contents of p16ink4a, ASPP1, Fas and GRIM-19 were lower than those in normal tissue, and mRNA contents of p16ink4a, ASPP1, Fas and GRIM-19 were negatively correlated with miRNA-21 contents; after miRNA-21 mimics were transfected, mRNA contents of p16ink4a, ASPP1, Fas and GRIM-19 significantly decreased, and after miRNA-21 inhibitors were transfected, mRNA contents of p16ink4a, ASPP1, Fas and GRIM-19 significantly increased.Conclusion:miRNA-21 contents in cervical cancer tissue significantly increase; downstream target genes of this miRNA may be apoptotic genes p16ink4a, ASPP1, Fas and GRIM-19.