Anti-nasopharyngeal carcinoma effect in vivo and in vitro of cytotoxicity T lymphocyte induced by Ad5-latent membrane protein 2A / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To study the biological characteristics of cytotoxicity T lymphocyte (CTL) induced by dendritic cell (DC) transfected with the Epstein-Barr virus latent membrane protein 2A (EBV-LMP2A) recombinant adenovirus. To establish nasopharyngeal carcinoma (NPC) animal models expressing LMP2A and investigate the anti-tumor effect of LMP2A specific CTL in vivo.</p><p><b>METHODS</b>The mononuclear cells were isolated from human peripheral blood mononuclear cells (PBMC) and cultured with the cytokines [granulocyto-monocyte colony stimulating factor (GM-CSF), interleukin-4 (IL-4) and tumor necrosis factor-alpha TNF-alpha]. The expression of surface markers on mature DC was detected by fluorescence activated cell sorter FACS. Mature DC were transfected with LMP2A recombinant adenovirus. Under the help of interleukin-2 (IL-2), LMP2A specific CTL were induced by coculturing LMP2A-transfected DC with autologous PBMC. The population of CTL was detected by FACS. NPC animal models were constructed by implanting CNE cells expressing LMP2A subcutaneously into BALB/c nude mice. After intra-tumoral injection of LMP2A specific CTL, the size of tumor was measured. The tumors were removed after 30 d and subjected to histological examination.</p><p><b>RESULTS</b>Mature DC displaying typical characteristics of morphology and phenotype were obtained from monocytes cultured in the medium containing GM-CSF, IL-4 and TNF-alpha. The LMP2A specific CTL induced by transfected DC were composed of mainly CD4+ and CD8+ T cells. The NPC animal models were constructed three weeks after implanting CNE cells. The study in vivo indicated that the tumors treated with LMP2A specific CTL grew slowly compared with control. Tumor volume of treated groups was significantly smaller than that of controls. The histological sections showed local necrosis and infiltration of lymphocyte in tumor tissue.</p><p><b>CONCLUSIONS</b>Typically mature DC could be generated in vitro by culturing monocytes with the cytokines. LMP2A-specific CTL could be induced by LMP2A transfected DC in vitro. NPC mice models could be constructed by implanting CNE cells. LMP2A specific CTL could inhibit the growth of implanted tumor and produce an anti-tumor effect in vivo.</p>

Expression of HBcAg in eukaryotic cells by retroviral vector mediated gene transfer / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To construct recombinant retroviral vector expressing HBcAg in eukaryotic cells.</p><p><b>METHODS</b>The HBV core gene fragment was amplified by using PCR from pADR which contains complement nucleotide sequence of HBV subtype adr and cloned into retroviral expression plasmid pLXSN, then transfected into packing cell (PT67) with lipofec AMINE. After 2-3 weeks selection with G418, large colonies were isolated and transferred to individual plates. Virus-containing medium was collected and used to infect NIH3T3, EL4 and mouse bone marrow derived dendritic cells(DC). DNA was extracted from infected cells and tested by PCR. Indirect immunofluorescence and FACS were used to detect the expression of HBcAg. Cell mediated immunity of immunized C57BL/6 mice with transduced DC was analyzed.</p><p><b>RESULTS</b>The insertion of HBV core gene fragment in the recombinant retroviral plasmid was confirmed by PCR as well as enzyme digestion with EcoR1 and BamH2. The viral titer reached 3 x 10(5) CFU/ml. The result of PCR showed that the HBV core gene had been integrated into the genome of infected NIH3T3 cells. Indirect immunofluorescence and FACS analysis showed that HBcAg had been expressed in these cells. HBcAg specific CTL responses could be generated in mice immunized with retrovirus transduced DC.</p><p><b>CONCLUSIONS</b>HBV core gene had been integrated into eukaryotic cells with retroviral vector and target gene had been expressed efficiently. These results may have some impact on gene therapy of chronic hepatitis B.</p>