.tk/mcp-1 Fusion Experimental Study Of Gene Therapy For Ovarian Cancer

OBJECTIVE: TO construct a bicistronic retroviral vector containing HSV-tk and MCP-1 gene. To construct retroviral expressing vectors containing HSV-tk single gene and MCP-1 single gene,respectively.METHODS: The total RNA was extracted from human peripheral blood mononuclear cell (PBMC) with isolation regents RNA and the cDNA of human MCP-1 was synthesized through reverse transcription. The MCP-1 gene was amplified by PCR and the purified PCR products was ligated into pUCm-T vector and transformed into DH5 α E.coli bacteria. The white clones were selected and the recombinant plasmid was purified, which was further identified by restriction endonucleases and sequence. The tk fragment was digested from the plasmid of pWZLneoCDglyTK, while the internal ribosome entry site(IRES) fragment was digested from the plasmid of MINV. The tk and MCP-1 fragment were linked by IRES and then was subcloned into corresponding restriction sites of pLXSN by the methods of directional cloning.The ligated product was thansformed into DH5 α E.coli copetent bacteria. The positive clones were randomly selected and the recombinant plasmids were purified,which was further identified by restiction endonuclease again. The tk fragment was digested from the plasmid of pWZLneoCDglyTK, while the MCP-1 fragment from pUCm-T-MCP-1. These two fragments were purified and ligated into the linear vectors pLXSN which were digested by the same two endonucleases. Their transformation, selection and identification afterwards were as the same as HSV-tk/MCP-1 fusion gene expression vector. Then the expression vections containing different goal genes were generated by the methods above.RESULTS: By the methods of restriction digestion and PCR, we confirmed that the length ,position and orientation of inserted genes and fusion genes were all correct. The retroviral expressing vectors of pLXSN/HSV-tk, pLXSN/MCP-1, pLXSN/tk-MCP-1 were suessfully constructed.CONCLUSIONS: The successful construction of expressing vectors containing tk, MCP-1 or tk/MCP-1 may be beneficial for the gene therapy of ovarian cancer. It also can be used for expressing new fusion genes and studying novel bioloical activities of chemokine.OBJECTIVE: To determine the in vitro behavior of simultaneously expressed TK and MCP-1 in epithelial ovarian cancer cell line NuTu-19.METHODS: Retorviral expression vectors pLXSN/HSV-tk> pLXSN/MCP-1 and pLXSN/tk-MCP-1 were transfected into the packaging cell line PA317 by lipofectin-mediated gene transfer system. The virus particles con- taining HSV-tk and/or MCP-1 genes were collected to infect NuTu-19.Reverse transcription-polymerase chain reaction (RT-PCR) and Enzyme- linked immunosorbent assay(ELISA)were used to confirm the express of HSV-tk and MCP-1.Chemotactic activity of MCP-1 was measured by chemo- taxis assay.According to the results ,cell lines which could highly express either or both of these genes were named NuTu-19/HSV-tk, NuTu-19/MCP-l or NuTu-19/tk-MCP-l.The cell line NuTu-19/neo which was transfected by vector pLXSN was used as a control. Cell growth characters were studied in vitro. MTT method was adopted to determine the sensitivity of different genes modified cells to GCV (ganciclovir). Scanning electron micrography (SEM)was used to examine the morpholgy of NuTu-19/tk-MCP-l after the effect of GCV.RESULTS: Stable HSV-tk and MCP-1 expressions in the cell lines, NuTu-19/HSV-tk,NuTu-19/MCP-l and NuTu-19/tk-MCP-l were confirmed by RT-PCR and ELISA.The secreted MCP-1 in the NuTu-19/MCP-l and NuTu-19/ tk-MCP-1 cells culture supernatant possesses the chemotatic activity. No obvious changes in cell growth were observed in vitro,compared with the cell line NuTu-19.GCV has significant killing efficacy in vitro on NuTu-19/HSV-tk and NuTu-19/tk-MCP-l cells and the effect of GCV between these two cell lineshas no significant difference (P>0.05) . Apoptosis is the major death form of NuTu-19 following the effect of pLXSN/tk-MCP-1+GCV.CONCLUSIONS: The constructed bicistronic retroviral vector can express TK and MCP-1 genes effectively in NuTu-19 cell.NuTu-19/ tk-MCP-1 cells may be used as a experimental vaccine for tumor therapy, they are also valuable in producing large quantity of MCP-1. It also could be used as a novel approach for the treatment of ovarian cancer.OBJECTIVE: To investigate the in vitro killing effect and its mechanisms of tk/MCP-1 fusion gene induce macrophage and synergisti-cally GCV on NuTu-19 ovarian epithelial cancer cell.METHODS: Isolate rat Fischer344 spleen macrophage. MTT method was applied to investigate the tumoricidal effect of macrophage. MTT method was used to study the in vitro killing effect on NuTu-19/HSV-tk cell and NuTu-19/tk-MCP-l cell of GCV alone or jointly with macrophage. Flow cytometry method was applied to analyse apoptosis and the expression of CD25 or CD44v6 after the effects of NuTu-19/tk-MCP-l+GCV .RESULTS: The maximum killing ratio of macrophage on NuTu-19/MCP-l cell and NuTu-19/tk-MCP-lcell was 28% and 23%,respectively. There is no significant difference ( P>0.05 ) . The cell killing of NuTu-19/HSV-tk or NuTu-19/tk-MCP-l was observed after treatment with GCV. Bystander effect was seen in mixed culture of cells. Furthermore,macrophage had a singnificant synergistic effect on the growth inhibition of the same mixture of modified by tk/MCP-1 and unmodified NuTu-19 cells in combination with GCV (P<0.05) . Flow cytometry suggests that there is a greater degree of apoptotie ratio(13.48), S-phase block(52.92%) and the expression rate of CD25(38.82%) in the NuTu-19/tk-MCP-l cell than inNuTu-19/HSV-tk cell (9.50%>38.31% and 20.00%, respectively) following the treatment with GCV. The expression of CD44V6 in NuTu-19/ HSV-tk or NuTu-19/tk-MCP-l cell is significant lower than control NuTu-19/neo cell.CONCLUSION: Combined transfer of suicide gene and chemokine gene induce nonspecific and specific antitumor immunity lead to a significant...