ABSTRACT
Eukaryotic plasmid vectors encoding the tyrosine hydroxylase (TH) gene and GTP cyclohydrolase-1 (GCH) gene were constructed and introduced into immortalized fibroblasts obtained from SV40 large antigen (LT(AG)) transformed rat primary fibroblasts. TH and GCH positive clones were selected and identified by immunohistochemistry and RT-PCR, respectively. Hemi-parkinsonian rats created using 6-hydroxydopamine (6-OHDA) were used to assess the therapeutic effect created by the co-implantation of immortalized fibroblasts genetically modified by TH or GCH genes. Animal behavior was significantly improved two weeks following implantation and behavioral correction was maintained for over 14 weeks. Behavioral improvement was paralleled by exogenous TH gene expression, identified by TH immunohistochemistry and RT-PCR analyses. The transplanted cells survived for at least 38 weeks as demonstrated by fibronectin immunohistochemical staining. Tumor formation or host reaction was not seen, although TH expression was negative for 20 weeks after the implantation. This work demonstrates that the co-transplantation of immortalized fibroblasts genetically modified by TH and GCH genes may be developed as a valuable approach to the treatment of Parkinson's disease.
Subject(s)
Biopterins/analogs & derivatives , Cell Transplantation/methods , GTP Cyclohydrolase/genetics , Genetic Therapy/methods , Parkinson Disease/therapy , Tyrosine 3-Monooxygenase/genetics , Animals , Behavior, Animal , Biopterins/biosynthesis , Brain/enzymology , Cell Line, Transformed/transplantation , Denervation , Fibroblasts/cytology , Fibroblasts/transplantation , Gene Expression Regulation, Enzymologic , Oxidopamine , Rats , Skin/cytology , SympatholyticsABSTRACT
<p><b>OBJECTIVE</b>To investigate the effects of direct intratumor injection of the packaged cells with retroviral vector carrying human endostatin (hEN) on the growth inhibition of B16 melanoma in C57/BL6 mice.</p><p><b>METHODS</b>Retroviral vector, pLNC-hEN, was constructed with modified and identified hEN gene. The cell line, PA317, was used to establish ecotropic virus producing cells by transfecting and packing with pLNC-hEN. Then the cells were injected directly into the tumor in C57/BL6 mice bearing B16 melanoma, established by intra-cutaneous injection of B16 cell suspension. The tumor size was measured at different intervals to observe the antitumor effect. Micro-vessel density (MVD) in the tumor tissue was evaluated by immunohistological examination to count the apoptotic cells by TUMEL staining.</p><p><b>RESULTS</b>Tumor with diameter of 2 - 3 mm was observed in all mice after 7 - 9 days. The average tumor volume on D3, D5, D7 and D9 after gene transfection was 4.67 +/- 1.1, 22.25 +/- 13.06, 84.17 +/- 43.5 and 155.08 +/- 81.1 mm(3) in the gene therapy group but 136.17 +/- 30.61, 390.17 +/- 220.47, 1 021.67 +/- 537.4 and 2 920.2 +/- 220.01 mm(3) in the control group, the difference of which was statistically significant. The average MVD in the gene therapy and control groups were 8 +/- 2.28 and 28.17 +/- 5.31 while the average apoptotic cell number in the two groups were 23.33 +/- 3.83 and 2.33 +/- 1.21, both of which were statistically significant.</p><p><b>CONCLUSION</b>The direct injection of packaged cells carrying hEN gene is able to inhibit the growth of micro-blood vessels and promote tumor cell apoptosis, which ultimately inhibits the growth of B16 melanoma.</p>
