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GENETHERAPY FOR MALIGNANTBRAIN TUMOR.-Improvement of transduction rate of recombinant retrovirus by modification of cell cycle, bystander effect-

恶性脑肿瘤的基因治疗。-通过改变细胞周期、旁观者效应提高重组逆转录病毒的转导率-

基本信息

批准号：
06671406
负责人：
MIYAGI Koichi
金额：
$ 1.22万
依托单位：
UNIVERSITY OF THE RYUKYUS
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

To improve the efficacy of HSVtk/ganciclovir system gene therapy, modification of proliferating pool and cell cycle, and bystander effect are studied. We confirmed the proleferating activity correlated to the transduction rate of recombinant retrovirus from producer cell to glioma cell.Ganciclovir were more cytotoxic to the cells their doubling time is short and high ratio of PCNA positive cell. Trichostatin A has a cell cycle regulation activity. Removing of trichostatin from culture media increased the S-phase glioma cell.Bystander effect also influenced the cytotoxicity of ganciclovir. The mechanism of bystander effect was considered to be due to the transfer of ganciclovir-metabolites derived from thymidine kinase (tk) -positive cells to the adjacent tk-negative cells via Gap-Junctional Intercellular Communication (GJIC). The purpose of this study is to clarify whether the compounds which are found to regulate the GJIC,Promote or inhibit the bystander effect. If the GJIC has a main … More role on bystander effect, these chemicals might have the influence on the bystander effect.GJIC is composed of 6 connexin43s on astrocyte. However, the connexin43 quantity was much smaller in human malignant glioma cell than that in rat normal fibroblast (NRF) by western immunoblot.Connexin43 was identified in mouse fibroblast NRF on the cell surface even in the state of semiconfluent by immunolight microscopic studies using anti-connexin43 monoclonal antibody. Contrary, connexin43 was not detected on the cell surface of malignant glioma.Connexin43 in the PG13, human glioma cells are mainly localized intra-cytoplasmic rather than at the boundaries of cells.Result of GJIC by Lucifer yellow fluorescent dye microinjection method, GJIC was apparent on NRF.On the contrary, GJIC on RBR17T and HBR96T wss one-third and one-tenths of NRF respectively. GJIC on PG13 was not apparent.Down-regulation of GJIC by phorbol was noted only GJIC positive NRF and RBR17T significantly.Despite the absence or small amount of GJIC in PG13 and glioma cell, we confirmed the bystander effect between human glioma cells and tk positive autologous glioma cells or PG13 (that hold HSVtk gene, but not the retroviral vector-producing cells) when these cells were treated with ganciclovir. Therefore, GJIC does not play a major role in the development of the bystander effect of PG13 or HSVtk gene transduced glioma cell on HSVtk negative glioma cell.We examined the effects of chemicals or anti-connexin43 monoclonal antibody that modulate GJIC on the bystander effect. When dexamethasone or beta-carotene was added during the coculture of glioma cell with autologous HSVtk positive glioma cell or PG13, dexamethasone and beta-carotene had no consistent promoting effect on bystander effect. When anti-connexin43 monoclonal antibody was added during the coculture of glioma cell with autologous tk positive glioma cell, anti-connexin43 monoclonal antibody did not inhibit bystander effect.Our results suggest : 1st.There is bystander effect between glioma cell and tk positive cell. This mechanism was noted even between cells from different species. 2nd.Cx 43 was mainly intracytoplasmic in glioma cell. Because the small amount of GJIC on glioma cells, functionally the GJIC does not play a major role in the development of the bystander effect. 3rd.Dexamethasone and beta-carotene have a cytostatic effect.However dexamethasone and beta-carotene had no consistent promoting effect on the bystander effect. We therefore conclude that enhancers of GJIC such as dexamethasone and beta-carotene have no benefit on the HSVtk/GCV gene therapy.This is the first report to directly demonstrate the effects of chemicals and anti-connexin43 monoclonal antibody that modulate GJIC on the bystander effect. Less

为了提高HSVtk/更昔洛韦系统基因治疗的疗效，研究了增殖池和细胞周期的修饰以及旁观者效应。我们证实了增殖活性与重组逆转录病毒从产生细胞到胶质瘤细胞的转导率有关。更昔洛韦对细胞有较强的细胞毒性，增殖时间短，PCNA阳性细胞比例高。曲古霉素A具有细胞周期调节活性。从培养基中去除曲古斯汀使s期胶质瘤细胞增多。旁观者效应也影响更昔洛韦的细胞毒性。旁观者效应的机制被认为是由于胸苷激酶（tk）阳性细胞产生的更昔洛韦代谢物通过间隙连接细胞间通讯（GJIC）转移到邻近的tk阴性细胞。本研究的目的是阐明所发现的调节GJIC的化合物是促进还是抑制旁观者效应。如果GJIC在旁观者效应中起主要作用，那么这些化学物质可能对旁观者效应有影响。GJIC由星形胶质细胞上的6个连接蛋白43s组成。western免疫印迹法发现，人恶性胶质瘤细胞中connexin43的表达量明显低于正常成纤维细胞（NRF）。使用抗Connexin43单克隆抗体，免疫光镜下观察发现，即使在半流畅状态下，小鼠成纤维细胞表面的NRF中也存在Connexin43。相反，恶性胶质瘤细胞表面未检测到connexin43。在PG13中的Connexin43，人胶质瘤细胞主要定位于细胞质内而不是细胞边界。路西法黄荧光染料显微注射法检测GJIC，在NRF上有明显的GJIC。相反，RBR17T和HBR96T的GJIC分别为NRF的三分之一和十分之一。PG13上的GJIC不明显。仅GJIC阳性NRF和RBR17T显著下调GJIC。尽管PG13和胶质瘤细胞中缺乏或少量GJIC，但我们证实，当更昔洛韦处理人类胶质瘤细胞和tk阳性的自体胶质瘤细胞或PG13（含有HSVtk基因，但不含逆转录病毒载体细胞）时，这些细胞之间存在旁观者效应。因此，GJIC在PG13或HSVtk基因转导的胶质瘤细胞对HSVtk阴性胶质瘤细胞的旁观者效应的发展中并不起主要作用。我们检测了调节GJIC的化学物质或抗connexin43单克隆抗体对旁观者效应的影响。当胶质瘤细胞与自体HSVtk阳性胶质瘤细胞或PG13共培养时，添加地塞米松或β -胡萝卜素对旁观者效应的促进作用不一致。在胶质瘤细胞与自体tk阳性胶质瘤细胞共培养时加入抗connexin43单克隆抗体，抗connexin43单克隆抗体不抑制旁观者效应。我们的研究结果表明：1。胶质瘤细胞与tk阳性细胞之间存在旁观者效应。甚至在不同物种的细胞之间也发现了这种机制。第二。cx43主要存在于胶质瘤细胞的胞浆内。由于GJIC作用于胶质瘤细胞的量很小，在功能上GJIC在旁观者效应的发展中并不起主要作用。第三。地塞米松和胡萝卜素有细胞抑制作用。地塞米松和β -胡萝卜素对旁观者效应的促进作用不一致。因此，我们得出结论，GJIC增强剂如地塞米松和β -胡萝卜素对HSVtk/GCV基因治疗没有益处。这是第一次直接证明化学物质和抗connexin43单克隆抗体调节GJIC对旁观者效应的影响。少