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PHARMACEUTICAL STUDY FOR THERAPY OF LUNG DISEASES USING HUMAN SOD GENE TRANSFORMED CELLS

使用人SOD基因转化细胞治疗肺部疾病的药物研究

基本信息

批准号：
07557313
负责人：
OKUMURA Katsuhiko
金额：
$ 0.9万
依托单位：
DEPARTMENT OF HOSPITAL PHARMACY,SCHOOL OF MEDICINE,KOBE UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

Purpose. The purposes of this work were to construct a secretable SOD protein using recombinant DNA technics and to evaluate the anti-inflammatory effects of SOD delivered by genetically modified skin fibroblasts and lung epithelial cells in vitro and in vivo.Methods. To secrete SOD protein into extracellular space, we constructed the plasmid with interleukin-2 signal peptide and human SOD (pRc/CMV-ILSOD). Rat skin fibroblasts and lung epithelial cells were transfected with pRc/CMV-ILSOD including secretable SOD-coding cDNA.The effects of host and transformants on oxidativestress using the xanthine/xanthine oxidase (X/XO) system were examined by two in vitro models to study the autocrine and paracrine SOD action. The anti-inflammatory effects by transplantation of host and transformants were evaluated in 3 kinds of acute inflammation models, carrageenin-induced paw edema, liquid nitrogen-induced edema and paraquat induced lung damage, in rats.Results. The transformants (ILSOD cells) se … More creted SOD protein into the extracellular space, and the extracellular SOD activity in ILSOD cells cultures was significantly increased in comparison with that in host cell cultures. ILSOD cells diminished the cytotoxic activity by X/XO,in autocrine and paracrine fashions. These protective effects of ILSOD cells against X/XO-induced cytotoxicity correlated well with the decreaase in lipid peroxidation in the damaged cells. The in vivo study showed that transplantation of ILSOD cell suspensions into the hind paw in rats inhibited carrageenin-induced paw edema for at least 7 days, and the degrff and the durability of these inhibitory effects were dependent on the number of ILSOD cells transplanted. These inhibitory effects of ILSOD cell suspensions were reduced by coadministration of hSOD antiserum. The healing of paw edema caused by carrageenin was markedly enhancec by transplantation of ILSOD cells into the edematous hind paw. And transplantation of ILSOD cell suspensions into the subcutaneous tissue in rats inhibited liquid nitrogen-induced edema. Furthermore, tansplantation of ILSOD cell suspensions into the thorax in rats inhibited liquid paraquat-induced lung damage.Conclusions. The results suggested that genetically modified skin fibroblasts and lung epithelial cells area suitable delivery system for obtaining efficient and continuous supply of SOD at the target site, and this strategy may be useful as a drug delivery system for other therapeutic proteins. Less

目的。本研究目的是利用重组DNA技术构建可分泌SOD蛋白，并在体外和体内评价转基因皮肤成纤维细胞和肺上皮细胞传递SOD的抗炎作用。为了将SOD蛋白分泌到细胞外，我们将白细胞介素-2信号肽和人SOD （pRc/CMV-ILSOD）构建质粒。用pRc/CMV-ILSOD转染大鼠皮肤成纤维细胞和肺上皮细胞，其中含有可分泌的sod编码cDNA。利用黄嘌呤/黄嘌呤氧化酶（X/XO）体系，通过两种体外模型研究了宿主和转化体对氧化应激的影响，研究了自分泌和旁分泌SOD的作用。采用角叉菜胶致大鼠足跖水肿、液氮致大鼠足跖水肿和百草枯致大鼠肺损伤3种急性炎症模型，评价宿主和转化体移植的抗炎作用。转化子（ILSOD细胞）在胞外空间产生了更多的SOD蛋白，与宿主细胞培养相比，ILSOD细胞培养的胞外SOD活性显著提高。ILSOD细胞以自分泌和旁分泌方式通过X/XO降低细胞毒活性。ILSOD细胞对X/ xo诱导的细胞毒性的这些保护作用与受损细胞中脂质过氧化的减少密切相关。体内研究表明，将ILSOD细胞悬浮液移植到大鼠后爪，可以抑制卡拉胶诱导的足跖水肿至少7天，并且这种抑制作用的降解程度和持续时间取决于移植ILSOD细胞的数量。同时加入hSOD抗血清后，ILSOD细胞悬液的抑制作用减弱。将ILSOD细胞移植至肿胀的后爪，可明显促进卡拉胶所致足跖水肿的愈合。ILSOD细胞悬液皮下移植对液氮诱导的水肿有抑制作用。此外，ILSOD细胞悬浮液胸腔移植对百草枯所致大鼠肺损伤有抑制作用。结果表明，基因修饰的皮肤成纤维细胞和肺上皮细胞是获得有效和持续供应的超氧化物歧化酶的合适的递送系统，这种策略可能有助于作为其他治疗性蛋白的药物递送系统。少