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

目的.本研究旨在利用重组DNA技术构建分泌型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分泌的SOD蛋白的抗炎作用。为了将SOD蛋白分泌到细胞外，我们构建了含IL-2信号肽和人SOD的质粒pRc/CMV-ILSOD。将含有分泌型SOD编码cDNA的pRc/CMV-ILSOD转染大鼠皮肤成纤维细胞和肺上皮细胞，通过两种体外模型研究宿主和转化子对黄嘌呤/黄嘌呤氧化酶（X/XO）系统氧化应激的影响，以探讨SOD的自分泌和旁分泌作用。采用角叉菜胶致大鼠足跖肿胀、液氮致大鼠足跖肿胀和百草枯致大鼠肺损伤3种急性炎症模型，评价宿主和转化子移植的抗炎作用。转化体（ILSOD细胞） ...更多信息 ILSOD细胞培养液中的SOD活性明显高于宿主细胞培养液。ILSOD细胞以自分泌和旁分泌方式降低X/XO的细胞毒活性。ILSOD细胞对X/XO诱导的细胞毒性的保护作用与损伤细胞内脂质过氧化反应的降低密切相关。在体研究表明，ILSOD细胞悬液移植于大鼠足爪可抑制角叉菜胶所致的足爪肿胀，其抑制作用至少持续7天，且抑制作用的强弱和持续时间与移植细胞的数量有关。ILSOD细胞悬液的这些抑制作用可通过共同给予hSOD抗血清而减弱。将ILSOD细胞移植到角叉菜胶引起的足肿胀的后爪中，可明显促进足肿胀的愈合。将ILSOD细胞悬液移植到大鼠皮下，可抑制液氮诱导的水肿。此外，ILSOD细胞悬液胸腔内移植可抑制百草枯所致的肺损伤。结果表明，基因修饰的皮肤成纤维细胞和肺上皮细胞是一种有效的、持续的SOD靶向给药系统，可作为其他治疗性蛋白质的给药系统。少

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