Effects of secretable SOD delivered by genetically modified cells on xanthine/xanthine oxidase and paraquat-induced cytotoxicity in vitro.
Effects of secretable SOD delivered by genetically modified cells on xanthine/xanthine oxidase and paraquat-induced cytotoxicity in vitro.
复制标题
转基因细胞分泌的 SOD 对黄嘌呤/黄嘌呤氧化酶和百草枯诱导的体外细胞毒性的影响。
DOI：
10.1248/bpb.22.846
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发表时间：
1999
影响因子：
2
通讯作者：
K. Okumura
中科院分区：
文献类型：
--
作者：
F. Komada;K. Nishiguchi;Y. Tanigawara;S. Iwakawa;K. Okumura
设计了一种新的分泌型超氧化物歧化酶（SOD）真核表达载体，将人SOD cDNA与人白细胞介素2（IL-2）基因的1个连接氨基酸和信号肽DNA序列（IL-SOD（2）cDNA）融合表达。通过PCR技术构建ILSOD（2）cDNA，将其克隆到pRc/CMV质粒的多克隆位点（pRc/CMV-ILSOD（2））。将pRc/CMV-ILSOD（2）脂质体转染大鼠肺上皮样细胞（L2细胞）和大鼠皮肤成纤维细胞（FR细胞）。IS（2）-L2细胞（转染pRc/CMV-ILSOD（2）的L2细胞）和IS（2）-FR细胞（转染pRc/CMV-ILSOD（2）的FR细胞）的细胞外SOD活性比宿主细胞高2-3倍。首先，我们研究了这些转化细胞（IS（2）-L2和IS（2）-FR细胞）分泌的胞外SOD对胞外超氧阴离子（黄嘌呤/黄嘌呤氧化酶; X/XO处理）诱导的正常细胞毒性的保护作用。与宿主细胞相比，这些转化细胞对X/XO诱导的细胞毒性的敏感性显著降低。尽管IS（2）-L2和IS（2）-FR细胞的条件培养基对X/XO诱导的细胞毒性有保护作用，但宿主细胞（L2和FR细胞）的条件培养基对X/XO诱导的细胞毒性没有显著影响。此外，转化细胞的条件培养基比宿主细胞更有效地对抗氧化应激条件下正常细胞的脂质过氧化。其次，我们通过百草枯处理在细胞内空间产生超氧阴离子。转化细胞对百草枯诱导的细胞毒性比宿主细胞更敏感。加入过氧化氢酶后，这些转基因细胞对百草枯的敏感性变得与宿主细胞相当。这些结果表明，对细胞外的超氧阴离子诱导的细胞毒性的分泌型SOD基因的转染的保护作用，虽然没有观察到这种保护作用对百草枯治疗产生的细胞内的细胞毒性。
We designed a new eukaryotic expression vector for secretable superoxide dismutase (SOD), which expresses human SOD cDNA by fusing it to 1 connecting amino acid and the signal peptide DNA sequence of the human interleukin-2 (IL-2) gene (IL-SOD(2) cDNA). The ILSOD(2) cDNA constructed by PCR-based gene expression was ligated into the multicloning site of the pRc/CMV plasmid (pRc/CMV-ILSOD(2)). Rat lung epithelial-like cells (L2 cells) and rat skin fibroblasts (FR cells) were transfected with pRc/CMV-ILSOD(2) by lipofection. The extracellular SOD activities of IS(2)-L2 cells (L2 cells transfected with pRc/CMV-ILSOD(2)) and IS(2)-FR cells (FR cells transfected with pRc/CMV-ILSOD(2)) were 2-3 times higher than those of host cells. Initially, we investigated the protective effect of extracellular SOD secreted from these transformed cells (IS(2)-L2 and IS(2)-FR cells) on extracellular superoxide anion (xanthine/xanthine oxidase; X/XO treatment)-induced cytotoxicity in normal cells. The sensitivities of these transformed cells to X/XO-induced cytotoxicity was decreased significantly as compared with that of host cells. Although, the conditioned medium from IS(2)-L2 and IS(2)-FR cells protected against X/XO-induced cytotoxicity, the conditioned medium from host cells (L2 and FR cells) showed no significant effects on X/XO-induced cytotoxicity. Furthermore, the conditioned medium from transformed cells was more effective than that of host cells against lipid peroxidation by normal cells under conditions of oxidative stress. Second, we generated superoxide anions in the intracellular space by paraquat treatment. The transformed cells were more sensitive to paraquat-induced cytotoxicity than host cells. Following addition of catalase, the sensitivity of these genetically modified cells to paraquat became equivalent to that of host cells. These results indicated a protective effect of transfection with secretable SOD genes against extracellular superoxide anion-induced cytotoxicity although no such protective effect was observed against the intracellular cytotoxicity generated by paraquat treatment.
DOI：
10.3109/10715769109145859
发表时间：
1991
期刊：
Free radical research communications
影响因子：
--
作者：
J. Doroshow;S. Akman;S. Esworthy;Fong-Fong Chu-Fong;T. Burke
通讯作者：
J. Doroshow;S. Akman;S. Esworthy;Fong-Fong Chu-Fong;T. Burke