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突变型与野生型TNF论文

2014-04-23 01:27 【关键词】 ,肿瘤
Apoptosis of tumor cells induced by mutant and wild type TNFα
　　【Abstract】 AIM: To compare the apoptosisinduced ability of recombinant human mutant type 471 rhTNFα (mt 471rhTNFα) with that of wild type rhTNFα (wt rhTNFα) and to study the apoptotic mechanism induced by mt 471rhTNFα. METHODS: The apoptosis of ZR751 cells, a breast cancer cell line, induced by mt 471rhTNFα or wt rhTNFα was analyzed and compared by 20 g/L agarose gel electrophoresis and flow cytometry techniques. The activation profiles of nuclear factor NFκB in ZR751 cells untreated and treated by mt 471rhTNFα or wt rhTNFα were detected using Trans AMTM NFκB p65 kit based on ELISA for further study of the apoptotic mechanism induced by mt 471rhTNFα. RESULTS: The results from 20 g/L agarose gel electrophoresis of genomic DNA indicated that ZR751 cells treated by mt 471rhTNFα provided a typical apoptotic ladder pattern of DNA fragmentation, whereas weakened ladders were observed in ZR751 cells treated by wt rhTNFα. Flow cytometry showed that the cell apoptotic rate in mt 471rhTNFα treated group was 43%, but 25% in wt rhTNFα treated group. The mt 471rhTNFα held a strong apoptosisinducible ability. The results of DNAbinding activity of NFκB showed that NFκB activation induced by wt rhTNFα was significantly higher than that of mt 471rhTNFα when the protein concentration of mt 471rhTNFα or wt rhTNFα reached 50 μg/L (P=0.002). CONCLUSION: The apoptosisinduced ability of mt 471rhTNFα is superior to that of wt rhTNFα. One of the possible reasons for the enhanced apoptosisinduced ability may be that NFκB activation is inhibited in tumor cells treated with mt 471rhTNFα.
　　【Keywords】 neoplasms; mutant type 471rhTNFα; wild type rhTNFα; NFkappaB; apoptosis
　　【摘要】 目的： 比较重组人突变型471rhTNFα（mt 471rhTNFα）与野生型rhTNFα（wt rhTNFα）诱导肿瘤细胞发生凋亡的能力，并对mt 471rhTNFα诱导肿瘤细胞发生凋亡的作用机制进行初步研究. 方法： 以乳腺癌细胞系ZR751细胞为靶细胞，应用基因组DNA琼脂糖凝胶电泳及流式细胞技术分析mt 471rhTNFα与wt rhTNFα诱导肿瘤细胞凋亡的情况；利用以ELISA为基础的Trans AMTM NFκB p65试剂盒检测经mt 471rhTNFα或wt rhTNFα处理的ZR751细胞核因子NFκB的活化情况，以便对其诱导凋亡的机制进行初步的研究. 结果： 20 g/L琼脂糖凝胶电泳显示，mt 471rhTNFα处理组的ZR751细胞基因组DNA呈现明显的ladder状分布，wt rhTNFα处理组的“ladder”条带明显减弱；流式细胞仪分析显示，mt 471rhTNFα诱导的细胞凋亡峰面积高于wt rhTNFα处理组. NFκB活化检测结果显示，当两型rhTNFα浓度增高到50 μg/L时，wt rhTNFα处理组的NFκB的活化量明显高于同浓度的mt 471rhTNFα处理组（P=0.002）. 结论： mt 471rhTNFα诱导肿瘤细胞凋亡的能力明显优于wt rhTNFα；肿瘤细胞内NFκB活化明显受抑是mt 471rhTNFα诱导凋亡能力增强的主要原因之一.
　　【关键词】 肿瘤；突变型471rhTNFα；野生型rhTNFα； NFκB；细胞凋亡
　　0引言
　　人肿瘤坏死因子α（tumor necrosis factor alpha, TNFα），主要由巨噬细胞产生，是具有多种生物学活性的细胞因子. mt 471rhTNFα是删除wt TNFαN端的7个氨基酸，并将Pro8Ser9Asp10置换为Arg8Lys9Arg10［1］的突变体. 这种突变并不改变TNFα分子的高级结构，仍然形成具有生物活性状态的三聚体，抗肿瘤作用增强且毒副作用降低，极具开发价值. 我们在获得mt 471rhTNFα重组蛋白的基础上［2-4］，进行新的研究，为mt 471rhTNFα的临床前研究提供实验依据.