【摘要】 目的 构建CD59突变的重组体，研究Hela细胞中CD59基因突变引起肿瘤凋亡相关分子caspase3表达的变化。方法 采用重组聚合酶链反应定点诱变技术，将CD59的第39～41位氨基酸突变为色氨酸，克隆入pALTERMAX质粒，采用阳离子脂质体法将重组质粒转染Hela细胞。G418筛选稳定表达细胞克隆，用免疫荧光、ELISA、流式细胞术筛选出高表达突变CD59的细胞株，用免疫组化法检测转染前后Hela细胞内caspase3表达的变化。结果 酶切鉴定和序列测定均证实成功构建了CD59氨基酸突变的重组质粒。转染突变CD59后Hela细胞内caspase3表达明显减少，与转染正常CD59的Hela细胞比较差异有显著意义（t=2.846,P<0.01)。结论caspase3表达变化可能是CD59引起肿瘤逃逸的另一途径。
【关键词】 抗原 CD59 点突变 肿瘤逃逸 基因重组 Hela细胞 Caspase 3
［ABSTRACT］ Objective To construct mutant CD59 molecular, and study the alleosis in the expression of caspase3 correlative with tumor apoptosis brought about by CD59 mutation in Hela cells. Methods Using polymerase chain reaction sitedirected mutagenesis, the amino acid of the 39th and the 41th mutated to tryptophanes. Mutant CD59 DNA inserted into the vector pALTERMAX and transfected into the Hela cells via lipofectamine method. Stable expression clones were selected by the addition of G418. The G418resistant clones which expressed relatively high levels of mutant CD59 were sorted by immunofluorescence method, ELISA and flow cytometry. The expression of caspase3 in Hela cells before and after transfection was tested immunohistochemically. Results Recombinant plasmids of pALTERMAXCD59 had been successfully constructed according to sequence of enzyme digestion analysis and fragment investigation. The amount of caspase3 in Hela cells which were transfected was obviously lower than that in Hela cells which were transfected with nomal CD59, and the difference was obvious (t=2.846,P<0.01). Conclusion Another way by which CD59 leads the tumor escaping may be the expression changing of caspase3.
［KEY WORDS］ Antigens, CD59; Point mutation; Tumor escaping; Mutagenesis; Hela cells; Caspase3
众所周知，肿瘤的免疫逃逸是导致治疗方案失败的重要原因[1]。至今为止，国内外学者对肿瘤逃逸的机制已有多方面的阐述。近年来，国外学者在肠道、卵巢及前列腺等多种实体肿瘤细胞中发现有CD59分子的高表达。CD59是一种补体调节蛋白,其以糖基磷脂酰肌醇（GPI）锚着于多种细胞膜表面[4]，通过阻止补体攻膜复合物(MAC)的装配保护宿主细胞免受补体介导的溶细胞作用[5,6]。近年来的研究显示，CD59与肿瘤的生长失控和转移密切相关[2,3]，但有关其与肿瘤逃逸的相关性研究目前尚未有定论。本文通过研究caspase3的表达变化，探讨CD59与肿瘤逃逸的相关活性位点，为肿瘤的靶向治疗提供新的思路。
1 材料和方法
1.1 材料
pALTERMAX、pALTERCD59由美国哈佛大学惠赠, EcoR Ⅰ酶、DNA聚合酶、T4连接酶均购自大连TaKaRa公司，细菌菌株E.coli.JM109为本室保存，小牛血清为天津灏洋生物制品科技责任有限公司产品，Trypsin（1∶250）购自SinoAmerican Biotec公司，RPMI 1640培养基、G418选择抗生素、LipofectamineTM2000均为Invitrogen公司产品，鼠抗人CD59单抗为本实验室制备，羊抗鼠IgG2aFITC、羊抗人CD59单抗和HRP标记兔抗山羊IgG（H+L）均购自北京中山生物技术有限公司，荧光抗体CD59FITC购自COULTER 公司，caspase3免疫组化试剂盒购自武汉博士德生物工程有限公司，Hela细胞由本实验室保存。设计两对寡核苷酸引物，通用引物：pT7，T3；突变引物：M/F，M/R。以上引物均购自上海生物工程有限公司。
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