hape反义核酸的构建及其对细胞辐射效应的影响

目的克隆人ＡＰ内切核酸酶基因（ｈａｐｅ）ｃＤＮＡ全长编码序列，研究ｈａｐｅ反义核酸对细胞辐射敏感性和Ｈ２Ｏ２细胞毒的影响。方法反转录ＰＣＲ克隆人ｈａｐｅｃＤＮＡ，应用基因重组技术构建真核反义表达载体，用Ｌｉｐｏｆｅｃｔａｍｉｎｅ介导基因转染，细胞克隆形成法分析细胞存活。结果从人胚肺（ＨＥＬ）细胞中克隆出ｈａｐｅｃＤＮＡ全长编码序列，构建了ｈａｐｅ的真核反义表达质粒ｐＣＩＮ／ＡＳｈａｐｅ并转染ＨＯＣ８细胞，研究表明ｈａｐｅ反义核酸，提高了ＨＯＣ８细胞对γ射线和Ｈ２Ｏ２损伤的敏感性。结论利用分子生物学技术手段，阻断细胞ＤＮＡ修复过程中的某一环节，是细胞辐射或化疗药物增敏的一种有效途径。

Construction of eukaryotic vector expressing antisense RNA of human DNA repair enzyme APE and its effects on cellular radiation response

Objective To clone the cDNA encoding human apurinic/apyrimidinic endonuclease gene(hape), and to study the effects of its antisense nucleic acids on cytotoxicity of ionizing radiation and H 2O 2. Methods hape cDNA was cloned by RT CPR. hape antisense RNA expression vector was constructed by inserting hape cDNA in reverse orientation into the mammalian cell expression vector pCI neo. Gene transfection was mediated by lipofectamine. The cytotoxicity was assayed by means of cellular colony forming ability. Results 0.96kb of cDNA encoding hape was cloned from human embryonic lung (HEL) cells. Eukaryotic vector(pCIN/AShape) expression antisense hape RNA was constructed to transfect HOC8 cells. Stable HOC8 cell transfectants expressing hape antisense RNA were more sensitive to ionizing radiation and hydrogen peroxide than the control cells or the pCI neo transfectants. Conclusion HAPE plays an important role in cellular protection against a wide range of cytotoxic DNA damaging agents by its DNA repair or redox activities. hape locus is a potential target for the sensitization of radio or chemo therapy of tumors.