RNA干扰MeCP2对肺癌细胞A549增殖带来的影响的论文.docVIP

　　RNA干扰MeCP2对肺癌细胞A549增殖带来的影响的论文 【摘要】 目的 研究甲基化结合蛋白mecp2对肺癌细胞a549增殖的影响。方法 针对genebank中mecp2基因的不同靶点，设计并合成两条dna干扰序列，克隆到真核表达载体psilencer2.1u6，构建能够转录形成mecp2shrna的重组质粒psilencermecp2，经pcr、测序鉴定正确后，稳定转染a549细胞，在mrna和蛋白水平观察两重组质粒对mecp2的干扰作用，mtt法检测对细胞增殖能力的影响。结果 成功构建两个针对mecp2不同靶序列的shrna真核表达质粒，两质粒在mrna和蛋白水平可以使mecp2表达明显下调，稳定转染两质粒的a549细胞增殖能力明显减弱。结论 甲基化结合蛋白mecp2参与了肺癌细胞的恶性增殖。 【关键词】 rna干扰；mecp2;肺癌 effects of inhibition of methylcpginding protein 2 by rna interference on proliferation of lung carcinoma cell a549 qiu xiaohua1,guo lingling2, duan fengying3, ent of molecular medicine laboratory, the second affiliate hospital of nanchang university, nanchang 330006, china； 2. department of respiration, the peoples hospital of jiangxi province, 3. department of respiration, the second affiliated hospital of nanchang university, 4. department of oncologyabstract：objective to study the effects of mecp2 in proliferation of lung cancer cell lines a549.methods by the sequence of human mecp2 gene in genebank, the oligonucleotides encoding shrna targeting the mecp2 gene d psliencer2.1u6, confirmed by pcr and dna sequencing analysis， the rebinant plasimd ine tm2000. the expression of mecp2 at mrna and protein ined by rtpcr and tt assay. results the tids targeting the mecp2 gene ecp2 in mrna and protein levels ecp2 inhibited the proliferation of a549 cell significantly. conclusion the mecp2 protein plays an important role in the proliferation of lung cancer cell. key ecp2; lung cancer dna甲基化可使抑癌基因转录失活，从而导致肿瘤的发生[1]，在此过程中，甲基化结合蛋白mecp2 （methylcpgbinding protein 2）起着关键作用［2］。.rna干扰是近年来发展起来的一项基因阻断技术，利用小片断双链rna（double strands rna，dsrna）进行转录后基因沉默，能够迅速降解靶基因［3］。本课题为了研究mecp2蛋白在肺癌细胞恶性增殖中的作用，针对mecp2基因不同靶点，重组构建了能够转录形成发夹状mecp2shrna的真核表达质粒psilencermecp2，然后稳定转染肺癌细胞a549。观察mecp2表达的降低对肺癌细胞a549恶性增殖生物学特性的影响。 1 材料与方法 1.1 材料 psilencer2.1u6线性化的质粒购于美国ambion 公司；mecp2shrna的转录模版由上海生工合成；t4dna连接酶、rtpcr试剂盒、质粒去内毒素小提取试剂盒购于美国promga公司；jm109感受态大肠杆菌购于博达泰克公司；a549细胞为南昌大学二附院医学分子重点实验室保存；脂质体2000购于invitrogen 公司；mecp2一抗