摘要
目的:
构建特异性BCR/ABL的siRNA真核细胞表达载体并转染人类红白血病细胞株K562,了解重组载体对K562细胞BCR/ABL融合基因的干扰效果以及对其生长增殖影响,为进一步探索BCR/ABL基因功能,RNA干扰(RNAi)分子靶向基因治疗慢性髓细胞性白血病(CML)提供一种新的手段和理论基础。
方法:
1.根据GenBank数据库提供的BCP/ABL基因核苷酸序列,按照Tuschl设计原则,选择设计双链小干扰RNA(small interfering RNA,siRNA),再转化为能表达其小发卡结构RNA(Small hairpin RNAs,sh RNA)的DNA序列,并与pTER质粒定向连接,构建受控于人RNA聚合酶Ⅲ启动子H1的真核表达载体pTER117、pTER363,经限制性内切酶酶切和DNA测序进行鉴定。
2.通过脂质体Lipofectamine 2000瞬时转染K562细胞24小时,RT-PCR和细胞化学染色检测BCR/ABL mRNA和P210蛋白。在瞬时转染的基础上,利用该载体带有的药物抗性筛选系统进行稳定转染,由于该载体带有受四环素(tet)调控的开关基因,才使筛选出依赖P210蛋白生长的重组载体和空载体的阳性K562细胞克隆成为可能,采用
Objective : To construct eukaryotic expression vector of siRNA specific for BCR/ABL as a molecular target tool to cleave BCR/ABL mRNA in chronic myelogenous leukemia cell, to initially investigate the effect of recombinant plasmid on BCR/ABL mRNA and P210 protein expression of K562 cells, to provide the experimental evidence and a new tool to further explore the function of BCR/ABL fusion gene and the feasibility of its gene therapy.Methods: Genome sequences of BCR/ABL fusion gene was retrieved from Genbank, siRNA (small interfering RNA) was designed according to the Tuschl's principle of RNAi-based medicine, and was converted into cDNA coding expression of shRNA (small hairpin RNAs) of siRNA for BCR/ABL fusion gene. The cDNA was synthesized and inserted into plasmid pTER. The pTER117 and pTER363 of recombinant plasmid being eukaryotic expression vector was controlled by the H_1 promoter of RNA polymerase III, and identified by the restriction map and the sequence analysis. The recombinant plasmid not only had the screening resisting antibiotics, it's expression but also are induced by tetracycline (tet). The plasmid was transiently transfected into K562 cells for 24 hours by Lipofectamine 2000, expression of BCR/ABL mRNA was assayed by
RT-PCR; P210 protein by immunohistochemistry. After steadily transfection, their positive mono-cell clones being resistant to Zeocin are isolated. TaqMan Real-time quantitative RT-PCR (RQ-PCR) and Western blot respectively detected expression of BCR/ABL mRNA and P210 protein. Trypaum blue dying was used to the number of cell and analyzed the proliferation of K562 cells. Cell apoptosis was observed by flow cytometer (FCM).Results:1. The pTER117 and pTER363 of recombinant plasmid identificated by the restriction map and the sequence analysis completely coincided with the designs.2. The recombinant plasmid was transiently transfected into K562 cells by Lipofectamine 2000, and tetracycline induced it's expression for 24 hours, pTER117 decreased the mRNA level of BCR/ABL 52%, reduced P210 protein 47% in K562 cells; pTER363 decreased the mRNA level of BCR/ABL 43%, reduced P210 protein 40% in K562 cells. But the control groups have no the effects on K562 cells.3. On the base of the transiently transfection, the recombinant plasmid was steadily transfected into K562 cells by Lipofectamine 2000. Their positive mono-cell clones being resistant to Zeocin are isolated. The proliferation of K562 cells were remarkably inhibited by the recombinant plasmid induced gene expression by tetracycline. Tetracycline induced it s expression for 48h, 72h after the steady transfection, pTER117 decreased the
mRNA level of BCR/ABL 90%, 91. 5% by RQ-PCR respectively; pTER363 decreased the mRNA level of BCR/ABL 82%, 84%. P210 protein were almost measured in K562 cells by Western blot. FCM analysis showed that the recombinant plasmid induced apoptosis in K562 cells, Apoptosis rate were respectively 34.4%, 58.1% in K562 cells treated by pTER117 for 48h, 72h, apoptosis rate 31.8%, 54.6% by pTER363. But the control groups have no these influence on K562 cells.4. These data also displayed that the effects of RNAi of pTERll? had more powerful than that of pTER363.Conclusions:The siRNA eukaryotic expression vector against BCR/ABL mRNA has been successfully constructed, and effectively inhibits the expression of BCR/ABL in K562 cells.
引文
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