M1-GS RNA靶向作用于K562细胞的研究  

Study on the targeting effects of M1-GS RNA on K562 cells

作　　者：陈波斌[1] 林果为[1] 陆华中[2] 范华骅[2] 高砾[2] 

机构地区：[1]复旦大学附属华山医院血液科,上海200040 [2]上海市血液中心

出　　处：《中华血液学杂志》2004年第9期552-555,共4页Chinese Journal of Hematology

摘　　要：目的　探讨带有导引序列的核糖核酸酶P亚单位M1RNA(M1 GSRNA)转染白血病细胞株K5 6 2细胞后对bcr abl融合基因mRNA及其表达产物的作用效应。方法　含有针对bcr ablmR NA的M1 GSRNA表达质粒 pAVGS4 ,以X tremeQ2介导转染K5 6 2细胞 ,设置空载体组作为对照 ,分别在转染后 2 4 ,4 8,72和 96h收集细胞 ,以Trizol试剂抽提总RNA ,通过RT PCR检测转染后不同时间bcr ablmRNA的变化 ;在转染后 4 8和 96h ,抽提总蛋白质 ,通过Westernblot检测癌基因的表达产物P2 10在不同时间的变化。利用半固体琼脂培养方法观察pAVGS4对K5 6 2细胞集落形成的影响。结果　pAVGS4转染K5 6 2细胞后 2 4 ,4 8,72和 96hRT PCR结果显示 :在转染后 2 4h ,随时间的推移 ,实验组细胞内bcr ablmRNA含量下降近 1个对数级 ,72和 96h实验组细胞内bcr ablmRNA的含量接近 ,而对照组无明显改变。Westernblot结果显示 :实验组在 4 8h的灰度值是同期对照组的 10 .4 % ,96h时为 6 .7%。K5 6 2细胞集落形成分析显示 96h后集落抑制率达 81.3%。结论　M1 GSRNA可以有效、特异地破坏bcr ablmRNA ,显著下调P2 10的表达 ,抑制K5 6 2集落形成。Objective To determine the effects of M1-GS RNA(M1 RNA) on bcr-abl mRNA and oncoprotein after M1 RNA with guide sequence (M1-GS RNA) targeting the oncogene was transf ected into K562 cells. Methods pAVGS4(an eukaryocyte expression vec tor containing M1-GS RNA sequence) and pNAV-1 (as the control) were transfecte d into K562 cells by X-tremeGENE Q2. Total RNA was extracted at 24, 48, 72 and 96 hours after transfection. Then RT-PCR was done to compare the products at di fferent time point. After collecting pAVGS4-transfected cells and the control c ells at 48 and 96 hours after transfection, total protein was extracted and quan tified. Change of P210 was determined by Western blot. Colony formation was anal yzed at 96 hours after transfection. Results RT-PCR based on tran sfected cells at different time point showed that the amount of bcr-abl mRNA began to decrease at 24 hours and reduced to 9.2% and 2.5% respectively at 48 and 72 hours after transfection. Western blot showed that the expression of P2 10 in the pAVGS4 group reduced to 10.4% of the control at 48 hours and 6.7% of the control at 96 hours after transfection. The inhibition rate of colony format ion was 81.3% after K562 cells were transfected by pAVGS4. Conclusion pAVGS4 can efficiently destroy bcr-abl mRNA in K562 cells. The transcript le vel of bcr-abl mRNA was reduced with the time after transfection. The expressi on of P210 was decreased significantly at 48 and 96 hours after transfection. K5 62 cell colony formation was prominently inhibited.

关 键 词：K562细胞 总RNA 转染 BCR-ABL GS 集落形成 靶向作用 表达产物 表达质粒 核糖核酸酶 

分 类 号：R733.7[医药卫生—肿瘤]