摘要
目的研究反义RNA对β地中海贫血红系细胞IVS2654C→T(β654)突变mRNA异常剪接的纠正作用。方法构建特异性针对β654mRNA前体异常剪接位点的反义RNA表达载体,和不针对上述位点的反义对照载体,转染培养的β654红系细胞后,抽提总RNA;以RTPCR法作mRNA定量,检测正常和异常剪接的β珠蛋白mRNA产物的比例[β/(β+β)];以珠蛋白肽链体外生物合成分析红系细胞中β和α珠蛋白肽链合成的比例(β/α)。结果在RNA水平,β654/β654纯合子的β/(β+β)值由0.19(转染后第0天)上升到0.58(第8天),β654/βA杂合子由0.45(0天)上升到0.83(第8天);在珠蛋白肽链水平,β654/β654细胞的β/α值由0.16(0天)上升到0.52(第8天),β654/βA细胞由0.39(0天)上升到0.84(第8天)。反义RNA处理βA/βA红系细胞以及对照RNA处理这3类红系细胞,对其mRNA剪接和珠蛋白肽链合成均影响不大。结论特异性反义RNA能抑制红系细胞β654mRNA前体的异常剪接,部分恢复其正常剪接途径。
Objective To investigate the restoration of correct splicing of β thalassemia allele (IVS2 654C→T,β 654 ) in cultured erythroid cells by antisense RNA.Methods Vectors transcribing an antisense RNA that was targeted against the aberrant splice sites of β 654 pre transcript(pCMVA) and transcribing a control antisense fragment (pCMVC) were constructed. The total RNA was extracted at given time point after transfection, and the effect of antisence RNA was studied by RT PCR mediated mRNA quantitative assay as well as globin chain microbiosynthesis. Results The antisense fragment transcribed from pCMVA effectively improved the β 654 splicing pattern in cultured erythroid cells. The level of correctly spliced transcript increased from 0.19(day 0 after transfection) to 0.58 (day 8) in β 654 /β 654 homozygous erythroid cells, and from 0.45(day 0) to 0.83(day 8) in β 654 /β A heterozygous erythroid cells, as determined by the ratio of normally spliced β globin transcript over total β globin transcript. Correspondingly, the ratios of globin chain biosynthesis(β/α) increased from 0.16(day 0) to 0.52( day 8) in β 654 /β 654 erythroid cells, and from 0.39(day 0) to 0.84(day 8) in β 654 /β A erythroid cells. Antisense RNA has no significant effect on the splicing pattern in β A /β A erythroid cells. The splicing pattern in transfected cells with pCMVA showed significant changes compared to that in untransfected cells and in transfected cells with pCMVc. Conclusions The antisense RNA transcribed from the mammalian expression vector pCMVA could efficiently and specifically suppress the aberrant splicing pattern of β 654 mutant pre transcript and restore the correct splicing pathway in vivo, leading to improvement of globin chain biosynthesis in thalassemic cells.
出处
《上海医学》
CAS
CSCD
北大核心
1999年第2期78-82,共5页
Shanghai Medical Journal
基金
国家自然科学基金
上海生命科学研究中心研究基金