可随HeLa细胞传代而传递的反义RNA真核表达系统

An Antisense RNA Expression Vector Transmitted from One Passage to the Next in HeLa Cells

为研究反义ＲＮＡ表达载体在细胞内的稳定性，构建了一个特异性针对β地中海贫血基因ＩＶＳ－２－６５４Ｃ→Ｔ（β６５４）突变ｍＲＮＡ前体异常剪接位点的反义ＲＮＡ表达载体ｐＣＭＶＡ．ｐＣＭＶＡ转染β６５４ＨｅＬａ细胞后，通过ＲＮＡ定量检测反义片段对β６５４ｍＲＮＡ异常剪接的纠正作用；再从转染后传代５次并冰冻保存１年的ＨｅＬａ细胞中回收反义表达载体，转染另外的β６５４ＨｅＬａ细胞，同样检测它对β６５４ｍＲＮＡ异常剪接的纠正作用．结果显示该载体在细胞传代前后均能阻断β６５４异常剪接，部分恢复其正常剪接途径：用回收的ｐＣＭＶＡ转染β６５４ＨｅＬａ细胞后，正常剪接的βｍＲＮＡ水平［β／（β＋β＊）］由０．０５上升到处理后１５ｄ的０．４８，而２种对照质粒处理后对这一比值影响不大．表明ｐＣＭＶＡ可在ＨｅＬａ细胞中随着细胞传代而传递下去。

To investigate the structural and functional stability of an antisencse RNA expression vector in β 654 HeLa cells during the cell subculture,a vector expressing the antisense RNA which targeted against the aberrant splice sites of β thalassemia allele IVS 2 654 C→T(β 654 ) pre mRNA named pCMVA,was constructed in pcDNA3 1.The vector was transfected into HeLa β 654 cells by lipid mediated DNA transfection method,and the effect of the antisense RNA was identified by RT PCR mediated mRNA quantitative assay.After the β 654 HeLa cells carrying pCMVA were subcultured 5 times and frozen over one year,the vector was recovered and then was transfected into another passage of β 654 HeLa cells,and the effect of the antisense vector was identified.The results showed that antisense RNA transcribed from pCMVA and restored pCMVA could both efficiently suppress the aberrant splicing pattern of β 654 mutant pre mRNA,and restore the correct splicing pathway in β 654 HeLa cells.The level of normally spliced mRNA [β/(β+β *)] increased from 0 05 (before treatment)to 0 48 on the 15th day after transfected with the recovered vector pCMVA.Correspondingly,the values of β/(β+β *) were 0 11 and 0 08 on the 15th day after transfected with the recovered control vectors pCMVS which expressed an antisense RNA fragment not targeting against β 654 gene,and pCMVI which expressed the RNA fragment covering the aberrant splice sites of β 654 gene,respectively.The data indicated that the vector p CMVA could be transmitted from one passage to the next in HeLa cells without changing its construction or losing its function after frozen over one year,which may have potential interests in the clinical application of β thalassemia treatment.