中国循环杂志
中國循環雜誌
중국순배잡지
CHINESE CIRCULATION JOURNAL
2014年
6期
461-465
,共5页
于海云%姚艳%张银辉%龚菁%黄健%浦介麟
于海雲%姚豔%張銀輝%龔菁%黃健%浦介麟
우해운%요염%장은휘%공정%황건%포개린
HERG%无义突变%PTC124%遗传性心律失常
HERG%無義突變%PTC124%遺傳性心律失常
HERG%무의돌변%PTC124%유전성심률실상
HERG%Nonsense mutation%PTC124%Inherited arrhythmia
目的:通过对HERG基因无义突变体应用不同剂量PTC124和G418来观察其通道功能的恢复情况,并探讨药物的作用机制。
<br> 方法:制备HERG基因R1014X突变体,将野生型(WT)HERG及R1014X分别瞬时转染至HEK293细胞,转染24 h后将其置入含不同浓度的PTC124和G418的培养液中孵育24 h。逆转录聚合酶链反应法检测各组细胞HERG信使核糖核酸(mRNA)的表达,蛋白印迹法检测野生型和R1014X给药前后通道蛋白的表达,全细胞膜片钳技术记录通道电流以评价HERG基因编码通道的功能性表达。
<br> 结果:与野生型相比,R1014X突变体编码的通道蛋白的蛋白条带变短,蛋白量及mRNA水平无明显差异(P>0.05)。药物PTC124和G418均能恢复R1014X完整蛋白的表达,并呈剂量依赖性变化(P<0.05),PTC124的作用弱于G418(P<0.05)。药物干预后,R1014X突变体HERG通道的功能得到不同程度的恢复,其最大尾电流密度由(4.75±0.88) pA/pF分别增加至(9.62±0.73) pA/pF (PTC124,P<0.05)和(22.57±2.26) pA/pF (G418,P<0.05),G418作用使通道的半激活电压得到恢复(P<0.05),差异均有统计学意义。
<br> 结论:药物PTC124和G418能不同程度恢复HERG基因无义突变的结构和功能性表达,其恢复HERG基因的蛋白表达呈剂量依赖性变化。
目的:通過對HERG基因無義突變體應用不同劑量PTC124和G418來觀察其通道功能的恢複情況,併探討藥物的作用機製。
<br> 方法:製備HERG基因R1014X突變體,將野生型(WT)HERG及R1014X分彆瞬時轉染至HEK293細胞,轉染24 h後將其置入含不同濃度的PTC124和G418的培養液中孵育24 h。逆轉錄聚閤酶鏈反應法檢測各組細胞HERG信使覈糖覈痠(mRNA)的錶達,蛋白印跡法檢測野生型和R1014X給藥前後通道蛋白的錶達,全細胞膜片鉗技術記錄通道電流以評價HERG基因編碼通道的功能性錶達。
<br> 結果:與野生型相比,R1014X突變體編碼的通道蛋白的蛋白條帶變短,蛋白量及mRNA水平無明顯差異(P>0.05)。藥物PTC124和G418均能恢複R1014X完整蛋白的錶達,併呈劑量依賴性變化(P<0.05),PTC124的作用弱于G418(P<0.05)。藥物榦預後,R1014X突變體HERG通道的功能得到不同程度的恢複,其最大尾電流密度由(4.75±0.88) pA/pF分彆增加至(9.62±0.73) pA/pF (PTC124,P<0.05)和(22.57±2.26) pA/pF (G418,P<0.05),G418作用使通道的半激活電壓得到恢複(P<0.05),差異均有統計學意義。
<br> 結論:藥物PTC124和G418能不同程度恢複HERG基因無義突變的結構和功能性錶達,其恢複HERG基因的蛋白錶達呈劑量依賴性變化。
목적:통과대HERG기인무의돌변체응용불동제량PTC124화G418래관찰기통도공능적회복정황,병탐토약물적작용궤제。
<br> 방법:제비HERG기인R1014X돌변체,장야생형(WT)HERG급R1014X분별순시전염지HEK293세포,전염24 h후장기치입함불동농도적PTC124화G418적배양액중부육24 h。역전록취합매련반응법검측각조세포HERG신사핵당핵산(mRNA)적표체,단백인적법검측야생형화R1014X급약전후통도단백적표체,전세포막편겸기술기록통도전류이평개HERG기인편마통도적공능성표체。
<br> 결과:여야생형상비,R1014X돌변체편마적통도단백적단백조대변단,단백량급mRNA수평무명현차이(P>0.05)。약물PTC124화G418균능회복R1014X완정단백적표체,병정제량의뢰성변화(P<0.05),PTC124적작용약우G418(P<0.05)。약물간예후,R1014X돌변체HERG통도적공능득도불동정도적회복,기최대미전류밀도유(4.75±0.88) pA/pF분별증가지(9.62±0.73) pA/pF (PTC124,P<0.05)화(22.57±2.26) pA/pF (G418,P<0.05),G418작용사통도적반격활전압득도회복(P<0.05),차이균유통계학의의。
<br> 결론:약물PTC124화G418능불동정도회복HERG기인무의돌변적결구화공능성표체,기회복HERG기인적단백표체정제량의뢰성변화。
Objective: To study the pharmaceutical effects of PTC124 and G418 at different doses on rescuing the nonsense mutation of HERG gene channels with its mechanism.
<br> Methods: The wide type (WT) and R1014X mutant of HERG gene were transiently transfected into HEK293 cells for 24 hours, and the cells were cultured with pharmaceuticals of PTC124 and G418 at different concentrations respectively for 24 hours. HERG mRNA expression was examined by RT-PCR, HERG WT and R1014X mutant protein expressions were detected by Western blot analysis, HERG channel function was evaluated by patch clamping recorded current lfow.
<br> Results: Compared with WT HERG, the R1014X mutant showed shorter protein zone in HERG channel protein, while the expressions of mRNA and protein were similar between WT and mutant,P>0.05. Both PTC124 and G418 may recover the entire protein expression of R1014X mutant in a dose-dependent model,P<0.05, and the effect of PTC124 was weaken than that of G418,P<0.05. With pharmaceutical intervention, the R1014X mutant of HERG channel function may recover at different degrees, the maximal tail current densities of R1014X channels increased from (4.75±0.88 ) pA/pF to (9.62±0.73) pA/pF by PTC124,P<0.05 and to (22.57±2.26) pA/pF by G418,P<0.05. The G418 recovered the semi-activation voltage of HERG channels,P<0.05.
<br> Conclusion: Both pharmaceuticals of PTC124 and G418 could recover the structure and function of HERG channel in nonsense mutation at different degree; they recover HERG channel protein expression in a dose-dependent model.