CAJ | 학술논문

目的：构建靶向小鼠TNF-α基因的RNA干扰慢病毒载体，为RNA干扰基因治疗提供基础。方法：设计、合成3组靶向TNF-α基因的特异小干扰RNA （ siRNA）：siRNA1、siRNA2、siRNA3及阴性对照siRNA，体外转染小鼠巨噬细胞株RAW264.7，用real-time PCR和ELISA法检测其对经脂多糖刺激的RAW264.7细胞表达TNF-α、IL-1β、IL-6的影响。筛选出特异且抑制性高的siRNA，根据其序列设计合成寡核苷酸链，构建表达短发卡RNA（ shRNA）的重组慢病毒质粒并测序，经鉴定质粒与包装质粒共转染293 T细胞生产慢病毒颗粒，计算病毒颗粒滴度。结果：①siRNA1、siRNA2、siRNA3组的TNF-αmRNA相对表达量分别为0.24±0.01、0.16±0.02、0.19±0.01，与阴性对照0.95±0.02相比差别均具有统计学意义（ F＝531.3， P＜0.001）；与阴性对照相比，mRNA表达抑制率分别为74.26％、83.09％、79.93％。siRNA1、siRNA2、siRNA3及阴性对照组的IL-1βmRNA或IL-6 mRNA相对表达量之间差别无统计学意义（F＝0.981，P＝0.9807＞0.05；F＝0.739，P＝0.5867＞0.05）。②siRNA1、siRNA2、siRNA3的TNF-α蛋白表达分别为（23.95±1.21）、（17.27±1.46）、（19.07±1.57） ng／ml 与阴性对照的（35.37±2.93）ng／ml相比，差别均具有统计学意义（F＝18.1，P＝0.0006＜0.001）；与阴性对照相比，TNF-α蛋白表达抑制率分别为32.29％、51.16％、46.08％。③以siRNA2序列设计、合成寡核苷酸链，构建重组慢病毒穿梭质粒，其PCR产物电泳结果为343 bp，空载体PCR产物306 bp；DNA测序显示pGCSIL-GFP-shRNA测序反应中断，但已显示部分插入序列。④转染后的293 T细胞，生长良好，荧光表达强，慢病毒滴度为2×106 TU／μl。结论：靶向小鼠TNF-α基因RNAi慢病毒载体构建成功。
목적：구건파향소서TNF-α기인적RNA간우만병독재체，위RNA간우기인치료제공기출。방법：설계、합성3조파향TNF-α기인적특이소간우RNA （ siRNA）：siRNA1、siRNA2、siRNA3급음성대조siRNA，체외전염소서거서세포주RAW264.7，용real-time PCR화ELISA법검측기대경지다당자격적RAW264.7세포표체TNF-α、IL-1β、IL-6적영향。사선출특이차억제성고적siRNA，근거기서렬설계합성과핵감산련，구건표체단발잡RNA（ shRNA）적중조만병독질립병측서，경감정질립여포장질립공전염293 T세포생산만병독과립，계산병독과립적도。결과：①siRNA1、siRNA2、siRNA3조적TNF-αmRNA상대표체량분별위0.24±0.01、0.16±0.02、0.19±0.01，여음성대조0.95±0.02상비차별균구유통계학의의（ F＝531.3， P＜0.001）；여음성대조상비，mRNA표체억제솔분별위74.26％、83.09％、79.93％。siRNA1、siRNA2、siRNA3급음성대조조적IL-1βmRNA혹IL-6 mRNA상대표체량지간차별무통계학의의（F＝0.981，P＝0.9807＞0.05；F＝0.739，P＝0.5867＞0.05）。②siRNA1、siRNA2、siRNA3적TNF-α단백표체분별위（23.95±1.21）、（17.27±1.46）、（19.07±1.57） ng／ml 여음성대조적（35.37±2.93）ng／ml상비，차별균구유통계학의의（F＝18.1，P＝0.0006＜0.001）；여음성대조상비，TNF-α단백표체억제솔분별위32.29％、51.16％、46.08％。③이siRNA2서렬설계、합성과핵감산련，구건중조만병독천사질립，기PCR산물전영결과위343 bp，공재체PCR산물306 bp；DNA측서현시pGCSIL-GFP-shRNA측서반응중단，단이현시부분삽입서렬。④전염후적293 T세포，생장량호，형광표체강，만병독적도위2×106 TU／μl。결론：파향소서TNF-α기인RNAi만병독재체구건성공。
Objective:To construct recombinant lentiviral vectors harboring interference RNA ( RNAi ) targetting murine TNF-αgene,so as to lay the foundation on the RNAi gene therapy.Methods: Three small interfering RNA ( siRNA) sequences targeting murine TNF-αgene ( siRNA1,siRNA2,siRNA3) and negative-control siRNA were designed and synthesized.The inhibition effects of siRNAs on TNF-α,IL-1βand IL-6 secretion of LPS-stimulated RAW264.7 macrophages were observed using real-time PCR and ELISA methods.DNA oligo was designed and synthesized according to the most effective siRNA 2 sequence.The recombinant lentiviral shuttle plasmid expressing short hairpin RNA ( shRNA) was constructed and sequenced.The lentiviral shuttle plasmids with packaging plasmids were transfected into 293T cells to produce lentiviral particles.Results: ①The TNF-αmRNA relative expression levels of siRNA1, siRNA2 and siRNA3 were 0.24±0.01,0.16±0.02,0.19±0.01 respectively,significantly lower than that of negative control (0.95± 0.02) (F=531.3,P<0.001).The inhibition rates at mRNA level were 74.26%,83.09%,79.93%,respectively comparing with negative control.No significance was observed in IL-1βor IL-6 mRNA relative expression change after TNF-αsiRNA transfection ( P>0.05).②The TNF-αprotein expression levels of siRNA1,siRNA2 and siRNA3 were (23.95±1.21),(17.27±1.46),(19.07± 1.57)ng/ml respectively,significantly lower than that of negative control (35.37±2.93)ng/ml (F=18.1,P=0.000 6<0.001).The inhibition rates of protein expression were 32.29%, 51.16%, 46.08%, respectively comparing with negative control.③The PCR product electrophoresis showed that recombinant vectors yielded 343 bp fragments,non-constructed vectors yielded 306 bp fragments.DNA sequencing partially showed insertion sequence.④Lentiviral particles were obtained by transfecting 293T cells with recombinant lentiviral shuttle plasmids and lentiviral packaging plasmids.Cells grew well during virus production with strong fluorescence expression.The titer of concentrated virus was 2×106 TU/μl.Conclusion:The lentiviral vector harboring RNAi targeting murine TNF-αgene has been successfully constructed.