天津医药
天津醫藥
천진의약
TIANJIN MEDICAL JOURNAL
2014年
2期
113-115
,共3页
崔丽%李广平%富华颖%王兴华%焦占全
崔麗%李廣平%富華穎%王興華%焦佔全
최려%리엄평%부화영%왕흥화%초점전
Toll样受体4%RNA干扰%RNA,小分子干扰%肌,平滑,血管%逆转录聚合酶链反应%免疫印迹法
Toll樣受體4%RNA榦擾%RNA,小分子榦擾%肌,平滑,血管%逆轉錄聚閤酶鏈反應%免疫印跡法
Toll양수체4%RNA간우%RNA,소분자간우%기,평활,혈관%역전록취합매련반응%면역인적법
Toll-like receptor 4%RNA interference%RNA,small interfering%muscle,smooth,vascular%reverse tran-scriptase polymerase chain reaction%immunoblotting
目的：设计合成有效的靶向Toll样受体4（TLR4）基因的小干扰RNA（siRNA）表达载体，并筛选出TLR4基因稳定沉默的血管平滑肌细胞（SMC）。方法依据RNA干扰（RNAi）序列设计原则，以TLR4基因为靶基因，合成3对小发夹RNA（shRNA）寡核苷酸链，经退火、与线性化pSilence 2.1-U6 neo质粒连接，酶切及测序进行鉴定。脂质体法转染血管SMC，新霉素（G418）加压筛选并收集稳定表达质粒的SMC。RT-PCR及Western blot法检测收集的SMC中TLR4 mRNA和蛋白表达，以确定siRNA对TLR4的抑制效率。结果测序鉴定插入的发夹样序列正确，成功构建了TLR4基因siRNA表达载体；并获得了稳定沉默TLR4的血管SMC。RT-PCR及Western blot证实RNA干扰TLR4基因后，SMC中TLR4 mRNA和蛋白表达均明显减低，其中pSilence2.1-siTLR4-1的抑制作用最强。结论成功构建了靶向TLR4基因的siRNA表达载体，并有效抑制血管SMC中TLR4表达。
目的：設計閤成有效的靶嚮Toll樣受體4（TLR4）基因的小榦擾RNA（siRNA）錶達載體，併篩選齣TLR4基因穩定沉默的血管平滑肌細胞（SMC）。方法依據RNA榦擾（RNAi）序列設計原則，以TLR4基因為靶基因，閤成3對小髮夾RNA（shRNA）寡覈苷痠鏈，經退火、與線性化pSilence 2.1-U6 neo質粒連接，酶切及測序進行鑒定。脂質體法轉染血管SMC，新黴素（G418）加壓篩選併收集穩定錶達質粒的SMC。RT-PCR及Western blot法檢測收集的SMC中TLR4 mRNA和蛋白錶達，以確定siRNA對TLR4的抑製效率。結果測序鑒定插入的髮夾樣序列正確，成功構建瞭TLR4基因siRNA錶達載體；併穫得瞭穩定沉默TLR4的血管SMC。RT-PCR及Western blot證實RNA榦擾TLR4基因後，SMC中TLR4 mRNA和蛋白錶達均明顯減低，其中pSilence2.1-siTLR4-1的抑製作用最彊。結論成功構建瞭靶嚮TLR4基因的siRNA錶達載體，併有效抑製血管SMC中TLR4錶達。
목적：설계합성유효적파향Toll양수체4（TLR4）기인적소간우RNA（siRNA）표체재체，병사선출TLR4기인은정침묵적혈관평활기세포（SMC）。방법의거RNA간우（RNAi）서렬설계원칙，이TLR4기인위파기인，합성3대소발협RNA（shRNA）과핵감산련，경퇴화、여선성화pSilence 2.1-U6 neo질립련접，매절급측서진행감정。지질체법전염혈관SMC，신매소（G418）가압사선병수집은정표체질립적SMC。RT-PCR급Western blot법검측수집적SMC중TLR4 mRNA화단백표체，이학정siRNA대TLR4적억제효솔。결과측서감정삽입적발협양서렬정학，성공구건료TLR4기인siRNA표체재체；병획득료은정침묵TLR4적혈관SMC。RT-PCR급Western blot증실RNA간우TLR4기인후，SMC중TLR4 mRNA화단백표체균명현감저，기중pSilence2.1-siTLR4-1적억제작용최강。결론성공구건료파향TLR4기인적siRNA표체재체，병유효억제혈관SMC중TLR4표체。
Objective To construct vectors expressing small interfering RNA targeting the Toll like receptor-4 (TLR4) gene and obtain TLR4 knock downed vascular smooth muscle cells (SMC). Methods Three small hairpin RNA (shRNA) targeting the TLR4 gene were designed, synthesized and cloned into the pSilence 2.1-U6 neo vector. Positive clones were verified with double enzyme digestion and sequencing. Then the recombinants were transfected to SMC by the cationic lipid method respectively.SMC were stably transfected with an expression plasmid and screened by G418. TLR4 mRNA and protein expression were detected by RT-PCR and Western blot methods. Results The pSilence2.1-siTLR4 ex-pression vectors were successfully constructed and a TLR4 knock-downed SMC cell line was established. RT-PCR and Western blot analysis confirmed that the expression of TLR4 was significantly down-regulated in the infected SMC cell line, and pSilence2.1-siTLR4-1was the most efficacious recombinant vector.Conclusion Recombinant vectors carrying shRNA targeting the TLR4 gene were successfully constructed and the TLR4 expression in vascular SMCs was inhibited.
