中南大学学报（医学版）
中南大學學報（醫學版）
중남대학학보（의학판）
JOURNAL OF CENTRAL SOUTH UNIVERSITY (MEDICAL SCIENCES)
2014年
4期
338-343
,共6页
谭思创%陈志康%曹小霞%俏程%张蔚林%曾庆仁%谭斯品
譚思創%陳誌康%曹小霞%俏程%張蔚林%曾慶仁%譚斯品
담사창%진지강%조소하%초정%장위림%증경인%담사품
Dystrophin Dp71%shRNA载体%干扰效率
Dystrophin Dp71%shRNA載體%榦擾效率
Dystrophin Dp71%shRNA재체%간우효솔
Dystrophin Dp71%shRNA plasmid%inhibition effciency
目的：构建有效针对人Dystrophin Dp71基因的短发夹RNA(short hairpin RNA，shRNA)真核表达载体，并验证其干扰效果。方法：设计合成3对针对人Dystrophin Dp71基因的和1对无同源性的siRNA片段，在两端和中间加上酶切位点和Loop环。将合成的DNA片段插入到干扰载体pRNAT-U6.1/Neo中，经过酶切和测序验证，成功构建人Dp71基因的shRNA和空白对照载体。将各干扰载体和空白载体转染人正常胃黏膜上皮细胞(gastric epithelial cells，GES-1)和人支气管上皮细胞(human bronchial epithelium，HBE)，Western印迹检测Dp71 shRNA真核表达载体的干扰效率。结果：酶切和测序验证均表明各Dp71-shRNA载体构建成功。将空载体及各Dp71 shRNA载体分别转染GES-1和HBEC，和空白细胞对照及shRNA空白载体组相比，3组Dp71-shRNA能够明显抑制Dp71蛋白表达，但是3组质粒的抑制效率有一定的差异，以Dp71 shRNA2对Dp71表达的干扰效率最强。结论：成功构建了3个有效针对人Dystrophin Dp71基因的shRNA干扰载体，3组质粒都能有效地抑制Dp71在GES-1和HBEC中的表达，其中以Dp71-shRNA2的抑制效率最高。
目的：構建有效針對人Dystrophin Dp71基因的短髮夾RNA(short hairpin RNA，shRNA)真覈錶達載體，併驗證其榦擾效果。方法：設計閤成3對針對人Dystrophin Dp71基因的和1對無同源性的siRNA片段，在兩耑和中間加上酶切位點和Loop環。將閤成的DNA片段插入到榦擾載體pRNAT-U6.1/Neo中，經過酶切和測序驗證，成功構建人Dp71基因的shRNA和空白對照載體。將各榦擾載體和空白載體轉染人正常胃黏膜上皮細胞(gastric epithelial cells，GES-1)和人支氣管上皮細胞(human bronchial epithelium，HBE)，Western印跡檢測Dp71 shRNA真覈錶達載體的榦擾效率。結果：酶切和測序驗證均錶明各Dp71-shRNA載體構建成功。將空載體及各Dp71 shRNA載體分彆轉染GES-1和HBEC，和空白細胞對照及shRNA空白載體組相比，3組Dp71-shRNA能夠明顯抑製Dp71蛋白錶達，但是3組質粒的抑製效率有一定的差異，以Dp71 shRNA2對Dp71錶達的榦擾效率最彊。結論：成功構建瞭3箇有效針對人Dystrophin Dp71基因的shRNA榦擾載體，3組質粒都能有效地抑製Dp71在GES-1和HBEC中的錶達，其中以Dp71-shRNA2的抑製效率最高。
목적：구건유효침대인Dystrophin Dp71기인적단발협RNA(short hairpin RNA，shRNA)진핵표체재체，병험증기간우효과。방법：설계합성3대침대인Dystrophin Dp71기인적화1대무동원성적siRNA편단，재량단화중간가상매절위점화Loop배。장합성적DNA편단삽입도간우재체pRNAT-U6.1/Neo중，경과매절화측서험증，성공구건인Dp71기인적shRNA화공백대조재체。장각간우재체화공백재체전염인정상위점막상피세포(gastric epithelial cells，GES-1)화인지기관상피세포(human bronchial epithelium，HBE)，Western인적검측Dp71 shRNA진핵표체재체적간우효솔。결과：매절화측서험증균표명각Dp71-shRNA재체구건성공。장공재체급각Dp71 shRNA재체분별전염GES-1화HBEC，화공백세포대조급shRNA공백재체조상비，3조Dp71-shRNA능구명현억제Dp71단백표체，단시3조질립적억제효솔유일정적차이，이Dp71 shRNA2대Dp71표체적간우효솔최강。결론：성공구건료3개유효침대인Dystrophin Dp71기인적shRNA간우재체，3조질립도능유효지억제Dp71재GES-1화HBEC중적표체，기중이Dp71-shRNA2적억제효솔최고。
Objective: To construct effective short hairpin RNA (shRNA) recombinant plasmids targeting humanDystrophin Dp71 gene, and evaluate their interference effciency. Methods: hTree pairs of siRNA sequences targeting human Dp71 gene and one pair of control siRNA sequence were designed, synthesized, and then inserted into the pRNAT-U6.1/Neo vector. hTe shRNA recombinant vectors were evaluated by enzyme digestion and sequencing. Dp71-shRNA and control shRNA plasmids were transfected into human normal gastric epithelial cells (GES-1) and humanbronchial epithelium (HBE). Western blot was used to evaluate its interfering effciency. Results: Restriction enzyme digestion and sequencing showed that the Dp71-shRNA vectors were successfully constructed. Western blot displayed that Dp71 protein expression was reduced to a signiifcant degree atfer transfection with the 3 Dp71-shRNA plasmids, and Dp71-shRNA2 plasmid inhibit the Dp71 expression most effciently. Conclusion: Dp71-shRNA vectors have been successfully constructed. The 3 Dp71-shRNA plasmids can inhibit Dp71 expression in GES-1 and HBEC, with Dp71-shRNA2 plasmid displaying the highest inhibition effciency.
