中华临床医师杂志（电子版）
中華臨床醫師雜誌（電子版）
중화림상의사잡지（전자판）
CHINESE JOURNAL OF CLINICIANS(ELECTRONIC VERSION)
2013年
5期
2033-2037
,共5页
陈丽%邓勇志%徐俊文%王倩%杨雪峰
陳麗%鄧勇誌%徐俊文%王倩%楊雪峰
진려%산용지%서준문%왕천%양설봉
1-磷脂酰肌醇3-激酶%肌细胞,平滑肌%shRNA%SMHC增强子/SM22α启动子%KDR增强子/启动子
1-燐脂酰肌醇3-激酶%肌細胞,平滑肌%shRNA%SMHC增彊子/SM22α啟動子%KDR增彊子/啟動子
1-린지선기순3-격매%기세포,평활기%shRNA%SMHC증강자/SM22α계동자%KDR증강자/계동자
1-phosphatidylinositol 3-kinase%Myocytes,smooth muscle%shRNA%SMHC e/SM22αp%KDR e/p
目的构建靶向血管平滑肌细胞和内皮细胞磷脂酰肌醇3激酶（ phosphatidylinositol 3-kinase， PI3K）基因的短发卡干扰RNA（short hair-pin RNA，shRNA）表达载体，研究其对上述细胞PI3K基因的靶向沉默作用，探索抑制移植血管狭窄的基因防治手段。方法根据Genbank中大鼠PI3K p110β亚单位编码基因Pik3cb序列设计并合成两条shRNA寡核苷酸片段，退火形成双链并克隆导入载体pGenesil-10，经荧光定量PCR方法筛选一条较合适的shRNA转录模板；合成血管平滑肌细胞特异性SMHC增强子/SM22α启动子序列和血管内皮细胞特异性KDR增强子/启动子序列，将该增强子/启动子片段亚克隆至pGenesil-10-Pik3cb-shRNA上，构建并鉴定重组质粒载体SMHCe/SM22αp-pGenesil-10-Pik3cb-shRNA（简称SM22αe/p shRNA）和KDRe/p-pGenesil-10-Pik3cb-shRNA（简称KDR e/p shRNA）；将重组质粒载体转染血管平滑肌细胞，分别于转染24 h、48 h、72 h后，采用实时荧光定量PCR检测Pik3cb基因mRNA的相对表达量。结果荧光定量PCR检测转染Pik3cb-shRNA-1组与转染Pik3cb-shRNA-2组比较，前者细胞Pik3cb mRNA相对表达量降低更为明显，且两组均较对照组mRNA表达明显减少（ P＜0.05）；酶切鉴定重组质粒载体SM22αe/p shRNA和KDR e/p shRNA构建成功；质粒转染细胞后，Pik3cb基因mRNA相对表达量SM22αe/p质粒组较阴性对照组均有所降低，且转染24 h后，SM22αe/p质粒组与空白对照、CMV质粒组比较Pik3cb基因mRNA相对表达量明显降低，差异均有统计学意义（ P＜0.05）。结论选择Pik3cb-shRNA-1作为转录模板，能够成功构建SM22αe/p shRNA和KDR e/p shRNA质粒载体，且特异性启动子SM22α介导的靶向大鼠Pik3cb基因的shRNA质粒载体可有效沉默靶基因在血管平滑肌细胞中的表达。
目的構建靶嚮血管平滑肌細胞和內皮細胞燐脂酰肌醇3激酶（ phosphatidylinositol 3-kinase， PI3K）基因的短髮卡榦擾RNA（short hair-pin RNA，shRNA）錶達載體，研究其對上述細胞PI3K基因的靶嚮沉默作用，探索抑製移植血管狹窄的基因防治手段。方法根據Genbank中大鼠PI3K p110β亞單位編碼基因Pik3cb序列設計併閤成兩條shRNA寡覈苷痠片段，退火形成雙鏈併剋隆導入載體pGenesil-10，經熒光定量PCR方法篩選一條較閤適的shRNA轉錄模闆；閤成血管平滑肌細胞特異性SMHC增彊子/SM22α啟動子序列和血管內皮細胞特異性KDR增彊子/啟動子序列，將該增彊子/啟動子片段亞剋隆至pGenesil-10-Pik3cb-shRNA上，構建併鑒定重組質粒載體SMHCe/SM22αp-pGenesil-10-Pik3cb-shRNA（簡稱SM22αe/p shRNA）和KDRe/p-pGenesil-10-Pik3cb-shRNA（簡稱KDR e/p shRNA）；將重組質粒載體轉染血管平滑肌細胞，分彆于轉染24 h、48 h、72 h後，採用實時熒光定量PCR檢測Pik3cb基因mRNA的相對錶達量。結果熒光定量PCR檢測轉染Pik3cb-shRNA-1組與轉染Pik3cb-shRNA-2組比較，前者細胞Pik3cb mRNA相對錶達量降低更為明顯，且兩組均較對照組mRNA錶達明顯減少（ P＜0.05）；酶切鑒定重組質粒載體SM22αe/p shRNA和KDR e/p shRNA構建成功；質粒轉染細胞後，Pik3cb基因mRNA相對錶達量SM22αe/p質粒組較陰性對照組均有所降低，且轉染24 h後，SM22αe/p質粒組與空白對照、CMV質粒組比較Pik3cb基因mRNA相對錶達量明顯降低，差異均有統計學意義（ P＜0.05）。結論選擇Pik3cb-shRNA-1作為轉錄模闆，能夠成功構建SM22αe/p shRNA和KDR e/p shRNA質粒載體，且特異性啟動子SM22α介導的靶嚮大鼠Pik3cb基因的shRNA質粒載體可有效沉默靶基因在血管平滑肌細胞中的錶達。
목적구건파향혈관평활기세포화내피세포린지선기순3격매（ phosphatidylinositol 3-kinase， PI3K）기인적단발잡간우RNA（short hair-pin RNA，shRNA）표체재체，연구기대상술세포PI3K기인적파향침묵작용，탐색억제이식혈관협착적기인방치수단。방법근거Genbank중대서PI3K p110β아단위편마기인Pik3cb서렬설계병합성량조shRNA과핵감산편단，퇴화형성쌍련병극륭도입재체pGenesil-10，경형광정량PCR방법사선일조교합괄적shRNA전록모판；합성혈관평활기세포특이성SMHC증강자/SM22α계동자서렬화혈관내피세포특이성KDR증강자/계동자서렬，장해증강자/계동자편단아극륭지pGenesil-10-Pik3cb-shRNA상，구건병감정중조질립재체SMHCe/SM22αp-pGenesil-10-Pik3cb-shRNA（간칭SM22αe/p shRNA）화KDRe/p-pGenesil-10-Pik3cb-shRNA（간칭KDR e/p shRNA）；장중조질립재체전염혈관평활기세포，분별우전염24 h、48 h、72 h후，채용실시형광정량PCR검측Pik3cb기인mRNA적상대표체량。결과형광정량PCR검측전염Pik3cb-shRNA-1조여전염Pik3cb-shRNA-2조비교，전자세포Pik3cb mRNA상대표체량강저경위명현，차량조균교대조조mRNA표체명현감소（ P＜0.05）；매절감정중조질립재체SM22αe/p shRNA화KDR e/p shRNA구건성공；질립전염세포후，Pik3cb기인mRNA상대표체량SM22αe/p질립조교음성대조조균유소강저，차전염24 h후，SM22αe/p질립조여공백대조、CMV질립조비교Pik3cb기인mRNA상대표체량명현강저，차이균유통계학의의（ P＜0.05）。결론선택Pik3cb-shRNA-1작위전록모판，능구성공구건SM22αe/p shRNA화KDR e/p shRNA질립재체，차특이성계동자SM22α개도적파향대서Pik3cb기인적shRNA질립재체가유효침묵파기인재혈관평활기세포중적표체。
Objective To construct expression vectors expressing short hairpin RNA ( shRNA ) sections targeting phosphatidylinositol 3-kinase(PI3K)gene expression in vascular smooth muscle cell (VSMC)and vascular endothelia cell ( VEC) ,to explore the silencing effect of PI 3K gene in these cells ,and provide a gene therapy strategy to prevent angiostegnosis after bypass grafting .Methods Two of the sense and antisense RNA oligonucleotide strands targeting Pik3cb mRNA were designed and synthesized individually according to the sequence of the rat Pik3cb,then they were annealed to form double strands and then cloned into pGenesil -10, the sequences were examined all corrected as design .One effective shRNA was selected for this study by real time polymerase chain reaction ( RT-PCR ) .SMHC enhancer/SM22αpromoter sequence and KDR enhancer/promoter sequence were synthesized respectively in VSMC and VEC and then cloned into pGenesil -10-Pik3cb-shRNA.These sequences were constructed and identified ,named SMHCe/SM22αp-pGenesil-10-Pik3cb-shRNA( SM22αe/p shRNA) and KDRe/p-pGenesil-10-Pik3cb-shRNA(KDR e/p shRNA).The VSMC was transfected by recombinant plasmid vector ,and the PI3K mRNA level was detected by RT-PCR at 24 h,48 h and 72 h,respectively.Results Expression of the Pik3cb in Pik3cb-shRNA-1 group was lower than Pik3cb-shRNA-2 group and these two groups were also lower than that of the control group(P<0.05).SM22αe/p shRNA and KDR e/p shRNA were successfully constructed and identified by restriction endonuclease digestion .Expression of the Pik3cb gene was lower after 24 h in SM22αe/p groups compared with vacuity contrast group and CMV group (P<0.05).Conclusions The Pik3cb-shRNA-1 sequence was selected to be transcription template and then SM 22αe/p shRNA and KDR e/p shRNA were successfully constructed.The shRNA plasmid vector targeting on rat Pik 3cb gene should be effectively downregulated the expression of Pik3cb mRNA by SM22αpromoter in VSMC.
