中华核医学与分子影像杂志
中華覈醫學與分子影像雜誌
중화핵의학여분자영상잡지
Chinese Journal of Nuclear Medicine and Molecular Imaging
2012年
2期
95-99
,共5页
田菊%王志刚%任建丽%张清凤%刘利
田菊%王誌剛%任建麗%張清鳳%劉利
전국%왕지강%임건려%장청봉%류리
造影剂%基因%穿膜肽%转染%内皮细胞%超声检查
造影劑%基因%穿膜肽%轉染%內皮細胞%超聲檢查
조영제%기인%천막태%전염%내피세포%초성검사
Contrast media%Genes%Cell-penetrating peptide%Transfection%Endothelial cells%Ultrasonography
目的 探讨P-选择素靶向载基因及穿膜肽超声造影剂的制备及转染缺氧损伤型人脐静脉内皮细胞(HUVEC)的可行性.方法 采用机械振荡法及碳二亚胺法制备P-选择素靶向载绿色荧光蛋白基因及穿膜肽超声造影剂,检测其形态分布、浓度、粒径,采用激光共聚焦显微镜观察基因和穿膜肽的分布,用荧光分光光度法计算基因及穿膜肽的包封率.体外培养HUVEC,用过氧化氢处理制备HUVEC缺氧模型,并分为靶向和非靶向载基因及穿膜肽造影剂组进行转染实验.用荧光显微镜观察绿色荧光蛋白的表达情况,流式细胞仪检测转染率,并用SPSS 17.0统计软件进行t检验及直线相关分析.结果 制备的P-选择素靶向载基因及穿膜肽超声造影剂平均粒径约(2.15±0.36) μm,计数为(1.58±0.23) ×107个/ml.激光共聚焦显微镜下观察到基因和穿膜肽均匀分布在造影剂壁上,基因和穿膜肽的包封率分别为28％[y=0.932x -0.09(r =0.993,P＜0.05)]和25％ [y =5.875x -0.81(r =0.987,P＜0.05)].转染24 h后,荧光显微镜下观察到靶向和非靶向载基因及穿膜肽造影剂组均有绿色荧光蛋白的表达,流式细胞仪检测靶向组和非靶向组平均转染率分别约为( 18.74±0.47)％和(15.34±0.22)％(t=10.923,P＜0.001).结论 P-选择素靶向载基因及穿膜肽超声造影剂能够转染缺氧损伤的HUVEC,这为靶向基因投递提供了新的思路.
目的 探討P-選擇素靶嚮載基因及穿膜肽超聲造影劑的製備及轉染缺氧損傷型人臍靜脈內皮細胞(HUVEC)的可行性.方法 採用機械振盪法及碳二亞胺法製備P-選擇素靶嚮載綠色熒光蛋白基因及穿膜肽超聲造影劑,檢測其形態分佈、濃度、粒徑,採用激光共聚焦顯微鏡觀察基因和穿膜肽的分佈,用熒光分光光度法計算基因及穿膜肽的包封率.體外培養HUVEC,用過氧化氫處理製備HUVEC缺氧模型,併分為靶嚮和非靶嚮載基因及穿膜肽造影劑組進行轉染實驗.用熒光顯微鏡觀察綠色熒光蛋白的錶達情況,流式細胞儀檢測轉染率,併用SPSS 17.0統計軟件進行t檢驗及直線相關分析.結果 製備的P-選擇素靶嚮載基因及穿膜肽超聲造影劑平均粒徑約(2.15±0.36) μm,計數為(1.58±0.23) ×107箇/ml.激光共聚焦顯微鏡下觀察到基因和穿膜肽均勻分佈在造影劑壁上,基因和穿膜肽的包封率分彆為28％[y=0.932x -0.09(r =0.993,P＜0.05)]和25％ [y =5.875x -0.81(r =0.987,P＜0.05)].轉染24 h後,熒光顯微鏡下觀察到靶嚮和非靶嚮載基因及穿膜肽造影劑組均有綠色熒光蛋白的錶達,流式細胞儀檢測靶嚮組和非靶嚮組平均轉染率分彆約為( 18.74±0.47)％和(15.34±0.22)％(t=10.923,P＜0.001).結論 P-選擇素靶嚮載基因及穿膜肽超聲造影劑能夠轉染缺氧損傷的HUVEC,這為靶嚮基因投遞提供瞭新的思路.
목적 탐토P-선택소파향재기인급천막태초성조영제적제비급전염결양손상형인제정맥내피세포(HUVEC)적가행성.방법 채용궤계진탕법급탄이아알법제비P-선택소파향재록색형광단백기인급천막태초성조영제,검측기형태분포、농도、립경,채용격광공취초현미경관찰기인화천막태적분포,용형광분광광도법계산기인급천막태적포봉솔.체외배양HUVEC,용과양화경처리제비HUVEC결양모형,병분위파향화비파향재기인급천막태조영제조진행전염실험.용형광현미경관찰록색형광단백적표체정황,류식세포의검측전염솔,병용SPSS 17.0통계연건진행t검험급직선상관분석.결과 제비적P-선택소파향재기인급천막태초성조영제평균립경약(2.15±0.36) μm,계수위(1.58±0.23) ×107개/ml.격광공취초현미경하관찰도기인화천막태균균분포재조영제벽상,기인화천막태적포봉솔분별위28％[y=0.932x -0.09(r =0.993,P＜0.05)]화25％ [y =5.875x -0.81(r =0.987,P＜0.05)].전염24 h후,형광현미경하관찰도파향화비파향재기인급천막태조영제조균유록색형광단백적표체,류식세포의검측파향조화비파향조평균전염솔분별약위( 18.74±0.47)％화(15.34±0.22)％(t=10.923,P＜0.001).결론 P-선택소파향재기인급천막태초성조영제능구전염결양손상적HUVEC,저위파향기인투체제공료신적사로.
Objective To prepare an anti-P-selectin targeted ultrasound contrast agent carrying genes and cell-penetrating peptides (CPP) and to investigate its feasibility of delivery to hypoxic human umbilical vein endothelial cells (HUVEC).Methods Anti-P-selectin targeted ultrasound contrast agent carrying a green fluorescent protein gene (pEGFP-N1) and CPP was prepared by mechanical vibration and carbodiimide techniques.The appearance,distribution,concentration and diameter of the ultrasound contrast agent were measured.The gene and CPP distribution on the agent was investigated using confocal laser scanning microscopy (CLSM).The efficiency of the ultrasound contrast agent to carry the gene and CPP was investigated by fluorospectrophotometry.HUVEC were cultured in vitro and hypoxic HUVEC were prepared using hydrogen peroxide (H2O2 ).Hypoxic HUVEC were randomly assigned targeted ultrasound contrast agents and non-targeted ultrasound contrast agents for transfection.The transfection effect of green fluorescent protein in the two groups was observed using fluorescence microscopy and flow cytometry.T-test and linear correlation analysis were used for statistical analysis.Results The average diameter of anti-P-selectin targeted ultrasound contrast agents carrying gene and CPP was (2.15 ±0.36) μm and the concentration was ( 1.58 ± 0.23) × 107/ml.The results of CLSM showed that gene and CPP were distributed on the shell of the agent.The gene encapsulation efficiency was 28％ (y =0.932x - 0.09,r =0.993,P ＜ 0.05 ),and the CPP encapsulation efficiency was 25％ (y =5.875x -0.81,r =0.987,P ＜0.05).EGFP expression was observed using fluorescence microscopy in targeted ultrasound contrast agents and non-targeted ultrasound contrast agents.The average transfection efficiencies of targeted ultrasound contrast agents and nontargeted ultrasound contrast agents were ( 18.74 ± 0.47 ) ％ and ( 15.34 ± 0.22) ％ after 24 h ( t =10.923,P ＜ 0.001 ).Conclusions The in vitro studies showed that the targeted ultrasound contrast agent could significantly enhance the transfection efficiency in hypoxic HUVEC.This may be a new idea for gene therapy.
