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
1期
10-15
,共6页
兰晓莉%覃春霞%田月丽%夏晓天%何勇%袁辉%张永学
蘭曉莉%覃春霞%田月麗%夏曉天%何勇%袁輝%張永學
란효리%담춘하%전월려%하효천%하용%원휘%장영학
基因,报告%雌激素受体配体结合域%放射性核素显像%雌二醇%同位素标记%大鼠
基因,報告%雌激素受體配體結閤域%放射性覈素顯像%雌二醇%同位素標記%大鼠
기인,보고%자격소수체배체결합역%방사성핵소현상%자이순%동위소표기%대서
Genes,reporter%Estrogen receptor ligand binding domain%Radionuclide imaging%Estradiol%Isotope labeling%Rats
目的 在体外细胞摄取实验及在体显像基础上,评价新型核素报告基因系统人ER配体结合域( hERL)/核素标记雌二醇(E2)应用的可行性,为其用于基因治疗的监测提供依据.方法 以内部核糖体进入位点序列(IRES)方式构建携带hERL和治疗VEGF165的重组质粒pDC316-hERL-IRES-VEGF165(简写为EIV),将其用腺病毒包装构建重组腺病毒复合体(Ad-EIV).采用悬浮贴壁法从SD大鼠股骨和胫骨原代提取骨髓MSCs并培养.将Ad-EIV和脂质体2000包裹重组质粒(LipoEIV)分别转染MSCs,利用RT-PCR和Western blot,对hERL和VEGF165 mRNA和蛋白质水平的表达分别进行检测.测定Ad-EIV组、Lipo-EIV组和未转染组MSCs对125I-E2在不同时间(1、3、6、9、12和24h)的摄取率.将转染Ad-EIV的MSCs注射至大鼠左前肢、未转染MSCs注射至右前肢后1d,注射16α-18 F-17β-E2(18F-FES)进行micro PET/CT活体显像.2组间均数比较采用t检验,相关性研究采用Pearson相关分析.结果 Ad-EIV转染MSCs后RT-PCR检测示hERL和VEGF165 mRNA的表达随着感染滴度的增加而增加,且呈正相关(r2分别为0.953和0.966,P均＜0.05);感染复数(MOI)=25、50、75和100时,其mRNA表达高于Lipo-EIV组.Western blot检测蛋白质水平的表达也得到同样的结果.Ad-EIV组和Lipo-E1V组MSCs对125I-E2的摄取率均随时间延长逐渐增高,24h最高,分别达到( 10.94 ±0.30)％和(8.93±0.18)％;各个时间点2种载体转染组均明显高于未转染对照组(3.54％～5.52％;t值分别为15.489～26.560、10.523～24.204,P均＜0.05),而Ad-EIV组摄取率高于Lipo-EIV组(t=4.132～16.168,P均＜0.05).Micro PET/CT大鼠活体显像显示注射Ad-EIV转染的MSCs之左前肢放射性浓聚明显高于对侧.结论 报告基因hERL可以通过腺病毒转染、脂质体包裹等多种形式转染细胞并成功表达,应用核素标记E2可以对其进行探测和显像.应用腺病毒转染报告基因的方式转染率更高.
目的 在體外細胞攝取實驗及在體顯像基礎上,評價新型覈素報告基因繫統人ER配體結閤域( hERL)/覈素標記雌二醇(E2)應用的可行性,為其用于基因治療的鑑測提供依據.方法 以內部覈糖體進入位點序列(IRES)方式構建攜帶hERL和治療VEGF165的重組質粒pDC316-hERL-IRES-VEGF165(簡寫為EIV),將其用腺病毒包裝構建重組腺病毒複閤體(Ad-EIV).採用懸浮貼壁法從SD大鼠股骨和脛骨原代提取骨髓MSCs併培養.將Ad-EIV和脂質體2000包裹重組質粒(LipoEIV)分彆轉染MSCs,利用RT-PCR和Western blot,對hERL和VEGF165 mRNA和蛋白質水平的錶達分彆進行檢測.測定Ad-EIV組、Lipo-EIV組和未轉染組MSCs對125I-E2在不同時間(1、3、6、9、12和24h)的攝取率.將轉染Ad-EIV的MSCs註射至大鼠左前肢、未轉染MSCs註射至右前肢後1d,註射16α-18 F-17β-E2(18F-FES)進行micro PET/CT活體顯像.2組間均數比較採用t檢驗,相關性研究採用Pearson相關分析.結果 Ad-EIV轉染MSCs後RT-PCR檢測示hERL和VEGF165 mRNA的錶達隨著感染滴度的增加而增加,且呈正相關(r2分彆為0.953和0.966,P均＜0.05);感染複數(MOI)=25、50、75和100時,其mRNA錶達高于Lipo-EIV組.Western blot檢測蛋白質水平的錶達也得到同樣的結果.Ad-EIV組和Lipo-E1V組MSCs對125I-E2的攝取率均隨時間延長逐漸增高,24h最高,分彆達到( 10.94 ±0.30)％和(8.93±0.18)％;各箇時間點2種載體轉染組均明顯高于未轉染對照組(3.54％～5.52％;t值分彆為15.489～26.560、10.523～24.204,P均＜0.05),而Ad-EIV組攝取率高于Lipo-EIV組(t=4.132～16.168,P均＜0.05).Micro PET/CT大鼠活體顯像顯示註射Ad-EIV轉染的MSCs之左前肢放射性濃聚明顯高于對側.結論 報告基因hERL可以通過腺病毒轉染、脂質體包裹等多種形式轉染細胞併成功錶達,應用覈素標記E2可以對其進行探測和顯像.應用腺病毒轉染報告基因的方式轉染率更高.
목적 재체외세포섭취실험급재체현상기출상,평개신형핵소보고기인계통인ER배체결합역( hERL)/핵소표기자이순(E2)응용적가행성,위기용우기인치료적감측제공의거.방법 이내부핵당체진입위점서렬(IRES)방식구건휴대hERL화치료VEGF165적중조질립pDC316-hERL-IRES-VEGF165(간사위EIV),장기용선병독포장구건중조선병독복합체(Ad-EIV).채용현부첩벽법종SD대서고골화경골원대제취골수MSCs병배양.장Ad-EIV화지질체2000포과중조질립(LipoEIV)분별전염MSCs,이용RT-PCR화Western blot,대hERL화VEGF165 mRNA화단백질수평적표체분별진행검측.측정Ad-EIV조、Lipo-EIV조화미전염조MSCs대125I-E2재불동시간(1、3、6、9、12화24h)적섭취솔.장전염Ad-EIV적MSCs주사지대서좌전지、미전염MSCs주사지우전지후1d,주사16α-18 F-17β-E2(18F-FES)진행micro PET/CT활체현상.2조간균수비교채용t검험,상관성연구채용Pearson상관분석.결과 Ad-EIV전염MSCs후RT-PCR검측시hERL화VEGF165 mRNA적표체수착감염적도적증가이증가,차정정상관(r2분별위0.953화0.966,P균＜0.05);감염복수(MOI)=25、50、75화100시,기mRNA표체고우Lipo-EIV조.Western blot검측단백질수평적표체야득도동양적결과.Ad-EIV조화Lipo-E1V조MSCs대125I-E2적섭취솔균수시간연장축점증고,24h최고,분별체도( 10.94 ±0.30)％화(8.93±0.18)％;각개시간점2충재체전염조균명현고우미전염대조조(3.54％～5.52％;t치분별위15.489～26.560、10.523～24.204,P균＜0.05),이Ad-EIV조섭취솔고우Lipo-EIV조(t=4.132～16.168,P균＜0.05).Micro PET/CT대서활체현상현시주사Ad-EIV전염적MSCs지좌전지방사성농취명현고우대측.결론 보고기인hERL가이통과선병독전염、지질체포과등다충형식전염세포병성공표체,응용핵소표기E2가이대기진행탐측화현상.응용선병독전염보고기인적방식전염솔경고.
Objective To evaluate the feasibility of a new reporter gene/probe system,namely human ER ligand binding domain (hERL)/radionuclide labeled estradiol,and to provide basis for its monitoring gene and cell therapy from in vitro cellular uptake study and in vivo imaging experiment.Methods Recombinant plasmid pDC316- hERL -internal ribosome entry site-VEGF165 ( pDC316-hERL-IRES-VEGF165,or EIV) and recombinant Ad-EIV were constructed,which carried a reporter gene (hERL) and a therapeutic gene (VEGF165 ) through IRES.Adenovirus was used as a vector.MSCs were obtained from tibias and femurs of rat,and cultured normally.Ad-EIV and EIV coated with lipofectamine 2000 (Lipo-EIV) were transfected into MSCs.RT-PCR and Western blot were performed to detect the expression of hERL and VEGF165 from mRNA and protein level.The cellular uptake values of 125I labeled estradiol ( 125I-E2 ) were measured in Ad-EIV,Lipo-EIV and non-transfected MSCs at different incubation time ( 1,3,6,9,12 and 24 h).Ad-EIV transfected MSCs were injected into the left upper limb of rats,and non-transfected MSCs into the right upper limb as self-control.Micro PET/CT images were obtained after 1 d of transfection.Ttest and Pearson linear correlation analysis were used to analyze the data.Results After transfected with Ad-EIV,mRNA and protein expressions of hERL and VEGF165 in MSCs were increased with adenovirus multiplicity of infection ( MOI),and positive correlation could be seen ( r2 =0.953 and 0.966,both P ＜0.05).The expressions of mRNA and protein in Ad-EIV group were higher than those of Lipo-EIV transfected MSCs.Time-dependent accumulation of 125I-E2 was observed in the Ad-EIV group and Lipo-EIV group,and the highest uptake rates occurred at 24 h,with peak values of ( 10.94 ± 0.30) ％ and (8.93 ± O.18)％,respectively.Higher cellular uptakes could be seen at all time points in the Ad-EIV group than those of the Lipo-EIV group (t =4.132-16.168,all P ＜0.05).Moreover,the cellular uptakes with two vectors were significantly higher than those of non-transfected control cells at all the tested time points ( t =15.489- 26.560 for Ad-EIV and t =10.523 - 24.204 for Lipo-EIV,respectively,all P ＜ 0.05 ).Compared with the right upper limb,significantly higher radioactivity could be seen in the left side where Ad5-EIV transfected MSCs were injected.Conclusions Reporter gene hERL could be transfected into the cells via adenovirus and liposome as vectors.Its successful expression in cells could be detected and imaged by radiolabled estradiol.The reporter gene/probe system hERL/radiolabled estradiol might be used as a novel reporter gene/probe system for monitoring gene and cell therapy.
