两种核素报告基因系统用于监测治疗基因VEGF165表达的基础研究
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摘要
目的：将人血管内皮生长因子165(human vascular endothelial growth factor 165, hVEGF165)基因分别与报告基因GGC肽(diglycylcysteine,双甘氨酰半胱氨酸)基因序列及突变型单纯疱疹病毒Ⅰ型胸苷激酶基因(mutant herpes simplex virus type 1 thymidine kinase, HSV1-sr39tk)通过基因工程技术连接起来,构建重组腺病毒载体Ad5-VIE及Ad5-SIV,通过一系列体外实验及部分体内实验,初步探讨以GGC肽编码序列及HSV1-sr39tk作为报告基因监测治疗基因VEGF基因表达的可行性,并且对两种报告基因显像的优缺点进行分析。
方法：
1.GGC/99mTc-GH报告基因系统监测治疗基因VEGF165表达的研究
1)重组腺病毒Ad5-VIE的构建及鉴定
pcDNA3-VEGF165质粒线性化后,化学合成GGC肽基因序列,之后将GGC肽基因序列连于VEGF165基因C端,通过内部核糖体切入位点(internal ribosomal entry site,IRES)技术将增强型绿色荧光蛋白(enhanced green fluoresce protein, EGFP)基因连接至下游,最后构建成重组腺病毒载体(Ad5-VIE)。
2)重组腺病毒(Ad5-VIE)感染大鼠骨髓间充质细胞后摄取99mTc-GH的实验研究
Ad5-VIE转染大鼠骨髓间充质细胞后48h,每孔加入含有99mTc-GH的培养基,37℃条件下按照实验要求进行孵育不同时间。分别进行时间相关(MOI=100,孵育时间分别为30min,60min,90min,120min)及感染复数(Multiplicity of Infection, MOI=0,10,25,50,100,孵育时间为2h)相关摄取实验。
3)实时定量PCR (quantitative real-time PCR, qRT-PCR)鉴定重组腺病毒感染MSCs后VEGF165及EGFP的表达
Ad5-VIE以不同滴度(MOI=0,10,25,50,100)转染大鼠骨髓间充质细胞后48h,提取每孔总RNA逆转录后进行qRT-PCR扩增实验,检测VEGF165及EGFP在mRNA水平上的表达。
4)半定量Western-blot;检测病毒转染后VEGF165蛋白的表达
Ad5-VIE不同MOI(0,10,25,50,100IU/per cell)转染MSCs后48h,提取每孔细胞胞浆蛋白,进行Western-blot蛋白印迹实验,将Western-blot结果与不同MOI细胞摄取结果进行对比分析,从而判断GGC肽与VEGF165蛋白的表达相关性。
5)报告基因体内显像的初步应用研究：
将2.5×108IU重组腺病毒Ad5-VIE与Ad5-EGFP转染大鼠骨髓间充质细胞中(1×107cells),转染24h后,胰酶消化后收集细胞,生理盐水制成250ul细胞悬液,将其多点注射于SD大鼠下肢中,48h后采用低能针孔准直器进行SPECT显像。
2.报告基因系统HSVl-sr39tk/FIAU监测治疗基因VEGF165表达的研究
1)重组腺病毒Ad5-SIV的构建及鉴定。
通过IRES技术将报告基因HSV1-sr39tk与治疗基因VEGF165连接起来,构建重组腺病毒载体Ad5-SIV。并且通过PCR等方法来鉴定其构建成功与否。
2)Ad5-SIV感染MSCs后摄取131I-FIAU的实验研究。
Ad5-SIV以不同滴度(MOI=0,10,25,50,50,75,100)转染MSCs后48h,与含131I-FIAU的培养基孵育3h,测量其摄取率；同时以MOI=50感染MSCs,测量不同时间(0,30,60,90,120,150,180min,240min摄取率。
3)qRT-PCR鉴定重组腺病毒感染MSCs后HSVl-sr39tk,及VEGF165 mRNA的表达
Ad5-SIV以不同滴度(MOI=0,10,25,50,75,100)转染MSCs后48h,提取每孔总蛋白逆转录后进行QRT-PCR实验,检测HSV1-sr39tk mRNA及VEGF165 mRNA的含量。
4)酶联免疫吸附实验(Enzyme-Linked Immunosorbent Assay, ELISA)检测不同感染复数Ad5-SIV转染MSCs后细胞培养上清中VEGF的含量
Ad5-SIV以不同滴度(MOI=0,10,25,50,75,100)转染MSCs后48h,提取每孔细胞培养上清,2000rpm离心20min,按照ELISA试剂盒说明书进行操作,检测转染不同感染复数病毒后细胞培养上清中VEGF的含量,并且将ELISA结果与细胞摄取结果做对比分析,从而检测报告基因HSV1-sr39TK酶活性与VEGF表达之间的关系。
结果：
1.GGC/99mTc-GH报告基因系统监测治疗基因VEGF165表达的研究
1)Ad5-VIE重组腺病毒的构建及鉴定
重组质粒PDC316-VEGFGGCmotif-IRES-EGFP经过PCR、酶切及测序等手段均证实其序列正确,包装生成的重组腺病毒Ad5-VIE毒种经PCR实验能扩增出预期条带,经纯化后物理滴度为2×1011(v.p/ml),感染滴度为(TCID50法)为4.5×109IU/ml。
2)Ad5-VIE感染MSCs后摄取99mTc-GH实验
时间相关摄取实验结果显示转染了包含目的基因重组腺病毒的MSCs细胞对99mTc-GH的摄取率逐渐增高,至120min时达到7.72±0.22%；转染了对照病毒Ad5-EGFP各时间点摄取值均处于较低水平。滴度相关摄取实验结果显示细胞对99mTc-GH的摄取率随病毒滴度的增加而逐渐增加(r2=0.86,P<0.05)
3)Ad5-VIE感染MSCs后VEGF165及EGFP的mRNA表达
不同滴度感染后,VEGF165及EGFP的mRNA含量随病毒感染滴度增加而增加,二者之间呈线性正相关(r2分别为0.91和0.90,P<0.05),并且VEGF165及EGFP的mRNA表达之间亦具备显著正相关(r2=0.99, P<0.05)。
4)Ad5-VIE感染后半定量western-blot检测VEGF165的表达
半定量western-blot结果显示Ad5-VIE以不同滴度感染MSCs48h后,细胞胞浆中VEGF165蛋白表达逐步上升,而在对照病毒Ad5-EGFP组及空白对照组未见表达。VEGF165相对量与细胞摄取率均随病毒滴度增高逐步增高,二者呈正相关(r2=0.90,P<0.05),说明VEGF165蛋白与GGC肽表达之间具有较好的相关性。
5)报告基因显像体内显像的初步研究
体内显像可见大鼠尾静脉注射99mTc-GH后,双侧肾脏及左侧下肢(感染Ad5-VIE细胞组)5min立即显影并且显影清晰,随着显像剂排出,膀胱显影逐渐清晰,左侧下肢显影逐渐模糊,2h左右,显影近乎消失。而在转染Ad5-EGFP细胞组,MSCs细胞组,生理盐水组未见明显显影。
2.报告基因系统HSV1-sr39tk/FIAU.监测治疗基因VEGF165表达的研究
1)Ad5-SIV重组腺病毒的构建及鉴定
重组质粒PDC316-sr39tk-IRES-VEGF 165经过PCR,酶切及测序均证实其序列正确,并且包装生成的重组腺病毒Ad5-SIV毒种经PCR实验能扩增出预期条带,经纯化后物理滴度为2×1011(v.p/ml),感染滴度为(TCID50法)为7.9×109IU/ml。
2)Ad5-SIV感染MSCs后摄取实验
随着时间延长,转染Ad5-SIV组细胞摄取率逐步升高,150min时达平台期,此时摄取率为20.06±1.33%。而对照组摄取一直处于低水平。病毒感染滴度相关摄取实验结果显示,细胞摄取率随病毒滴度的增加而逐渐增加(r2=0.89,P<0.05)。
3)Ad5-SIV感染MSCs后VEGF165及EGFP的mRNA表达
不同滴度感染后,HSV1-sr39tk及VEGF 165 mRNA含量随病毒感染滴度增加而增加,二者之间呈线性正相关(r2分别为0.97和0.96,P＜0.05),并且HSV1-sr39tk及VEGF 165 mRNA表达之间亦具备显著正相关(r2=0.94,P<0.05)。
4)Ad5-SIV感染后ELISA检测VEGF165的表达
ELISA结果证实VEGF165蛋白分泌随病毒滴度增大而增加(r2=0.99,P<0.05)。将VEGF165蛋白分泌结果与病毒滴度相关摄取结果做相关性分析,可以反映VEGF蛋白分泌与HSV1-sr39TK蛋白酶活性之间的关系,二者之间具有较好的相关性(r2=0.84,P<0.05)。
结论：本研究成功构建了重组腺病毒Ad5-VIE及Ad5-SIV,并且通过体外研究证实转染了目的病毒的MSCs细胞对报告探针99mTc-GH及131I-FIAU的摄取均与治疗基因VEGF165表达呈正相关。从而使利用报告基因系统来检测治疗基因的表达成为可能。
Objective Two recombinant adenovial vectors Ad5-VIE and Ad5-SIV which contain therapeutic gene and report gene were constructed to evaluate the feasibility of monitoring therapeutic gene vascular endothelial growth factor 165(VEGF165) gene with report gene GGCmotif sequence and mutant herpes simplex virus type 1 thymidine kinase(HSV1-sr39tk) gene.
Methods
1. Study of monitoring the expression of therapy gene VEGF165 via GGC/99mTc-GH report gene/probe system
1) Construction and identification of recombinant adenovial vector Ad5-VIE
GGC (diglycylcysteine) motifs were positioned at the C end of VEGF165 gene after the linearization of pcDNA3-VEGF165 plasmid. A replication-defective adenovirus vector Ad5-VEGF165GGCmotif-IRES-EGFP (Ad5-VIE) was constructed, carrying a CMV early promoter driving the expression of VEGF165 gene, GGC motifs and enhanced green fluoresce protein (EGFP), with the aid of an internal ribosomal entry site (IRES)。
2) cellular uptake of99mTc-GH
MSCs were infected with Ad5-VIE about 48h, then we perform the time-based (MOI=100, Incubation time:30min,60min,90min,120min)cellular uptake of 99mTc-GH and MOI based(MOI=0,10,25,50,100,incubation time:2h) cellular uptake study.
3) Quantitative Real-time PCR (qRT-PCR) analysis for VEGF165 and EGFP mRNA.
MSCs was infected with different infection units (MOI=0,10,25,50,100),48h later, The total cellular RNA was extracted, then qRT-PCR was performed to detect the VEGF165 mRNA and EGFP mRNA.
4) Semi-quantitative western-blot anlysis for VEGF165 protein
MSCs was infected with different infection units (MOI=0,10,25,50,100),48h later, the cytoplasmic protein was extracted from each well for Semi-quantitative western-blot study, The correlation analysis was performed between the cellular uptake of 99mTc-GH, which indirectly showed the expression of GGCmotifs and the expression of VEGF165 protein.
5) Preliminary study of reporter gene imaging
MSCs (1×107cells) was infected with 2.5×108 IU Ad5-VIE and Ad5-EGFP,24h after transfection, cells were collected after trypsin digestion, then suspended with 250μl saline. The cells suspension was multi-point injected in the SD rats lower limbs, then the SPECT imaging was peformed with the low-energy pinhole collimator 48h later.
2. Study of monitoring the expression of therapy gene VEGF165 via HSVl-sr39tk/FIAU report gene/probe system
1) Construction and identification of recombinant adenovial vector Ad5-SIV
With the aid of an internal ribosomal entry site (IRES) technique, a replication-defective adenovirus vector Ad5-SIV was constructed, carrying a CMV early promoter which driving the expression of HSV1-sr39tk gene and VEGF165 gene. PCR was performed to identify the adenovirus vector.
2) Cellular uptake of131I-FIAU
MSCs was infected with increasing multiplicities of infection (MOI= 0,10,25,50,75 and 100) of Ad5-SIV or Ad5-EGFP (used as a negative control). Forty-eight hours after infection, the culture medium was replaced by medium containg 131I-FIAU. After incubation for 3 hour, the radioactive medium was collected and the cells were then harvested and lysed with 1N NaOH in counting tube. Subsequently, the extracellular radioactivity in the medium and the intracellular radioactivity in cells lysate were determined by a gamma counter and corrected for decay. Meanwhile, the MSCs were infected with same MOI(MOI=50),and incubated with culture medium containing 131I-FIAU for 30min,60min,90min,120min,3h and 4h to determine the time course of131I-FIAU accumulation after adenovirus infection.
3) qRT-PCR analysis for HSV1-sr39tk and VEGF165 mRNA.
MSCs was infected with different MOI (MOI=0,10,25,50,75, 100IU/per cell),48h later, The total cellular RNA was extracted and qRT-PCR was performed to detect the HSV1-sr39tkmRNAandVEGF165 mRNA.
4) Enzyme-Linked Immunosorbent Assay (ELISA) anlysis for VEGF165 protein
MSCs was infected with different infection units (MOI=0,10,25,50,75, 100IU/per cell),48h later, the culture medium was collected and centrifuged to remove all cellular fragments.ELISA was carried out according the manufacturer's introductions. Finally the absorbance of each well was determined within 30min using a microplate reader set to 450 nm. Finally, we compared the protein expression of VEGF165 and HSV1-sr39TK by analyzing the data obtained from ELISA and cellular uptake study.
Results
1. Study of monitoring the expression of therapy gene VEGF165 via GGC/99mTc-GH report gene/probe system
1) Construction and identification of recombinant adenovial vector Ad5-VIE
Plasmid PDC316-VEGFGGCmotif-IRES-EGFP was identified by PCR, corresponding endonucleases digestion and sequencing. The recombinant adenovirus Ad5-VIE was identified by PCR, and the results showed the specific target band. The titer of purified adenovirus stock was 2×1011VP/ml (OD260 method) and 4.5×109IU/ml (classical TCID50 method)
2) Cellular uptake of99mTc-GH
Time dependent uptake showed that in Ad5-VIE infected MSCs, uptake rate of 99mTc-GH increased graduately over time, and the highest uptake occurred at 120min with a peak uptake rate of 7.72±0.22%. But in the control groups, the uptake of Ad5-EGFP infected cells at a low level all the time. The titers dependent uptake showed that uptake rate of 99mTc-GH in Ad5-VIE infected MSCs increased with the increasing viral titer (r2=0.86, P<0.05).
3) qRT-PCR analysis for VEGF165 and EGFP mRNA.
The qRT-PCR showed that VEGF165 mRNA and EGFP mRNA expression increased with increasing virus titers (r2=0.91 and r2=0.90, p<0.05). Furthermore there was a good correlation between VEGF165 mRNA and EGFP mRNA (r2=0.99,P<0.05).
4) Semi-quantitative western-blot anlysis for VEGF165 protein
Semi-quantitative western-blot results showed that in the Ad5-VIE groups, expression of cytoplasmic VEGF165 protein increased graduately; while in the Ad5-EGFP group and uninfected control group, VEGF165 protein did not express. The relative amount of VEGF165 and the cell uptake rate increased with the titer gradually increased, a good correlation exited between expression of VEGF165 protein and GGC motifs (r2=0.90, P <0.05).
5) Preliminary study of reporter gene imaging
In vivo imaging shows that after intravenous injection of 99mTc-GH, the bilateral renal and left lower extremity (infection with Ad5-VIE cell group) could be imaging clearly and immediately. With the imaging agent discharge, bladder imaging progressively clearly, the left lower limbs imaging gradually blurred,2h or so, the imaging almost disappeared. In cells transfected with Ad5-EGFP group, MSCs cells group, no significant imaging emerged.
2. Study of monitoring the expression of therapy gene VEGF165 via HSVl-sr39tk/FIAU report gene/probe system
1) Construction and identification of recombinant adenovial vector Ad5-SIV
Plasmid PDC316-sr39tk-IRES-VEGF165 was identified by PCR, corresponding endonucleases digestion and sequencing. The recombinant adenovirus Ad5-SIV was identified by PCR, and the results showed the specific target band. The titer of purified adenovirus stock was 2×1011VP/ml (OD260 method) and 7.9×109IU/ml (classical TCID50 method)
2) Cellular uptake of131I-FIAU
The time dependent cellular uptake studies showed that uptake rates increased rapidly between 30min and 150min and reached a plateau after 150min,at 150min point, the uptake rates were 20.06±1.33%. The uptake rates of 131I-FIAU with Ad5-SIV-infected cells were significantly higher than those with Ad5-EGFP-transfected groups at all time points (t=12.978-38.253).The MOI dependent cellular uptake studies showed that the cellular uptake of 131I-FIAU increased with increasing virus titer (R2=0.89, P<0.05)
3) qRT-PCR analysis for HSV1-sr39tk and VEGF165 mRNA.
HSV1-sr39tk mRNA and VEGF165 mRNA could express successfully after MSCs infected with Ad5-SIV at a series MOI (0,10,25,50,75,100 IU/per cell). Additionally, HSV1-sr39tk mRNA and VEGF165 mRNA expression increased with increasing virus titer (R2=0.97 and R2=0.96, p<0.05). Moreover, the real-time PCR results showed a good correlation between HSV1-Sr39tk mRNA and VEGF165 mRNA (R2=0.93,P<0.05)
4) ELISA anlysis for VEGF165 protein
The data obtained from ELISA showed a good correlation between VEGF protein and adenovirus titer (R2=0.99, P<0.05) after the MSCs was infected with different MOI (0,10, 25,50,75,100 IU/per cell) for 48h., furthermore, VEGF protein secretion was correlated well with the uptake rate of 131I-FIAU (R2=0.84, P<0.05)
Conclusion:The recombinant adenovirus Ad5-VIE and Ad5-SIV was successfully constructed, and the in vitro study confirmed that expression of therapeutic gene VEGF165 correlated significantly with the report gene GGC motifs sequence and HSV1-sr39tk gene. This provided a theoretical basis for monitoring therapeutic gene with nuclear reporter gene imaging.
方法：
1.GGC/99mTc-GH报告基因系统监测治疗基因VEGF165表达的研究
1)重组腺病毒Ad5-VIE的构建及鉴定
pcDNA3-VEGF165质粒线性化后,化学合成GGC肽基因序列,之后将GGC肽基因序列连于VEGF165基因C端,通过内部核糖体切入位点(internal ribosomal entry site,IRES)技术将增强型绿色荧光蛋白(enhanced green fluoresce protein, EGFP)基因连接至下游,最后构建成重组腺病毒载体(Ad5-VIE)。
2)重组腺病毒(Ad5-VIE)感染大鼠骨髓间充质细胞后摄取99mTc-GH的实验研究
Ad5-VIE转染大鼠骨髓间充质细胞后48h,每孔加入含有99mTc-GH的培养基,37℃条件下按照实验要求进行孵育不同时间。分别进行时间相关(MOI=100,孵育时间分别为30min,60min,90min,120min)及感染复数(Multiplicity of Infection, MOI=0,10,25,50,100,孵育时间为2h)相关摄取实验。
3)实时定量PCR (quantitative real-time PCR, qRT-PCR)鉴定重组腺病毒感染MSCs后VEGF165及EGFP的表达
Ad5-VIE以不同滴度(MOI=0,10,25,50,100)转染大鼠骨髓间充质细胞后48h,提取每孔总RNA逆转录后进行qRT-PCR扩增实验,检测VEGF165及EGFP在mRNA水平上的表达。
4)半定量Western-blot;检测病毒转染后VEGF165蛋白的表达
Ad5-VIE不同MOI(0,10,25,50,100IU/per cell)转染MSCs后48h,提取每孔细胞胞浆蛋白,进行Western-blot蛋白印迹实验,将Western-blot结果与不同MOI细胞摄取结果进行对比分析,从而判断GGC肽与VEGF165蛋白的表达相关性。
5)报告基因体内显像的初步应用研究：
将2.5×108IU重组腺病毒Ad5-VIE与Ad5-EGFP转染大鼠骨髓间充质细胞中(1×107cells),转染24h后,胰酶消化后收集细胞,生理盐水制成250ul细胞悬液,将其多点注射于SD大鼠下肢中,48h后采用低能针孔准直器进行SPECT显像。
2.报告基因系统HSVl-sr39tk/FIAU监测治疗基因VEGF165表达的研究
1)重组腺病毒Ad5-SIV的构建及鉴定。
通过IRES技术将报告基因HSV1-sr39tk与治疗基因VEGF165连接起来,构建重组腺病毒载体Ad5-SIV。并且通过PCR等方法来鉴定其构建成功与否。
2)Ad5-SIV感染MSCs后摄取131I-FIAU的实验研究。
Ad5-SIV以不同滴度(MOI=0,10,25,50,50,75,100)转染MSCs后48h,与含131I-FIAU的培养基孵育3h,测量其摄取率；同时以MOI=50感染MSCs,测量不同时间(0,30,60,90,120,150,180min,240min摄取率。
3)qRT-PCR鉴定重组腺病毒感染MSCs后HSVl-sr39tk,及VEGF165 mRNA的表达
Ad5-SIV以不同滴度(MOI=0,10,25,50,75,100)转染MSCs后48h,提取每孔总蛋白逆转录后进行QRT-PCR实验,检测HSV1-sr39tk mRNA及VEGF165 mRNA的含量。
4)酶联免疫吸附实验(Enzyme-Linked Immunosorbent Assay, ELISA)检测不同感染复数Ad5-SIV转染MSCs后细胞培养上清中VEGF的含量
Ad5-SIV以不同滴度(MOI=0,10,25,50,75,100)转染MSCs后48h,提取每孔细胞培养上清,2000rpm离心20min,按照ELISA试剂盒说明书进行操作,检测转染不同感染复数病毒后细胞培养上清中VEGF的含量,并且将ELISA结果与细胞摄取结果做对比分析,从而检测报告基因HSV1-sr39TK酶活性与VEGF表达之间的关系。
结果：
1.GGC/99mTc-GH报告基因系统监测治疗基因VEGF165表达的研究
1)Ad5-VIE重组腺病毒的构建及鉴定
重组质粒PDC316-VEGFGGCmotif-IRES-EGFP经过PCR、酶切及测序等手段均证实其序列正确,包装生成的重组腺病毒Ad5-VIE毒种经PCR实验能扩增出预期条带,经纯化后物理滴度为2×1011(v.p/ml),感染滴度为(TCID50法)为4.5×109IU/ml。
2)Ad5-VIE感染MSCs后摄取99mTc-GH实验
时间相关摄取实验结果显示转染了包含目的基因重组腺病毒的MSCs细胞对99mTc-GH的摄取率逐渐增高,至120min时达到7.72±0.22%；转染了对照病毒Ad5-EGFP各时间点摄取值均处于较低水平。滴度相关摄取实验结果显示细胞对99mTc-GH的摄取率随病毒滴度的增加而逐渐增加(r2=0.86,P<0.05)
3)Ad5-VIE感染MSCs后VEGF165及EGFP的mRNA表达
不同滴度感染后,VEGF165及EGFP的mRNA含量随病毒感染滴度增加而增加,二者之间呈线性正相关(r2分别为0.91和0.90,P<0.05),并且VEGF165及EGFP的mRNA表达之间亦具备显著正相关(r2=0.99, P<0.05)。
4)Ad5-VIE感染后半定量western-blot检测VEGF165的表达
半定量western-blot结果显示Ad5-VIE以不同滴度感染MSCs48h后,细胞胞浆中VEGF165蛋白表达逐步上升,而在对照病毒Ad5-EGFP组及空白对照组未见表达。VEGF165相对量与细胞摄取率均随病毒滴度增高逐步增高,二者呈正相关(r2=0.90,P<0.05),说明VEGF165蛋白与GGC肽表达之间具有较好的相关性。
5)报告基因显像体内显像的初步研究
体内显像可见大鼠尾静脉注射99mTc-GH后,双侧肾脏及左侧下肢(感染Ad5-VIE细胞组)5min立即显影并且显影清晰,随着显像剂排出,膀胱显影逐渐清晰,左侧下肢显影逐渐模糊,2h左右,显影近乎消失。而在转染Ad5-EGFP细胞组,MSCs细胞组,生理盐水组未见明显显影。
2.报告基因系统HSV1-sr39tk/FIAU.监测治疗基因VEGF165表达的研究
1)Ad5-SIV重组腺病毒的构建及鉴定
重组质粒PDC316-sr39tk-IRES-VEGF 165经过PCR,酶切及测序均证实其序列正确,并且包装生成的重组腺病毒Ad5-SIV毒种经PCR实验能扩增出预期条带,经纯化后物理滴度为2×1011(v.p/ml),感染滴度为(TCID50法)为7.9×109IU/ml。
2)Ad5-SIV感染MSCs后摄取实验
随着时间延长,转染Ad5-SIV组细胞摄取率逐步升高,150min时达平台期,此时摄取率为20.06±1.33%。而对照组摄取一直处于低水平。病毒感染滴度相关摄取实验结果显示,细胞摄取率随病毒滴度的增加而逐渐增加(r2=0.89,P<0.05)。
3)Ad5-SIV感染MSCs后VEGF165及EGFP的mRNA表达
不同滴度感染后,HSV1-sr39tk及VEGF 165 mRNA含量随病毒感染滴度增加而增加,二者之间呈线性正相关(r2分别为0.97和0.96,P＜0.05),并且HSV1-sr39tk及VEGF 165 mRNA表达之间亦具备显著正相关(r2=0.94,P<0.05)。
4)Ad5-SIV感染后ELISA检测VEGF165的表达
ELISA结果证实VEGF165蛋白分泌随病毒滴度增大而增加(r2=0.99,P<0.05)。将VEGF165蛋白分泌结果与病毒滴度相关摄取结果做相关性分析,可以反映VEGF蛋白分泌与HSV1-sr39TK蛋白酶活性之间的关系,二者之间具有较好的相关性(r2=0.84,P<0.05)。
结论：本研究成功构建了重组腺病毒Ad5-VIE及Ad5-SIV,并且通过体外研究证实转染了目的病毒的MSCs细胞对报告探针99mTc-GH及131I-FIAU的摄取均与治疗基因VEGF165表达呈正相关。从而使利用报告基因系统来检测治疗基因的表达成为可能。
Objective Two recombinant adenovial vectors Ad5-VIE and Ad5-SIV which contain therapeutic gene and report gene were constructed to evaluate the feasibility of monitoring therapeutic gene vascular endothelial growth factor 165(VEGF165) gene with report gene GGCmotif sequence and mutant herpes simplex virus type 1 thymidine kinase(HSV1-sr39tk) gene.
Methods
1. Study of monitoring the expression of therapy gene VEGF165 via GGC/99mTc-GH report gene/probe system
1) Construction and identification of recombinant adenovial vector Ad5-VIE
GGC (diglycylcysteine) motifs were positioned at the C end of VEGF165 gene after the linearization of pcDNA3-VEGF165 plasmid. A replication-defective adenovirus vector Ad5-VEGF165GGCmotif-IRES-EGFP (Ad5-VIE) was constructed, carrying a CMV early promoter driving the expression of VEGF165 gene, GGC motifs and enhanced green fluoresce protein (EGFP), with the aid of an internal ribosomal entry site (IRES)。
2) cellular uptake of99mTc-GH
MSCs were infected with Ad5-VIE about 48h, then we perform the time-based (MOI=100, Incubation time:30min,60min,90min,120min)cellular uptake of 99mTc-GH and MOI based(MOI=0,10,25,50,100,incubation time:2h) cellular uptake study.
3) Quantitative Real-time PCR (qRT-PCR) analysis for VEGF165 and EGFP mRNA.
MSCs was infected with different infection units (MOI=0,10,25,50,100),48h later, The total cellular RNA was extracted, then qRT-PCR was performed to detect the VEGF165 mRNA and EGFP mRNA.
4) Semi-quantitative western-blot anlysis for VEGF165 protein
MSCs was infected with different infection units (MOI=0,10,25,50,100),48h later, the cytoplasmic protein was extracted from each well for Semi-quantitative western-blot study, The correlation analysis was performed between the cellular uptake of 99mTc-GH, which indirectly showed the expression of GGCmotifs and the expression of VEGF165 protein.
5) Preliminary study of reporter gene imaging
MSCs (1×107cells) was infected with 2.5×108 IU Ad5-VIE and Ad5-EGFP,24h after transfection, cells were collected after trypsin digestion, then suspended with 250μl saline. The cells suspension was multi-point injected in the SD rats lower limbs, then the SPECT imaging was peformed with the low-energy pinhole collimator 48h later.
2. Study of monitoring the expression of therapy gene VEGF165 via HSVl-sr39tk/FIAU report gene/probe system
1) Construction and identification of recombinant adenovial vector Ad5-SIV
With the aid of an internal ribosomal entry site (IRES) technique, a replication-defective adenovirus vector Ad5-SIV was constructed, carrying a CMV early promoter which driving the expression of HSV1-sr39tk gene and VEGF165 gene. PCR was performed to identify the adenovirus vector.
2) Cellular uptake of131I-FIAU
MSCs was infected with increasing multiplicities of infection (MOI= 0,10,25,50,75 and 100) of Ad5-SIV or Ad5-EGFP (used as a negative control). Forty-eight hours after infection, the culture medium was replaced by medium containg 131I-FIAU. After incubation for 3 hour, the radioactive medium was collected and the cells were then harvested and lysed with 1N NaOH in counting tube. Subsequently, the extracellular radioactivity in the medium and the intracellular radioactivity in cells lysate were determined by a gamma counter and corrected for decay. Meanwhile, the MSCs were infected with same MOI(MOI=50),and incubated with culture medium containing 131I-FIAU for 30min,60min,90min,120min,3h and 4h to determine the time course of131I-FIAU accumulation after adenovirus infection.
3) qRT-PCR analysis for HSV1-sr39tk and VEGF165 mRNA.
MSCs was infected with different MOI (MOI=0,10,25,50,75, 100IU/per cell),48h later, The total cellular RNA was extracted and qRT-PCR was performed to detect the HSV1-sr39tkmRNAandVEGF165 mRNA.
4) Enzyme-Linked Immunosorbent Assay (ELISA) anlysis for VEGF165 protein
MSCs was infected with different infection units (MOI=0,10,25,50,75, 100IU/per cell),48h later, the culture medium was collected and centrifuged to remove all cellular fragments.ELISA was carried out according the manufacturer's introductions. Finally the absorbance of each well was determined within 30min using a microplate reader set to 450 nm. Finally, we compared the protein expression of VEGF165 and HSV1-sr39TK by analyzing the data obtained from ELISA and cellular uptake study.
Results
1. Study of monitoring the expression of therapy gene VEGF165 via GGC/99mTc-GH report gene/probe system
1) Construction and identification of recombinant adenovial vector Ad5-VIE
Plasmid PDC316-VEGFGGCmotif-IRES-EGFP was identified by PCR, corresponding endonucleases digestion and sequencing. The recombinant adenovirus Ad5-VIE was identified by PCR, and the results showed the specific target band. The titer of purified adenovirus stock was 2×1011VP/ml (OD260 method) and 4.5×109IU/ml (classical TCID50 method)
2) Cellular uptake of99mTc-GH
Time dependent uptake showed that in Ad5-VIE infected MSCs, uptake rate of 99mTc-GH increased graduately over time, and the highest uptake occurred at 120min with a peak uptake rate of 7.72±0.22%. But in the control groups, the uptake of Ad5-EGFP infected cells at a low level all the time. The titers dependent uptake showed that uptake rate of 99mTc-GH in Ad5-VIE infected MSCs increased with the increasing viral titer (r2=0.86, P<0.05).
3) qRT-PCR analysis for VEGF165 and EGFP mRNA.
The qRT-PCR showed that VEGF165 mRNA and EGFP mRNA expression increased with increasing virus titers (r2=0.91 and r2=0.90, p<0.05). Furthermore there was a good correlation between VEGF165 mRNA and EGFP mRNA (r2=0.99,P<0.05).
4) Semi-quantitative western-blot anlysis for VEGF165 protein
Semi-quantitative western-blot results showed that in the Ad5-VIE groups, expression of cytoplasmic VEGF165 protein increased graduately; while in the Ad5-EGFP group and uninfected control group, VEGF165 protein did not express. The relative amount of VEGF165 and the cell uptake rate increased with the titer gradually increased, a good correlation exited between expression of VEGF165 protein and GGC motifs (r2=0.90, P <0.05).
5) Preliminary study of reporter gene imaging
In vivo imaging shows that after intravenous injection of 99mTc-GH, the bilateral renal and left lower extremity (infection with Ad5-VIE cell group) could be imaging clearly and immediately. With the imaging agent discharge, bladder imaging progressively clearly, the left lower limbs imaging gradually blurred,2h or so, the imaging almost disappeared. In cells transfected with Ad5-EGFP group, MSCs cells group, no significant imaging emerged.
2. Study of monitoring the expression of therapy gene VEGF165 via HSVl-sr39tk/FIAU report gene/probe system
1) Construction and identification of recombinant adenovial vector Ad5-SIV
Plasmid PDC316-sr39tk-IRES-VEGF165 was identified by PCR, corresponding endonucleases digestion and sequencing. The recombinant adenovirus Ad5-SIV was identified by PCR, and the results showed the specific target band. The titer of purified adenovirus stock was 2×1011VP/ml (OD260 method) and 7.9×109IU/ml (classical TCID50 method)
2) Cellular uptake of131I-FIAU
The time dependent cellular uptake studies showed that uptake rates increased rapidly between 30min and 150min and reached a plateau after 150min,at 150min point, the uptake rates were 20.06±1.33%. The uptake rates of 131I-FIAU with Ad5-SIV-infected cells were significantly higher than those with Ad5-EGFP-transfected groups at all time points (t=12.978-38.253).The MOI dependent cellular uptake studies showed that the cellular uptake of 131I-FIAU increased with increasing virus titer (R2=0.89, P<0.05)
3) qRT-PCR analysis for HSV1-sr39tk and VEGF165 mRNA.
HSV1-sr39tk mRNA and VEGF165 mRNA could express successfully after MSCs infected with Ad5-SIV at a series MOI (0,10,25,50,75,100 IU/per cell). Additionally, HSV1-sr39tk mRNA and VEGF165 mRNA expression increased with increasing virus titer (R2=0.97 and R2=0.96, p<0.05). Moreover, the real-time PCR results showed a good correlation between HSV1-Sr39tk mRNA and VEGF165 mRNA (R2=0.93,P<0.05)
4) ELISA anlysis for VEGF165 protein
The data obtained from ELISA showed a good correlation between VEGF protein and adenovirus titer (R2=0.99, P<0.05) after the MSCs was infected with different MOI (0,10, 25,50,75,100 IU/per cell) for 48h., furthermore, VEGF protein secretion was correlated well with the uptake rate of 131I-FIAU (R2=0.84, P<0.05)
Conclusion:The recombinant adenovirus Ad5-VIE and Ad5-SIV was successfully constructed, and the in vitro study confirmed that expression of therapeutic gene VEGF165 correlated significantly with the report gene GGC motifs sequence and HSV1-sr39tk gene. This provided a theoretical basis for monitoring therapeutic gene with nuclear reporter gene imaging.
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