【作者】 沈艳霞；

【导师】 张永学；

【作者基本信息】 华中科技大学 ， 影像医学与核医学， 2008， 博士

【摘要】 目的:将编码GGC肽(diglycylcysteine,双甘氨酰半胱氨酸)的基因序列与人生长抑素2a型受体(hSSTr2a)基因的膜外N端开放阅读框相连接,构建融合受体重组腺病毒载体Ad5-GGC-hSSTr2a-IRES-EGFP。通过体外受体结合试验、细胞摄取试验、核素报告基因显像等一系列体外与体内实验研究,探讨以GGC序列为报告基因进行核素报告基因显像的可行性,以期建立一种新型的核素报告基因显像系统。方法:1.重组腺病毒载体Ad5-GGC-hSSTr2a-IRES-EGFP的构建与表达鉴定。采用RT-PCR方法扩增hSSTr2a基因全长cDNA序列,将GGC肽的基因序列(亲水序列P1,疏水序列P2)与hSSTr2a的膜外N端开放阅读框相连接,通过一系列基因工程技术构建同源重组腺病毒骨架质粒pAdEasy-1/GGC-hSSTr2a-IRES-EGFP (pAd-SIG-P1, P2)。提取pAd-SIG-P1和pAd-SIG-P2质粒并转化大肠杆菌DH5a感受态细胞,酶切线性化后经脂质体(Lipofectamine2000)介导转染HEK293细胞,同源重组产生融合受体重组腺病毒载体Ad5-GGC-hSSTr2a-IRES-EGFP ( Ad5-P1/STR ,Ad5-P2/STR)。经PCR鉴定后进行大量扩增和纯化,采用PCR、荧光显微术鉴定目的基因的表达,并测定病毒感染滴度。2. Ad5-GGC-hSSTr2a-IRES-EGFP在人乳腺癌MCF-7细胞内的表达。以MOI = 50的重组腺病毒(Ad5-P1/STR, Ad5-P2/STR)感染人乳腺癌细胞MCF-7后,分别在24h、48h、72h和96h采用荧光显微术与流式细胞术检测细胞内EGFP的表达。研究99mTc-HYNIC-TOCA与受感染MCF-7细胞的体外受体结合特性。并利用99mTc-GH放射性摄取实验研究GGC肽在MCF-7细胞的表达。3. Ad5-GGC-hSSTr2a-IRES-EGFP在荷瘤裸鼠体内的报告基因显像。建立荷人乳腺癌裸鼠模型(双侧上肢荷瘤),以重组腺病毒Ad5-P1/STR、Ad5-P2/STR、Ad5-EGFP及Ad5-TIS(Ad5-HSV1-TK-IRES-hSSTr2a)对照性注射于两侧瘤体内(1×109IU),于感染48h后分别进行以99mTc-GH和99mTc-HYNIC-TOCA为显像剂的核素报告基因SPECT显像以及GFP活体荧光显像。同时以1×108～3×109 IU梯度滴度的重组腺病毒分组感染两侧瘤体,48小时后进行核素报告基因SPECT显像。通过感兴趣区(region of interest, ROI)技术进行半定量分析,计算肿瘤/非肿瘤比值(T/NT)。显像完毕后进行生物学分布实验,测定离体肿瘤标本的放射性计数,计算显像剂在裸鼠体内各脏器及肿瘤组织的克组织百分摄取率(%ID/g)。分别取感染Ad5-P1/STR和Ad5-P2/STR两侧肿瘤组织固定保存,以免疫组织化学技术检测病毒感染后肿瘤组织内hSSTr2a蛋白的表达。结果:1.重组腺病毒载体Ad5-GGC-hSSTr2a-IRES-EGFP的构建与表达鉴定。重组腺病毒载体Ad5-GGC-hSSTr2a-IRES-EGFP(Ad5-P1/STR,Ad5-P2/STR)构建正确,PCR可特异性扩增出hSSTr2a片段(1212bp),荧光显微镜下显示出绿色荧光蛋白EGFP (enhanced green fluorescent protein)的表达。测得Ad5-P1/STR和Ad5-P2/STR的TCID50值均为8.2×109/mL。2. Ad5-GGC-hSSTr2a-IRES-EGFP在人乳腺癌MCF-7细胞内的表达。重组腺病毒以MOI=50感染MCF-7后,细胞内EGFP的表达随时间的延长而逐渐增强,至感染后48h,显微镜下绿色荧光最强且集中,流式细胞术检测其EGFP表达率为99.6%。72h后荧光逐渐减弱,流式细胞术检测EGFP表达率亦逐渐减低。以MOI=50的重组腺病毒感染后,MCF-7细胞对99mTc-GH的摄取率随时间延迟而逐渐增高,30min后重组腺病毒感染组与Ad5-EGFP感染的对照组细胞的摄取率有显著性差异( Ad5-P1/STR vs Ad5-EGFP : t=4.267~39.728 , Ad5-P2/STR vs Ad5-EGFP :t=4.362~9.455 , P均<0.01 )。而Ad5-P1/STR组与Ad5-P2/STR组无显著性差异(Ad5-P1/STR vs Ad5-P2/STR:t=0.347~1.083,P>0.05)。以MOI=0～100的重组腺病毒感染MCF-7后,细胞对99mTc-GH的摄取率均随感染滴度的增加而逐渐增加,且细胞对99mTc-GH的摄取率与Ad-P1/STR或Ad-P2/STR的感染滴度之间存在良好的相关性,R2分别为0.8997和0.9242 (P均<0.01)。3. Ad5-GGC-hSSTr2a-IRES-EGFP在荷瘤裸鼠体内的报告基因显像。活体GFP荧光显像可见,感染Ad5-P1/STR、Ad5-P2/STR和Ad5-EGFP的肿瘤部位均可见绿色荧光,而感染Ad5-TIS的肿瘤部位未见绿色荧光。体内放射性核素显像可见,感染重组腺病毒Ad5-P1/STR和Ad5-P2/STR的肿瘤部位可被99mTc-GH显像,而感染Ad5-EGFP的肿瘤部位无明显显像剂浓聚,其5h的T/NT比值有显著性差异(tP1, EGFP=4.780, tP2, EGFP=8.981, P均<0.05)。感染Ad5-P1, P2/STR的肿瘤部位在99mTc-GH显像与99mTc-HYNIC-TOCA显像中均可见显影,而感染Ad5-TIS的肿瘤部位仅在99mTc-HYNIC-TOCA显像中显影。以不同滴度的重组腺病毒感染肿瘤后显像提示,肿瘤组织对显像剂的摄取随感染滴度的增高而逐渐增加,且平均% ID/g值与Ad5-P1/STR或Ad5-P2/STR感染滴度的对数值呈线性相关(相关系数R2分别为0.8458和0.8948,P均<0.05)。而感染Ad5-P1/STR和Ad5-P2/STR的两组肿瘤的平均% ID/g值之间无显著性差异(t=0.039~2.314,P均>0.05)。免疫组织化学检测显示,感染Ad5-P1/STR和Ad5-P2/STR的肿瘤组织内均可检测到hSSTr2a的表达。结论:含有GGC肽和hSSTr2a基因序列的融合受体重组腺病毒载体Ad5-P1/STR和Ad5-P2/STR构建成功。重组腺病毒感染荷瘤裸鼠后,GGC肽在其体内的表达可被核素显像所显示,肿瘤组织对显像剂的摄取与病毒感染滴度具有良好的相关性。GGC序列有望作为新型报告基因用于核素报告基因显像。更多还原

【Abstract】 Objective To construct the recombinant adenoviral vectors Ad5-GGC-hSSTr2a-IRES-EGFP by fusing the GGC (diglycylcysteine) motifs on the open reading frame of extracellular N-terminus of human somatostatin receptor subtype 2a gene. A series of experiments in vitro and in vivo such as characteristics of receptor-binding, uptake kinetics and radionuclide reporter rene imaging were performed to investigate the feasibility of GGC motifs used for radionuclide reporter gene imaging as a reporter gene.Methods1. Construction and the expression of recombinant adenoviral vectors encoding GGC and human somatostatin receptor subtype 2aThe full length of hSSTr2a cDNA was amplified from plasmid pGEM-T/hSSTr2a by RT-PCR, and its open reading frame of extramembranous N-terminal was combined with the GGC motifs (hydrophilic motif P1, or hydrophobic motif P2). A series of gene engineering techniques were performed to construct homologous recombinant adenovirus plasmids pAdEasy-1/GGC-hSSTr2a-IRES-EGFP (pAd-SIG-P1, P2). The recombinant plasmids pAd-SIG-P1 and pAd-SIG-P2 were extracted and transformed into competent E coli DH5α. After been linearized, the recombinant plasmids were transfected into HEK 293 cells by lipofectamine 2000 to generate recombinant adenoviral vectors Ad5-GGC-hSSTr2a- IRES-EGFP (Ad5-P1/STR, Ad5-P2/STR). After the amplification and purification of the recombinant adenovirus, PCR and fluorescence microscopy detection were performed to confirm the expression of hSSTr2a and enhanced green fluorescent protein (EGFP). TCID50 of Ad5-P1/STR and Ad5-P2/STR were detected.2. Expression of the recombinant adenovirus Ad5-GGC-hSSTr2a-IRES-EGFP in MCF-7 cells.The human breast cancer cells MCF-7 were infected with the recombinant adenovirus (Ad5-P1/STR, Ad5-P2/STR) at a wide range multiplicity of infection (MOI) from 0 to 100 infectious units (IU/per cell). The fluorescence microscopy and flow cytometry were used to validate the expression of enhanced green fluorescent protein (EGFP) at 24h, 48h, 72h and 96h after infection. Then the characteristics of receptor-binding of 99mTc- hydrazinonicotinyl-Tyr3-octreotate (99mTc-HYNIC-TOCA) to MCF-7 cells infected with the recombinant adenovirus were studied. And the protein expression of GGC motifs in MCF-7 cells was determined by experiments of the radioactive uptake rate of 99mTc-GH in MCF-7 cells.3. Reporter gene imaging using the recombinant adenovirusAd5-GGC-hSSTr2a-IRES-EGFP in nude mice bearing MCF-7 tumors. BALB/c A nude mice bearing MCF-7 tumor (both upper limbs) were injected with 1×109 IU of Ad5-P1/STR, Ad5-P1/STR or Ad5-TIS (Ad5-HSV1-TK-IRES-hSSTr2a). 48 hours later, the whole-body GFP fluorescence tomography and radionuclide reporter gene SPECT imaging were performed using 99mTc-GH and 99mTc-HYNIC-TOCA. On the other hand, we infected both MCF-7 tumors of nude mice with recombinant adenovirus at a multiplicity infection from 1×108 to 1×109 IU, and then performed reporter gene SPECT imaging. Semiquantitative analysis was studied to evaluate the tumor/non-tumor ratios (T/NT) with region of interest (ROI). Bio-distribution of the probe in each organ and tumor of mice was analyzed withγcounter ex vivo. Immunohistochemistry technique was used to validate the expression of hSSTr2a in tumors infected with Ad5-P1/STR and Ad5-P2/STR, separately. Results1. Construction and the expression of recombinant adenoviral vectors encoding GGC and human somatostatin receptor subtype 2aThe recombinant adenoviral vectors Ad5-GGC-hSSTr2a-IRES-EGFP (Ad5-P1/STR, Ad5-P2/STR) were constructed correctly. The expression of hSSTr2a could be identified by PCR, and the expression of EGFP could be identified by fluorescence microscopy. TCID50 of both Ad5-P1/STR and Ad5-P2/STR were 8.2×109/mL.2. Expression of the recombinant adenovirus Ad5-GGC-hSSTr2a-IRES-EGFP in MCF-7 cells.After infected with the recombinant adenovirus at a MOI of 50, the expression level of EGFP in MCF-7 cells increased gradually with time. At 48 hours after infection, the green fluorescent in MCF-7 was detected strongest and localized by fluorescence microscopy, and the expression rate was determined 99.6% by flow cytometry. 72 hours later, the green fluorescent in MCF-7 tapered, and its expression rate also decreased. After infected with the recombinant adenovirus at a MOI of 50 infectious units, the radioactive uptake rate of 99mTc-GH in MCF-7 cells increased gradually with time. There was significant difference between experimental group (infected with Ad5-P1/STR or Ad5-P2/STR) and control group (infected with Ad5-EGFP) after 30 min (Ad5-P1/STR vs Ad5-EGFP: t=4.267~39.728, P<0.01; Ad5-P2/STR vs Ad5-EGFP: t=4.362~9.455, P <0.01). But there was no significant difference between Ad5-P1/STR and Ad5-P2/STR (Ad5-P1/STR vs Ad5-P2/STR:t=0.347~1.083,P>0.05). Infected with the recombinant adenovirus at a wide range MOI from 0 to 100 infectious units, the radioactive uptake rate of 99mTc-GH increased gradually with increasing viral titer. Furthermore, the uptake rate of 99mTc-GH correlated significantly with the viral titer (R2=0.8997 for Ad5-P1/STR; R2=0.9242 for Ad5-P2/STR, P both<0.01). 3. Reporter gene imaging using the recombinant adenovirusAd5-GGC-hSSTr2a-IRES-EGFP in nude mice bearing MCF-7 tumors. Green fluorescence could be shown in tumors that infected with Ad5-P1/STR, Ad5-P2/STR and Ad5-EGFP in the whole-body GFP fluorescence tomography, but there was no green fluorescence in tumors that infected with Ad5-TIS. Radionuclide reporter gene imaging in vivo showed tumors that infected with Ad5-P1/STR or Ad5-P2/STR. However, tumors infected with Ad5-EGFP could not be shown. There was significant difference between their T/NT at 5h (tP1, EGFP=4.780, P<0.05; tP2, EGFP=8.981, P<0.05). Tumors infected with Ad5-P1, P2/STR could be shown in the SPECT imaging with 99mTc-GH or 99mTc-HYNIC-TOCA. However, tumors infected with Ad5-TIS only could be shown in the SPECT imaging with 99mTc-HYNIC-TOCA. Radioactivity uptake of 99mTc-GH in tumors increased with increasing infection titer of adenovirus. The mean %ID/g of tumors positively correlated with the logarithm of infection titer of Ad5-P1/STR or Ad5-P2/STR (R2P1=0.8458, P<0.05; R2P2=0.8948, P<0.05). There was no significant difference between %ID/g of tumors that infected with Ad5-P1/STR and Ad5-P2/STR respectively (t=0.039~2.314, P both>0.05). The expression of hSSTr2a could be detected in tumors infected with Ad5-P1/STR or Ad5-P2/STR.Conclusions: The recombinant adenovirus vectors Ad5-P1/STR and Ad5-P2/STR were constructed successfully. Infected with the recombinant adenovirus, the expression of GGC peptide in nude mice could be clearly imaged with radionuclide imaging in vivo. The GGC motifs could be used in radionuclide reporter imaging as a new reporter gene.更多还原