| 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. |
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