Study On The Uptake Dynamics Of HSV1-tk/FIAU Reporter Gene Imaging System In Neonatal Cardiac Myocytes In Vitro

ObjectiveReporter gene imaging is a promising approach for noninvasive monitoring of cardiac gene therapy. In the present study, the recombinant plasmid and adenoviral vector carrying reporter gene, herpes simplex virus type 1 thymidine kinase(HSV1-tk), were constructed and transducted into neonatal cardiac myocytes, and a series of in vitro studies on the uptake dynamics were carried out on the cells transferred to compare both vectors for cardiac reporter gene imaging.Methods1. The construction and identification of recombinant plasmid and adenovirus vectorThe recombinant plasmid was constructed by inserting HSV1-tk gene into multiple cloning sites of the vector plasmid pDC316, with cytomegalovirus (CMV) as a promoter. It was used as a shuttle plasmid to cotransfect with adenovirus skeletal plasmid in the 293 cells, where the recombinant adenovirus, Ad5-tk, was packaged. It was finished with the cooperation of Vector Gene Technology Company Ltd. in Beijing.2. The culture of neonatal cardiac myocytesThe neonatal Sprague–Dawley rats (1-3 days) ventricular tissue was obtained and cut into small pieces. Subsequently, the cardiac myocytes were dispersed by incubation at 37°C in phosphate buffered saline (PBS) containing 1mg/ml collagenase type II and 10mg/ml bovine serum albumin(BSA) for 5 hours. Then,the cells were collected by centrifugation and pre-plated for about 1 hours to minimize fibroblast contamination. The cardiac myocytes were cultured in the Dulbecco's modified Eagle's medium nutrient mixture F-12 ham (DMEM/F12) supplemented with 10% newborn bovine serum (NBS) at 37°C in a humidified 5% CO2 atmosphere to permit cell adherence and growth. Cell morphology was observed under the invert microscope.3. Cardiac myocytes transfection by liposome-coated plasmid The liposome-coated plasmid, pDC316-tk/lipoplex, was prepared according to the recommended procedure provided by liposome manufacturer. When the cells were approximately 90% confluence, pDC316-tk/lipoplex was added into each well. After incubation at 37°C in 5% CO2 for 4 h, the transfer medium was removed and replaced with growth medium. The transferred cells were cultured further for 24 h.4. Cardiac myocytes infection by recombinant adenovirusWhen the cardiac myocytes were approximately 90% confluence, the cells were counted to calculate the volume of Ad5-tk with multiplicity of infection (MOI) of 20,40,80. The diluted virus was added into each well and replaced with growth medium after 4h. The transferred cells were cultured further for 24 h.5. The radioiodination of FAUUsing solid phase oxidation with Iodogen, FAU was labeled with [131]I. The product, [131I] FIAU, was purified on a reverse-phase Sep-Pak C-18 column and the labeling efficiency was then assayed. Moreover, the product was identified for the radiochemical purity assessment. To study the stability in vitro, the [131I]FIAU was incubated at 37°C for 4h,12h,24h before the aliquots were removed for radiochemical purity analysis.6. Comparison of cellular uptake of [131I]FIAU and gene expression in cardiac myocytes transferred by adenovirus or liposome vectorThe uptake experiment was carried out after the cardiac myocytes were transferred for 24 h, and the uptake efficiency was compared between the two different vectors; The expression of HSV1-tk in the cells transferred was detected in the mRNA and protein level by semi-quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and immunocytochemical staining;The viability of transferred cardiac myocytes were evaluated by MTT assay.Results1. The recombinant plasmid, pDC316-tk, was identified by PCR,restriction enzyme digestion and sequencing. The titer of packaged recombinant adenovirus, Ad5-tk, was 1.1×1012 v.p./ml, and its infection titer was 1.6×1010 IU/ml determined by TCID50 method.2. The cardiac myocytes obtained from single collagenase digestion had strong viability with cross-linking with each other and some beat in a cluster at 24h. Several clusters reached the same frequency at 48h. 3. FAU could be labeled efficiently by solid phase oxidation with Iodogen. The radiolabeling efficiency of [131I]FIAU was 53.82%±2.05% (n=5) and the radiochemical purity of peak fractions was 94.85%±1.76%(n=5). For the stability analysis, the radiochemical purity of [131]FIAU maintained above 90% after incubated at 37°C for 4h,12h and 24h.4. Significant, time-dependent increase of the accumulation of [131I]FIAU was observed in both Ad5-tk group and pDC316/lipoplex group, and the highest uptake rate occurred at 5h, with peak values of 12.55%±0.37% and 2.09%±0.34% respectively. However, it also indicated that greater accumulation of [131I]FIAU for Ad5-tk infected cells compared with pDC316/lipoplex transfected ones occurred for all the time points (P<0.01). After transferred for 24h, the cardiac myocytes showed significant expression of HSV1-tk mRNA, while the control groups showed negative. Moreover, the exogenous gene expression in the adenovirus vector-infected cardiac myocytes was significantly higher than in the pDC316-tk/lipoplex-transducted ones. Immunocytochemistry showed that the transferred cardiac myocytes successfully expressed the target protein, and the positive rate was (81.70±0.40)% and (22.06±0.32)% respectively, with significant difference between them(P<0.01). The MTT assay indicated that the viability of cardiac myocytes infected by Ad5-tk with higher title decreased more significantly than the pDC316-tk/lipoplex transfected ones.ConclusionBoth vectors can transfer the reporter gene HSV1-tk into the cardiac myocytes successfully, and the expressed exogenous protein can form a functional enzyme as to uptake the reporter probe much efficiently. But the transferred cardiac myocytes mediated by the adenovirus vector act even more efficiently than the ones mediated by liposome-coated plasmid with higher uptake rate. Thus the adenovirus is competent for the gene transfer in cardiac reporter gene imaging. It provides a theoretical foundation for the cardiac imaging of reporter gene in vivo, and may be used for the noninvasive monitoring of myocardial gene therapy.