Experimental Study Of Cardiac Reporter Gene Imaging With FIAU

ObjectiveThe aim of this study was to evaluate the possibility of HSV1-tk which was transduced by an adenoviral vector, and radiolabled FIAU as a marker gene / marker substrate for imaging reporter gene expression in the heart. In this study, we radiolabeled ¹²⁵I-FIAU, cultured myocardial cells, constructed recombinant adenovirus carrying HSV1-tk gene. The susceptibility to the vector, ¹²⁵I-FIAU uptake ratios and the relations to MOIs and times were evaluated through a series of tests.Methods1. FAU was labeled with ¹²⁵I-NaI and the biodistribution of ¹²⁵I-FIAU in the normal mice was to evaluate. FAU was radiolabeled with ¹²⁵I-NaI by Iodogen iodization. The product was purified through Sep-Pak C-18 reverse phase chromatograph column. Radiochemical purity and the stability in vitro were measured by TLC-SG. We studied the biodistribution of ¹²⁵I-FIAU in the normal mice, and the distributions in cardiac muscle and blood should be emphasized.2. To culture normal SD neonatal rat myocardial cells. To culture purified neonatal rat myocardial cells by the simplified and modified methods. The neonatal rat heart tissue was digested once in collagenase typeâ…¡including BSA. The purified cadiocytes were obtained by differential adhesion after 1 hour. All of these steps were carried out in 25-37℃. We evaluated the survival rate through staining the cell with typsin on the day of 5th, 10th, 15th, 20th, 25th, 30th, and then observed cell form under the inverted biological microscop and detected the purity of the cadiocytes which were cultured for 48 hours by the method of immunohistochemistry.3. To construct the Ad-CMV-HSV1-tk recombinant. The compounds of Ad-CMV-HSV1-tk were recombinated by using HSV1-tk as a reporter gene, CMV as a promoter and adenoviral as a vector. This part of the research was collaborated with Vector Gene Company LTD.4. Study viability of the myocardical cells after Ad5-tk infection and the relations between ¹²⁵I-FIAU uptake and MOIs or time. HSV1-tk was transferred into myocardial cells after the fact that cells was infected by Ad -CMV-HSV1-tk. MTT test was used to evaluate the viability of the myocardial cells, and the relation between cell viability and the MOIs was analysised. RT-PCR test was used to identify HSV1-tk expression. Cultural medium of myocardial cells was changed to those including ¹²⁵I-FIAU at the time of 24 hours after being transferred. The uptake rates at the different times of 30min, 1h, 2h, 3h, 4h and 5h were observed. The relationship between uptake rates and different times or different MOIs was evaluated. Results1. FAU was effectively radiolabeled with ¹²⁵I-NaI by Iodogen iodization. The radiochemistry purity of product which was purified through Sep-Pak the C-18 reverse phase chromatograph column was 98.60%±0.52%. ¹²⁵I-FIAU was steady and the radiochemistry purity was exceeded 95% from 0.5 h to 24h in PBS and fresh normal human serum at the temperature of 37℃. The biological distribution experiment of ¹²⁵I-FIAU in the normal Kunming mice suggested that the distribution in blood was washed out quickly, the kidney was the main metabolism organ in which biodistribution of ¹²⁵I-FIAU was highest at the beginning. Little distribution of ¹²⁵I-FIAU was observed in cardiac muscle after 24h.2. The neonatal rat heart tissue was digested once in collagenase typeâ…¡including BSA. The heart tissue was digested almost completely at the 6th hour. The survival rate was more than 95% on the day of 5th, 10th, 15th, 20th, 25th and 30th. Under the inverted biological microscop, we observed the cells connecting each other and beating after 24 hours. The purity of the cadiocytes which were cultured for 48 hours was (97.1±1.9)% by the method of immunohistochemistry.3. Collaborated with Vector Gene Company LTD., the identification of pDC316-tk was correct and we obtained Ad–CMV–HSV1-tk recombinant of which v.p. was 1.1×1012 and TCID was 1.6×10¹⁰.4. MTT test suggested that relation between the MOIs and cell viability after Ad5-HSV1-tk infection was negative. RT-PCR showed that HSV1-tk gene expression in myocardial cells after transferring the HSV1-tk gene by adenovirus. RT-PCR demonstrated the susceptibility of myocardial cells to adenovirus. The statistic analysis showed that uptake ratio was positive related to MOI and time.Conclusions¹²⁵I-FIAU which had ideal biodistribution in normal mice could be radiolabeled by Iodogen iodization and be purified through Sep-Pak C-18 reverse phase chromatograph column. It was possible to get purer neonatal rat myocardial cells which survived long enough for many researches by the simplified and modified methods. Using an adenoviral vector, HSV1-tk can be successfully expressed in myocardial cell in vitro, yielding high uptake of radiolabled FIAU. Therefore, cardiac reporter gene imaging could be achievement by using adenovirus as vector, HSV1-tk as reporter gene and ¹²⁵I-FIAU as imaging agent.