Comparative study of cellular kinetics of reporter probe [(131)I]FIAU in neonatal cardiac myocytes after transfer of HSV1-tk reporter gene with two vectors.
OBJECTIVE
Reporter gene imaging is a promising approach for noninvasive monitoring of cardiac gene therapy. In this study, HSV1-tk (herpes simplex virus type 1 thymidine kinase) and FIAU (2'-fluoro-2'-deoxy-1-beta-d-arabinofuranosyl-5-iodouracil) were used as the reporter gene and probe, respectively. Cellular uptakes of radiolabeled FIAU of neonatal rat cardiac myocytes transferred with HSV1-tk were compared between two vectors, adenovirus and liposome. The aims of this study were to choose the better vector and to provide a theoretical basis for good nuclide images.


METHODS
Neonatal cardiac myocytes were obtained from rat heart by single collagenase digestion. HSV1-tk inserted into adenovirus vector (recombinant adenovirus type 5, Ad5-tk) and plasmid (pDC316-tk) coated with Lipofectamine 2000 (pDC316-tk/lipoplex) were developed; thus, HSV1-tk could be transferred into neonatal cardiac myocytes. FAU (2'-fluoro-2'-deoxy-1-beta-d-arabinofuranosyluracil) was labeled with (131)I, and the product was assessed after purification with reversed-phase Sep-Pak C-18 column. The uptake rates of [(131)I]FIAU in the transferred cardiac myocytes at different times (0.5, 1, 2, 3, 4 and 5 h) were detected. Furthermore, mRNA expression and protein expression of HSV1-tk were detected by semiquantitative reverse-transcriptase polymerase chain reaction and immunocytochemistry.


RESULTS
FAU could be labeled with (131)I, and the labeling efficiency and radiochemical purity rates were 53.82+/-2.05% and 94.85+/-1.76%, respectively. Time-dependent increase of the accumulation of [(131)I]FIAU was observed in both the Ad5-tk group and the pDC316/lipoplex group, and the highest uptake rate occurred at 5 h, with peak values of 12.55+/-0.37% and 2.09+/-0.34%, respectively. Greater uptakes of [(131)I]FIAU in Ad5-tk-infected cells compared with pDC316/lipoplex-transfected ones occurred at all the time points (t=12.978-38.253, P<.01). The exogenous gene expression by polymerase chain reaction in adenovirus vector-infected cardiac myocytes was significantly higher than that in pDC316-tk/lipoplex-transducted ones (semiquantitative analysis, 3.11+/-0.14 versus 1.60+/-0.05, P<.01). Immunocytochemistry showed that the transferred cardiac myocytes successfully expressed the target protein, and the positive rates were 81.70+/-0.40% in Ad5-tk and 22.06+/-0.32% in liposome (P<.01).


CONCLUSIONS
Both adenovirus and liposome could transfer reporter gene into cardiac myocytes successfully, and the expressed exogenous protein could form functional enzymes efficiently. However, the adenovirus vector acted more efficiently than did liposome, with a higher uptake rate of the reporter probe. Thus, adenovirus is competent for gene transfer in cardiac reporter gene imaging.
Lan
Xiaoli
X
Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. lxl730724@hotmail.com
Yin
Xiaohua
X
Wang
Ruihua
R
Liu
Ying
Y
Zhang
Yongxue
Y
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D000831:Animals, Newborn; D001086:Arabinofuranosyluracil / Q000031:analogs & derivatives* / Q000493:pharmacokinetics; D002478:Cells, Cultured; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D018259:Herpesvirus 1, Human / Q000201:enzymology*; D007150:Immunohistochemistry; D007457:Iodine Radioisotopes; D007553:Isotope Labeling; D008081:Liposomes; D032383:Myocytes, Cardiac / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013937:Thymidine Kinase / Q000235:genetics*
D007457:Iodine Radioisotopes; D008081:Liposomes; D001086:Arabinofuranosyluracil; C043457:fialuridine; D013937:Thymidine Kinase