腺病毒载体介导的HSV1-tk报告基因感染心肌细胞后摄取^(125)I-FIAU的实验研究

Uptake of reporter probe ^(125)I-FIAU in neonatal cardiac myocytes after adenoviral transfer of herpes simplex virus type I thymidine kinase reporter gene

携带HSV1-tk的重组腺病毒(Ad5-tk)以感染复数(MOI)为20、40、80的病毒滴度感染体外培养的乳鼠原代心肌细胞;通过四甲基偶氮唑蓝(MTT)比色法检测感染后心肌细胞活力;应用半定量逆转录-聚合酶链反应(RT-PCR)检测基因转导后心肌细胞内HSV1-tkmRNA的表达情况,并研究对报告探针125I-FIAU的摄取情况。结果显示:不同滴度Ad5-tk感染组心肌细胞存活率与时间均呈负相关,且病毒滴度越大,心肌细胞存活率越低;心肌细胞感染Ad5-tk后24h,RT-PCR即可检测到HSV1-tkmRNA的表达,且MOI=40感染组的表达量高于MOI=20感染组(P<0.01);心肌细胞感染不同滴度Ad5-tk后对125FIAU的摄取与时间呈正相关,且MOI=40感染组在各时间点的摄取率高于其余各组(P<0.01)。表明腺病毒载体介导的报告基因HSV1-tk进入心肌细胞后,能表达生成有活性的磷酸化酶HSV1-TK,使心肌细胞对核素标记报告探针125I-FIAU有明确的摄取,摄取程度与时间呈正相关,且感染滴度为40时摄取率最高,这为活体报告基因心脏显像提供了细胞水平依据。

Cardiac myocytes were obtained from neonatal rat heart and cultured. HSVI-tk, chosen as the reporter gene, was inserted into adenovirus vector （Ad5-tk）, and the recombinant adenovirus infected neonatal cardiac myocytes with multiplicity of infection （MOI） of 20, 40 and 80. MTT assay was used to detect the viability of infected cardiac myocytes, and semi-quantitative reverse transcriptase polymerase chain reaction （RT-PCR） was applied to determine HSV1-tk mRNA expression in the infected cells. The accumulation of 125I-FIAU into infected cardiac myocytes was detected as uptake rate. The survival rate of infected cells had negative correlation with time, and it decreased with higher viral multiplicity of infection （MOI）. After transferred for 24 h, the cardiac myocytes showed significant expression of HSVI-tk mRNA, while the control groups showed negative. Moreover, the exogenous gene expression in the infected ceils at MOI 40 was significantly higher than in the ones at MOI 20 （P〈0.01）. The cardiac myocytes infected with increasing titer of Ad5-tk showed positive correlation between uptake rate and time, and the uptake rate of 125I-FIAU in the MOI 40 group was significantly higher than all the other groups （P〈0.01）. The HSV1-tk reporter gene could be transferred into the neonatal cardiac myocytes successfully with adenovirus vector, and encoded for HSV1-TK catalyzing phosphorylation so as to accumulate the marker substrates 125I-FIAU. The uptake rates, correlated to the uptake time, were the highest with transferred titer 40 at all the time points. This provides some cellular evidence for cardiac imaging of reporter gene in vivo.