新型PET心肌灌注显像剂18F-MyoZone对大鼠心肌细胞的摄取作用及其机制

Mechanism of uptake and retention of 18F-MyoZone in cardiomyocytes:A novel PET myocardial perfusion imaging agent

目的探讨新型18F标记PET心肌灌注显像剂18F-MyoZone的心肌细胞摄取及滞留机制。方法①抑制线粒体呼吸链酶活性机制的研究:使用线粒体呼吸链酶复合体Ⅰ(MC-Ⅰ)活性测定试剂盒测定不同浓度的19F-MyoZone溶液(起始浓度15 μmol/L,3倍稀释,稀释12个点)测定19F-MyoZone抑制线粒体呼吸链酶活性的半数抑制率(IC50)。②18F-MyoZone与MC-Ⅰ结合的放射自显影研究:采用大鼠心肌组织切片,分别与生理盐水和4种已知MC-Ⅰ抑制剂(4 μmol/L鱼藤酮、4 μmol/L 19F-Flurpiridaz、4 μmol/L 19F-MyoZone及4 μmol/L哒螨灵)孵育,再加入18F-MyoZone进行放射自显影研究,检测18F-MyoZone是否可与心肌细胞内MC-Ⅰ特异性结合;再将大鼠心肌组织切片与不同浓度的鱼藤酮或19F-MyoZone溶液(抑制剂浓度采用0、20 μmol/L、2 μmol/L、200 nmol/L及20 nmol/L)孵育30 min后,加入18F-MyoZone再孵育30 min,清洗切片后储磷屏显影10 min,获取曝光后的显影图像,计算抑制剂各浓度的抑制率。③鱼藤酮抑制心肌细胞对18F-MyoZone的摄取研究:采用新生大鼠原代心肌细胞,以不同浓度的19F-MyoZone或鱼藤酮(起始浓度10 μmol/L,3倍稀释,稀释12个点)孵育15 min,完成后加入18F-MyoZone(约17 kBq)50 μl,孵育30 min。收集裂解液,对其进行放射性计数,计算鱼藤酮的IC50。④18F-MyoZone心肌细胞的外流实验:采用新生大鼠原代心肌细胞,加入18F-MyoZone(约37 kBq)500 μl孵育30 min后清洗再孵育,依照各时间点(0、10、20、30、60、90、120、150 min)依次收集细胞上清液及裂解液并测定放射性计数,计算心肌细胞外流的放射性占比。结果酶活性研究显示19F-MyoZone可有效抑制线粒体呼吸链酶复合物的活性(IC50为229.9 nmol/L),并呈剂量依赖关系;放射自显影研究显示MyoZone可以特异性结合于MC-Ⅰ,结合位点与抑制剂鱼藤酮、哒螨灵及19F-Flurpiridaz一致。鱼藤酮抑制心肌细胞对18F-MyoZone的摄取研究显示,18F-MyoZone可被大鼠心肌细胞摄取,随着鱼藤酮抑制浓度的增加,心肌细胞中的放射性摄取值逐渐减少,抑制剂的IC50为7 nmol/L;心肌细胞外流实验显示,18F-MyoZone可被大鼠心肌细胞摄取并滞留,在0~30 min外流逐渐增加,60~150 min保持稳定,滞留量约为总摄取量的20%。结论 18F-MyoZone可与大鼠心肌细胞内的MC-Ⅰ特异性结合,并可在心肌细胞内长时间滞留。18F-MyoZone是极具研究价值的MC-Ⅰ抑制剂类心肌灌注显像剂。

Objective To investigate the mechanism of uptake and retention of a novel PET myocardial perfusion imaging agent 18F-MyoZone in cardiomyocytes.Methods 1) Mechanism of inhibition of mitochondrial respiratory chain enzyme activity:With mitochondrial respiratory chain enzyme complex Ⅰ (MC-Ⅰ) activity assay kit,a sequence of 19F-MyoZone solution (start at 15 μmol/L,3 times dilution,12 points) was interacted with MC-Ⅰ to detect the half inhibition rate (IC50) of 19F-MyoZone inhibiting mitochondrial respiratory chain activity.2) Autoradiography experiment of 18F-MyoZone combining with MC-Ⅰ:Myocardial tissue sections of neonatal rat were hatched with normal saline and 4 known MC-Ⅰ inhibitors [rotenone (4 μmol/L),19F-Flurpiridaz (4 μmol/L),19F-MyoZone (4 μmol/L) and pyridaben (4 μmol/L)],and then 18F-MyoZone was added to autoradiography for detecting whether 18F-MyoZone can specifically bind the cardiomyocytes MC-Ⅰ;and then the myocardial tissue sections of rat were hatched for 30 min with different concentrations of rotenone or 19F-MyoZone solution (0,20 μmol/L,2 μmol/L and 200 nmol/L,20 nmol/L),then the 18F-MyoZone was added and hatched for 30 min again,developing for 10 min with phosphor storage screen after cleaning the slice,and analyzing and calculating the inhibition rate of each inhibitor concentration.3) Experiment of rotenone inhibitting the uptake of 18F-MyoZone by cardiomyocytes:Neonate rats’ primary cardiomyocytes were cultured for 15 min with different concentrations of 19F-MyoZone or equivalent rotenone (start at 10 μmol/L,3 times dilution,12 points),then 50 μl of 18F-MyoZone (about 17 kBq) was added and culturing for 30 min.The lysate was then collected,the radioactivity was counted and the IC50 of rotenone was calculated.4) Outflow experiment of 18F-MyoZone from cardiomyocytes:Cultured neonate rats’ primary cardiomyocytes were interacted with 500 μl of 18F-MyoZone (about 37 kBq) for 30 min,then the cell supernatant and lysate were separated to do photon counts at the time points of 0,10,20,30,60,90,120 and 150 min.The ratio of cardiomyocyte outflow rate was then calculated.Results Enzyme activity studies showed that 19F-Myo Zone may effectively inhibit the activity of MC-Ⅰ(IC50=229.9 nmol/L) in a dose dependent manner.MyoZone could specifically bind to MC-Ⅰwith binding sites in accordance with the inhibitors rotenone,pyridaben and 19F-Flurpiridaz.Experiment of rotenone inhibitting the uptake of 18F-MyoZone by cardiomyocytes showed that 18F-MyoZone could be absorbed by rat’s cardiomyocytes,and with the increase of rotenone concentration,the radio uptake of cardiomyocytes decreased gradually with inhibitor IC50 as 7 nmol/L.Outflow experiment showed that rat’s cardiomyocytes could uptake 18F-MyoZone and stably detain over time.The outflow rate increased gradually within 0-30 min,and then maintained constantly from 60 min to 150 min.The amount of retention was about 20% of the entire uptake.Conclusions 18F-MyoZone may specifically bind MC-Ⅰand detain for a long time in rat’s cardiomyocytes.18F-MyoZone is a valuable myocardial perfusion imaging agent with great research value.