基于iPS技术探索肿瘤靶向药物心肌毒性的实验研究

Experimental Study on Cardiotoxicity of Tumor Targeting Drugs Based on iPS Technology

酪氨酸激酶抑制剂(tyrosine kinase inhibitors,TKIs)的心血管副作用越来越得到人们的重视。人诱导多能干细胞(hiPSCs)在体外可分化为各种类型的体细胞,且来源充足,为药物早期毒性评价提供了一个较理想的细胞模型。该研究通过TKIs类药物舒尼替尼干预人诱导性多能干细胞来源心肌细胞(human induced pluripotent stem cell-derived cardiomyocytes,hiPSC-CMs),观察其心肌毒性。体外培养结合化学诱导定向分化得到hiPSC-CMs,实验分为对照组以及5.6μmol/L舒尼替尼(IC50浓度,CCK8实验结果)干预24 h和48 h组。通过免疫荧光观察HIF-1α的表达、细胞缺氧状态及线粒体膜电位变化;通过透射电镜观察线粒体结构变化;通过流式细胞术探索线粒体膜电位变化与细胞凋亡情况;通过Western blot技术检测各组HIF-1α蛋白的表达。结果显示,与对照组相比,观察到舒尼替尼干预组线粒体结构出现明显损伤,线粒体膜电位受到破坏,出现明显凋亡和坏死现象,且细胞处于缺氧状态,HIF-1α被过度激活。总之,舒尼替尼可引起心肌细胞线粒体结构破坏、细胞缺氧及凋亡和坏死,并且可诱导HIF-1α过度激活。

Increasing attention has been paid to the cardiovascular side effects of TKIs(tyrosine kinase inhibitors).Human induced pluripotent stem cells can differentiate into various types of somatic cells in vitro and have a rich resource,which provides an ideal choice for early drug toxicity evaluation.In this study,the cardiotoxicity of sunitinib was observed in sunitinib-exposed hiPSC-CMs(human induced pluripotents stem cell-derived cardiomyocytes).Cultured in vitro and chemically induced,hiPSC-CMs were divided into control group,5.6μmol/L sunitinib(IC50 concentration,determined by CCK8 method)intervention group for 24 h and 48 h group.The expression of HIF-1α,the state of hypoxia and the change of mitochondrial membrane potential were evaluated by immunofluorescence;the structural changes of mitochondria were observed by transmission electron microscope;mitochondrial membrane potential and cell apoptosis were examined by flow cytometry.The results showed that sunitinib induced significant mitochondrial structure damage,mitochondrial membrane potential dissipation,apparent hypoxia,and elevated HIF-1αexpression,which was paralleled with overt apoptosis and necrosis.In summary,sunitinib can cause mitochondrial structural damage,hypoxia,apoptosis and necrosis,as well as hyperactivation of HIF-1αin hiPSC-CMs.