目的 建立一種基于心臟類(lèi)器官模型的場(chǎng)電位測(cè)定方法，用于臨床前藥源性心臟毒性評(píng)價(jià)。方法 自組織法體外構(gòu)建人誘導(dǎo)多能干細(xì)胞（hiPSC）的心臟類(lèi)器官模型，顯微鏡下觀察形態(tài)，培養(yǎng)至第18天（D18）免疫熒光法檢測(cè)成纖維細(xì)胞標(biāo)志物波形蛋白（VIM）、內(nèi)皮細(xì)胞標(biāo)志物（CD31）、心肌細(xì)胞標(biāo)志物肌鈣蛋白T（cTnT）的表達(dá)；選用鉀通道阻滯劑E-4031作為陽(yáng)性藥評(píng)估方法的有效性，待心臟類(lèi)器官狀態(tài)穩(wěn)定（D18≤培養(yǎng)時(shí)間≤ D30）、選取信號(hào)強(qiáng)度穩(wěn)定且持續(xù)時(shí)間超過(guò)3 d的類(lèi)器官分為對(duì)照組（不加藥）、E-4031（0.5 μmol·L^-1）組，加藥前1 h進(jìn)行培養(yǎng)基更換，加藥后使用微電極陣列技術(shù)對(duì)場(chǎng)電位進(jìn)行檢測(cè)，記錄波形和電生理參數(shù)；應(yīng)用建立的模型對(duì)美西律（3、10、30 μmol·L^-1）潛在的心律失常風(fēng)險(xiǎn)進(jìn)行評(píng)估。結(jié)果 自D1起，hiPSC濃縮聚集呈球狀，形成擬胚體，隨著誘導(dǎo)分化時(shí)間延長(zhǎng)，細(xì)胞球直徑逐漸增加，球體邊緣逐漸清晰；D8，球體產(chǎn)生自發(fā)性跳動(dòng)； D18，心臟類(lèi)器官狀態(tài)穩(wěn)定，細(xì)胞球體直徑大小趨于穩(wěn)定，直徑為（1 499.03± 101.60） μm，形狀規(guī)則，邊緣光滑清晰，搏動(dòng)頻率規(guī)律，可達(dá)（33.39± 8.14）次·min^-1。VIM、CD31、cTnT在心臟類(lèi)器官內(nèi)高度表達(dá)，到達(dá)預(yù)期成熟特征。與對(duì)照組相比，加入0.5 μmol·L^-1的E-4031 1 h時(shí)，T波明顯延長(zhǎng)； 1 h起場(chǎng)電位持續(xù)時(shí)間（FPD）變化率顯著升高（P＜0.05、0.01），校正場(chǎng)電位持續(xù)時(shí)間（FPDc）的變化率顯著升高（P＜0.05）；搏動(dòng)周期增加，3 h時(shí)達(dá)到較高幅度，尖峰振幅的變化率呈現(xiàn)出下降趨勢(shì)，但差異均不顯著。類(lèi)器官暴露于3、10 μmol·L^-1美西律后，與對(duì)照組相比T波均無(wú)明顯位移，當(dāng)美西律濃度達(dá)30 μmol·L^-1時(shí)無(wú)場(chǎng)電位信號(hào)。與對(duì)照組相比，3 h時(shí)10 μmol·L^-1的美西律組FPDc變化率顯著增加（P＜0.05）；搏動(dòng)周期變化率在美西律濃度為10 μmol·L^-1時(shí)存在明顯變化，3 h時(shí)達(dá)到較高幅度，存在顯著性差異（P＜0.001）； 3 μmol·L^-1的美西律組尖峰振幅的變化率表現(xiàn)出明顯的下降趨勢(shì)，5 h時(shí)存在顯著性降低（P＜0.05），當(dāng)美西律濃度達(dá)到10 μmol·L^-1時(shí)，尖峰振幅的變化率愈加明顯，1、3、5 h時(shí)呈現(xiàn)顯著性下降（P＜0.05）。結(jié)論 建立的心臟類(lèi)器官的場(chǎng)電位檢測(cè)方法可以用于評(píng)估藥物引起的心臟毒性，可在藥物研發(fā)早期提供較為準(zhǔn)確的數(shù)據(jù)支持。

Objective A field potential measurement method based on a heart organoid model was established for preclinical druginduced cardiotoxicity evaluation. Methods Human induced pluripotent stem cells (hiPSCs) were used to construct a heart organoid model in vitro by self-organization. The morphology was observed under a microscope. On day 18 (D18) of culture, the expression of fibroblast marker vimentin (VIM), endothelial cell marker CD31, and cardiomyocyte marker cardiac troponin T (cTnT) was detected by immunofluorescence. Potassium channel blocker E-4031 was used as a positive drug to evaluate the effectiveness of the method. When the heart organoids were in a stable state (D18 ≤ culture time ≤ D30), organoids with stable signal intensity and duration over three days were divided into a control group (without drug) and an E-4031 (0.5 μmol·L^-1) group. The culture medium was changed 1 h before drug administration. Field potentials were detected using microelectrode array technology after drug administration, and waveforms and electrophysiological parameters were recorded. The potential arrhythmogenic risk of mexiletine (3, 10, 30 μmol·L^-1) was evaluated using the established model. Results From D1, hiPSCs condensed and aggregated into spherical structures, forming embryoid bodies. As the induction differentiation time increased, the diameter of the cell spheres gradually increased, and the edges of the spheres became clearer. On D8, the spheres began to beat spontaneously. On D18, the heart organoids were in a stable state, the diameter of the cell spheres tended to be stable, with a diameter of (1 499.03 ± 101.60) μm, regular shape, smooth and clear edges, and regular beating frequency, reaching (33.39 ± 8.14) beats·min^-1. VIM, CD31, and cTnT were highly expressed in the heart organoids, reaching the expected mature characteristics. Compared with the control group, when 0.5 μmol·L^-1 E-4031 was added for 1 h, the T wave was significantly prolonged; the field potential duration (FPD) change rate significantly increased from 1 h (P < 0.05, 0.01), and the corrected field potential duration (FPDc) change rate significantly increased (P < 0.05); the beating period increased, reaching a higher amplitude at three hours, and the peak amplitude change rate showed a downward trend, but the differences were not significant. After exposure to 3 and 10 μmol·L^-1 mexiletine, there was no significant displacement of the T wave compared with the control group. When the mexiletine concentration reached 30 μmol·L^-1, there was no field potential signal. Compared with the control group, the FPDc change rate in the 10 μmol·L^-1 mexiletine group significantly increased at 3 h (P < 0.05); the beating period change rate showed a significant change at a mexiletine concentration of 10 μmol·L^-1, reaching a higher amplitude at 3 h, with a significant difference (P < 0.001); the peak amplitude change rate in the 3 μmol·L^-1 mexiletine group showed a significant downward trend, with a significant decrease at five hours (P < 0.05). When the mexiletine concentration reached 10 μmol·L^-1, the peak amplitude change rate became more obvious, showing significant decreases at 1, 3, and 5 h (P < 0.05). Conclusion The field potential detection method of heart organoids can be used to evaluate drug-induced cardiotoxicity and provide relatively accurate data support in the early stage of drug development.

R965

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