北京生物醫(yī)學(xué)工程

機械牽張對人誘導(dǎo)多能干細胞分化來源的心肌細胞成熟的影響

Effect and mechanism of mechanical stretch on maturation of induced pluripotent stem cell derived cardiomyocytes

作者：戴越周帆鄭建偉穆軍升伯平尤斌

單位：首都醫(yī)科大學(xué)附屬北京安貞醫(yī)院心臟外科（北京100029）<br />解放軍總醫(yī)院第三醫(yī)學(xué)中心超聲科(北京 100039)<br />陽光融和醫(yī)院心臟中心(山東濰坊 261205)<br />通信作者: 穆軍升，主任醫(yī)師，教授，博士研究生導(dǎo)師，留法博士。E-mail: [email protected]

關(guān)鍵詞： CDM3；人誘導(dǎo)多能干細胞分化來源心肌細胞；機械牽張；Piezo1

分類號：R318.01

出版年·卷·期（頁碼）：2023·42·2（130-137）

摘要：

目的通過使用標準的化學(xué)定義和基于小分子誘導(dǎo)方案（chemically defined medium, 3 components, CDM3）獲得人誘導(dǎo)多能干細胞分化來源的心肌細胞（human induced pluripotent stem cell-derived cardiomyocytes, hiPSC-CMs），進一步對其施加機械牽張，探討機械牽張對hiPSC-CMs成熟的影響及潛在機制。方法復(fù)蘇、培養(yǎng)和鑒定hiPSCs后，將hiPSCs接種到覆有基質(zhì)膠的傳統(tǒng)培養(yǎng)皿上。24 h后在熒光顯微鏡下通過DAPI熒光觀察細胞生長情況，并通過OCT4熒光進行hiPSCs干性鑒定，在細胞匯合度達到80％時更換CDM3分化培養(yǎng)基，分化6 d后將獲得的hiPSC-CMs分為對照組和機械牽張組，拉伸結(jié)束后重新種板培養(yǎng)24 h后在熒光顯微鏡下通過DAPI熒光觀察細胞生長情況，通過cTnT和MLC2V熒光進行hiPSC-CMs心肌細胞標志物鑒定，Piezo1熒光進行潛在機制研究。結(jié)果 hiPSCs與傳統(tǒng)培養(yǎng)基共培養(yǎng)24 h免疫熒光顯示：OCT4發(fā)出綠色熒光，hiPSCs保持干性。DAPI發(fā)出藍色熒光：細胞呈克隆生長，細胞形態(tài)均一。機械牽張結(jié)束后重新種板培養(yǎng)，24 h后免疫熒光顯示機械牽張組較對照組心肌標志物cTnT和MLC2V表達增高(P<0.05)；機械牽張組較對照組機械敏感型離子通道Piezo1蛋白表達增高(P<0.05)。結(jié)論機械牽張可以促進人誘導(dǎo)多能干細胞分化來源心肌細胞的成熟，這可能與Piezo1蛋白表達增高有關(guān)。

Objective Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were obtained using standard chemical definitions and small molecule induction protocol (CDM3), and further mechanical stretching was applied to them to explore the effect of mechanical stretch on hiPSC-CMs maturation and the potential mechanism. Methods After recovery, culture, and identification of hiPSCs, hiPSCs were inoculated on traditional culture dishes covered with matrigel. After 24 h, the cell growth was observed by DAPI fluorescence under a fluorescence microscope, and dry identification of hiPSCs was performed by OCT4 fluorescence. CDM3 differentiation medium was replaced when the cell connexion reached 80%. After 6 days of differentiation, hiPSC-CMs obtained were divided into control group and mechanical stretch group. After the stretching, Piezo1 fluorescence was used to investigate the potential mechanism of Piezo1 fluorescence. The growth of the cells was observed by DAPI fluorescence under a fluorescence microscope. The hiPSC-CMs myocardial markers were identified by cTnT and MLC2V fluorescence. Results Immunofluorescence of hiPSCs co-cultured with traditional medium for 24 h showed that OCT4 emitted green fluorescence and hiPSCs remained dry. DAPI gave off blue fluorescence: the cells were cloned and homogeneous. After the mechanical stretching was over, the plates were re-cultured. After 24 h, immunofluorescence showed that the expressions of cTnT and MLC2V in mechanical stretch group were higher than those in control group (P<0.05). The protein expression of Piezo1 in the mechanically sensitive ion channel was increased in the mechanical stretch group compared with that in the control group (P<0.05). Conclusions Mechanical extension may stimulate the maturation of human induced pluripotent stem cell-derived cardiomyocytes by facilitating the increased expression of the protein Piezo1.

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