自体骨髓间充质干细胞移植对急性心肌梗死后心律失常影响的实验研究

Effect of bone mesenchymal stem cells transplantation on malignant ventricular arrhythmia induced by electrophysiological stimulation in a mini-swine model of acute myocardial infarction

目的观察自体骨髓间充质干细胞（MSC）移植对小型猪心肌梗死后心律失常的影响，并研究其作用机制。方法利用介入方法制作小型猪心肌梗死模型并进行自体MSC移植。MSC移植组12头和心肌梗死对照组10头，分别于移植2h及4周后通过电生理程序刺激，观察室性心动过速（室速）的发生情况。于建模4周后，通过膜片钳技术研究移植后MSC在心肌环境下的离子通道表达和梗死区域心电异质性变化。结果（1）建模后2hMSC移植组9头（75％）、对照组9头（90％）诱发出室速，两组间差异无统计学意义（P=0．445）。木后4周MSC移植组3头（25％）、对照组8头（80％）可诱发出持续性单形性室速（P=0．012）。（2）MSC移植组的心外膜细胞（Epi）、心内膜细胞（Endo）和中层细胞（M）INa峰值电流密度分别为（-12．43±3．04）pA／pF、（-14．04±3．82）pA／pF和（-29．26±5．70）pA／pF，对照组梗死边缘区的Epi、Endo和M层INa峰值电流密度分别为（-8．47±3．34）pA／pF、（-9．71±3．38）pA／pF和（-18．98±4．05）pA／pF；MSC移植组在三层心肌的表达具有异质性，与对照组相比差异具有统计学意义（P〈0．05）。（3）MSC移植未室速组MSC的Epi、Endo和M层INa失活半数电压分别为（-93．1±13．8）mV、（-95．2±15．5）mV和（-103．4±8．7）mV，MSC移植室速组分别为（-126．2±10．9）mV、（-106．7±11．9）mV和（-105．4±11．0）mV，心肌梗死室速组分别为（-129．1±10．9）mV、（-112．2±9．9）mV和（-109．7±9．3）mV，MSC移植室速组和心肌梗死室速组相比各层差异无统计学意义（P〉0．05），和MSC移植未室速组相比各层差异有统计学意义（P〈0．05）。（4）多元logistic回归分析表明INa失活半数电压（RR=1．449，95％CI 1．276-2．079，P=0．029）、INa峰值密度（RR=1．092，95％CI 1．008-1．917，P=0．012）是影响心肌梗死室性心律失常的独立危险因素。结论自体MSC移植致心律失常可能性较小，且有抑制梗死后心律失常发生的作用。自体MSC在心肌内可分化成为具有心肌细胞离子通道特性的类心肌细胞，其离子通道分化程度可能是影响室性心律失常发生的主要机制。

Objective To investigate the effects of autologous bone mesenchymal stem cells（MSC） transplantation on malignant arrhythmia induced by electrophysiological （EP） stimulation and cardiomyocyte ion channels remodeling in a minl-swine model of acute myocardial infarction （AMI）. Methods Immediately after AMI （LAD occluded for 120 min） , MSC （10 × 10^7, labeled by colloidal gold and cocultivated with 5-azacytidine, 5-aza, n = 12） or equal volume saline （n = 10） were injected through overthe-wire （OTW） balloon in LAD at distal over D1. EP stimulation is performed after 2 hours and 4 weeks in both groups to induce arrhythmia. The variance of heterogeneity of sodium currents （ INa ） and INa steady-state inactivation curves in different zones of infracted wall were investigated by patch clamp technology and the relationship between ionic channel and ventrieular arrhythmia is analyzed. Results EP induced malignant ventricular arrhythmia （VT） rate was similar （MSC 75% vs. saline 90% , P =0.455） at 2 hours post AMI and was significantly lower in MSC group （25% vs. 80%, P =0. 012） at 4 weeks post AMI. The Peak INa current densities of the Endo, Media and Epi were significantly lower in MSC group [ （ - 14. 04 ±3.82） pA/pF, （ - 29.26 ± 5.70） pA/pF, （ - 12.43 ± 3.04） pA/pF] compared those in saline group [ （ - 9.71 ± 3.38 ） pA/pF,（ -18.98 ±4.05） pA/pF,（ -8.47 ±3.34） pA/pF, all P 〈0.05]. The INa steady-state inactivation curves of the Epi, Endo and Media in mini-swine with VT in MSC group [ （ - 126. 2 ± 10.9） mV, （ - 106.7 ± 11.9） mV, （ - 105.4 ± 11.0） mV ] were similar as those in saline group with VT [ （ - 129.1 ± 10.9） mV, （ - 112.2 ± 9.9） mV, （ - 109.7 ± 9.2） mV, all P 〉 0.05 ] while significantly lower compared to MSC group without VT [ （ -93.1 ± 13.8） mV, （ -95.2 ± 15.5） mV, （ - 103.4 ±8.7） mV, all P 〈0.05]. The multiple logistic regression analysis showed that INa current density（ RR = 1. 449, 95% CI 1. 276-2. 079, P = 0. 029） and INa steady-state inactivation curves（ RR = 1. 092, 95% CI 1. 008- 1. 917,P = 0. 012） were the independent factors for reduced VT. Conclusions Autologous MSC attenuated malignant ventrieular arrhythmia induced by EP at 4 weeks in mini-swine with AMI which might due to altered eardiomyocyte ion channels remodeling induced by MSC.