心房肌细胞移植修复重建窦房结功能的实验研究

Transplanted neonatal cardiomyocytes as a potential biological pacemaker in canines with sick sinus syndrome

目的通过制备犬的病态窦房结（病窦）综合征模型，研究心房肌细胞移植修复重建窦房结功能，构建生物起博器的可行性。方法用射频消融（n=5）及化学消融（n=3）的方法制备犬的病窦综合征模型。采用酶法及差速贴壁法分离纯化新生犬的右心房细胞（包括窦房结），4，6-二脒基-2-苯基吲哚（DAPI）标记细胞核，移植前制备成单个心肌细胞悬液备用。在模型制备后4周，用微量注射器将1 ml细胞悬液多点注射至实验组的窦房结及其周围区域，对照组则单纯注射1 ml低糖培养液（L-DMEM）培养液，手术前后行电生理监测及组织学检查。结果消融窦房结后，出现了稳定的窦性心动过缓[（110±10）次／min]。细胞移植后第5天实验组体表心电图显示较前明显增快，为（150±12）次／min左右，电生理检测窦房结恢复时间（SNRT）、校正窦房结恢复时间（CSNRT）及窦房传导时间（SACT）均有明显缩短，微电极记录显示其自发性搏动频率快于对照组。而对照组自始至终无明显改变。HE染色及免疫荧光检测均显示移植细胞与宿主细胞间连接蛋白43（Cx43）及N-钙黏素（N-cadherin）的表达。结论心房肌细胞移植构建生物起搏器具有可行性，提供了生物介入治疗的新途径。

Objective The study aims to prove the hypothesis that transplanted neonatal cardiomyocytes can improve pacing function in canine sick sinus syndrome models, Methods Canine sick sinus syndrome models were made by catheter （ n = 5 ） and chemical ablation （ n = 3 ） of sinus nodes, Neonatal atrial cardiomyocytes including sinus nodal cells were dissociated and labeled with DAPI, Then the cells were injected into sinus node areas of canine sick sinus syndrome models. The improvement of pacing function was evaluated by electrocardiography and microelectrode studies, Immunofluorescence was used to assess the survival of donor cells and expression of gap junction between donor and host cardiomyocytes, Results Sinus bradycardia appeared after ablating sinus nodes. Five days after neonatal atrial cardiomyocytes transplanted, the heart rate was significantly faster than before [ （ 150±12 ） bpm vs （ 110± 10） bpm,P 〈 0, 05]. Eleetrophysiology demonstrated that SNRT, CSNRT, SACT are significantly shortened, Microeletrode recordings showed that spontaneously beating rate was higher than the eontrol groups [ （ 150±11 ） bpm vs （ 117±16 ） bpm, P 〈 0, 05）], Morever, eonnexin43 and N-cadherin positive junetions between donor and host cardiomyoeytes were identified by immunofluorescenee examination in eelltransplanted groups. Conehlslons Neonatal atrial cardiomyocytes are able to survive and integrate into the host myocardium, and show an improvement of pacing function in canines with sick sinus syndrome,