乳鼠心肌细胞联合人工基质移植改善大鼠心肌梗死后心功能的实验研究

Implantation of neonatal cardiomyocytes plus artificial matrix improve heart function in a rat infarct model

目的探讨体外构建液态组织工程学心肌(engineering heart tissue,EHT)的方法,以及其移植于大鼠心肌梗死区域后是否能存活并改善心脏功能。方法结扎雌性Sprague-Dawley(SD)大鼠左冠状动脉,制作心肌梗死模型。分离、标记及培养新生1～3天SD乳鼠心肌细胞。心肌梗死后合格的动物模型随机分成EHT组(n=12)、单纯心肌细胞组(n=12)、单纯基质组(n=12)、对照组(n=11)。移植术后4周,用超声心动图及离体心脏灌流2种手段评价心功能,标本取材做病理学检查及Y染色体多聚酶链检测(polymerase chain reaction,PCR)。结果PCR检测证实EHT组和单纯细胞组移植细胞存活,但病理学检查表明EHT组的移植细胞排列更紧密,细胞间有连接蛋白形成。超声心动图显示EHT组的左室缩短分数最高(22.82±3.44)%,高于细胞组(20.55±4.11)%、人工基质组(17.05±4.57)%和对照组(19.80±3.98)%,P=0.012。心脏灌流模型提示在左心室球囊容积为0.06、0.08、0.10ml时,左心室发展压及dp/dt差异有统计学意义,EHT组最佳(P<0.05)。结论体外成功构建液态EHT后,可以通过注射方法移植于大鼠心肌梗死区域。液态EHT移植后较单纯细胞移植组织形态更佳、更能改善梗死心脏的功能。

Objective To investigate wheather injectable engineering heart tissue （EHT） can survive and improve heart function after transplantation into infarct area. Methods Ventricular cardiomyocytes from 1-3 day-old Sprague-Dawley（SD） rats were isolated by using trypsin method, and then labeled and cultured. The left coronary of female SD rats was ligated to create a myocardial infarct model. Three weeks later, the qualified animals were randomized into four groups： EHT group （n = 12）, which were transplanted with both cardiomyocytes and matrix;cell transplantation group （n = 12） ;matrix group （n = 12）, control （n = 11 ）. Four weeks after implantation, echocardiography and Langendoff model were used to assess heart function, and then the hearts were harvested for pathological examination. Meanwhile polymerase chain reaction （PCR） was performed to detect SRY gene on Y chromosome. Results The grafted cells were identified in both EHT and cell transplantation group by either pathology or PCR. But in EHT group, transplanted cells formed more condensed tissue, and produced definite connected protein. Data of fraction shortness from echocardiography are showed as follows ： EHT group, （22. 82 ± 3.44） % ; ceil transplantation group, （20.55 ± 4. 11 ） %, matrix group, （ 17.05 ± 4.57）% ; control, （ 19.80 ± 3.98 ）% （P = 0. 012）. Langendoff examination revealed significant differences among four groups when left ventricular balloon volume was at the level of 0. 06 ml, 0. 08 ml and 0. 10 ml, in which EHT group had the highest developed pressure and dp/dt. Conclusion It is feasible to fabricate injectable EHT in vitro. The fabricated EHT could survive in the myocardial infarct area after transplantation in a rat model and improve heart function due to better histological configuration.