hRAMP1修饰的骨髓间充质干细胞移植对兔球囊损伤血管再狭窄及心功能的影响

Effects of hRAMP1 modified mesenchymal stem cells on restenosis and heart function in rabbit modelof carotid angioplasty and myocardial infarction

目的观察受体活性修饰蛋白1（hRAMP1）修饰的骨髓间充质干细胞（MSC）移植对血管再狭窄及心肌梗死后兔心功能的影响及其基因修饰MSC治疗的安全性。方法建立兔心肌梗死早期再灌注模型并行右侧颈动脉球囊损伤（简称双模型），携带增强型绿色荧光蛋白（EGFP）的腺病毒转染MSC，实验按数字随机分为hRAMP1转染MSC移植组（hRAMP1-MSC组，n,=24），空病毒转染MSC移植组（MSC组，n=24）和PBS移植对照组（对照组，n=24）组，细胞移植后28d免疫荧光检测心肌和血管组织MSC归巢和分化及存活情况，免疫组织化学检测心肌血小板-内皮细胞黏附分子（CD31）及血管α-平滑肌抗体（α-SMA）和增殖细胞核抗原表达（PCNA），HE染色对心肌和血管组织形态学检测，TTC检测心肌梗死面积，超声心动图测定心功能。结果细胞移植28d，损伤血管内膜有EGFP标记的MSC，EGFP表达位置检测到CD31表达，并沿损伤内膜分布，对照组无EGFP表达；与对照组比较，hRAMP1-MSC和MSC组新生内膜面积（0．15±0．05和0．33±0．08比0．77±0．11）与新生内膜和中膜面积比（0．24±0．07和0．51±0．12比1．09±0．23）均明显低（均P〈0．05），尤以hRAMP1-MSC组显著（均P〈0．05）；损伤血管增生内膜细胞α-SMA表达阳性，PCNA表达率在对照组最高，MSC组次之，hRAMP1-MSC组最低（0．627±0．049、0．366±0．013和0．120±O．028，均P〈0．05）。与对照组比较，细胞移植后28dhRAMP1-MSC组和MSC组梗死交界区毛细血管密度增加（44．2±3．8和22．3±1．7比9．3±3．6，均P〈0．05）；心功能均得到改善（射血分数：60．6％±1．5％和50．8％±3．2％比38．2％±2．O％，均P〈0．05）；心肌梗死面积缩小（20．7％±1．4％和33．2％±3．7％比35．6％±2．7％，均P〈0．05），尤以hRAMP1-MSC组显著。结论hRAMP1基因修饰MSC较单纯MSC移植更能改善梗死后心脏功能，促进损伤血管内皮修复，而且显示出更强地抑制血管平滑肌细胞增殖作用；外源基因转染不影响MSC向内皮细胞分化潜能。

Objective To observe the effects of mesenchymal stem cells （MSC） modified by humanreceptor activity-modifying protein 1 （hRAMP1） on restenosis and cardiac function post-myocardial infarction （MI） and explore the therapeutic safety for gene modification of MSC. Methods The doubleinjury rabbit model with MI reperfusion and sacculus damaged atherosclerotic carotid were established according to the previous study. MSC were transfected with adenovirus vector with enhanced green fluorescence protein （EGFP） and then introduced into rabbit model. The animals were randomly divided into Ad-EGFP-hRAMP1-MSC transfection group （ hRAMP1-MSC group, n = 24） and Ad-EGFP-MSC transfection group （MSC group, n = 24）, and PBS transfection group （C group, n = 24）. At Day 28 posttransplantation, the injured carotid arteries and infarction myocardium were harvested to detect the expression of EGFP-positive MSC and assess organization morphology by hematoxylin and eosin, triphenyltetrazolium chloride or immunohistochemical stains and heart function by echocardiography. Results On flow cytometry. most cells expressed CD29 and CD90 while few ones expressed CD45. MSC with EGFP and a continuous expression of CD31 were found in intima of damaged carotid of both hRAMP1-MSC and MSC, but the expression of EGFP was not found in the control group. At Day 28 post-transplantation, the improvement of heart function and the decrease of infarct size were found in the hRAMP1-MSC and MSC groups compared with that in the control group（EF： 60.6%± 1.5%,50.8%±3.2% vs 38.2%±2.0%, infarct size： 20.7% ±1.4% ,33.2%±3.7% vs 35.6%±2. 7% , all P 〈0. 05）, especially much higher in hRAMP1MSC group. At Day 28 post-transplantation, the area of intima hyperplasia and the rate of neointima and media in the hRAMP1-MSC group were lower than those in the MSC and C groups（0. 15 ±0. 05 and 0. 33±0. 08 vs 0. 77 ±0. 11,0. 24 ±0. 07 and 0. 51 ±0. 11 vs 1.09 ±0. 23, all P 〈0. 05 ）. Also the expression of α-SMA was found in the hyperplasia intima.The expression of proliferating cell nuclear antigen significantly decreased in the hRAMP1-MSC group than those of the MSC and control groups （0. 120 ±0. 028 vs 0. 366 ± 0. 013 and 0. 627 ± 0. 049, both P 〈 0. 05 ）. And it was much lower in the MSC group than that in the control group. Conclusions Compared with MSC alone, hRAMPl-modified MSC have the potency not only of more improvement on cardiac function and the recovery of damaged endothelium, but also of significant inhibition of the proliferation of vascular smooth muscle cells. The recombinant hRAMP1 adenovims vectors do not affect the differentiation potential MSC into endothelial cells.