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腺病毒介导RAMP1修饰MSCs移植对兔心肌梗死后心肌新生血管形成的影响
The effect of MSCs transfected with hRAMP1 transplantation on rabbits angiogenesis in myocardial infarction

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DOI:
作者:
龙仙萍1，赵然尊1，石蓓1，许官学1，沈长银1，王正龙1，喻田2
LONG Xian-ping1，ZHAO Ran-zun，SHI Bei 1，XU guan-xue
作者单位:
1第一附属医院心血管内科，2医学与分子生物学研究中心
1Deparment of Cardiology，The First Affiliated Hospital，2 research center of Medical and molecular biology，Zun yi Medical College，Zun yi
关键词:
心肌梗死；间充质干细胞；受体活性修饰蛋白-1；血管形成；心功能
myocardial infarction； mesenchymal stem cells； Receptor activity modified protein-1(RAMP1) ；angiogenesis；cardiac function
摘要:
目的 hRAMP1修饰的MSCs移植治疗急性心肌梗死模型兔后，观察其对心肌梗死后兔新生血管及心功能的影响。 方法 密度梯度离心结合贴壁法培养兔的MSCs，取第3～4代（P3～4）MSCs用于实验；应用带EGFP报告基因的hRAMP1重组腺病毒载体和空病毒载体转染MSCs，建立心肌梗死兔模型，实验随机分Ad-EGFP-hRAMP1-MSCs移植组（hRAMP1-MSCs组，n=12）、Ad-EGFP -MSCs移植组（MSCs组，n=12）和等体积的PBS液注射组（对照组，n=12）；模型建立后40分钟经局部注射等体积MSCs或PBS至心肌梗死交界区。分别于细胞移植前及移植后1d、7d、14d和28d用ELISA法测定血清VEGF浓度；细胞移植后4周，超声心动仪检测兔心脏功能，HE染色评估心肌病理学改变、TTC染色测定心肌梗死面积、免疫组织化学染色检测梗死交界区心肌CD31的表达并计数新生毛细血管密度。 结果 培养细胞经流式细胞仪鉴定显示绝大部分细胞表达CD29和CD90，少量细胞表达CD45；心肌梗死模型建立后外周VEGF浓度水平增加，尤以hRAMP1-MSCs组在细胞移植后1d、7d和14 d VEGF 浓度增加明显， MSCs组次之；细胞移植后4周，hRAMP1-MSCs组心肌纤维化程度、心肌梗死面积均明显小于MSCs组和对照组（P＜0.05），MSCs组次之，免疫组化染色显示hRAMP1-MSCs组新生毛细血管密度明显高于MSCs组和对照组（P＜0.05），而MSCs组又优于对照组（P＜0.05）。 结论 hRAMP1基因修饰MSCs移植较单纯MSCs移植更能增加梗死交界区心肌新生血管密度，改善梗死后心脏的功能。
Objective: To observe the effect of MSCs transplantation modified by hRAMP1 gene on angiogenesis and cardiac function of acute myocardial infarction rabbit model. METHODS: MSCs were collected through density gradient centrifugation and adherent culture, P3-4 of MSCs was used in the experiment. MSCs were transfected with adenovirus vector with enhanced green fluorescent protein (EGFP), or with both EGFP and hRAMP1. Rabbit model with acute myocardial infarction were made according to the previous study. All animals were randomly divided into three groups: Ad-EGFP-hRAMP1-MSCs transplantation group (hRAMP1-MSCs group, n=12)，Ad-EGFP-MSCs transplantation group (MSCs group, n=12), and isovolumetric PBS administration group (control group, n=12). We administrated the same volume of MSCs or PBS into the junctional area of infracted myocardium after building the rabbit model 40 minutes. At the different time-points of cell transplantation after 1d, 7d, 14d and 28d, the serum level of VEGF was measured by ELISA. At the fourth weeks after cell transplantation, we determined the cardiac function by echocardiography, myocardial pathological changes by HE staining, myocardial infarction size by TTC staining; the expression of CD31 in the junctional area of infracted myocardium was examined by immunohistochemistry staining and the density of new blood capillaries was counted. RESULTS: Results Flow cytometry demonstrated that most MSCs expressed CD29 and CD90, very few cells expressed CD45. Peripheral levels of VEGF were increased in myocardial infarction model. In the MSCs group, VEGF levels were increased at 1d, 7d and 14d after cell transplantation, whereas VEGF levels in the hRAMP1-MSCs group were markedly increased. At 4th weeks after cell transplantation, Myocardial fibrosis and myocardial infarct size in hRAMP1-MSCs group were lower significantly than that in MSCs group and control group (P＜0.05）, and that much lower in MSCs group compared with control group. Capillary density detected by immunohistochemistry staining in hRAMP1-MSCs group was markedly higher than that in MSCs group and control group(P＜0.05）, and that much higher in MSCs group compared with control group(P＜0.05). CONCLUSION: Transplantation of MSCs transfected with hRAMP1 can improve angiogenesis and cardiac function in the infarcted area of the hearts of rabbi.

 

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