肾上腺髓质素基因转染增强骨髓间充质干细胞移植对心肌梗死大鼠心室重构及心功能的影响

Adrenomedullin gene transfection enhances the therapeutic effects of transplantation of bone marrow mesenchymal stem cells on cardiac function and ventricle remodeling in acute myocardial infarction rats

目的:研究肾上腺髓质素(ADM)基因转染增强骨髓间充质干细胞(MSCs)移植对心肌梗死大鼠心室重构及心功能的治疗作用。方法:贴壁法体外分离、扩增培养大鼠骨髓间充质干细胞,用X-gal染色测含ADM基因的重组腺病毒(Ad-ADM)对MSCs感染率,ELISA检测Ad-ADM转染后细胞上清液ADM的含量。通过结扎左冠状动脉前降支制备大鼠心肌梗死模型;以DAPI标记细胞通过心肌局部注射移植MSCs;采用生理记录仪测量大鼠的心功能,荧光显微镜观察移植细胞在心脏存活及分布,免疫组化检测ADM在心肌梗死区的表达及心梗区血管密度;天狼猩红染色检测胶原含量,用偏振光显微镜分析Ⅰ/Ⅲ型胶原比率。结果:重组腺病毒对MSCs的转染率与病毒感染复数(MOI)具有量效关系,MOI为150时细胞的感染率达95.4%;转染Ad-ADM后,MSCs可有效表达ADM,7 d时达到表达高峰,与对照组相比明显增加[(26.53±1.42vs1.34±0.08)ng/L,P<0.05],15 d后与空白对照组无差别[(2.20±1.44vs1.52±0.33)ng/L,P>0.05];在受体大鼠的心脏切片上有DAPI标记的移植细胞的存活;与对照组及其它组相比,ADM基因修饰的MSCs组心肌梗死区ADM的表达增加;与对照组比较,单纯细胞移植组心梗区新生血管密度增高(P<0.01);而与单纯MSCs移植组及单纯Ad-ADM注射组比较,ADM基因修饰的MSCs移植组梗死区血管密度增高更明显(P<0.05);单纯MSCs移植组能降低梗死区Ⅰ/Ⅲ型胶原比率(P<0.01),并能改善左室功能;与单纯细胞组比较ADM基因修饰的MSCs移植组梗死区Ⅰ/Ⅲ型胶原比率降低更明显(P<0.05),而左室功能改善更多。结论:ADM转染的MSCs移植可能通过局部ADM表达的增加,增强MSCs移植的血管新生作用及降低梗死区Ⅰ/Ⅲ型胶原比率,从而增强单纯MSCs细胞移植改善心功能的作用。

AIM： To investigate adrenomedullin gene transfection enhances the therapeutic effects of homogeneous transplantation of bone marrow mesenchymal stem cells （MSCs） on cardiac function and ventricle remodeling in acute myocardial infarction rats. METHODS： MSCs were isolated and expanded using the preplating method. The infection efficiency of adenovirus vector to MSCs was tested by X - gal staining. Ad - ADM expression in MSCs and its secretion in culture medium were measured by ELISA. The left anterior descending branch of rats was ligated to establish a myocardial infarction model. The MSCs were labeled by DAPI, and were directly implanted into the acute infarct site via focal injection. Four weeks later, cardiac function was evaluated using physiological recorder. Hearts were harvested and sliced to be analyzed by immunohistochemistry （ factor Ⅷ and ADM） and the DAPI - labeled cells were identified. Sirius red staining was used to identify interstitial collagen on slides. Analysis of collagen type Ⅰ and Ⅲ was performed using a polarized filter on sections stained for collagen with Sirius red, and the ratio of collagen type Ⅰ and Ⅲ were detected. RESULTS ： With X - gal staining, MSCs were effectively transfected by adenovirus in vitro. The transfection efficiency showed the dose - effect relationship with multiplicities of infection （MOI）. When MOI was 150, the infection efficiency was 95.4%. The expression of ADM was traced in culture medium and expressed in the time - dependent manner. A maximum production of ADM was observed at 7 d after infection [ （26. 53 ± 1.42 ） ng/L vs （ 1.34 ± 0.08 ） ng/L, P 〈 0. 05 ] , and ADM secretion reduced to normal level at 15 d [ （2. 20 ± 1.44） ng/L vs （ 1.52 ± 0. 33 ） ng/L, P 〉 0. 05 ]. DAPI - labeled MSCs transplantation was found in the hearts of the recipients. Immunohistochemical studies demonstrated that intense immunostaining for ADM was higher in Ad - ADM plus MSCs group, compared to other groups. Compared with control, MSCs transplantation significantly increased capillary density in infarct area （ P 〈 0.01 ）. A combination of Ad - ADM trensfection and MSCs transplantation demonstrated a further increase in capillary density compared with Ad - ADM or MSCs alone. MSCs trans- plantation decreased the ratio of collagen type ⅠandⅢ, obviously improved the left ventricular functions. Furthermore the combination treatment resulted in further decrease in the ratio of collagen type Ⅰ and Ⅲ, and significantly improved the left ventricular functions. CONCLUSION： Ad - ADM transfection enhances the angiogenic potency of MSCs transplantation and decreases the ratio of collagen type Ⅰ and Ⅲ through increasing ADM expression in infarct area, thus contributes to reverse the ventricular remodeling and improves the cardiac function.