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目的建立重组人血管内皮生长因子(VEGF)165基因转染大鼠骨髓间充质干细胞(MSC)的方法,探讨该细胞心肌移植对缺血性心脏病心功能及血管新生的影响,并比较联合治疗与单独基因或细胞治疗的疗效差异。方法采用密度梯度离心-贴壁培养法获取Wistar近交系大鼠MSC,用脂质体将pcDNA3.1-hVEGF165转入该细胞,通过ELISA、RT—PCR和Western印迹检测后者VEGF的表达情况；以Wistar近交系大鼠建立心肌缺血模型,随机分成4组(每组12只),心肌梗死模型建立2周后,联合组在心肌梗死区移植转染VEGF165基因的MSC,细胞组移植等量的MSC,基因组注射脂质体-pcDNA3.1-VEGF165DNA复合物,对照组注射等容积培养液；另取12只未结扎冠脉的大鼠为假手术组。细胞移植4周后,用Buxco系统检测心功能；然后处死动物,取心肌标本,测量心肌梗死面积；用5-溴脱氧尿嘧啶(Brdu)、肌钙蛋白T免疫组化双染法确定移植细胞的存活与分化；用Ⅷ因子染色法检测血管新生,RT-PCR法检测VEGF165基因的体内表达情况。结果(1)pcDNA3.1-hVEGF165基因通过脂质体转染大鼠MSC后获稳定表达；(2)移植4周后,联合组心肌梗死面积(27．8％±3．0％)明显低于细胞组(37．0％±10．1％)与基因组(37．1％±5．2％,均P<0．05),心功能改善优于细胞组与基因组；(3)联合组心肌梗死区毛细血管密度(每视野40．2个±5．5个)高于细胞组(27．2个±6．3个,P<0．01)和对照组(18．5个±5．8个,P<0．01),较基因组(35．8个±7．7个)亦有增加的趋势(P=0．189)；(4)Brdu、肌钙蛋白T双染示各治疗组心肌梗死区心肌细胞数量不同程度的多于对照组；(5)联合组VEGF基因的体内表达(hVEGFmRNA相对含量0．18±0．04)高于基因组(0．10±0．03,P<0．01)。结论转染VEGF基因的MSC移植可使鼠冠脉结扎造成的心肌梗死面积缩小、心功能明显改善,其疗效优于单独应用基因或细胞治疗,为缺血性心脏病的细胞基因联合治疗提供了理论依据。
Objective To establish a method of transfection of rat bone marrow mesenchymal stem cells (MSC) with recombinant human vascular endothelial growth factor (VEGF) 165 gene to investigate the effect of myocardial transplantation on the cardiac function and angiogenesis of ischemic heart disease. Efficacy of treatment versus single gene or cell therapy. Methods Wistar inbred rat MSCs were obtained by density gradient centrifugation-adherent culture. The pcDNA3.1-hVEGF165 was transfected into the cells by lipofectamine. The expression of VEGF was detected by ELISA, RT-PCR and Western blotting The model of myocardial ischemia in Wistar rats was established and randomly divided into 4 groups (12 in each group). After establishing myocardial infarction model for 2 weeks, the combined group was transplanted with MSCs transfected with VEGF165 gene in myocardial infarction area. The same amount of MSCs was injected into the lipofectamine-pcDNA3.1-VEGF165 DNA complex. The control group was injected with equal volume of culture fluid. Twelve non-ligated coronary arteries were used as sham-operated group. After 4 weeks of cell transplantation, heart function was detected by Buxco system. Then the animals were sacrificed and the myocardial samples were taken to measure the area of myocardial infarction. The transplanted cells were identified by double staining with Brdu and Troponin T The survival and differentiation of VEGF165 gene were detected by Ⅷ factor staining. The expression of VEGF165 gene in vivo was detected by RT-PCR. Results (1) After transfection of pcDNA3.1-hVEGF165 gene into rat MSCs, the stable expression of pcDNA3.1-hVEGF165 gene was achieved. (2) After 4 weeks of transplantation, the myocardial infarct size (27.8% ± 3.0%) in the combined group was significantly lower (37.1% ± 5.2%, both P <0.05) in the cell group (37.0% ± 10.1%) and the improvement of cardiac function was superior to the cell group and the genome; (3) the combination group Capillary density in myocardial infarction area (40.2 ± 5.5 per field) was higher than that in cell group (27.2 ± 6.3, P <0.01) and control group (18.5 ± 5 (P = 0.189, P <0.01), and there was a trend of increase compared with the genome (35.8 ± 7.7) (P = 0.189). (4) Brdu and troponin T double staining (5) The expression of VEGF gene in the combination group (relative content of hVEGF mRNA 0.18 ± 0.04) was higher than that of the control group (0.10 ± 0.03, P <0.01). Conclusion Transplantation of vascular endothelial growth factor (VEGF) -transfected MSCs can reduce myocardial infarction area caused by coronary artery ligation and significantly improve cardiac function, which is superior to the single gene or cell therapy alone. It provides a combination of gene therapy for the treatment of ischemic heart disease Theoretical basis.