血小板源性生长因子B基因转染抑制缺血缺氧诱导心肌细胞凋亡

摘要

背景：血小板源性生长因子B(platelet-derived growth factor-B，PDGF-B)是一种有效的促血管生长因子，9型腺相关病毒(adeno-associated virus serotype，rAAV9)对心肌细胞具有良好的靶向亲和力，是目前缺血性心脏疾病基因治疗的理想载体。　　目的：探索rAAV9介导PDGF-B基因(rAAV9-PDGF-B)体外转染乳鼠心肌细胞，抵抗缺血缺氧诱导心肌细胞凋亡的作用。　　方法：分离培养原代乳鼠心肌细胞，分别将感染复数为1×105，1×106及1×107 MOI携带rAAV9-PDGF-B基因的重组9型腺相关病毒及空病毒(rAAV9-eGFP)转染乳鼠心肌细胞，逐日在荧光显微镜下观察eGFP的阳性表达，采用流式细胞仪测定rAAV9载体的转染效率，Western blot和免疫荧光鉴定PDGF-B蛋白表达情况；并于rAAV9-eGFP及rAAV9-PDGF-B(107感染复数)转染第5天时，建立体外心肌细胞缺血缺氧损伤模型，通过 TUNEL法检测心肌细胞凋亡数目，Western Blot 检测 Bax及 Caspase-3凋亡相关蛋白的表达，研究PDGF-B基因过表达抗心肌细胞凋亡的作用及可能机制。　　结果与结论：rAAV9载体可有效转染乳鼠心肌细胞，eGFP和PDGF-B蛋白可在心肌细胞中高效正确表达，其表达强度随着转染时间和转染复数的增加而逐渐增强，第5天趋于稳定，此时各组转染率相比差异均有显著性意义(P<0.01)。rAAV9-PDGF-B组心肌细胞凋亡率低于rAAV9-eGFP组(P<0.05)，且rAAV9-PDGF-B组凋亡相关蛋白Bax和Caspase-3的表达与rAAV9-eGFP组比均显著下调(P <0.05)。结果表明，PDGF-B基因过表达可有效减轻缺血缺氧诱导的心肌细胞凋亡，其机制通过抑制Bax和Caspase-3蛋白表达而发挥作用，为rAAV9-PDGF-B载体用于缺血性心脏疾病的基因治疗提供依据。%BACKGROUND:Platelet-derived growth factor-B (PDGF-B) is an effective pro-angiogenic growth factor, and adeno-associated virus type 9 (rAAV9) has a strong cardiomyocyte targeting affinity, which is an ideal vehicle for ischemic heart disease gene therapy. OBJECTIVE:To explore the PDGF-B gene transfection of in vitro neonatal rat myocardial cells mediated by rAAV9 against ischemia and hypoxia-induced cardiomyocytes apoptosis. METHODRat neonatal myocardial cells were isolated and cultured, and then transfected by rAAV9-PDGF-B and empty virus, rAAV9 with enhance green fluorescent protein (eGFP), under multiplicity of infection (MOI) of 105, 106 and 107, respectively. We observed the expression of eGFP under fluorescence microscopy every day, and used flow cytometry to measure transfection efficiency of vector rAAV9. Western blot and immunofluorescence were used to evaluate protein expression of PDGF-B. Myocardial ischemia and hypoxia injury model was established in vitro on the 5th day of transfection of rAAV9-eGFP and rAAV9-PDGF-B with 107 MOI. The number of myocardial apoptosis was measured by TUNEL assay. Western blot was employed to detect the protein expression of Bax and Caspase-3 which were related apoptosis, and the effect and its possible mechanism of PDGF-B gene overexpression against myocardial apoptosis were explored. RESULTS AND CONCLUSION:rAAV9 vector can efficiently transfect neonatal rat myocardial cells. eGFP and PDGF-B protein expressed in myocardial cells correctly and efficiently, and the expression intensity increased gradual y with the increasing of time course and MOI. The expression became stable on the 5th day, and the transfection efficiency showed significant difference among these groups (P<0.01). Myocardial apoptosis rate was significantly reduced in the rAAV9-PDGF-B group than the rAAV9-eGFP group (P<0.05), and protein levels of Bax and Caspase-3 in the rAAV9-PDGF-B group were significantly lower than those of the rAAV9-eGFP group (P<0.05). These data indicate that overexpression of PDGF-B gene can effectively reduce ischemia and hypoxia-induced myocardial apoptosis, and the possible mechanism might be by inhibiting Bax and Caspase-3 protein expression, which can provide evidence of rAAV9-PDGF-B vector in the gene therapy of ischemic heart diseases.