Platelet-derived growth factor-B gene transfection reduces ischemia and hypoxia-induced myocardial apoptosis

doi:10.3969/j.issn.2095-4344.2014.38.005

Abstract

Abstract:

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.
METHODS: Rat 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 gradually 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.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

Key words: tissue engineering, adenoviridae, viruses, anoxia, apoptosis

CLC Number:

R318

Chen Bang-dang, Chen Xiao-cui, Ma Yi-tong, Yang Yi-ning, Ma Xiang, Liu Fen.

Platelet-derived growth factor-B gene transfection reduces ischemia and hypoxia-induced myocardial apoptosis

[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(38): 6090-6098.

Figures/Tables 3

2.1 心肌细胞的培养及纯度鉴定取培养2 d的细胞，用心肌特异性肌钙蛋白T进行免疫荧光鉴定，于荧光倒置显微镜下观察计数，红色荧光标记的是心肌细胞,蓝色荧光标记视野下所有的细胞核，计数后，心肌细胞的纯度可达到98%以上(图1A)，400倍荧光显微镜下可以清晰看到心肌细胞的肌丝结构(图1B)，说明采用胰蛋白酶结合胶原酶消化心肌组织、二次差速贴壁及添加BrdU的分离培养方法可获得高纯度心肌细胞，有效抑制非心肌细胞污染。 2.2 rAAV9-eGFP载体转染效率用感染复数为1×105、1×106和1×107 MOI转染心肌细胞后，倒置荧光显微镜下观察发现eGFP的表达随着感染复数的增加和转染时间的延长而逐渐增强(图2)。在病毒转染1 d后可见少量心肌细胞表达，转染两三天表达eGFP阳性的细胞逐渐增加，随着时间的延长，eGFP阳性的细胞逐渐增加，转染五六天荧光表达的细胞数及强度趋于稳定，转染第9天仍有表达，但随着细胞的衰老表达强度略有减弱。收集1×105、1×106和1×107 MOI的rAAV9-eGFP转染第5天的心肌细胞，流式细胞仪测定其转染效率分别为(8.23±0.49)%、(19.60±0.65)%、(60.90±1.13)%，见图3，各组相比差异有显著性意义(P < 0.01)，表明rAAV9-eGFP载体对心肌细胞有较高的亲和力，可高效转染乳鼠心肌细胞。 2.3 Western Blot检测PDGF-B蛋白表达结果分别收集1×105、1×106和1×107 MOI的rAAV9-PDGF-B转染第5天时的心肌细胞，提取细胞总蛋白，Western Blot检测发现PDGF-B蛋白的表达随着感染复数的增加而逐渐增强(图4A，B)，与内参GAPDH蛋白相比，其表达分别为(31.51±4.80)%，(54.86±5.22)%，(86.51±5.56)%，各组相比差异有显著性意义(P < 0.01)。 1×107 MOI的rAAV9-PDGF-B转染1-5 d的心肌细胞，PDGF-B蛋白的表达水平随着转染时间的延长而逐渐增强(图4C，D)，与内参GAPDH相比，其表达分别为(28.45±2.88)%，(46.25±2.84)%，(57.79±2.93)%，(71.92± 2.25)%，(86.51± 5.56)%，各组相比差异有显著性意义(P < 0.05)，表明rAAV9-PDGF-B载体转染心肌细胞后可高效正确表达PDGF-B蛋白。 2.4 免疫荧光鉴定PDGF-B蛋白表达结果取转染后第5天的心肌细胞进行检测，在倒置荧光显微镜下观察，红色荧光为表达PDGF-B的心肌细胞，用DAPI(蓝色)复染细胞核，经计算约55%的心肌细胞表达PDGF-B蛋白(图5)。 2.5 心肌细胞凋亡检测结果取缺血缺氧培养24 h的心肌细胞，用TUNEL染色检测细胞凋亡数目，其中红色标记凋亡的细胞核，蓝色标记所有的细胞核，红色与蓝色可叠加为粉红色的计数为凋亡的细胞(图6)，计数后，rAAV9- PDGF-B组心肌细胞凋亡率为(20.10±2.50)%，而rAAV9-eGFP组为(30.00±3.40)%，PDGF-B的过表达可使心肌细胞凋亡率减少33%，两组相比差异有显著性意义 (P < 0.05)，说明PDGF-B蛋白表达可有效抵抗缺血缺氧诱导心肌细胞凋亡的作用。 2.6 细胞凋亡蛋白检测结果 Western blot检测各组细胞Bax表达，经分析正常对照组Bax表达为(9.54±1.01)%，eGFP组为(20.90±2.67)%、PDGF-B组为(12.10±2.08)%。与正常对照相比，PDGF-B组Bax水平无明显差异，与eGFP组相比Bax水平显著下调(P < 0.05)。 Caspase-3表达变化趋势与Bax基本一致，其表达对照组为(7.85±0.66)%，eGFP组为(12.99±1.92)%，而PDGF-B组为(8.82±0.67)%，与eGFP组相比Caspase-3的表达明显下调(P < 0.05)(图7)。结果表明，PDGF-B蛋白表达可有效抑制促凋亡蛋白Bax和Caspase-3的表达。"

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