Treatment of spinal cord injury by transfection of double gene recombinant adenovirus vector into rat bone marrow mesecnhymal stem cells

doi:10.3969/j.issn.2095-4344.2014.23.007

Abstract

Abstract:

BACKGROUND: Brain-derived neurotrophic factor (BDNF) has widespread effects on dopaminergic neurons, cholinergic neurons and other neurons, which can promote more differentiation of stem cells into neuron-like cells. Hypoxia inducible factor-1α (HIF1α) can improve tissue and cell viability in the ischemic environment and maintain the local microenvironment of hemangioblasts, which has a more far-ranging physiological role than genes.

OBJECTIVE: By constructing double gene recombinant adenovirus vector with BDNF and three points mutant HIF1α to complete the transfection of adenovirus vector into rats mesenchymal stem cells and then study the promoting nerve regeneration and angiogenesis effect of these two genes to spinal cord injury in vitro in constant oxygen conditions.

METHODS: (1)We finished the site-directed mutagenesis of 402, 564 and 803 amino acids in human HIF1α gene CDS region by PCR method and we finished recombination of mutation posterior HIF1α gene and BDNF into adenovirus pAdEasy-1 system. Packaging viral and titration determination were also finished. (2)Four kinds of virus fluids and blank group were selected for subsequent experiments. We observed transfection efficiency after transfection of virus into bone marrow mesenchymal stem cells and detected the expressions of BDNF and HIF1α mRNA and protein in transfection cells. (3) The protein expression of vascular endothelial growth factor, downstream formation vascular gene of HIF1α, in cells in all groups was detected by western blot assay.

RESULTS AND CONCLUSION: (1)The site-directed mutagenesis of 402, 564 and 803 amino acids to alanine in coding sequence region was successful. The construction of four kinds of adenoviral recombinants was successful and identification of packaging was completed. (2)The level of BDNF gene mRNA and protein expression in experimental and positive control 1 groups was significantly higher than that in the other groups (P < 0.05). The level of HIF1α mRNA and protein expression in experimental and positive control 2 groups was significantly higher than that of the other groups (P < 0.05). The level of vascular endothelial growth factor protein was also increased in the experimental and positive control 2 groups, which was significantly different from other groups (P < 0.05). These findings indicate that the BDNF and HIF1α proteins are largely and efficiently expressed in constant oxygen conditions after single vector double gene adenovirus system transfection into mesenchymal stem cells and high-efficiency expression of vascular endothelial growth factor protein is promoted so that this is a new direction for treatment of spinal cord injury by gene therapy combined with cell transplantation.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: stem cells, bone marrow, spinal cord injuries, nerve growth factor, vascular endothelial growth factors, adenoviruses

CLC Number:

R394.2

Xu Yan-mei, Sun Fei, Hui Chun-ying, Wang Wei. Treatment of spinal cord injury by transfection of double gene recombinant adenovirus vector into rat bone marrow mesecnhymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(23): 3645-3652.

Figures/Tables 4

2.1 测序及腺病毒载体鉴定基因测序显示低氧诱导因子1α基因编码区第402位脯氨酸由CCA突变成GCA(丙氨酸)，第564位脯氨酸由CCC突变成GCC(丙氨酸)，第803位天冬酰胺由ATT突变成AGC丙氨酸(说明：第803位天冬酰胺测序的是反义链，所以天冬酰胺正义链应为AAT，而丙氨酸正义链应为GCT)(图1)。凝胶电泳成像显示Ad-BDNF- IRES-HIF-1αmu经PacⅠ酶切后出现30 kb和3 kb两个条带，提示重组双基因单腺病毒载体构建成功(图2)。 2.2 腺病毒包装及滴度测定重组病毒体经Lipofectamine 2000转染HEK293A细胞后均出现细胞病变效应(Cytopathic effect，CPE)，具体体现在细胞触角回收、肿胀变圆，一部分细胞脱落，悬浮于视野中(图3A)。包装成功的病毒液采用终点稀释实验法(End-Point Dilution Assay)进行病毒滴度测定(图3B)，病毒滴度检测结果依次为实验组(1.6×108 pfu/mL)，阳性对照组1(1.9× 108 pfu/mL)，阳性对照组2(1.4×108 pfu/mL)和阴性对照组(2.0×108 pfu/mL)，均符合后续转染实验要求。 2.3 病毒转染骨髓间充质干细胞表达结果包装成功的两组阳性对照及一组阴性对照病毒液在荧光显微镜下均观察到较强绿色荧光表达，转染效率较高(图4A-C)；根据对照组转染效果进行实验组病毒液的转染，48 h后可见大鼠骨髓间充质干细胞发生细胞病变效应，转染效率依然很高(图4D)。 2.4 RT-PCR结果脑源性神经生长因子 mRNA表达：实验组、阳性对照组1相对A值分别为0.81±0.06，0.75±0.07，两组比较差异无显著性意义(P > 0.05)；而另外3组脑源性神经生长因子mRNA的表达量非常低，几乎可以忽略(P > 0.05)；实验组、阳性对照组1两组表达量均明显高于其他3组 (P < 0.05，图5A)。低氧诱导因子1α mRNA表达：实验组、阳性对照组2表达量分别为0.69±0.05，0.73±0.08，两组之间比较差异无显著性意义(P > 0.05)；而另外3组低氧诱导因子1α mRNA的表达量基本没有(P > 0.05)；实验组、阳性对照组2两组表达量均明显高于其他3组(P < 0.05，图5B)。"

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