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- 2015
突变型低氧诱导因子1α加速骨缺损部位新血管生成的实验观察
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Abstract:
摘要：目的 通过突变低氧诱导因子1α(hypoxia inducible factor 1, HIF1α)基因的3个氨基酸位点研究该基因在常氧条件下骨缺损区域对血管新生效应的影响。方法 定点突变HIF1α编码区(coding sequence, CDS)402、564和803位3个氨基酸后将基因重组入腺病毒pAdEasy-1系统并测定滴度，同理包装未突变组和空病毒组；以3种病毒液连同空白组共分4组进行后续实验(A组：含突变HIF1α基因病毒液，B组：含未突变HIF1α基因病毒液，C组：空病毒液，D组：空白组)；将病毒液转染入兔骨髓间充质干细胞(mesenchymal stem cells, MSCs)内，通过示踪因子人源化绿色荧光蛋白(human renilla reniformis green fluorescent protein, hrGFP)观察病毒转染效率，检测各组转染细胞中HIF1α基因mRNA和蛋白表达情况；制作兔桡骨缺损动物模型，将含目的基因的病毒液按上述分组转染MSCs后作为种子细胞移植到多孔纳米磷灰石-硅灰石生物活性玻璃陶瓷(apatite-wollastonite magnetic bioactive glass-ceramic, A-W MGC)载体中搭建人工组织工程骨架载体并植入体内，于术后8周处死各组动物，取植入区域组织做新血管生成检测。结果 CDS区第402、564和803位氨基酸均定点突变为丙氨酸；3种腺病毒重组体构建成功并鉴定完毕；A、B两组HIF1α mRNA表达量明显高于C、D两组，差异具有统计学意义(P<0.05)，而A、B两组之间及C、D两组之间比较，差异无统计学意义(P>0.05)；A组HIF1α蛋白表达量明显高于其他3组，差异具有统计学意义(P<0.05)，而B、C、D 3组之间比较差异无统计学意义(P>0.05)；A组转染到动物体内后可见明显成血管效果，其他3组未见缺损区域有血管新生。结论 ①3点突变后HIF1α基因能够在常氧条件下大量且高效表达；②3点突变后HIF1α基因能够在体内局部形成有效的血管网络。
ABSTRACT: Objective To investigate the functions of triple point-mutants of hypoxia-inducible factor 1α (HIF1α) in angiogenesis in bone defect region under normoxic conditions. Methods Triple point-mutations (the 402, 564 and 803 amino acids) in HIF1α coding sequence (CDS) were induced. The triple mutant of HIF1α(402/564/803) was inserted into the adenovirus pAdEasy-1 system to complete viral packaging and titer measurements. The wild-type HIF1α gene and the empty adenovirus vector were packaged in the same way. For the in vitro experiment, the rabbit bone marrow mesenchymal stem cells (MSCs) were divided into four experimental groups: A, MSCs infected with viral solution containing mutant HIF1α; B, MSCs infected with viral solution containing wild-type HIF1α; C, MSCs infected with viral solution without any HIF1α; and D, MSCs without viral infection. The efficiency of infection was observed by the expression of human renilla reniformis green fluorescent protein (hrGFP). The expression levels of HIF1α mRNA and protein in infected cells in each experimental group were measured. For the in vivo experiment, the MSCs were divided into the same four groups and infected with the virus solutions from each group and cultured under normoxic conditions. At 72h after the infection, the MSCs were used as seed cells and transplanted into Apatite-wollastonite magnetic bioactive glass-ceramic (AW MGC) vector to construct artificial tissue-engineering scaffolds, and then the scaffolds were implanted into the in vivo rabbit radial bone defect model. The animals from each group were sacrificed 8 weeks after the surgery and the tissues from the implantation region were
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