人骨形态发生蛋白7基因转染对兔髓核细胞增殖和细胞外基质合成的影响(英文)

Effect of human bone morphogenetic protein-7 gene transfection on proliferation of rabbit nucleus pulposus cells and synthesis of extracellular matrix

背景:以往的研究表明人骨形态发生蛋白7(hBMP-7)能够有效促进细胞外基质合成,修复受损的椎间盘基质,恢复椎间盘高度。因此有希望用于控制和逆转椎间盘退变。然而重组蛋白半衰期短,生物学活性低,退变椎间盘中很难保持骨形态发生蛋白7浓度。基因治疗能够有效预防这些缺陷。目的:观察人骨形态发生蛋白7基因转染对原代培养的兔髓核细胞生物学活性的影响,为人骨形态发生蛋白7基因治疗椎间盘退变的体内实验研究奠定基础。设计、时间及地点:随机对照,细胞学体外实验。于2005-12/2006-09在解放军第二军医大学长海医院全军胸心外科研究所实验室完成。材料:4周龄新西兰大耳白兔6只,体质量约500g。Ad-hBMP7由长海医院胸心外科研究所构建。方法:麻醉后处死白兔,提取髓核,依次经过链霉蛋白酶、Ⅱ型胶原酶和Ⅱ型DNA酶在37℃条件下消化4h,细胞接种于培养皿内,孵箱内培养,7d后每周更换培养液2次。将细胞随机分为3组,用整合有人骨形态发生蛋白7基因的腺病毒感染髓核细胞,整合有Lac-Z基因的腺病毒感染的髓核细胞以及未转染的髓核细胞作为对照组。主要观察指标:以反转录-聚合酶链反应和Western-blot的方法在不同水平检测其表达情况,以四甲基偶氮唑盐的方法观察其对细胞增殖能力的影响,以改进的二甲基亚甲蓝色比色法和ELISA法观测人骨形态发生蛋白7基因转染对细胞合成蛋白多糖和Ⅱ型胶原能力的影响。结果:鉴定确定人骨形态发生蛋白7基因为正向插入腺病毒载体,无突变。原代培养的兔髓核细胞形态与文献报道一致。腺病毒载体介导的人骨形态发生蛋白7基因能够高效转染髓核细胞,并表达人骨形态发生蛋白7,同时促进了髓核细胞增殖以及合成蛋白多糖和Ⅱ型胶原,较对照组有显著性差异(P<0.05)。结论:腺病毒介导的人骨形态发生蛋白7基因具有逆转兔椎间盘退变的能力。

BACKGROUND： Previous studies demonstrated that recombinant human bone morphogenetic protein-7 （hBMP-7） can effectively promote the synthesis of extracellular matrix, repair of damaged disc matrix, restore of degenerative disc height. It is hoped that BMP-7 can be used to control and reverse the intervertebral disc degeneration. However, because of the short half-life of recombinant protein and low biological activity, it is difficult to maintain BMP-7 high concentrations on degenerative disc. Gene therapy can prevent these defects effectively. OBJECTIVE： To study the effect of hBMP-7 gene transfection on biological activity of primary cultured rabbit nucleus pulposus cells in vitro, to determine the feasibility of hBMP-7 gene which will on gene therapy research of intervertebral disc degeneration, and to provide basis for further study. DESIGN, TIME AND SETTING： A randomized and controlled observation was performed at the Institute of Cardiothoracic Surgery of Changhai Hospital from December 2005 to September 2006. MATERIALS： Six healthy New Zealand white rabbits of either gender, averaging 4 weeks old and weighing 500 g, were used in this study, and Ad-hBMP7 was constructed by the Cardiothoracic Surgery Institute of Changhai Hospital. METHODS： After rabbits sacrifice under aseptic condition, the nucleus pulposus was got. After digested with Pronase, type II collagenase and type II DNAase （4 hours, 37 ~C）, the cells were seeded in the Petri dishes and were kept in the incubator. After 7 days and then twice a week, the media were refreshed. The nucleus pulposus cells were infected by adenovirus integrated with hBMP7 gene. The cells which were transfected by adenovirus vector for Lac-Z gene and which were not transfected were adopted as control. MAIN OUTCOME MEASURES： The expression of hBMP-7 was determined by RT-PCR and Western blot. The effect of hBMP-7 on cell proliferation was surveyed by M]-I. Furthermore, the effect of hBMP-7 gene on synthesis of proteoglycan and type II collagen was detected by modified dimethylmethylene blue method and ELISA, respectively. RESULTS： Gene sequencing and PCR showed that hBMP-7 gene was inserted correctly and no mutation happened. The primary cultured rabbit nucleus pulposus cells were identical with those reported in literature. After Ad-hBMP7 transduct into the nucleus pulposus cells, high level of hBMP-7 expression was observed and lasted over 3 weeks. Also hBMP-7 gene can promote cell proliferation and synthesis of proteoglycan and type II collagen with significant difference compared with control groups （P〈 0.05）. CONCLUSION： hBMP-7 gene mediated by adenovirus can be the target gene for the further study on gene therapy of intervertebral disc degeneration.