【作者】 郑文杰；

【导师】 周跃；

【作者基本信息】 第三军医大学 ， 外科学， 2008， 博士

【摘要】 脊柱融合术被广泛应用于退行性病变、创伤、肿瘤等各种脊柱疾患的治疗中,是脊柱外科手术中最常用技术之一。但仍有最多高达45%的患者发生假关节形成,同时自体髂骨植骨增加了手术创伤并带来了相关并发症。进一步提高脊柱融合率和寻找更为合适的移植替代物,成为脊柱外科的研究热点。在对成骨的分子机制的研究过程中,逐渐发现了一系列生物因子对诱导成骨细胞分化和促进局部骨形成有着重要的作用,这些因子被称为骨诱导因子。许多骨诱导因子被发现、研究并应用于脊柱融合术。BMPs是现在研究最多的骨诱导生长因子。研究证实rhBMP-2的应用增加了脊柱融合几率并减少了自体骨移植所带来的手术创伤。但rhBMP-2价格昂贵并有明确的剂量依赖性,常需要使用毫克级别的重组蛋白才能达到满意的临床疗效。LIM矿化蛋白(LIM mineralization protein, LMP)是近期发现的诱导成骨细胞分化成熟的胞内细胞信号因子,可促进成骨细胞分化和骨形成,在骨的矿化过程中发挥重要作用。研究证实,LMP体外和体内可以诱导多种细胞内BMP-2、BMP-4、BMP-6、BMP-7以及TGF-β等多种骨诱导因子的分泌,同时提高成骨相关细胞对外源性BMPs的反应性,招募周围细胞参与成骨。相关对比研究发现,在诱导体内异位成骨和脊柱融合的动物实验中,LMP-1的效能强于BMP-2。将LMP作为目的基因的局部基因治疗中发现:不需要转染每一个受体细胞,也不需要长时间的表达就可以产生较强的成骨效应,故LMP较BMPs等更适合于脊柱融合的治疗。上述研究表明LMP-3和LMP-1可以作为BMPs类理想的替代物,有望避免了大剂量单一应用BMPs的不便之处,并产生更大的诱导成骨效应。迄今已分离出3种LMP基因型,Boden等对LMP同源蛋白的结构和功能进行了系列研究,发现:hLMP-1长457个氨基酸,包含1个PDZ功能区(N端)和3个LIM功能区(C端),hLMP-2长423个氨基酸,包含完整的PDZ功能区和LIM功能区,hLMP-3长153个氨基酸,包含1个完整PDZ功能,LIM功能区缺失。研究证实,hLMP-2拥有完整的LIM功能区但没有促进成骨作用,hLMP-3没有LIM功能区却具备与hLMP-1类似的诱导成骨能力。对LMP的分析揭示:LMP-1和LMP-3共有一个40aa的独特区域显示出与成骨作用密切相关。LMP作为胞内信号转导分子,目前均采用质粒、腺病毒等基因治疗的方式导入编码cDNA以过度表达的方式实现其研究和应用。基因治疗导入效率偏低,且存在安全问题,有引起插入突变及细胞恶性转化的潜在危险。构建具有入胞转导能力并保持成骨活性的LMP重组蛋白,有望大大拓宽LMP的应用范围,并使LMP的实际临床应用成为可能。1988年Green和Frankle发现人免疫缺陷病毒(HIV-1)的反式激活蛋白(tat)能介导自细胞外转运至细胞内的跨膜转运,后续对tat蛋白的结构研究发现tat49-57(RKKRRQRRR)9肽序列是其主要功能区,也是保持tat蛋白功能活性的最小片断。在分子生物学和细胞生物学的研究中,陆续发现了一系列短肽具有相似的跨膜转运功能,这类短肽被统称为蛋白转导域(protein transduction domains,PTDs)。PTD转导技术可介导包括蛋白在内的多种生物大分子的转运,且不受细胞类型的限制,并有望保持被转运分子的生物活性,在生物细胞学研究和药物开发等领域有着广泛的应用前景。PTD介导的入胞转导存在明显的优势:(1)PTD能快速有效的将携带物质跨膜转运;(2)对活体细胞干扰小,绝大部分PTD复合物在一定浓度内基本无毒性;(3)应用上操作简单,转导效率高,大多情况下能保持携带物质的生物学活性,低浓度即可发挥有效生物学效能。如前所述, TatPTD具有高效的蛋白转导的作用,我们设想将tat49-57 (RKKRRQRRR)9肽序列与LMP-3进行连接,构建TAT/LMP-3融合蛋白,利用TatPTD将LMP-3介导进入靶细胞内。骨髓间充质干细胞(mesenchymal stem cells, MSCs)具有多向分化的潜能,是组织工程常用的种子细胞,其本身亦可向成骨细胞分化,具有来源广泛、取材方便、增殖和分化能力强、能与宿主骨良好结合的优点。本实验中采用重组蛋白与人MSCs共培养的方法来验证Tat序列诱导入胞的能力和TAT/LMP-3融合蛋白促成骨分化的活性。同时,我们制备重组蛋白LMP-3作为对照。本课题的研究主要包括以下三个部分,所用到的主要方法及结果如下:第一部分TAT/LMP-3融合蛋白的原核表达、纯化及多克隆抗体制备1.人工合成针对大肠杆菌表达体系优化的LMP-3编码基因序列,通过PCR方法分别获得TAT/LMP-3融合基因和LMP-3基因并引入酶切位点。通过酶切、连接构建原核表达载体,命名为pET43.1a-TAT/LMP-3和pET43.1a- LMP-3。测序结果提示无PCR引起的突变,基因无移码,质粒构建成功。2.将构建载体于BL21(DE3)工程菌中进行原核表达,优化表达条件后目的蛋白表达以可溶性表达为主,将表达产物上清经Ni-NTA树脂纯化后,获得重组蛋白TAT/LMP-3和LMP-3,纯度均在90%以上。3.将重组蛋白LMP-3免疫家兔获得多抗血清,经饱和硫酸铵法纯化获得多克隆抗体。ELISA法检测其对TAT/LMP-3和LMP-3的效价均在1:4000以上,western-blot分析提示抗体与重组蛋白有较好的抗原抗体反应性和一定特异性。第二部分人骨髓间充质干细胞的分离、培养和鉴定1.本部分实验中通过梯度离心法结合贴壁培养法分离获得人骨髓间充质干细胞(hMSCs),流式细胞仪分析第三代hMSCs细胞表面标志表达情况,结果阳性表达CD29、CD105,CD34和CD45表达为阴性,细胞纯度达到95%以上。2.在成骨诱导培养液(含地塞米松10-8mol/L、β-甘油磷酸钠10mmol/L、维生素C 50mg/L)诱导下,hMSCs碱性磷酸酶活性增高,免疫细胞化学染色检测到hMSCs内骨钙素阳性表达。14d后茜素红S染色提示钙结节形成,证实hMSCs在适当条件可诱导分化为成骨细胞。第三部分TAT/LMP-3融合蛋白诱导人骨髓间充质干细胞成骨分化的研究1.将TAT/LMP-3和LMP-3分别与人骨髓间充质干细胞共孵育,通过间接免疫荧光染色和western-blotting技术检测重组蛋白的入胞效应。结果TAT/LMP-3能以浓度/时间依赖的方式,高效快速的转导进入细胞。对照蛋白LMP-3不具备入胞转导能力。2.将纯化重组蛋白分别与人骨髓间充质干细胞共孵育,检测重组蛋白对人骨髓间充质干细胞的成骨分化诱导效能。结果提示: 100ng/ml的TAT/LMP-3与hMSCs共孵育,可诱导人骨髓间充质干细胞Ⅰ型胶原、碱性磷酸酶、骨钙素等成骨细胞标志基因的表达,培养14天后可观察到钙结节形成。半定量RT-PCR提示,与TAT/LMP-3共孵育后,人骨髓间充质干细胞BMP-2,4,6,7基因水平表达上调。更多还原

【Abstract】 Spinal fusion is one of the commonest procedures in spinal surgery, which is widely used to treat degenerative disease, trauma and tumor of the spine. However, pseudoarthrosis formation occurs in up to 45% patients following spinal fusion. Meanwhile, the use of autologous iliac bone graft in the procedure results in additional surgical trauma and certain complications. Hence, it has become a focus of interest to further increase the success rate of spinal fusion and find ideal grafting substitute.It has been found that some biological factors play important roles in inducing osteoblast differentiation and promoting local bone formation, and these factors are called osteoinductive factors. Many osteoinductive factors have been discovered, investigated and applied to spinal fusion procedures. BMPs are the osteoinductive factors that have been studied most intensively. It was confirmed that rhBMP-2 increases the success rate of spinal fusion and attenuates surgical trauma. Nevertheless, rhBMP-2 is costly and its efficacy is dose-dependent. Normally, several milligrams of rhBMP-2 are required to achieve satisfactory clinical results.LMP is a recently discovered intracellular signaling factor that can induce osteoblast differentiation and maturation as well as bone formation. It has been shown that LMP can induce the secretion of osteoinductive factors such as BMP-2, BMP-4, BMP-6, BMP-7 and TGF-βin vitro and in vivo, and improve the responsiveness of osteogenesis-related cells to exogenous BMPs, and recruit peripheral cells to form bone. Animal experiments demonstrated that LMP-1 is more potent than BMP-2 in inducing ectopic bone formation and spinal fusion. Local gene therapy using LMP as target gene resulted in significant osteogenic effect without needing transfection of all recipient cells or long-term expression of the target gene. Hence, LMP is superior to BMPs for use in spinal fusion. These findings demonstrate that LMP-3 and LMP-1 are ideal substitutes for BMPs, and that they avoid the pitfalls in the use of large doses of BMPs alone and show more potent action to induce osteogenesis.Three LMP genotypes have been identified. Boden et al investigated the structure and function of LMP homologous proteins. According to their findings, hLMP-1 comprises 457 amino acid residues, including one PDZ domain at its N-terminal and three LIM domains at its C-terminal, hLMP-2 comprises 423 amino acid residues, including a complete PDZ domain and LIM domains, and hLMP-3 comprises 153 amino acid residues, including a complete PDZ domain, but no LIM domain. Further studies demonstrated that hLMP-2 has complete LIM domains, but has no osteogenesis-promoting effect, and that hLMP-3 has no LIM domain, but has a potential to induce osteogenesis just like hLMP-1. It was further indicated that LMP-1 and LMP-3 have a unique domain comprising 40 amino acid residues and this domain is closely related to osteogenesis.LMP gene therapy is currently mediated by plasmid and adenovirus and LMP gene over-expression is desired. The efficiency of gene transfer is relatively low in gene therapy. Moreover, gene therapy may bring some safety concerns. For instance, gene therapy might cause insertion mutations and cell malignant transformation. Through constructing recombinant LMP protein with cell transduction capability and osteogenic activity, we hope to broaden the application of LMP, particularly, to use LMP clinically.In 1988, Green and Frankle et al discovered human immunodeficiency virus-1 (HIV-1) trans-activation protein (TAT) which mediates substance transportation across cell membrane. Further researches revealed that the TAT protein contains a major domain which is a sequence of nine amino acid residues, i.e., tat49-57(RKKRRQRRR), and that this domain is the smallest fragment to conserve the functional activity of the TAT protein. An array of short peptides have been found to have similar transmembrane transporting capability, and these peptides are collectively termed protein transduction domains (PTDs). PTD transduction technology can be applied to transporting multiple biological macromolecules such as protein, without influencing the biological activity of molecules transported. Moreover, this technology is not cell type-specific. Hence, PTD transduction technology may be used widely, for instance, in cell biology and drug development. PTD transduction exhibits obvious advantages: (1) PTD can transport substances across cell membrane quickly and efficiently; (2) most PTD complexes are not toxic within a certain concentration range and impact living cells insignificantly; (3) PTD transduction technology is easy to operate, and its transduction is efficient. Under most circumstances, the substances transported can have their biological activity not affected, and thus can exert their biological action under low concentrations.As mentioned above, TatPTD can deliver protein across cell membrane efficiently. We planned to fuse tat49-57(RKKRRQRRR) with LMP-3 and transport LMP-3 into target cells using TatPTD. Bone marrow mesenchymal stem cells (MSCs) exhibit the potential of multidirectional differentiation and are common seed cells in tissue engineering. MSCs can differentiate into osteoblasts, and they have many advantages, such as ample sources, being easy to prepare, and strong potential of proliferation and differentiation, as well as good compatibility with host bone. In the present study, we cultured human MSCs with TAT/LMP-3 fusion protein to investigate the transduction capability of the Tat sequence and the potential of TAT/LMP-3 fusion protein to promote osteogenic differentiation of human MSCs.Part 1. Prokaryotic expression and purification of TAT/LMP-3 fusion protein and preparation of polyclonal antibodies against TAT/LMP-3 fusion protein1. LMP-3 encoding sequence optimized for E. coli expression system was synthesized. TAT/LMP-3 fusion gene and LMP-3 gene were obtained by PCR amplification and prokaryotic expression vectors pET43.1a-TAT/LMP-3 and pET43.1a-LMP-3 were constructed. Sequencing results suggest successful plasmid construction, with no PCR caused mutations or frame shifts.2. The prokaryotic vectors were expressed by BL21(DE3) engineering bacteria. Under the optimized expression conditions, soluble expression of target proteins was dominated. The supernatant expression products were subjected to purification using Ni-NTA resin to obtain recombinant TAT/LMP-3 and LMP-3 proteins. The purity of the target proteins was higher than 90%.3. Rabbits were immunized with TAT/LMP-3 to obtain polyclonal antibody containing serum, and polyclonal antibodies were purified using saturated ammonium sulfate. ELISA indicated the titer of TAT/LMP-3 and LMP-3 to be higher than 1:4000, and Western blotting analysis suggested sound reactiveness of the antibody to TAT/LMP-3 and satisfactory specificity of the antibody.Part 2. Isolation, culture and identification of hMSCs 1. hMSCs were isolated and purified through Percoll density gradient centrifugation with the assistance of adherent culture, and hMSCs were subjected to flow cytometry for cell surface marker expression after two passages. The result indicates the cells were positive for CD29 and CD105, while negative for hematopoietic cell surface marker such as CD34 and CD45. The cell purity was higher than 95%.2. Under induction with the osteogenic medium containing dexamethasone (10-8 mol/L),β-sodium glycerophosphate (10mmol/L), and vitamin C (50mg/L), hMSCs showed an increase in the salkaline phosphatase activity. In addition, expression of osteocalcin in hMSCs was detected by immunocytochemical stain. After 14d of culture, alizarin red S staining suggested the formation of calcified nodules. These findings demonstrated that hMSCs can be induced to differentiate into osteoblasts under certain conditions.Part 3. TAT/LMP-3 fusion protein induces osteogenic differentiation of hMSCs1. hMSCs were cultured with TAT/LMP-3 and LMP-3, respectively. Endocytosis of TAT/LMP-3 was detected by indirect immunofluorescence method and Western blotting. The results suggested that TAT/LMP-3 can be transduced quickly and efficiently into the cell in a concentration- and time-dependent manner. The control protein LMP-3 cannot be transduced into the cell.2. hMSCs were cultured with TAT/LMP-3 and LMP-3, respectively. The efficacy of TAT/LMP-3 to induce osteogenic differentiation of hMSCs was assessed. The results suggested that 100ng/ml TAT/LMP-3 can induce hMSCs to express the marker genes of osteoblasts such as type I collagen, alkaline phosphatase, and osteocalcin. Calcified nodules were observed after 14d of culture. RT-PCR suggested that TAT/LMP-3 upregulated BMP-2, BMP-4, BMP-6, BMP-7 gene expression by hMSCs.更多还原