【作者】 崔少千；

【导师】 范广宇；

【作者基本信息】 中国医科大学 ， 外科学， 2007， 博士

【摘要】 目的脊柱融合术是脊柱外科最常见的手术之一,但融合失败率较高。目前设计的多种内固定器械完全可以保证脊柱的即刻稳定性,提供植骨融合所需的力学环境,但长期稳定还须依靠骨性融合。脊柱融合术中,自体骨移植通常被认为是“金标准”,但脊柱融合术的植骨需要量较大而自体骨来源有限,并且会对供体造成新的损害,常会伴有供区疼痛、神经损伤、骨折、感染等并发症;异体骨有免疫原性,而且存在传染疾病的潜在危险。影响脊柱骨性融合的因素有很多,许多研究希望通过寻找高效能的自体骨替代材料促进脊柱融合。由支架材料、种子细胞和骨诱导因子复合构建的组织工程骨作为一种理想的自体骨替代物,有希望通过骨引导和骨诱导方式促进脊柱融合,成为目前骨科研究热点之一。本实验利用腺病毒载体介导的BMP-2体外基因治疗策略,联合应用骨组织工程技术,将骨形态发生蛋白-2(BMP-2)基因导入兔骨髓基质干细胞(BMSCs),复合胶原基纳米骨(nHAC)体外构建组织工程骨,回植兔腰椎椎体间,观察椎体间融合效果,为探索一种可应用于促进脊柱融合,具有骨诱导生物活性的自体骨替代新材料奠定实验基础。方法1、腺病毒介导的BMP-2基因转染兔BMSCs及对其增殖、成骨分化的影响(1)携带BMP-2基因的重组腺病毒表达载体(Ad-BMP-2)的扩增、鉴定与滴定常规贴壁培养及传代人胚肾细胞(HEK293细胞);通过感染293细胞扩增、提纯Ad-BMP-2;通过PCR检测、基因测序鉴定;通过噬菌斑试验测定病毒滴度。(2)Ad-BMP-2体外转染兔BMSCs及转染后BMP-2表达检测全骨髓培养法分离培养兔BMSCs。通过成骨、成脂肪诱导鉴定BMSCs:①Von kossa染色观察矿化结节、钙钴法染色观察碱性磷酸酶(ALP)活性;②油红染色观察脂肪滴。Ad-BMP-2转染BMSCs,同等条件下β-半乳糖酐酶基因的腺病毒表达载体(Ad-Lacz)转染BMSCs,X-gal免疫染色检测转染效率;原位杂交和免疫组化检测转染后BMSCs内BMP-2基因在mRNA和蛋白水平的表达;Western Blot检测BMP-2分泌性蛋白的表达。(3)BMP-2基因转染对BMSCs增殖分化的影响通过流式细胞仪观察细胞周期的变化,分析转基因对BMSCs增殖的影响。通过酶标法检测ALP活性;免疫荧光检测骨钙素(OCN)、Ⅰ型胶原表达,分析转基因对BMSCs成骨分化的影响。2、BMP-2基因修饰的BMSCs复合nHAC体外构建组织工程骨胰酶消化收集Ad-BMP-2转染的BMSCs,均匀接种nHAC支架,复合培养。扫描电镜观察支架材料中细胞粘附、生长情况;同时能量谱仪检测细胞周围钙质沉积情况;Western Blot检测BMP-2表达情况。3、BMP-2基因修饰的BMSCs复合nHAC促进兔腰椎椎间融合的实验研究(1)实验动物分组新西兰大耳白兔随机分为5组,每组12只:①Ad-BMP-2转染的BMSCs+nHAC支架组(A组);②BMSCs+nHAC支架组(B组);③nHAC支架组(C组);④自体髂骨组(D组);⑤空白组(E组)。各组再均分为4、8、12周观察组。(2)建立兔腰椎椎体间融合动物模型及植骨(3)观察指标①X线检查分别于术后4、8、12周摄腰椎侧位X线片,观察新骨形成及椎间融合情况。②大体观察手法检测判断植骨融合情况,计算融合率。③生物力学检测第12周取融合节段标本,在生物力学实验机上进行抗压、抗弯、抗伸强度测试,比较各组融合节段生物学性能。④组织形态学观察第4、8、12周各组标本HE染色,光镜下观察各组融合区成骨情况。⑤组织计量学分析各组融合区新骨形成情况取4、8、12周的各组HE染色组织切片,显微镜下电脑摄取图像,图像分析软件统计新生骨在融合区域所占的面积百分比。⑥免疫组化染色观察融合区BMP-2表达、分布情况。⑦统计学方法数据采用SPSS11.5统计软件处理,方差分析比较组间差异,P＜0.05为差异有统计学意义。实验结果1、第一部分结果(1)Ad-BMP-2的鉴定通过提取DNA进行PCR扩增,琼脂糖凝胶电泳结果表明仅Ad-BMP-2转染组获得1.2 kb的基因片段,经基因测序证实与BMP-2cDNA序列一致。(2)BMSCs的培养观察原代细胞接种24～72h后,圆形细胞贴壁逐渐变化成短梭形和多角形,第7天贴壁细胞呈梭形,增殖活跃,第10天贴壁细胞融合率达80%以上,部分融合成片状,呈旋涡状生长,从整体来看大多数细胞沿胞体长轴呈有序排列。细胞传代后生长更加迅速,大多于传代后12h内贴壁,24h后迅速生长,7～10天后长满瓶底。(3)BMSCs的鉴定成骨诱导后细胞仍继续生长繁殖,呈现铺路石样外观,细胞间出现了较多的、散在的致密圆形团块;Von kossa染色显示细胞间出现了黑染的矿化结节;ALP钙钴法染色显示大多数的细胞胞浆呈棕黑色阳性着色。成脂肪诱导后,油红染色,细胞中出现橙红染色的脂肪滴。结果表明,细胞经诱导可向成骨细胞、脂肪细胞分化,具有多向分化潜能,证实培养细胞为BMSCs。(4)Ad-BMP-2转染BMSCs及转染后BMP-2表达检测对照Ad-Lacz转染BMSCs,X-gal免疫染色检测显示病毒转染效率达90%以上。BMP-2原位杂交显示,转基因BMSCs的胞浆中有棕黄色颗粒性阳性杂交信号:免疫细胞化学检测显示,转基因BMSCs的胞浆中有棕黄色的阳性信号;细胞培养液Western Blot检测显示仅转染组有BMP-2阳性条带。上述结果表明Ad-BMP-2可高效转染BMSCs,并有BMP-2基因在mRNA和蛋白水平的高表达。(5)Ad-BMP-2转染对BMSCs增殖及成骨分化的影响流式细胞仪检测G1期、S期细胞比例与空白对照组无差异,表明细胞DNA的合成以及细胞的增殖未因转染受影响。转基因细胞ALP活性较对照组明显增高;骨钙素、Ⅰ型胶原免疫荧光检测,见胞浆内有大量显绿色荧光的阳性物质,结果表明,转染后BMSCs向成骨细胞分化。2、第二部分结果扫描电镜结果表明,BMP-2基因修饰的BMSCs与nHAC复合培养,细胞在支架表面和孔隙内粘附、生长良好。能量谱仪检测到细胞部位含钙量较空白处明显增高,表明细胞向成骨方向分化。Western Blot检测显示Ad-BMP-2转染组有BMP-2蛋白表达,而Ad-Lacz转染组和未转染组呈阴性,表明BMP-2基因修饰的BMSCs与nHAC复合后,细胞功能未受影响,仍可较高水平表达BMP-2。3、第三部分结果X线检查,大体观察及组织形态学观察结果显示,12周时E组脊柱未融合,表明造模成功。A、B、D组完全融合,C组融合率为75%。组织计量学分析测量各时相修复性新骨面积,A组显著高于B、C组(P＜0.01),与D组无显著性差异(P＞0.05),表明A组较快的成骨速度,与自体骨无差异。生物力学检测结果显示,A组各项指标显著优于B、C组(P＜0.01),与D组无显著性差异(P＞0.05),表明A组良好成骨质量及生物力学性能。BMP-2免疫组化检测显示,术后4周A组软骨及骨痂内为强阳性反应,棕黄色颗粒状阳性物质分布于间充质细胞、成软骨细胞、成骨细胞内,成熟骨细胞未见明显的阳性反应。B、C组阳性细胞分布少,表达较弱。结果表明,BMP-2基因修饰的BMSCs在体内仍可持续稳定的表达、分泌BMP-2。结论1、在重组复制缺陷型腺病毒载体介导下BMP-2基因成功导入BMSCs,基因转染效率及表达水平较高。2、BMP-2基因修饰的BMSCs其增殖未因转染受影响,并可持续表达基因产物,诱导其向成骨细胞分化。3、nHAC支架具有良好的生物相容性,适于转基因细胞的粘附、生长和增殖;转基因细胞在支架环境中仍继续表达并分泌BMP-2,诱导BMSCs成骨分化。4、nHAC具有良好的可降解性、骨键合能力和骨传导性,是优选的骨组织工程支架材料。BMP-2基因修饰的BMSCs在体内保持存活并继续表达外源基因,同时促进植入细胞向成骨细胞转化,参与成骨,促进椎间融合。5、BMP-2基因修饰的BMSCs复合nHAC构建的组织工程骨是一种可应用于促进脊柱融合,具有骨诱导生物活性的自体骨替代新材料。更多还原

【Abstract】 PrefaceSpinal fusion has been one of the most popular procedures performed in spine surgery,however,some clinical investigations have shown a considerable rate of failure to achieve a solid bony union.The most common clinical approach for preventing nonunions has been the use of internal fixation,which provides the initial stability and mechanical environment for bony fusion.Despite the improved mechanical strength of the recent spinal instrumentations,spinal bony fusion is the objective.Traditional spinal fusion procedures use autogenous bone as a graft to provide the osteoinductive and osteoconductive components necessary for the formation of new bone at the operative site.Although autogenous bone graft is though to be a gold standard in the achievement of solid spinal fusion,there is frequently an inadequate supply of autogenous bone graft for performing multilevel spinal arthrodesis.In addition,the morbidity of autogenous bone graft harvest is reported to be as high as 30%,with the most frequent complications including chronic donor site pain,nerve injury,infection,fracture,hematoma,and increased operative time.The healing of a spine fusion is a multifactorial process,the donor site morbidity,limited supply,and imperfect success rate of autogenous bone graft has alerted researchers to search for new suitable alternatives.The ideal autograft alternative is a material that may be used entirely in place of autogenous bone graft to achieve the same or a better fusion success rate.The rationale underlying the use of a matrix/stem cells/growth factor combination as an alternative to autogenous bone is that the osteoconductive and osteoinductive components of such a combination will attract,support,and simulate host osteogenic cells to form new bone tissue.Currently,there has been a considerable clinical interest in the use of matrix/stem cells/growth factor combination for bone regeneration and osseous graft supplementation therapy.Accordingly,the current study introduced collagen based composite （nano-hydroxyapatite collagen,nHAC） as a scaffold to carry adenovirus-mediated bone morphogenetic protein-2（BMP-2） transfected rabbit bone marrow stromal cells （BMSCs） in vitro,and evaluated its effect on the relative success of gene therapy by means of a rabbit spinal-fusion experiment.Materials and Methods1、Adenovirus-mediated bone morphogenetic protein-2（BMP-2） transfected rabbit bone marrow stromal cells（BMSCs） in vitro（1） Production of the Adenoviral VectorTwo different adenoviral constructs were prepared for this study: Adenovirus-Lacz construct（Ad-Lacz） and adenovirus BMP-2 construct（Ad-BMP-2）. 293 cells were incubated in Dulbecco’s Modified Eagle Medium（DMEM） containing 10%fetal bovine serumand.Ad-BMP-2 were purified and detected by PCR.Titers were determined by standard plaque assay.（2） Ad-BMP-2 transfected rabbit BMSCs in vitroRabbit BMSCs were isolated and cultured by the method of complete bone borrow culture.BMSCs were identified by inducting multipotent differentiation.BMSCs differentiated into osteoblasts were assessed by Von Kossa staining to observe the mineralized nodules and standard alkaline phosphatase（ALP） assay.Adipocytes differentiation of BMSCs was assessed by Alizarin Red-S staining.Ad-BMP-2 transfected rabbit BMSCs.mRNA and protein expression of BMSCs was detected by BMP-2 hybridization in situ and immunohistochemical technique.Ad-Lacz transfected rabbit BMSCs in the same way.X-gal staining was used to detect the transfection rate and Western blot analysis was used to determine if BMSCs infected with Ad-BMP-2 produced BMP-2 protein.（3） The effect of BMP-2 gene transfection on BMSCs proliferation and differentiationThe change of BMSCs cell cycle after transfection was observed by flow cytometry.Alkaline phosphatase activity and staining,immunofluorescence analysis of osteocalcin and collagen I were used to analyze the effect of BMP-2 gene transfection on BMSCs proliferation and differentiation.2、BMSCs modified by BMP-2 gene was planted into the scaffold of nano-hydroxyapatite collagen（nHAC） to construct tissue engineering bone in vitroBMSCs transfected by Ad-BMP-2 were collected after they were trypsinized with trypsin.Then the BMSCs were planted into the scaffold of nHAC equably and were cultured at 37℃in a humidified atmosphere with 5%CO₂.The adhesion and growth of cells on the nHAC scaffold were observed by scanning electronic microscopy（SEM）.Energy spectrometer was used to detect the secretion of calcium around cells.Western blot analysis was used to determine the expression BMP-2 protein.3、BMSCs by Ad-BMP-2 Gene transfer combined nHAC scaffold for a rabbit Lumbar-Fusion Experiment（1） Surgical ProcedureSixty mature New Zealand white rabbits（weight 2.0-2.5kg） were divided into five groups:BMSCs transfected by Ad-BMP-2 with nHAC group（group A）,BMSCs not transfected by Ad-BMP-2 with nHAC group（group B）,nHAC group（group C）, autologous bone group（group D） and control group（group E）.Rabbits were anesthetized by abdominal injection of 1.5 ml/kg of 3%sodium pentobabital.After being placed in the prone position in sterile fashion,an anterior approach by posterior peritoneal was made,followed by developing to expose the vertebral body and intervertebral space of L4-5 or L5-6.After removing the nucleus pulposus,cartilage and annulus fibrosus and endplates,graft materials were placed and fixed.Penicillin was intramuscularly injected on postoperative day 3 for antibiosis.（2） Radiographic EvaluationThe L5-L6 spines from each group animals were examined by lateral plain radiographs sequentially at 4,8,and 12 weeks after surgery.Fusion and location of new bone formation were observed.（3） Biomechanical TestingBiomechanical testing to evaluate the solidity of the L5-L6 fusion site was performed by a three-point flexion-bending test.The biological character of fusion segment was compared and accounted.（4） Gross and Histological AnalysisThe specimens of the spinal segments of all the animals were harvested at 4,8,12 weeks postsurgery.They were inspected grossly first,and then sent for light microscopic examination.In the laboratory,they were stained with hematoxylin and eosin（HE stain）,and observed under a light microscope to examine for bony fusion. HE-stained specimens were analyzed by computational photogrammetry and the pixels of new form bone were calculated by image-analysis software.Immunohistochemical stain analysis was used to observe the expression of BMP-2.（5） Statistical AnalysisSPSS11.5 for windows was used for statistical analysis.Comparisons in each group were made using one-way analysis of variance.Significance for all tests was defined as P＜0.05.Results1、Part 1 （1） Determination of Ad-BMP-2DNA-PCR analysis showed that 1.2 kb gene segment was observed in Ad-BMP-2 tranfected group,whereas in Ad-Lacz transfected group or 293 cells not transfected group there were none.（2） BMSCs cultureAfter primary culture was sustained for 24-72 hours,the round cells displayed a polygonal morphology.At day 7-10 of the culture,the rate of proliferation accelerated to form the colony with approximately 80%confluency and at day 14-21,the cells grew circinately and fused to sheets.The cultured cells appeared to grow more rapidly after passages were done.（3） Determination of BMSCsThe cultured cells proliferated with cobblestone-like appearance after mineralized fluid was added into the medium,more dispersed compact round conglomeration could be observed among the cells.Von Kossa staining showed black mineralized nodule among the cells and standard alkaline phosphatase（ALP） assay displayed the positive brown-black stain in plasma.After adipocytes induction,orange-red stained fat globelet was observed by Alizarin Red-S staining.The cultured cells were identified as BMSCs because of the multipotent differentiation to blastocytes and adipocytes.（4） BMP-2 expression of BMSCs transfected by Ad-BMP-2Positive X-Gal staining was observed in histologic sections with more than 90% of tranfection rate.In the hybridization in situ and immunocytochemical analysis,we observed positive signal in the plasma of transfected BMSCs.In the Western blot analysis,positive signal lane of BMP-2 was observed in transfected group,whereas in the control group no signal was seen.It was proved that Ad-BMP-2 transfected BMSCs efficiently with high expression of mRNA and protein of BMP-2 gene.（5） The effect of BMP-2 gene transfection on BMSCs proliferation and differentiationFlow cytometry showed there was no difference of cell proportion of G1 phase and S phase between tansfected BMSCs group and the control group,which indicated that the synthesization of cell DNA and proliferation was not effected by transfection. Alkaline phosphatase activity of transfected cells was significantly higher than that in the control group.Positive green flurescent substance in plasma by immunofluorescence analysis of osteocalcin and positive expression of collagenⅠby immunocytochemical analysis indicated that transfected BMSCs differentiated to osteoblasts.2、Part 2BMSCs transfected by Ad-BMP-2 adhered to the surface and hole of the nHAC scaffold and grow well by scanning electronic microscopy（SEM） observation.Energy spectrometer detection showed that higher calcium content than that in the control.Positive expression BMP-2 protein were detected by Western blot analysis in Ad-BMP-2 transfected BMSCs group while negative in Ad-Lacz transfected BMSCs group and non-transfected BMSCs group,indicating that BMSCs transfected by Ad-BMP-2 was not effected by the nHAC.3、Part 3Radiographic evaluation,gross and histological analysis showed that all specimens were graded as nonunion in group E,complete fusion in group A,B,D and partial fusion（75%） in group C at 12 weeks.Computational photogrammetry demonstrated that the area of new formation bone in group A was obviously higher than that in group B,C.There was no difference on the area of new formation bone between group A and E,which indicated that bone fusion of group A is the same as the autografts.Biomechanical testing was performed to compare the stiffness of the specimens.The results showed that they were not significantly different between group A and E.Immunohistochemical analysis of BMP-2 showed strong positive reaction in group A and brown-yellow granule distributed in mesenchymal cells,chondrocytes, osteoblasts and bone cells,while weak positive reaction was observed in group B and C. It indicated that BMSCs transfected by Ad-BMP-2 could express BMP-2 persist and stably.Conclusions1、Adenovirus-mediated BMP-2 gene could be transfect to rabbit bone marrow stromal cells with high level transfection rate and expression.2、The proliferation of BMSCs transfected by Ad-BMP-2 was not effected by transfection,BMSCs could express gene products and differentiate to blastocytes.3、nHAC scaffold could facilitate bony ingrowth and biocompatible,which was in favor of the adhesion,growth and proliferation of trans-gene cells.Trans-gene cells could express BMP-2 and induct the differentiation to blastocytes of BMSCs.4、BMSCs transfected by Ad-BMP-2 could express extrinsic gene and promote the differentiation to blastocytes of planted BMCSs and bony fusion in rabbit.5、BMSCs transfected by adenovirus-mediated BMP-2 Gene combined collagen based composite（nHAC） scaffold was a new substitute of autografts with bioactivity of blastocytes induction.更多还原