电穿孔介导的基因治疗对下颌骨牵引成骨影响的初步研究

【作者】 黎德平；

【导师】 郭力； 吴国平；

【作者基本信息】 泸州医学院 ， 外科学， 2009， 硕士

【摘要】 牵引成骨技术(distraction osteogenesis,DO)是通过将骨段逐渐牵引获得新骨的一种新方法,其最大优点是避免了植骨和供区并发症,同时可牵引延长软组织。自1992年McCarthy首次将DO技术用于下颌骨畸形患者的治疗以来, DO技术已广泛用于颅颌面骨严重发育不足畸形及骨缺损的整复治疗。然而,DO的某些并发症(如骨再生不良、延迟愈合、不愈合),特别是其较长的疗程及牵引装置长时间留置面部或口腔内所引发的各种问题(如固定螺钉松脱、伤口感染、骨折等)成为临床应用和推广该项新技术的“瓶颈”和主要障碍。因此,医生和病人都欢迎新的技术来加速治疗进程。本研究以新西兰大白兔双侧下颌骨牵引为动物模型,着眼于利用电穿孔技术将重组质粒转染下颌骨牵引区,试图利用基因治疗的方法促进下颌骨牵引成骨,进行了一系列的探索。首先观察了电穿孔技术介导的pIRES-hVEGF165-EGFP转染对下颌骨DO过程中早期血管生成的影响,然后在此模型基础上进行了电穿孔技术介导的pIRES-hBMP2-VEGF165体内转染,观察其对下颌骨DO成骨的影响。简述如下:第一部分:电穿孔介导的pIRES-hVEGF165-EGFP对牵引成骨过程中早期血管生成的影响目的:探索电穿孔介导的重组质粒pIRES-hVEGF165-EGFP转染对下颌骨DO过程中早期血管生成的影响,为下颌骨牵引成骨的深入研究奠定基础。方法:选用新西兰大白兔30只,沿双侧下颌骨边缘切开皮肤,钝性分离附着下颌骨的肌肉,暴露下颌骨。于下颌骨颏孔处,用微型电动钻截开下颌骨,于下颌骨断开处安放2cm牵引器,固定,逐层缝合组织。于术后3天开始牵引,每天0.8mm ,连续牵引7天后,将兔随机分为3组:A组:质粒+电脉冲组,B组:质粒组, C组:生理盐水组。A、C组均施加电脉冲刺激)。C组织注射重组质粒而不施加电刺激。各组分别于注射后1天、3天、7天、14天处死动物,切取牵引区组织约0.4cm×0.4cm大小,进行组织学检查、电镜观察,CD34免疫组织化学染色进行微血管密度(microvessel density, MVD)检测。结果:电镜观察发现,A、B组血管内皮细胞呈增殖活跃状态:细胞呈梭状,具有多个突起,基底膜完整,胞浆内具有丰富的线粒体呈椭圆形或圆形;C组多数血管内皮细胞部分呈现退变和凋亡早期改变。免疫组化染色发现,转染后第1天血管壁内皮细胞浆CD34表达较弱;第3~14天,牵引区肉芽组织血管内皮细胞均出现CD34阳性表达。B组CD34阳性表达较C组强,A组CD34阳性表达较B组强,C组最弱(P<0.05),各组各时间点差异显著(P<0.05)。比较施加电穿孔的A组、C组和未施加电穿孔的B组,A组、B组的CD34表达持续阳性且呈上升趋势,而C组CD34阳性表达维持在第1天CD34阳性表达水平上平稳波动。结论:电脉冲介导的pIRES-hVEGF165-EGFP重组质粒体内转染能够促进牵引区早期微血管的生成,使局部血管增生、渗入,增加骨断端的血流量。提示其有刺激骨基质合成和诱导细胞增殖分化等活性功能,从而对调节和促进骨的生长和修复过程具有重要作用。第二部分:电穿孔介导的基因治疗对下颌骨牵引成骨影响的初步研究目的:探索电穿孔介导的基因治疗对下颌骨DO过程中牵引间隙新骨生成的影响,从而为促进下颌骨DO新骨生成,缩短牵引周期,减少并发症提供新思路。方法:选用新西兰大白兔50只,沿双侧下颌骨边缘切开皮肤,钝性分离附着下颌骨的肌肉,暴露下颌骨。于下颌骨颏孔处,用微型电动钻截开下颌骨,于下颌骨断开处安放2cm牵引器,固定,逐层缝合组织。于术后3天开始牵引,每天0.8mm ,连续牵引7天后,将兔随机分为5组:A组:在牵引区注射2μg(0.1μg/μl)重组质粒pIRES-hVEGF165-hBMP2。B组:在牵引区注射2μg ( 0.1μg/μl )重组质粒pIRES-hBMP2。C组:在牵引区注射2μg(0.1μg/μl)重组质粒pIRES-hVEGF165。D组:在牵引区注射2μg(0.1μg/μl)空质粒pIRES。E组:在牵引区注射相同剂量的生理盐水。A、B、C、D、E组均施加电穿孔刺激(电脉冲参数:电压300V,频率0.2Hz,平均脉宽2ms,6个脉冲,3个脉冲后更换正负极)。各组分别于固定期第1、2、4、8周行X线及QCT检查。选整个牵张间隙新生骨痂部分为兴趣区(regions of interest, ROI),测定骨密度。然后处死动物,取材测量牵引区新生骨的三点抗压强度;牵引区组织切片HE染色进行组织学检查和形态计量学分析。结果:A、B、C组各时点新生骨痂密度明显高于D、E组;各时点新生骨的三点抗压强度明显高于D、E组;组织学检查和形态计量学分析发现,A、B、C组与D、E组比较,间隙内有更多的新生血管、成骨细胞和间充质细胞等成分,新生骨量明显高于后者;新生骨小梁的宽度仍明显优于D、E组。结论:电脉冲介导的pIRES-hVEGF165-hBMP2重组质粒体内转染能够使牵引区获得较满意的骨再生和骨化成熟进程,其新骨骨化、改建过程超过对照组。提示,VEGF和BMP可作为下颌骨DO治疗的分子靶目标,联合应用BMP与VEGF,可能会实现成骨与血供的联合重建,并且使单一生长因子的效应放大,使骨愈合的速度加快。更多还原

【Abstract】 Distraction osteogenesis is a method of producing new bone directly from the osteotomy site by gradual traction of the divided bone fragments. The benefits of distraction include the avoidance of bone grafting and donor-site morbidity, and the concurrent expansion of the soft-tissue envelope. Since the first clinical report on the use of distraction osteogenesis to lengthen the human mandible in 1992, distraction osteogenesis has become a widely accepted approach in the treatment of severe craniofacial deformities and bone defects. However, one of the major disadvantages of this technique is the lengthy course of treatment required for distraction and consolidation, which may result in pin tract soft-tissue infection, bone infection, and psychological problems. In addition, fibrous nonunion still occurs from inadequate neovascularization (a significant problem in irradiated bone) or from unstable fixation resulting from secondary bone infection or device malposition. Consequently, both surgeons and patients would welcome any technical improvement to speed the treatment process.New Zealand rabbits bilateral mandibular DO model was employed in our studies. This study evaluates the effect of electroporation mediated transfecting recombinant plasmid on mandibular distraction at normal distraction rates. At first, we observed the effect of electroporation mediated transfecting recombinant plasmid pIRES-hVEGF165-EGFP on angiogenesis of the distraction area during early mandibular DO, and investigate the effect of electroporation mediated recombinant plasmid pIRES-hBMP2-VEGF165 transfect on osteogenesis of mandibular DO.PART I: The Effect of Electroporation Mediated Transfecting Recombinant Plasmid pIRES-hVEGF165-EGFP on Angiogenesis of The Distraction Area During Early Mandibular Distraction OsteogenesisObjective: To explore the effect of electroporation mediated transfecting recombinant plasmid pIRES-hVEGF165-EGFP on angiogenesis of the distraction area during early mandibular DO, for further study of distraction osteogenesis. Methods : Thirty New-Zeland rabbit employed submandible extraoral incisions to osteotomy and place the distaction devices. A mechanical burr supplemented by saline irrigation, can be used to interrupt the buccal cortex as well as the superior and inferior cortical borders of the mandible, The osteotomy is completed by inserting and rotation an osteotome to demonstrate separation of the bony segments. The arms of distractors are placed percutaneously outside the oral. Chose bicortical or moncortical bone screws to fix the distractor accoding to the rockiness and thickness of mandible. The arms kept outside through the anterior incision. The wound was irrigated with saline and closed with interrupted suture respectively. The latency period was 3 days. Activation of the device was commenced after the latency period and proceeds at the rate of 0.8mm per day for 7days. After the completion of activation, the device was maintained in position, and then the rabbits were randomly divided into 5 groups: group A: recombinant plasmid pIRES-VEGF165-EGFP was injected into the distraction area, after the completion of activation; group B: recombinant plasmid pIRES-VEGF165-EGFP was injected into the distraction area ; group C: normal saline (NS) group NS was injected into the distraction area. Afer injection group A and group C employed electroporation, but group B without electroporation, then the rabbits were sacrificed at 1d, 3d, 7d and 14d after injection respectively. Harvest the distraction area tissue for histological examination and electron microscope observe, immunohistochemical stain for CD34 and detection microvessel density. Results: Under electron microscope, generation of vascular endothelial cell(VEC) of group A and group B are active, VECs are spindle and have many ecptoma, basal membrane are integrity. Intracytoplasm, there are many ellipse or round chondriosome, majority of VEC in group C take on early change of cataplasia and apoptosis. Immunohistochemistry detection The immunochemical stain for CD34 showed it expressed weakly at the first day after transfection, from 3 to 14 days after transfection, CD34 of VECs in the distraction area expressed positively, it was stronger in group B than that of group C, and that in group A was the strongest(p<0.05), there are remarkable difference among three groups and different time respectively. Compare to each other, CD34 of VECs expressed continue positively with a tendency to rise in group A and B. But it fluctuate at the level of the frist day in group C. Conclusion: Electroporation-mediated transfecting recombinant plasmid could promote angiogenesis during early stage of mandibular DO. It could promote loca vascular proliferation and penetration, increase the blood flow of broken ends of fractured bone. It indicate that genen therapy can stimulate bone matrix synthesis and induce cell generation differentiation. Thus, it play an important role in regulate and promote growth and reparative process of bone.PART II: Primary Study of the Effect of Electroporation Mediated Gene Therapy on Rabbit Mandibular Distraction OsteogenesisObjective: To explore the effect of electroporation mediated gene therapy on the new bone formation in distraction area, and to find one potential approach to accelerating bone regeneration, to shorten the cycle of distraction. Methods:Thirty New-Zeland rabbit employed submandible extraoral incisions to osteotomy and place the distaction devices. A mechanical burr supplemented by saline irrigation, can be used to interrupt the buccal cortex as well as the superior and inferior cortical borders of the mandible, The osteotomy is completed by inserting and rotation an osteotome to demonstrate separation of the bony segments. The arms of distractors are placed percutaneously outside the oral. Chose bicortical or moncortical bone screws to fix the distractor accoding to the rockiness and thickness of mandible. The arms kept outside through the anterior incision. The wound was irrigated with saline and closed with interrupted suture respectively. The latency period was 3 days. Activation of the device was commenced after the latency period and proceeds at the rate of 0.8mm per day for 7days. After the completion of activation, the device was maintained in position, and then the rabbits were randomly divided into 5 groups: group A: recombinant plasmid 2μg ( 0.1μg/μl )pIRES-hVEGF165-hBMP2,was injected into the distraction area, after the completion of activation; group B: recombinant plasmid pIRES-hBMP2 was injected into the distraction area ; group C: recombinant plasmid pIRES-hVEGF165 was injected into the distraction area; group D: pIRES was injected into the distraction area, and group E: normal saline (NS) group NS was injected into the distraction area. After injection every group employed electroporation. then the rabbits were subjected to be examined by X ray and quantitative computed tomography (QCT), chose the distraction area as regions of interest(ROI), bone mineral density(BMD) of newly formed bone was detected at 1 week, 2 weeks, 4 weeks and 8 weeks after gene transfected. After examined the rabbits were sacrificed and harvest the distraction area tissue for detection crushing strength of three points of the newly formed bone. At the same time, all the specimen were taken for histology examination and morphometry analysis.Results: BMD of newly formed bone of the distraction area in group A, group B and group C were remarkable higher than those of group D and group E. The crushing strength of three points of the newly formed bone in group A, group B and group C were also remarkable higher than those of group D and group E. The findings of histology examination and morphometry analysis showed that compare to group D and group E, ther are more new vessels osteoblast and mesenchymal cells in group A, group B and group C, the volume of newly formed bone and the width of newly bone trabecular also higher than those of group D and group E. Conclusion: Electroporation-mediated transfecting recombinant plasmid pIRES-hVEGF165-hBMP2 could obtain satisfactory proceeding of osteogenesis and calcification, which surpassed that of control group. It indicate that VEGF and BMP may act as the molecule target of gene therapy for mandibular DO, combination of VEGF and BMP may be make osteogenesis and angiogenesis come true at the same time, further more, it may be magnify the effect of single growth factor, and promote growth and reparative process of bone.更多还原