【作者】 陈岗；

【导师】 吴海山； 吴宇黎； 李晓华； 钱其荣； 祝云利；

【作者基本信息】 第二军医大学 ， 外科学， 2010， 博士

【摘要】 第一部分：血小板衍化生长因子基因活化材料的制备及生物活性检测目的：构建含有人血小板衍化生长因子-B (hPDGF-B)基因片段的真核表达质粒,并利用兔成纤维细胞做生物活性检测。方法：应用基因重组技术和限制性内切酶双酶切构建并鉴定EGFP-N1-hPDGF-B真核表达载体,体外培养兔成纤维细胞,传代培养后用阳离子脂质体(LipofectAMINE):EGFP-N1-hPDGF-B为3uL：1μg的比例转染,G418筛选获得稳定转染的细胞。通过RT-PCR和荧光法检测PDGF-B表达水平；利用四甲基偶氮唑法检测PDGF-B的生物学活性绘制成纤维细胞生长曲线。结果：成功构建含EGFP-N1-PDGF-B基因的真核表达载体,经RT-PCR检测了PDGF-B在转录水平mRNA的表达情况,荧光法检测目标基因成功转染至靶细胞,转染后的成纤维细胞呈强阳性表达,并持续4周以上。结论：重组真核表达载体构建正确,并能在成纤维细胞中表达PDGF-B mRNA, PDGF-B基因转染可使成纤维细胞有效而稳定的表达活性PDGF-B而产生生物效应。对成纤维细胞有明显的促进生长作用。第二部分：血小板衍化生长因子基因活化材料在体实验研究的组织学分析目的：利用光镜和透射电镜等组织形态学观察手段,比较实验组与对照组在重建愈合过程中胶原表达情况和重建细胞显微结构的差异,以评价脂质体与携带血小板衍化生长因子基因片段的合成质粒共同构成的基因转染系统在兔前交叉韧带重建动物模型中对移植物愈合的促进作用。方法：成年新西兰兔48只,随机分为实验组和对照组。取半腱肌肌腱替代前交叉韧带制备重建动物模型,实验组在重建部位给予基因转染材料,对照组给予盐水。术后1个月,3个月和6个月,取出重建前交叉韧带标本,对其进行光学显微镜和电镜观察,比较胶原表达和细胞超微结构的变化。结果：光镜下观察发现,在不同时期,实验组的成纤维细胞数量、胶原含量、胶原纤维的直径以及胶原纤维的排列均好于对照组,实验组标本组织形态在6月时已十分接近正常前交叉韧带组织结构。电镜观察见各时期实验组的细胞增生现象比对照组活跃,提示修复细胞代谢更旺盛,纤维间隙内有较多基质成分,胞质中可见较为丰富的内质网结构和线粒体。结论：PDGF生长因子基因转染的重建韧带组织结构愈合好于对照组,提示以脂质体为载体携带PDGF-B基因的质粒在重建前交叉韧带愈合局部具有良好的原位转染生物学效能。具有一定的实际应用前景。第三部分血小板衍化生长因子基因活化材料在体实验生物力学测试目的：观察脂质体-PDGF-B基因转染系统对兔半腱肌肌腱替代前交叉韧带动物模型中重建韧带愈合强度的促进作用。方法：成年新西兰兔48只,随机分为实验组和对照组。取半腱肌肌腱替代前交叉韧带制备重建动物模型,实验组在重建部位给予基因转染材料,对照组给予盐水。术后1个月,3个月和6个月,取出重建前交叉韧带标本,对其作生物力学测试,记录最大断裂负荷(极限负荷)、断裂时最大拉伸位移,应力、应变及刚度,并作出以上数据随时间变化的关系图谱。结果：1.实验标本大体观察无异常,对照组重建韧带断裂1例,实验组未发现重建韧带断裂,动物模型重建手术总体成功率为97.9%。2.术后1个月时,两组标本的各项力学指标无显著性差异。术后第3月和第6月时,实验组重建韧带的最大断裂负荷和弹性模量两项性能显著大于对照组,p≤0.05,但两组的其它力学指标无显著性差异。结论：脂质体-PDGF基因转染系统对兔重建前交叉韧带愈合有明显的正性促进作用,可以有效提高韧带移植物在重建塑性过程中的生物力学性能。更多还原

【Abstract】 part 1:Synthesis of gene active material of human platelet-derived growth factor-B and it’s bioactivity testObjective:To construct the eukaryotic expression plasmid of human platelet-derived growth factor-B (hPDGF-B), and explore the ability of transfection of cultured rabbit Gingival Fibroblasts with platelet-derived growth factor-B in vitro.Methods:To construct and verify the eukaryotic expression vector for EGFP-N1-hPDGF-B by recombinant DNA technique and restriction enzyme.Rabbit gingival fibroblasts were cultured in vitro and the cultured gingival fibroblasts were transfected with the complexes of LipofectAMINE reagent and EGFP-N1-hPDGF-B in the ratio of 3:1 (μL:μg) in vitro. Positive clones were selected by G418 screening. The expression of hPDGF-B was detected by RT-PCR and fluorescence microscope. MTT test was done to examine the bioactivity of hPDGF-B and draw a growth curve of fibroblasts.Results:The eukaryotic vector containing EGFP-N1-PDGF-B was constructed successfully. The expression of PDGF-B on the level of mRNA was tested by PCR. The process that the target cell was transfected by target gene can be observed under fluorescent microscope. Fibroblasts cells showed prominently elevated mRNA expression of hPDGF-B and lasted over 4 weeks.Conclusions:The recombinant vector was constructed correctly, and the mRNA of rabbit hPDGF-B could be efficiently and stably expressed in Gingival Fibroblasts cells. Transfected Gingival Fibroblasts could effectively express the active hPDGF-B, take biological effects and apparently improve growth. fibroblastPart 2:The histological evaluations of experiment of reconstructive anterior cruciate ligament with PDGF gene material in vivoobjective To determine the difference between two groups on collagen expression and microstructural observation according to histomorphological method, including light microscope and transmission electron microscope. To evaluate the function of PDGF-B with lipofectMINE gene transfected system to improve the healing of transplant in the rabbit anterior cruciate ligament reconstructive model.Methods Twenty-four New Zealand adult rabbits were randomly divided into 2 groups, the experimental group and the control group. The rabbit anterior cruciate ligament reconstructive model was established according to the procedure as mentioned previously. PDGF-B was locally injected into the reconstructive location in the experimental group while only normal saline (NS) was administered in the control group. The samples were taken and studied under light microscope and electron microscope 1,3,6 months after operation respectively. Through which we can observe the changes of collagen expression and the microstrcture of cells.Results 1.The amounts of fibroblasts and collagen, the diameter and arrangement of collagen fiber in the experimental group are all superior to those in the control group. And the histological morphology was approaching the normal structure 6 month after operation under light microscope.2. The karyokinesis of cell miscrostructure was more active in the experimental group than that in the control group, indicating a prosperous recovery metabolism. There were plenty of endoplasm and mitochondria in cytoplast and copious extracellular matrix between fibers Conclusion The reconstructive tendon transfected by PDGF was better than that in the control group, indicating that PDGF gene plasmids using lipofectMINE as vector have favorable transfected biological effects in the healing location of resconstructive anterior cruciate ligament and have a specific practical prospect.Part 3:Biomechanical test of reconstructive anterior cruciate ligament graft with PDGF gene material in vivoObjective:To observe the liposome-PDGF gene transfected system’s function in improving the biomechanical strength of reconstructive ligament in animal models.Methods:Twenty-four New Zealand adult rabbits were randomly divided into two groups, the experimental group and the control group. Each group concludes 12 animals. The rabbit anterior cruciate ligament reconstructive model was established, PDGF-B was locally injected into the reconstructive location in the experimental group while only NS was administered in the control group. Specimens were obtained 1 month,3 months and 6 months after operation, to observe the tendon graft in general configuration, test their biomechanical indexes including the maximum broken load (peak load), the displacement of maximum load, and calculated stress, strain and stiffness, and form a time-dependent relationship chart according to data above.Results:1. The animals showed no obvious abnormality by observation. There is one case experiencing rupture of ligament in control group while no such case was found in the experimental group. The total successful rate of reconstructive surgery in animal models is 97.9%.2. There was no apparent difference between two groups on each biomechanical index one month after operation. The maximum broken load and stiffness of tendon graft in the experimental group was significantly higher than that in the control group, p≤0.05, while other biomechanical indexes between two groups show no difference 3 months and 6 months after operation respectively.Conclusion:Liposome-PDGF gene transfected system has a positive promoting effect in the wound healing of anterior cruciate ligament reconstruction in rabbits, and can enhance biomechanical efficiency in the process of reconstruction of ligament graft.更多还原