【作者】 朱国强；

【导师】 吴织芬；

【作者基本信息】 第四军医大学 ， 口腔临床医学， 2004， 博士

【摘要】 因牙周病而丧失的牙周组织的修复再生难度较大，因此要达到牙槽骨、牙骨质和牙周膜的完全功能性再生一直是牙周病学领域研究的热点。传统的牙周治疗只能部分修复牙周组织，近年来发展起来的引导性组织再生技术(GTR)虽可使一定的牙周组织获得再生，但总的看来，尚不能达到牙周组织完全再生的目的。将组织工程技术引入牙周病治疗的研究领域，为牙周组织再生带来新思路。现代的组织工程学包括种子细胞／支架材料、生长因子／支架材料和种子细胞／生长因子／支架材料三种构建方式。但生长因子复合入支架材料时一般需要高温和高浓度的表面活性剂，这会加快蛋白的降解，使其活力降低，重组蛋白的免疫毒性增加。复合入支架材料后由于湿度的变化和苛刻的微环境，生长因子可出现不可逆的聚合、脱氨基和氧化，因此需要大剂量的使用生长因子才能维持生长因子的生物学活性和持续释放，而生产这些蛋白价钱昂贵，且大剂量使用有很多安全危险：如蛋白可能扩散到血液中，在机体其他组织发挥生物学效应等。如果种子细胞在一定的时间内，在损伤组织局部高表达细胞因子，可以避免将蛋白复合入支架材料的上述缺点。因此我们把基因治疗技术与组织工程技术相结 第四军医大学博士学位论文合，成功地克隆了犬胰岛素样生长因子一1(IGF-l)编码区基因，构建两种真核质粒载体，利用荃因转染技术，将其转染入自体骨盆荃质细胞(MSCs)，赋予细胞新的生物学特性。将这种IGF一1基因修饰的MSCs与鸵鸟钙磷陶瓷骨复合，修复动物模型上的牙周组织缺损。探讨IGF-1墓因修饰的MSCs在牙周组织再生中的作用，为将基因治疗与组织工程联合应用于牙周组织缺损再生治疗莫定实验基础。为此，本研究进行了如下几方面的实验:1.新鲜犬左心室组织中提取总RNA，以RT一PCR方法获取IGF一1编码区 cDNA全序列，将其与真核表达载体peDNA3.1(+)连接，转化大肠杆 菌，将构建的重组表达载体peDNA3.1(+)一IGF一1侧序鉴定.结果重 组质粒peDNA3.1(+)一IGF一1的酶切图谱和序列分析结果与国外文献报 道一致.将PcDNA3.1(+)一IGF一1质粒双酶切，获得目的基因，·亚克 隆入真核表达载体PI双s2一EGFP质粒中，转化大肠杆菌，构建重组真 核表达载体pIRESZ一EGFP一IGF一1.结果酶切鉴定证实犬IGF-l成功的克 隆入PIRESZ一EGFp中。2.体外培养大鼠骨髓基质细胞(MSCs)，利用脂质体将 pIREsZ一EGFP·IGF-1基因转染至MsCs中，荧光显微镜、免疫组化检测 MSCs是否表达重组细胞因子，收集转染细胞的培养液上清，培养L929 细胞，流式细胞仪检测转染细胞的培养液上清对L929细胞周期的影响。 结果pIREsZ-EGFP-IGF一1转染阳性MsCs细胞，荧光显徽镜观察与免 疫组化证实转染细胞内有目的蛋白的高表达;转染目的荃因的MSCs 培养液存在有活性的生长因子，可明显促进L929细胞的增殖。3.培养狗自体MSCs细胞，利用脂质体将p1RESZ一EGFP.IGF一1基因转染 至MSCs中，经G418筛选后获得了稳定表达IGF一1蛋白的细胞克隆; 在体外培养扩增后，通过W七Stem blot、免疫组化、夹心EllSA检侧基 因转染对MSCs表达IGF一1的影响:通过检测IGF。1基因转染的MSCs 细胞的碱性磷酸醉活性，MTT检测转染细胞培养上清液对人牙周膜细 胞(PDLCs)增殖活性的影响，对该细胞的生物学特性进行了观察.结 果:荃因转染后的MSCs细胞形态没有明显变化，免疫组化和W粉怨m blot证实，转染细胞内有IGF一1蛋白的高表达。ELISA方法检侧显示 IGF·1能分泌至细胞外。IGF一1基因转染的MSCs细胞的孩性磷酸醉活 性较未转染细胞明显升高(p<0 .05).转染细胞培养上清液能明显促进 第四军医大学博士学位论文 PDLcs细胞的增殖(卜0.05)。4.通过免疫组化和图像分析的方法检侧IGF-l基因转染对MSCs合成血管 内皮细胞生长因子(VEGF)、骨形成蛋白一2(BMP一2)、骨涎蛋白(BSP)、 骨桥蛋白(OFN)、骨粘连素(ON)和I型胶原的形响。结果:IGF一1 基因转染后可促进MSCs合成BMP一、VEGF、ON、OPN和I型胶原 (p<0 .05)，但对合成BsP影响不显著(p>.05)，当与矿化液培养9d 后，IGF一1基因转染的MSCs与未转染的MSCs相比较可明显的促进 BSP的合成。5.将IGF一1基因转染的自体MSCs与鸵鸟钙磷陶瓷骨(OTBC)复合后， 体外培养，扫描电镜观察。将IGF一1基因转染后的自体MSCs细胞作为 种子细胞，与OTBC复合，植入狗的人工牙周组织缺损区。通过组织 学观察和组织学测盆，评价基因转染MSCs细胞对牙周组织再生的影 响，并以未转染的MSCs细胞和无细胞的支架材料作对照。结果:MScs 复合OTBC后生长良好，24h细胞已贴附、伸展，随着时间的增加，细 胞生长与增殖明显，并有大量细胞外基质分泌。证明IGF一1基因转染 MSCs与01，BC有较好的生物相容性。动物实验修复狗人工牙周组织缺 损，sw后，基因转染的MSCs细胞组可见近乎完全的牙周组织再生， 未见结合上皮长入、根吸收和骨粘连等不良愈合方式.未转染M更多还原

【Abstract】 Periodontal regeneration is expected to reconstitute the injured periodontal tissues physiologically and functionally, including cementum, periodontal ligament and alveolar bone. It is difficult for the traditional treatment to attain this goal. Although the guided tissue regeneration(GTR) achieved the periodontal regeneration, it can not fulfill this goal entirely. To treat the periodontitis with tissue engineering established the new field for the therapy of periodontal regeneration. Nowadays the form styles of tissue engineering have included cell/scaffolds, growth factors/ scaffolds and cells/ growth factors/ scaffolds. But the proteins have to expose in high temperature and high concentrations of surfactants when the protein incorporated into scaffolds. These conditions can promote the protein degradation, and in turn, decrease potency and increase risk of immune toxicity. Moreover, protein successfully incorporated in scaffolds may undergo irreversible aggregation, deamidation and oxidation because of elevated levels of moisture and evolution of harsh microclimate. So to maintain full bioactivity and to sustain the release of growth factors, we need a large amounts of recombinant cytokine. The proteins are expensive to manufacture. High-dose proteins bring us an additional safety risk: the cytokine may diffuse from the wound bed into the bloodstream. If the cells in the scaffolds can highly express the recombinant cytokine, the disadvantages of using the protein will be overcome. So wecombine gene therapy with tissue engineering. Dog’s Insulin-like growth factor I (IGF-1) gene was transfected into bone marrow stem cells (MSCs). The transfected MSCs with ostrich true bone ceramics(OTBC) were used to repair the periodontal defects. This study is to estimate the potential effects of IGF-1 gene transfected MSCs and to provide a new way for restoring the periodontal defects. This study has included five experiments:1. Total RNA was extracted from dog’s left ventricular myocardium. The desired DNA product was obtained from the total RNA by RT-PCR. The segment (about 297bp) was inserted into pcDNA3.1(+) vector and the inserted plasmid was transformed into E.coli DH5 a . The positive clone was analyzed by restriction endonuclease mapping and DNA sequencing. The restriction endonuclease map and sequence of dog IGF-I functional fragment were consistent with those of the published. The IGF-1 gene in the pcDNA3.1(+)-IGF-l were subcloned into pIRES2-EGFP expressing vector. The plasmid was transformed into E.coli DH5 a . Restrictive enzyme(Nhe I, EcoR I) digestion analysis showed that recombinant expression vector pIRES2-EGFP-IGF-l has been constructed successfully.2. pIRES2-EGFP-IGF-l was transfected into MSCs with lipofectamine. After 48h transfection, the Insulin-like growth factor I (IGF-1) expression in the transfected MSCs was detected under fluorescence microscope and by immunohistochemistry. The supernatant of the transfected MSCs cultured with L929 fibroblasts. The effect of the recombinant IGF-1 on the L929’s cell cycle was observed by flow cytometer analysis. Result: The transfected MSCs displaying green fluorescence were observed under fluorescence microscope. The immunohistochemical staining showed that positive reactant of brown-yellow can be seen in the cytoplasm of MSCs transfected by pIRES2-EGFP-IGF-l. The supernatant of the transfected MSCs can increase the L929’s proliferation.3. pIRES2-EGFP-IGF-l was transfected into MSCs with lipofectamine. Positive clones were selected with G418. The expression of IGF-1 protein in the MSCs was determined by immunohistochemistry and Western blot analysis. The IGF-1 in the supernatant of the transfected MSCs was detectedby sandwich-in ELISA. The transfected MSCs’ ALP activity were investigated. Add culture medium of stably transfected MSCs cells to periodontal ligament cells (PDLCs). The change of PDLCs’ proliferation was observed by MTT.The results of immunohistochemistry and Western blot analysis suggested that IGF-1-trnsfected MSCs could e更多还原