【作者】 金晗；

【导师】 孙宏晨； 杨柏；

【作者基本信息】 吉林大学 ， 口腔临床医学， 2014， 博士

【摘要】 由于先天缺陷、肿瘤、外伤及牙周炎等原因造成的骨缺损一直是国内外临床医生所面临的难题。自体骨移植是治疗骨缺损的“金标准”。但是由于自体骨移植物存在供区骨量有限，可能引起供区并发症等缺点，因而限制了它的应用。而异体骨移植物和异种骨移植物具有与宿主骨相似的物理和生物学性质，已经成功应用于许多矫形外科的手术中。然而，它们存在疾病传播和宿主的免疫排斥反应等问题限制了其应用。而合成的骨替代物由于不具有与天然骨相似的机械性能和骨诱导性，因此常常导致移植的失败。骨组织工程学的出现，为解决这些问题提供了一条充满希望的途径。近年来，基于基因和干细胞的治疗已经被用于骨组织工程学的研究，并取得了良好的成果。骨髓间充质干细胞（bone marrow mesenchymal stem cells, BMSCs）是目前骨组织工程学中应用最为广泛的种子细胞，它具有多向分化的潜能，在一定条件下可以分化为骨、软骨、肌肉、脂肪等组织。而骨形态发生蛋白2（bone morphogeneticprotein2, BMP-2）是目前已知的骨诱导能力最强的生长因子，它可以直接诱导间充质干细胞向成骨方向分化。因此，常常利用BMP-2基因转染BMSCs以提高其成骨分化能力。外源基因进入细胞通常需要基因载体，主要包括病毒载体和非病毒载体两类。病毒载体由于其较高的转染效率是目前应用最多的基因载体，但是作为一种改造的病毒，其价格昂贵，难以大规模生产，且存在安全隐患和免疫原性等问题，因此许多学者把注意力逐步转向对非病毒载体的研究。各种类型的聚乙烯亚胺（polyethylenimine, PEI）是最高效的非病毒载体之一，但是它仍然有许多缺点，如：无组织或细胞靶向性、相对于病毒载体转染效率较低以及具有较高的细胞毒性等。因此，为了克服PEI的这些缺点，需要对PEI进行改造。支架材料也是骨组织工程学的主要因素之一。常见的支架材料主要包括天然聚合物、陶瓷材料、合成聚合物和复合材料。但是目前已开发或应用的支架材料都存在一些问题，如：引起炎症反应，降解速率不匹配，缺少生物活性等。因此，一种不需要外源性支架的细胞膜片技术的出现了。它以一种无创的方式将细胞以膜状结构收集，完整的保留了细胞和细胞之间的连接和细胞外基质，以及相关蛋白和生物活性因子，为体内骨损伤的愈合提供了适宜的微环境。基于以上背景，本研究设计为以下三个部分：一、聚乙烯亚胺-藻酸盐/质粒复合物的制备与表征利用阳离子聚合物PEI与天然阴离子聚合物海藻酸钠（alginate）通过静电作用构建了PEI-alginate（PEI-al）纳米基因载体，并对其理化性质进行表征。结果显示：PEI-al纳米复合物的平均粒径为73.8nm，平均电势为+19.9mV；扫描电镜观察可见PEI-al纳米复合物为球形的单分散微粒，当PEI-al纳米复合物与BMP-2质粒（BMP-2plasmid, pBMP-2）结合后可发生部分聚集；通过纳米粒度仪测得PEI-al/pBMP-2的平均粒径增加到149.0nm，电势降低到+18.8mV。DNA凝胶阻滞实验结果表明当质量比达到1.5：1时，材料能够完全结合DNA，材料与基因的复合物为中性或是正电性。二、PEI-al/pBMP-2复合物体内外促成骨的作用的研究利用PEI-al/pBMP-2复合物转染MC3T3-E1细胞，通过realtime-PCR和茜素红染色研究其体外促成骨的作用；将PEI-al纳米复合物和PEI-al/pBMP-2复合物与明胶海绵支架分别复合后移植到大鼠的颅骨缺损内，观察它们体内促成骨的作用。结果显示：转染PEI-al/pBMP-2后，realtime-PCR检测成骨相关基因的表达都有上调，钙结节的形成量也明显多于转染了PEI-al/pEGFP的细胞；Micro-CT观察到PEI-al/pBMP-2组的大鼠颅骨缺损边缘不规则，内部有大量新生骨样组织，且明显多于PEI-al组；H&E染色结果也与Micro-CT相符， PEI-al/pBMP-2组缺损边缘有新生骨组织，缺损中心处也有一些编织状骨形成，而PEI-al组缺损处全部由纤维结缔组织修复。三、PEI-al/pBMP-2复合物修饰的BMSCs膜片体内外促成骨作用的研究利用PEI-al/pBMP-2复合物转染BMSCs后，在含有维生素C的培养基中继续培养10天，获得了能够分泌BMP-2蛋白的BMSCs细胞膜片。通过ELISA检测BMP-2分泌情况；通过realtime-PCR和碱性磷酸酶活性研究其体外促成骨的作用；将分泌EGFP蛋白的BMSCs细胞膜片（EGFP/CS）和分泌BMP-2蛋白的BMSCs细胞膜片（BMP/CS）分别移植到大鼠的颅骨缺损内，观察它们体内促成骨的作用。结果显示：在转染PEI-al/pBMP-2后，BMSCs能够持续分泌BMP-2蛋白达14天以上；realtime-PCR检测表明相关成骨基因的表达均有上调，这一结果说明分泌的BMP-2蛋白能够有效的诱导BMSCs细胞膜片向成骨方向分化；Micro-CT和组织学检测观察到BMP/CS组与其他组相比有效的促进了缺损处骨组织的再生。以上实验结果表明：PEI-al纳米复合物是一种高效的基因载体；利用PEI-al/pBMP-2修饰的BMSCs细胞膜片能够有效的促进骨缺损的修复。更多还原

【Abstract】 Bone loss caused by congenital defects, traumatic injury, cancer, reconstructivesurgery or periodontal disease is always the most difficult challenge both at home andabroad. Autologous bone graft is the so-called gold standard in transplantation surgery.But the use of autologous bone may be restricted because the amount of donor bone islimited and the complication in donor site. The strategies of allogenic or xenogenicare associated with problems such as the risk of disease transmission and graftrejection with a concomitant necessity for secondary surgery, and cost much.Synthetic grafts are important substitutes, but they fail to provide similar mechanicalproperties of human bone tissue, often causing failure in bone healing. Bone tissueengineering attempts to solve the referred problems mentioned above. Recently, gene-and stem cell-based therapy have been applied in bone tissue engineering andachieved some good clinical results.Bone marrow mesenchymal stem cells (BMSCs) have been widely used for bonetissue engineering. They potentially can be used to develop bone，cartilage，muscle oradipose. Bone morphogenetic proteins (BMPs) are well known as osteoinductivegrowth factors which play an important role in bone regeneration process. Theseproteins are capable of inducing the osteogenic differentiation of mesenchymal cells.So BMP-2gene has been frequently used to induce the osteogenic differentiation ofBMSCs.Gene vectors are required for the transfer of exogenous gene into cells, the mainapproach to gene delivery in gene therapy is viral vector systems due to theirrelatively high transfection efficiency. But viral-based gene vectors are a kind ofengineered viruses, so they have the drawbacks of high cost, immune response againstrepeated administration, oncogenicity and difficulty in large-scale production.Therefore, many scientists have shifted their interest to develop non-viral gene vectors.Different types of polyethyleneimine (PEI) are regarded as a kind of the most effective non-viral gene vectors. However, PEI has the disadvantage of no specifictissues and cells targeting, relative lower transgene efficiency compared with viralvectors and high cytotoxicity. In order to overcome the drawbacks, it is necessary tomodify PEI.Scaffold is also a main factor of bone tissue engineering, including naturepolymer, ceramics, synthesis polymer and composite material. However, the scaffoldsstill face the problem of causing inflammation, the degradation rate of biomaterial notmatch the regeneration rate of bone and the lack of biological activity. So the newertechnology of cell sheet without scaffold has emerged. The cells are collected as asheet, which maintains the connections between cells and ECM, and the relativeproteins and biological activity factors. It also can provide a suitablemicroenvironment for bone regeneration.Preparation and characterization of PEI-alginate/plasmid complexesPEI-al gene carriers were prepared by electrostatic interation between the cationicPEI and polyanionic alginate, and physicochemical properties were characterized. Theresults showed that the average particle size of PEI-al nanocomposites were73.8nm,and the zeta potential distribution was+19.9mV; SEM images showed that PEI-alnanocomposites were spherical and monodispersed particles while some aggregationoccured when PEI-al nanocomposites were blended with pBMP-2; DLS resultsshowed that the average particle size of PEI-al/pBMP-2complexes were increased to149.0nm, and the zeta potential distribution was decreased to+18.8mV; Agarose gelelectrophoresis result suggested the formation of neutral or positive complexesbetween PEI-al and pBMP-2, when the weight ratio reaches1.5.The effects of PEI-al/pBMP-2on bone formation in vitro and in vivoPEI-al/pBMP-2complexes were transfected into MC3T3-E1cell line in vitro,and the capability of inducing osteogenic differentiation was evaluated byrealtime-PCR and alizarin red stain. PEI-al/pBMP-2complexes and PEI-alnanocomposites were absorbed into gelatin sponges, respectively, and the gelatinsponges were implanted into the rat calvarial defects in vivo. The effect of boneregeneration was observed by Micro-CT and histologic analysis. The results showedthat the expression of osteogenesis-related gene was increased post-transfected withPEI-al/pBMP-2complexes, and the calcium nodules formation was much more inPEI-al/pBMP-2group than PEI-al/pEGFP group; Micro-CT anlysis indicated that in the PEI-al/pBMP-2group, there are irregular high density thing in the central of thedefect site and the edge of defect is irregular; And more new bone formation wasobserved in PEI-al/pBMP-2group than in the PEI-al group. HE staining showed thatthere are many bone cores in the PEI-al/pBMP-2group, while in the PEI-al groups alldefects were repaired with fibrous connective tissue.The effects of PEI-al/pBMP-2engineered BMSCs cell sheet on bone formation invitro and in vivoPEI-al nanocomposites carrying BMP-2gene could efficiently transfect BMSCs,and the BMP-2protein producing cell sheet was made by culturing the cells invitamin C containing medium for10days. To quantify the secretion of BMP-2protein,ELISA assay was performed; the capability of inducing osteogenic differentiation wasevaluated by realtime-PCR and ALP activity. EGFP producing cellsheet (EGFP/CS)and BMP-2producing cellsheet (BMP/CS) were implanted into the rat calvarialdefects in vivo, then the effect of bone regeneration was observed by Micro-CT andhistologic analysis. The results showed that the genetically engineered cells releasedthe BMP-2for at least14days; The expression of osteogenesis-related gene wasincreased, which demonstrated the released BMP-2could effectively induce the cellsheet osteogenic differentiation in vitro. Micro-CT and histologic analysis indicatedthat BMP/CS group was more efficient than other groups on promoting boneformation in the defect area.Our study demonstrated that PEI-al nanocomposites could efficiently transfectBMSCs and the BMP-2gene engineered cell sheet could effectively enhance boneformation.更多还原