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BMP2和BMP7基因共转染BMSC/PRP凝胶组织工程材料的构建及体内外实验研究
Preparation and in Vivo Study for Evaluating Biocompatibility and Osteogenesis of BMP2and BMP7Gene Co-transfected BMSC/platelet-rich Plasma Gel Complex as Biomateirals in Tissue Engineering
【作者】 宋佳；
【导师】 贾连顺；
【作者基本信息】 第二军医大学 ， 外科学， 2012， 博士
【摘要】 研究目的本课题拟制作并证明BMP2、BMP7基因共转染骨髓间充质干细胞/自体PRP凝胶复合组织工程骨在体内有良好的成骨能力和生物安全性，可以达到植骨融合材料的要求。建立可替代自体骨和其他骨移植替代物的组织工程骨。研究方法将两月龄的新西兰兔36只随机分为4组，每组9只。第一组为单纯植入PRP凝胶组；第二组为植入转染BMP2的BMSCs/PRP凝胶组织工程骨组；第三组为植入转染BMP7的BMSCs/PRP组织工程骨组；第四组为植入共转染BMP2，7的BMSCs/PRP凝胶组织工程骨组。获取、分离、培养、扩增兔骨髓间充质干细胞BMSCs并检测，第三代细胞保存并选取为待转染细胞。以pAd/CMV/V5-Dest为载体构建BMP2和BMP7腺病毒，将两种基因按照分组分别转染第三代兔BMSCs。从每只兔子的耳中央静脉抽取5ml全血，采用Lendersberg法制备得到PRP，并于-20℃保存。将BMP2和BMP7共转染的BMSCs和PRP混合后激活，制备得到BMP2、BMP7基因共转染骨髓间充质干细胞/自体富血小板凝胶支架组织工程骨，部分培养7天后对该组织工程骨进行电镜扫描观察其组织学和形态学。制备新西兰兔胫骨缺损模型，在胫骨平台下方约2cm处用钻头磨出大小约为3mm×5mm×7mm大小的单皮质骨缺损槽，按照分组分别植入材料，丝线间断缝合伤口后将动物分笼饲养。16周处死动物，观察组织工程骨的表面变化、骨痴形成、骨缺损修复情况及周围组织反应。进行X线和CT检查，观察各组骨愈合情况，行组织学染色观察及电镜观察等。研究结果BMP2、BMP7基因共转染骨髓间充质干细胞/自体富血小板凝胶支架组织工程骨在体外能良好表达相关基因和蛋白，植入兔体内后无免疫源性，具有良好的成骨作用。其在体内能稳定的表达BMP2与BMP7，较未转染BMP2与BMP7组有更好的骨诱导性能，且其较单基因转染组有更好的成骨活性，可成为替代自体骨和其他骨移植替代物的组织工程材料。研究结论1、以全骨髓贴壁法可以分离得到具备多向分化潜能的兔BMSCs；2、以pAd/CMV/V5-Dest为载体构建的BMP2腺病毒和BMP7腺病毒能够共转染兔BMSCs，并共同表达BMP2和BMP7；3、以Lendersberg法可以制备得到PRP，和BMP2、BMP7基因共转染BMSCs有很好的组织相容性；4、BMP2、BMP7基因共转染组，较其他组有更好的骨诱导活性和成骨能力，更能促进成骨相关基因和蛋白的表达。
【Abstract】 Objective The purpose of this study is to establish an tissue engineering bone withautologous platelet-rich plasma (PRP) gel as scaffold,combined with rabbit bonemarrow mesenchymal stem cells (BMSCs) coexpressed with BMP2and BMP7,andprove it’s good osteogenic capacity and bio-security in vivo,which can achieve therequirements of the interbody fusion materials and replace autologous bone and bone graftsubstitutes.Methods36New Zealand rabbits(two-month-old) were randomly divided into4groups of9. The first group implanted with PRP gel; the second group implanted withautologous platelet-rich plasma (PRP) gel combined with BMP2gene transfectedrabbit bone marrow mesenchymal stem cells (BMSCs); the third group implantedwith autologous platelet-rich plasma (PRP) gel combined with BMP2genetransfected rabbit bone marrow mesenchymal stem cells (BMSCs), the forth groupimplanted with autologous platelet-rich plasma (PRP) gel combined with BMP2,7gene co-transfected rabbit bone marrow mesenchymal stem cells (BMSCs).To obtain,isolate,culture and amplified rabbit bone marrow mesenchymal stem cells and check,select the third generation of cell to be transfected cells. To establish BMP2and BMP7adenovirus vector wiht pAd/CMV/V5-Dest as the carrier, and transfect the two genes into thethird generation of the rabbit BMSCs.5ml of whole blood was taken from ear central vein in each rabbit, PRP is prepared using theLendersberg method, and stored at-20℃.Mix and activate BMP2and BMP7co-transfected BMSCs and PRP, establish the tissueengineering bone with autologous platelet-rich plasma (PRP) gel combined withrabbit bone marrow mesenchymal stem cells (BMSCs) coexpressed with BMP2andBMP7,Part of which culture7days,then to observe the histological and morphological by scanning electron microscopy.Preparation of New Zealand rabbit lumbar defect model: drilling a single cortical bonedefect about2cm below the tibial plateau，the grind size is approximately3mm x5mmx7mm. Implant materials in the bone defect in accordance with the grouping.4，8and12week after surgery，we kill3rabbits in each group to observe changes in thetissue-engineered bone surface,callus formation,the repair of bone defect and surrounding tissuereaction. The remaining experimental animals were sacrificed16weeks after surgery,X-ray examination,CT scan, histological staining and electron microscopy are takento observe bone healing in each group.Results The tissue engineering bone we established with autologous platelet-richplasma (PRP) gel combined with rabbit bone marrow mesenchymal stem cells(BMSCs) coexpressed with BMP2and BMP7,can express genes and proteins related toosteogenesis adequately, without immunogenic,and has a good osteoinductive and osteogenesis invitro.It can express BMP2and BMP7stablely, and it has a better performance of boneinduction compared with untransfected group of BMP2and BMP7and other groups. Thetissue engineering materials we established can become the substitute to autologous bone andother bone graft materials.Conclusions1、Preparation of BMSCs by bone marrow adherent method was proper.The BMSCs obtained have multilineage differentiation potential.2、BMP2adenovirus and BMP2adenovirus vector in a pAd/CMV/V5-Dest could be transfected in rabbit BMSCs andco-expression of BMP2and BMP7.3、PRP prepared by Lendersberg method have goodhistocompatibility with BMP2, BMP7gene co-transfected BMSCs.4、BMP2, BMP7geneco-transfected group with better expression of osteoblast-related genes and proteins have betterosteoinductive activity and osteogenic capacity, compared with other groups.