bFGF真核表达载体构建后分泌表达促进颌骨种植体周围骨缺损的研究  

Repair of mandibular defects around implants by construction and expression of bFGF eukaryotic expression vector

作　　者：杨春艳[1] 刘阳[2] 张斌[1] 李春明[1] 徐旭光[1] 

机构地区：[1]哈尔滨医科大学附属第二医院口腔科,黑龙江哈尔滨150081 [2]哈尔滨医科大学附属第二医院普外科,黑龙江哈尔滨150081

出　　处：《哈尔滨医科大学学报》2013年第5期392-396,共5页Journal of Harbin Medical University

基　　金：黑龙江省自然科学基金面上项目(D200935)

摘　　要：目的构建研究碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)真核表达载体,并在体外进行表达和检测。同时利用bFGF基因修饰的组织工程骨移植修复颌骨种植体周围的骨缺损。方法从骨髓基质干细胞中分离总RNA,进行RT-PCR获得bFGF cDNA。测序证实其结果正确后,构建真核表达载体。并经BamHⅠ/EcoRⅠ双酶切、PCR及测序鉴定。利用Blast程序,搜索NCBI GenBank中与重组质粒(pVAX1-bFGF)中编码bF GF基因同源的序列。通过脂质体将重组质粒转染骨髓基质干细胞(bone marrow stromal cells,BMSCs),使用间接免疫荧光法进行表达产物检测。应用基因重组技术使bFGF重组质粒转染BMSCs,筛选出阳性细胞后,复合以多孔矿化骨移植修复颌骨种植体周围骨缺损。通过对缺损处形态及X线、组织学等检查进行评价。同时以外源性注射bFGF修复缺损组作为对照。结果 bFGF基因成功地克隆到真核表达载体pVAX1中;pVAX1-bFGF中编码bFGF的序列与基因库中的bFGF编码序列完全一致;间接免疫荧光表达产物检测结果显示转染重组质粒的细胞表面有绿色荧光。bFGF基因修饰的组织工程骨成骨能力较外源性注射bFGF组更强,周围的毛细血管大量增殖,骨髓腔通畅,骨缺损完全愈合。结论成功地构建人bFGF真核表达载体,并且此载体可以在体外进行表达。bFGF基因修饰的骨髓基质干细胞所形成的组织工程骨成骨能力更强,对修复颌骨种植体周围骨缺损极其适合。Objective To construct the eukaryotic expression vector of basic fibroblast growth factor gene and to observe its expression in vitro. To investigate the effect and practical value of repairing ＇the maxillary and mandibular of implant defect with tissue engineering of bone based on bone marrow stromal cells （BMSCs） of gene modificating. Methods Total RNA from BM- SCs was used to perform RT-PCR to acquire bFGF cDNA. After the sequence was confirmed by sequencing, the eukaryotic expression vector was constructed. It was confirmed by restrictive enzymes（BamH ｜/EcoR I ） digestion analysis, PCR and DNA sequencing. Similar nucleotid.e sequences encoding bFGF of pVAXI-bFGF were looked up by basic local alignment search toolmeans in NCBI GenBank. The pVAXI-bFGF was transfected into BMSCs by lipofectamine and the expressed product was detected by indirect immunofluorescence. Through the technology of gene recompose, BlfISCs were transfected by bFGF. After selecting the positive cells, the transfected BMSCs were composed with biotic support material. The BMSCs were transplanted to repair the maxillary and mandibular defect（ including the immediate implanting when was not enough）. The new bone was evaluated by gross inspection, X-ray examination and histologic observation after implantation. Results The recombinant expression vector pVAXI-bFGF was constructed successfully. The indirect immunofluorescence result showed green fluorescence on the membrane of transfected cells. The sequence of encoding bFGF of pVAX1- bFGF was ex- actly the same as the human bFGF in GenBank. The bone-formed ability of bone modified with basic fibroblast growth factor was stronger than pure biotic support material. There were a lot of capillaries around the bone defect. The bone defects hadbony union with trabeculate and corti- cal bone on the specimen with BMSCs. Conclusion The recombinant expression vector pVAXI-bFGF can be constructed successfully, and the constructed eukaryotic expression vector of pVAXI-bFGF can be expressed

关 键 词：碱性成纤维细胞生长因子基因 真核表达载体 种植体 骨缺损 

分 类 号：R782.7[医药卫生—口腔医学]