【作者】 康健；

【导师】 袁文；

【作者基本信息】 第二军医大学 ， 骨外科学， 2013， 博士

【摘要】 研究目的利用TGF-β3和BMP-7共转染兔骨髓间充质干细胞（BMSCs）并诱导其分化，以富血小板凝胶（PRG）为支架，构建可局部注射的组织工程髓核。研究方法以全骨髓贴壁法分离培养兔BMSCs，通过细胞表面标记物检测及中胚层细胞诱导分化，确定其为BMSCs。人工合成TGF-β3和BMP-7基因片段，并行基因测序确定序列。以pDC316-MCMV-EGFP为载体质粒， PPE3为骨架质粒，构建TGF-β3腺病毒（TGFβ3-pDC316-MCMV-EGFP）和BMP-7腺病毒(BMP7-pDC316-MCMV-EGFP)，通过Realtime PCR检测腺病毒中TGF-β3和BMP-7基因mRNA的表达。将培养的兔BMSCs传代后，分成五组，分别为：A.空白对照组；B. GFP免疫荧光对照组；C. TGF-β3基因单独转染组；D. BMP-7基因单独转染组；E. TGF-β3和BMP-7基因共转染组；以普通DMEM培养基培养14天后，以Western blot方法检测A、C、D、E组TGF-β3和BMP-7蛋白的表达情况。以Realtime PCR测定各组细胞的ACAN、Collagen I、Collagen II、CollagenX、SOX9基因的mRNA表达水平。以Lendersberg二次离心法制备PRP，与激活剂以10:1比例混合激活后，以3000rpm离心10min获得富生长因子上清，检测其中TGF-β1和PDGF-AB生长因子的浓度，再将PRP分别与兔BMSCs及TGF-β3和BMP-7基因共转染的BMSCs分别混合，培养14天后，以扫描电镜观察共转染组组织工程髓核结构及细胞在富血小板凝胶支架中的生长情况。研究结果以全骨髓贴壁法分离培养获得的细胞在特定诱导条件下具备向成骨细胞、成软骨细胞及脂肪细胞三种中胚层细胞分化的能力；流式细胞术检测证实获得的细胞存在CD29、CD105、CD166表面标记物的表达。TGF-β3和BMP-7基因测序证明合成基因序列正确无误。以pDC316-MCMV-EGFP载体质粒和PPE3骨架质粒构建的TGF-β3和BMP-7腺病毒扩增后滴度分别为：1.495×1010pfu/ml和1.185×1010pfu/ml。TGF-β3和BMP-7腺病毒DNA经PCR扩增后酶切电泳检测显示构建腺病毒包含TGF-β3和BMP-7基因片段。以TGF-β3和BMP-7重组腺病毒转染兔BMSCs后常规DMEM培养基培养14天，细胞形态出现明显变化，圆形及椭圆形细胞明显增多。Western blot方法检测TGF-β3和BMP-7蛋白表达水平明显增高。培养14天时，Realtime PCR检测ACAN、CollagenI、Collagen II、SOX9基因的表达水平较对照组明显升高（P<0.05），其中Collagen I和SOX9单独转染组和共转染组表达无明显差异（P>0.05），Collagen II共转染组表达较单独转染组明显增高（P<0.05）。TGF-β3单独转染组和共转染组的Collagen X基因表达较BMP-7单独转染组和对照组明显降低（P<0.05）。以Lenderberg法制备得PRP后，以激活剂激活获得的富血小板凝胶支架中TGF-β1和PDGF-AB浓度分别为351.03±11.15ng/ml和267.38±14.2ng/ml，明显高于兔全血中的浓度。扫描电镜观察结果示：凝胶内部网状结构的孔隙直径约在40μm至100μm之间，转基因BMSCs较为均匀的分布于凝胶中，多数细胞伸出大量指突到纤维骨架和凝胶孔隙之中。细胞在富血小板凝胶支架中生长良好。研究结论通过全骨髓贴壁法可以成功分离获得状态良好的兔BMSCs。以pDC316-MCMV-EGFP为载体可成功构建TGF-β3和BMP-7腺病毒，且其可转染兔BMSCs，获得TGF-β3和BMP-7蛋白的表达。TGF-β3和BMP-7腺病毒共转染兔BMSCs后，ACAN、Collagen II、SOX9基因表达均明显升高，而Collagen X基因表达明显降低，说明兔BMSCs向类髓核细胞方向分化。Lenderberg法制备的富血小板凝胶支架孔隙率及孔隙直径适合转基因兔BMSCs在其中正常生长和增殖。更多还原

【Abstract】 Objective The purpose of this study is to construct an injectable tissue engineering nucleuspulposus composed of Platelet-rich plasma gel(PRG) as scaffold and rabbit bone marrowmesenchymal stem cells (BMSCs) co-transfected byf adenoviral vectors with TGF-β3andBMP-7as seed cells.Methods Obtain the rabbit BMSCs with the method of direct way. Be sure of theBMSCs through the surface mark and induced differentiation into three differentmesoderm cells.Compose the TGF-β3and BMP-7gene fragment and make sure of their gene order.Establish the TGF-β3adenoviral vector(TGFβ3-pDC316-MCMV-EGFP) and BMP-7adenoviral vector(BMP7-pDC316-MCMV-EGFP) with plasmid pDC316-MCMV-EGFP.Determine the TGF-β3and BMP7expression in mRNA level through Realtime PCR.Divide BMSCs into five groups: A. blank control group; B. immunofluorescence controlgroup with GFP; C. singly transfected by TGF-β3adenovirus; D. singly transfected byBMP7adenovirus; E. co-transfected by both TGF-β3and BMP7adenovirus. After14days， measure the TGF-β3and BMP-7protein expression of group A、C、D、E and theexpression of ACAN、Collagen I、Collagen II、Collagen X and SOX9in mRNA levelthrough Western blot and Realtime PCR respcetively.Prepare the PRP by Lendersberg way. Obtain PRG by mixing PRP with activator. Obtaingrowth-factor-rich supernatant through centrifugalization. Then measure the concentrationof TGF-β1and PDGF-AB of it. Activating PRP by mixing with activator after combiningwith BMSCs and BMSCs co-transfected by both TGF-β3and BMP-7adenovirusseparately, and scan electron microscope (SEM) was used to discover the threedimensional structure of the tissue engineering nucleus pulposus and the growth conditionof co-transfected BMSCs at14days after activation.Result The cells got through the all bone marrow adherence method are able todifferentiate into at least three kinds of mesoderm cells: osteoblast, chondroblast andadipocyte. The cells also express cell surface marker of CD29, CD105, CD166accordingto flow cytometry detection. Gene order sequencing demonstrate that the gene fragments are correct. The tite ofadenoviral vector of TGF-β3and BMP-7are1.495×1010pfu/ml and1.185×1010pfu/mlseparately. The amplified DNA by PCR contain TGF-β3and BMP-7gene fragments testedby electrophoresis.14days after culture of Co-transfected BMSCs, the shape of most cells changed obviously.Western blot showed TGF-β3and BMP-7proteins at a higher level than BMSCs. Theexpression level of ACAN, Collagen I, Collagen II and SOX9gene were much higher thancontrol group(P<0.05), and the expression level of Collagen II in co-transfected group washigher compared to solo-transfected groups(P<0.05). The expression of Collagen X inTGF-β3transfected group and co-transfected group were obviously lower than BMP-7transfected group and control group(P<0.05).The concentration of TGF-β1and PDGF-AB in PRG are351.03±11.15ng/ml and267.38±14.2ng/ml separately, which are obviously higher than the concentration of them inrabbit blood. Scanning electron microscope showed that diameter of ventage in PRG wasbetween40μm to100μm, and co-transfected BMSCs were well-distributed in PRG byputting stylodes into the ventage. The cells growed well in PRG.Conclusion It is pertinent to prepare the BMSCs by direct way. Adenoviral vector ofTGF-β3and BMP-7could transfect the BMSCs and express the TGF-β3and BMP-7withpDC316-MCMV-EGFP. BMSCs transfected by TGF-β3and BMP-7get higher expressionof ACAN, Collagen I, SOX9and lower expression of Collagen X, what demonstrated thatboth TGF-β3and BMP-7are able to induce the BMSCs into nucleus pulposus-likecells(NPCs). PRG preparated through Lenderberg method had a proper structure forco-transfected BMSCs growth.更多还原