【作者】 林涛；

【导师】 杨述华；

【作者基本信息】 华中科技大学 ， 骨科学， 2014， 博士

【摘要】 第一章载体的构建、细胞的分离培养及转染目的构建过氧化物酶增殖物活化受体γ(Peroxisome pmlifemtor-activated receptor γ, PPARγ)基因的发夹状干扰RNA (shRNA)并携带盐酸强力霉素(Doxycycline hyclate, Dox)正向调控"Tet-On"系统的表达载体。转染大鼠BMSC,观察并比较不同浓度四环素诱导后BMSC中PPARy基因沉默情况,成骨及成脂分化情况。方法利用RNAi技术,以小鼠PPARy基因为靶基因,构建靶向小鼠PPARy基因的"Tet-On"慢病毒载体,除了携带目的基因PPARγ-shRNA外,还将携带四环素正向调控的"Tet-On"系统和红色荧光蛋白(RFP)基因标记。通过将测序正确的慢病毒载体包装并测定滴度后,转染大鼠BMSC,观察红色荧光蛋白的表达变化情况。RT-PCR (Real-time quantitative PCR)检测PPARγ-mRNA、成脂因子ADD1-mRNA和成骨因子Collagen I-mRNA的表达水平,Western blot检测PPARy蛋白、ADD1蛋白和I型胶原蛋白(Collagen I)的表达,从而确定转染后PPARγ基因的沉默效应。分别将转染的骨髓间充质干细胞(Transfected mesenchymal stem cells, tBMSC)向成骨诱导,通过转染前后碱性磷酸酶(ALP)定性检测成骨分化能力；向成脂分化方向诱导,通过转染前后油红-O染色对比成脂能力。结果设计并筛选出shRNA序列：GTCTGCTGATCTGCGAGCC.成功构建了PPARy基因的"Tet-On"’慢病毒载体,测量滴度为3.6×10E8TU/ml,转染大鼠BMSC后,与未转染BMSC比较,PPARy-mRNA24h (hours)抑制率为36%(P<0.01); ADD13d(days)下调表达32%(p<0.05),7d表达下调75%(p<0.01)；I型胶原3d表达上调37%(p<0.05),7d表达上调66%(p<0.01);48h PPARy蛋白抑制率为53%(p<0.01)。与未转染BMSC比较,转染的tBMSC成骨诱导9d后,在2-8μg/ml时随盐酸强力霉素(Doxycycline hyclate, Dox)的浓度增大ALP活性增强,在8μg/ml成骨能力最强,在10μg/ml时呈现下降趋势；成脂诱导14d后,OD值测定tBMSC组显著降低61%(p<0.01)。结论慢病毒转染大鼠BMSC转染效率高,转染的BMSC增殖能力强,成脂分化减弱,成骨分化增强,传代后PPARy沉默效应稳定。Dox对目的基因表达调控存在浓度依赖关系,在8μg/ml时Dox调控作用最强。第二章HA/PLLA涂层BCP支架材料制备、Dox微球的制备与支架复合目的采用将Dox包裹于微球中,再沉积于生物活性双相磷酸钙(Biphasic calcium phosphate, BCP)支架上,组成组织工程"BCP/Dox"缓释系统。方法采用的是有机泡沫浸渍法制备圆盘状2mmx5mm (直径R=5mm)BCP多孔支架,用羟基磷灰石／左旋聚乳酸(HA/PLLA)涂层两遍。通过双乳液法制各聚乙丙交酯-聚乙二醇(PLGA-mPEG, LA/GA=9:1)共聚物载Dox微球,将制备出的载药微球涂层BCP多孔支架上,傅里叶变换红外光谱(FTIR)和扫描电镜(SEM)对支架材料的表面结构和形态学进行分析,并进行体外PBS溶液中的释药实验。结果制备的的载Dox微球载药量为0.5%,包封率为24.7%；电镜显示微球尺寸在15-20μm,大小分布均匀,在孔径300-600μm的多孔HA/PLLA涂层BCP支架上容易粘附。体外PBS缓冲液中释放显示,前16h内有爆释现象,其中8h释放月32.6%,7d释放65.5%,7d后进入缓慢释放阶段,可控制释放约60d。结论BCP支架孔径为300-600μm、孔隙率90.8%-92.3%、力学性能4.8-5.73MPa,可以满足松质骨压缩强度的要求。载Dox微球大小均匀,可以良好地附着BCP多孔支架上,有良好缓释作用。第三章复合tBMSC的多孔BCP/Dox支架治疗大鼠骨缺损的研究目的将‘’BCP/Dox"支架缓释系统,复合转染的大鼠tBMSC后,植入骨缺损处。通过Dox缓慢持续释放,调控tBMSC在骨缺损处向成骨方向分化,作为骨修复材料治疗大鼠骨骨缺损,探讨骨缺损的靶向基因治疗新的方法和策略。方法体外实验利用BMSC和tBMSC对BCP/Dox支架在6h和24h时的粘附,细胞计数后验证支架的粘附性；通过1、3、5d的MTT实验去验证BCP/Dox支架对细胞增殖的影响；通过诱导9d时的ALP染色和11d时茜素红染色验证BCP/Dox支架对BMSC和tBMSC的成骨分化的影响；14d后油红-O染色验证支架材料对BMSC和tBMSC成脂分化的影响。体内验证BCP/Dox支架的急性毒性实验、致敏试验、溶血实验。实施异位成骨实验,采用SD-大鼠30只,制作右大腿后内侧肌袋,植入一片BCP/Dox支架材料,分别在1d、1w、2w、3w和4w分批处死各6只取材并进行HE染色,观察免疫反应和异位成骨情况。SD-大鼠45只,制作大鼠颅骨右后囟5mm圆形骨缺损模型,随机分为A组9只、B组18和C组18只,A组为空白对照；B植入P4代BMSC+BCP/Dox; C组植入P4代tBMSC+BCP/Dox。在4、8和12w分批处死,X线和Micro-CT分析支架的成骨量。结果在BCP/Dox支架的影响下,6h和24h后tBMSC组比BMSC组粘附率低,两组比较无统计学意义(p>0.05),24h后粘附率均在70%以上。增殖率在1d和3d各组无明显区别；5d后,tBMSC+BCP/Dox组和BMSC+BCP/Dox组增殖率低于BMSC组,无统计学意义(p>0.05)。5d后电镜扫描显示BCP/Dox支架细胞周围有细胞外基质及载Dox的PLGA-mPEG微球。三组细胞的ALP定量分析,6d和9d时,BMSC+BCP/Dox组较BMSC组有明显统计学意义(p<0.01); tBMSC+BCP/Dox组较BMSC+BCP/Dox组有明显统计学意义(p<0.01)；12d时,两组与对照组比较均有明显统计学意义(p<0.01)。钙结节染色,显示三组细胞外散布钙结节,tBMSC+BCP/Dox组>BMSC+BCP/Dox组>BMSC组。油红-O染色分析,诱导14d后,tBMSC+BCP/Dox组较BMSC+BCP/Dox组和BMSC组明显减少,有明显统计学意义(p<0.01)；BMSC+BCP/Dox组<BMSC组有统计学意义(p<0.05)。X线显示4w缺损边缘与材料边缘连接紧密,8w时两支架组可见缺损边缘与材料连接、分界不清,12w时tBMSC+BCP/Dox组骨缺损处显示更高的密度影,边缘不清；灰度分析显示,12w后BMSC+BCP/Dox组较BCP/Dox支架密度增高(p<0.05),tBMSC+BCP/Dox组较BMSC+BCP/Dox组密度增高(p<0.01)。Micro-CT显示12w时,tBMSC+BCP/Dox组较BMSC+BCP/Dox组和BCP/Dox支架骨量显著增高(p<0.01),BMSC+BCP/Dox组较BCP/Dox支架骨量显著增高(p<0.01)。结论BCP/Dox支架有良好的生物相容性,tBMSC在支架上具有良好的粘附、增殖和成骨分化能力。多孔BCP/Dox支架复合tBMSC具有良好的骨缺损修复能力。更多还原

【Abstract】 Part I The construction of lentiviral vector, BMSC separation and culture, gene transfectionObjective The short hairpin RNA (shRNA) eukaryotic expression vector targeting of the PPARy gene was constructed and transfected into cultured rat bone marrow stem cells (BMSC) via lentiviral expression vector, which also contains "Tet-On" expression system. The inhibition effects on PPARy gene and the ability to undergo osteogenic and adipogenic differentiation were determined under the different Dox concentration.Methods The PPARy gene of mouse was designed from the mouse PPARy mRNA sequence, which also contains "Tet-On" expression system and the gene of red fluorescence protein (RFP). The plasmid vector was isolated, purified and transfected into rat BMSC. To observe the expression of PPARy genes and RFP cultured in vitro. The expression of PPARy gene and protein, the adipogenic factors adipocyte determination and differentiation-dependent factor1(ADD1), the osteogenic genes encoding collagen I were measured by RT-PCR and Western blot assay following PPARy silencing. To observe tBMSC after the osteoinductive culture under the different Dox concentration by detecting changes of the activity of alkaline phosphatase(ALP) and the amount of calcium deposition as measured by Alizarin red Sstaining, and the lipid droplet count as measured by Oil red O staining.Results The shRNA target sequence (GTCTGCTGATCTGCGAGCC) was successful designed and cloned into "Tet-On" ukaryotic expression vector. After measuring virus titer (3.6x10E8TU/ml), BMSC were transfected and the RFP fluorescence intensity was observed under fluorescence microscope. RT-PCR and Western blotting showed that the expression level of PPARy was significantly inhibited by PPARy-shRNA compared with controls, the inhibition rate was36%at24h (P<0.01) and53%at48h (P<0.01). The mRNA levels of ADD1in tBMSC were32%(p<0.05) at3d and75%(p<0.01) at7d when compared with BMSC. The mRNA levels of collagen I in tBMSC were37%(p<0.05) at3d and66%(p<0.01) at7d when compared with BMSC. The tBMSC induced to differentiate into adipocytes for9days, the activity of ALP increased with the increase of concentration of doxycycline hyclate (Dox) between2μg/ml and8μg/ml, and decreased from10μg/ml. The OD values of the destained solution of Oil red O from tBMSC was61%when compared with BMSC (p<0.01) after14days of adipogenic induction.Conclusion The efficiencies were high with eukaryotic expression vector in tBMSC, the down regulation of PPARy by shRNA inhibited adipogenic differentiation and promoted osteogenic differentiation. The tBMSC had showed active proliferative capacity and inheritable stability of PPARy silencing after generation, the inhibition ratio relianced on the Dox concentration, the optimal Dox concentration for promote osteogenesis was8μg/ml. Part IIThe preparation of BCP scaffold coated with HA/PLLA nanomposites, the preparation of microspheres with Dox and loade the microspheres on the BCP scaffoldsObjective The purpose of this part is preparing the linear PLGA-mPEG microspheres contain Dox, the micropore was loaded on the pore wall of BCP scaffold coated with HA/PLLA nanomposites to produce a biomineralized materials with slow release system for bone tissue engineering.Methods The round disks2mm×5mm(R)multihole BCP scaffolds coated with two layer of HA/PLLA nanocomposites were prepared by the method of organic foam plastic. The linear PLGA-mPEG (PLGA-mPEG, LA/GA=9:1) microspheres loaded with Dox was prepared by multiple emulsion method. The microspheres were loaded on the BCP scaffolds coated with HA/PLLA. The structure and morphology of BCP/Dox scaffolds was analyzed by means of metallographic microscope (SEM) and Fourier transform infrared spectroscopy (FTIR). The encapsulation rate, drug content and the release rate of Dox were calculated in Phosphate Buffer solution (PBS) in vitro at37℃, and the results were statistically analyzed.Results The drug content was0.5%, the encapsulation rate was24.7%.The size of the microspheres was uniform and between15μm and20μm. The microspheres could be easy loaded on the BCP scaffolds with pore size between300μm and600μm. The drug release experiments in vitro showed that there was burst release phenomenon, especially in the first16h, the release percentage was32.6%in the first8h and65.5%in the first7d, and the stage of slow release started from7d, and continued for60d.Conclusion The pore size of BCP/Dox scaffolds between300μm and600μm, porosity between90.8%and92.3%, and compressive strength between4.8MPa and 5.73MPa, the scafford can meet essential requirements for bone tissue engineering and regeneration. The microspheres loaded with Dox had good slow release effect for a long time. Part Ⅲ Repair of rat bone defects by BCP/Dox scaffolds combine with tBMSC cellsObjective Making rat cranial bone defects model, and implanting the BCP/Dox scaffolds combined with tBMSC cells into the defect site. Dox releasing from BCP/Dox scaffolds regulated tBMSC promoted osteogenic differentiation by controlling release and target delivery, to explore a new method for the therapy of bone defect.Methods The effect of BCP/Dox scaffolds on tBMSC and BMSC adhesion was measured with cell counting; the proliferation was detected by MTT at1d,3d and5d; the osteogenic differentiation was detected by the qualitative analysis of ALP after the osteoinductive culture9d, and by alizarin staining to count calcium depositions after the osteoinductive culture11d. The adipogenic differentiation was detected by oil red staining after14d culture. The biocompatibility of BCP/Dox scaffolds in vivo was measured by the acute toxicity experiment, the sensitization test and the hemolysis test. The ectopic osteogenesis was investigated in the muscle of rat by the histological method, total30SD rats of6w, making a muscle bag in the medial posterior thigh of right side models and implanting a BCP/Dox scaffolds,6rats was sacrificed respectively in1d,7d,14d,21d and28d for HE to study the ectopic osteogenesis potential. Divided the45SD rats of6w in age into control group (9rats), BMSC+BCP/Dox group (18rats), and tBMSC+BCP/Dox group (18rats) random in each group. Making rat cranial bone defects model, and implanting the BCP/Dox scaffolds combined with tBMSC or BMSC into the defect site for the BMSC+BCP/Dox group and tBMSC+BCP/Dox groups. After operation, rats were sacrificed respectively in4,8and12weeks, to measure the bone mass by the X-ray and Micro-CT.Results The adherence rate of tBMSC on the surface of the BCP/Dox scaffolds was lower than BMSC at6h and24h, there were no significant differences between the two groups(p>0.05), and the adhesion rate was more than70%in the two groups. There was no significant differences for the proliferation rate at Id and3d, tBMSC+BCP/Dox group smaller than those in tBMSC+BCP/Dox group at5d, there were no significant differences between the two groups (p>0.05). The SEM showed that the adhesion and the proliferation rate of tBMSC on the surface of BCP/Dox scaffolds, and there were extracellular matrix and microspheres in the space surrounding the cells.The ALP activity was higher in the tBMSC+BCP/Dox group than other two groups (p<0.01) at6d and9d, and BMSC+BCP/Dox group>BMSC group (p<0.01), tBMSC+BCP/Dox group> BMSC+BCP/Dox group (p<0.01). The alizarin staining showed that the calcium nodule numbers were highest in tBMSC+BCP/Dox group and lowest in BMSC group. The quantitative analysis of Oil Red Ostaining extraction showed that tBMSC+BCP/Dox group less than BMSC groups (p<0.01) and BMSC+BCP/Dox group less than BMSC groups (p<0.05). X-ray showed that the sharp edges of defect in all groups at4w, and blurred edges of deceet in two BCP/Dox groups at8w, and bone fusion in tBMSC+BCP/Dox group at12w; The gray analysis showed that tBMSC+BCP/Dox group was higher than BMSC+BCP/Dox group (p<0.01), and BMSC+BCP/Dox group was higher than BCP/Dox group (p<0.05). The Micro-CT analysis showed that the bone mass were highest in tBMSC+BCP/Dox group and lowest in BCP/Dox group, there were statistical significance was found between groups (p<0.01).Conclusion The BCP/Dox scaffolds possessed good biocompatibility and high bone inductive potentiality, it can be used to repair bone defect combined with tBMSC cells.更多还原