【作者】 郑东；

【导师】 杨述华；

【作者基本信息】 华中科技大学 ， 外科学， 2007， 博士

【摘要】 [摘要]目的:通过对TGF-β3促进软骨分化的基础和应用的研究,探究将TGF-β3应用于临床的软骨修复的可能性,力图寻找生物细胞因子用于临床治疗软骨损伤成熟的方案。方法:首先,从原代培养骨髓细胞形成的单克隆中,应用显微操作技术挑取单克隆来源的MSCs,然后在有饲养层细胞(feeder cells FCs)的条件下,进行大量扩增,在传代至P20(Passage 20)时进行细胞表面标志(STRO-1、CD34、CD45、OCT-4、CD105、Nestin)的流式鉴定,并进行这种单克隆来源的MSCs向骨、软骨、肌腱和神经样细胞分化的实验。其次,在TGF-β3促进MSCs向软骨分化的不同时间点,通过Western Blot检测Erk1/2(extracellular signal-regulated kinase-1/2 Erk1/2)磷酸化的表达,然后再用Erk1/2磷酸化抑制剂U206处理诱导分化过程中的MSCs,RealTime-PCR检测软骨分化基因(Sry-trype HMG box protein 9)Sox9、胶原Ⅱ(collagen COLⅡ)和Aggrecan(Agc)的表达的变化;之后构建Smad4在磷酸化位点Thr277突变的表达载体,转染MSCs后在分化过程中检测上述基因表达的变化。再次,从大鼠的胚胎(胚芽)中提取TGF-β3基因全长,克隆到真核表达载体pIRES2–EGFP中,构建出表达载体pIRES2–EGFP-TGF-β3,转染骨髓基质干细胞(marrow stem cell MSCs),观察转染后MSCs向软骨细胞分化的效应。在体内实验中,将转染后的MSCs接种于载体脱钙骨基质(Decalcified bone matrix DBM)后移植于软骨缺损的动物模型中,观察转染后MSCs体内修复软骨的能力。接着,应用基因工程的方法改进自然TGF-β3蛋白,构建工程化TGF-β3的表达载体pIRES-EGFP -LAP -MMP-mTGF-β3,将基质金属蛋白酶(matrix metalloproteinase MMP)酶切位点分别插入到前体相关蛋白(Latent Associated Protein LAP)和成熟TGF-β3(mature TGF-β3 mTGF-β3)之间,并分别转染MSCs和软骨前体细胞(Chondrogenic progenitor Cells CPCs),检测上述转染后的两种细胞球团培养( pellet culture)过程中在有MMP酶和无MMP酶的条件下COLⅡ, Agc和基质金属蛋白酶组织抑制因子(Tissue inhibitor of metalloproteinase TIMP)的表达和软骨基质中蛋白聚糖Proteoglycan的积累。最后,人工合成GADD45-β的反义核苷酸,在TGF-β3促进MSCs向软骨细胞分化的后期进行干预,并检测GADD45-β的反义核苷酸干预后对软骨终末分化因子基质金属蛋白酶13(MMP-13)、血管内皮生长因子(vascular endothelial growth factor VEGF)和胶原Х(collagenХCOLХ)表达的影响,以及对软骨基质中proteoglycan积累的影响。结果:我们成功获得了由一个单克隆MSCs扩增出来的大量单克隆来源的MSCs,而且这种单克隆来源的MSCs能被成功的诱导向骨、软骨、肌腱和神经样细胞分化。Western blot检测结果显示Erk1/2的磷酸化在TGF-β3诱导MSCs向软骨分化的过程中随着时间增加而增加,同时Erk1/2磷酸化特异性的阻滞剂U206能够成功减弱TGF-β3诱导的软骨分化和软骨基质的合成;而Erk1/2磷酸化底物Smad4在Thr277上的突变亦能减弱TGF-β3诱导的软骨分化,效果与U206类似。接着,我们成功构建了真核表达载体pIRES2–EGFP-TGF-β3并在MSCs中获得了成功和高效的表达,在接下来的细胞球团培养(pellet culture)中,转染后的MSCs成功和高效被诱导向软骨细胞分化。在体内实验中,转染了pIRES2–EGFP-TGF-β3的MSCs以DBM为载体成功修复了动物模型中的大面积软骨缺损。接下来,我们还成功构建了工程化TGF-β3的真核表达载体pIRES-EGFP-LAP-MMP-mTGF-β3,并在MSCs和CPCs中成功获得了表达,而且转染后的MSCs和CPCs只在有MMP酶的条件下能被特异性的促进向软骨细胞分化。最后,GADD45-β反义寡核苷酸对GADD45-β表达的封闭能够成功阻滞TGF-β3诱导MSCs向软骨分化后期软骨细胞的终末分化,并维持和增加软骨基质的积累。结论:单克隆来源的MSCs能被成功的诱导向中胚层和外胚层的组织细胞分化,这直接证明了MSCs的“横向分化能力”,也获得了大量的可供利用的均质MSCs;Smad4基因在Thr277上的磷酸化是TGF-β3诱导软骨分化中Erk1/2信号途径促进软骨分化的关键;TGF-β3转染MSCs后能成功的应用于大面积软骨缺损的修复;经过基因工程改造后的工程化的TGF-β3蛋白则更能特异性修复受损的软骨;而GADD45-β是TGF-β3诱导软骨分化后软骨肥大和终末分化的关键因子,其反义寡核苷酸能有效阻止软骨的终末分化。更多还原

【Abstract】 Objective: To find a way of using TGF-β3 in clinical treatment of injury of cartilage by foundation and application research on chondrogenesis induced by TGF-β3, and it would be a beginning of using biological cytokines to repair cartilage in all kinds of orthopedic diseases. Method: Firstly, primary marrow cells were cultured with low density. When there were monoclones of marrow cells, the cells were picked out from one monoclone by micromanipulative technique. Then these cells from a monoclone were co-culture with the mouse embryonic fibroblasts which were cultured as feeder cells. When the cells coming from a monoclone generated to passage 20, these MSCs were identified by means of cell surface markers such as STRO-1、CD34、CD45、OCT-4、CD105 and Nestin. Then the identified MSCs were induced to differentiate to osteoblasts, chondrocyts, tendon cells and neurons. Secondly, the phosphorylation of Erk1/2 was checked by western blot during chondrogenesis induced by TGF-β3. Then U206 which was the specific inhibitor of phosphorylation of Erk1/2 was used during the chondrogenesis, and the expression of sox9, COLⅡand Agc was detected by RealTime-PCR. The expression vector of Smad4 with point mutation at Thr277 was constructed by overlap PCR technique, and the Smad4 with mutation was transfected into MSCs during the chondrogenesis induced by TGF-β3. Then the expression of sox9, COLⅡand Agc were detected by RealTime-PCR as same as above. At the same time the accumulation of proteoglycan was confirmed by TB staining respectly. Thirdly, the total length cDNA of rattus TGF-β3 was obtained by RT-PCR from rattus embryo(embryonic bud), then the cDNA was cloned into eukaryotic vector pIRES-EGFP for constucting the expression vector pIRES2–EGFP-TGF-β3. Then pIRES2– EGFP- TGF-β3 was transfected into MSCs, and the effect of transfection on chondrogensis was analyzed by measured the expression of COLⅡ, COLХ, Agc and the accumulation of proteoglycan. In vivo experiment, these genetically modified MSCs were co-cultrued with decalcifies bone matrixs for 7days, then the mixtures were transplanted to huge cartilage defect in animal model. The healing of cartilage defect was checked by macroscopic observations and histomorphological observations. Fourthly, engineering TGF-β3 was obtained by genetic engineering technique. The recombinant eukaryotic plasmid pIRES-EGFP-LAP-MMP-mTGF-β3 was constructed by inserting gene region of matrix metalloproteinase cutting site between latent associated protein(LAP) and mature TGF-β3(mTGF-β3). Then the pIRES-EGFP-LAP-MMP-mTGF-β3 was transfected into MSCs and chondrogenic progenitor cells(CPCs), and the expression of COLⅡ, Agc and tissue inhibitor of metalloproteinase(TIMP) was checked by RT-PCR when the both genetically modified cells above culture in medium with or without MMP. At last, the antisense oligodeoxyribonucleotide of GADD45-βwas synthetized, and it was used at terminal of chondrogenesis induces by TGF-β3. The expression of MMP-13, COLХand VEGF were checked by RealTime-PCR, and the accumulation of proteoglycan was evaluated by TB staining. Result: Firstly, We got plenty monocloned MSCs successfully by amplifying cells which came from one monclone in primary culture of marrow cells, and the monocloned MSCs could be induced differentiating to osteocytes, chondrocytes, tendon cells and neurons successfully. Secondly, western blot showed that phosphorylation of Erk1/2 increased during the chondrogensis induced by TGF-β3, and U206 could weaken the chondrogenesis. Furthermore, the mutation of Smad4 on Thr277 could take the same effect on chondrogenesis as U206 and decrease the expression of Sox9, COLⅡ, Agc and the accumulation of proteoglycan during the chondrogenesis induced by TGF-β3. Thirdly, the embryonic plasmid pIRES2–EGFP-TGF-β3 was constructed successfully, and plenty TGF-β3 was detected in the medium after MSCs were transfected by pIRES2– EGFP- TGF-β3. The genetically modified MSCs differentiated to chondrocyts and turned to chondral pellets by pellet culture successfully in vitro. In vivo, the genetically modified MSCs with DBM as carrier could repair huge cartilage defect more efficiently than nature MSCs with DBM as carrier or DBM without MSCs. Fourthly, eukaryotic expression vector of engineering TGF-β3 was constructed successfully, and the engineering protein was gotten by transfecting pIRES-EGFP-LAP-MMP-mTGF-β3 into CHO cells, and the expression of engineering TGF-β3 also had been detected after CPCs and MSCs was transfected with pIRES-EGFP-LAP-MMP-mTGF-β3. During the pellet culturing of CPCs and MSCs which were transfected with pIRES-EGFP-LAP-MMP-mTGF-β3, CPCs and MSCs could differentiate to chondrocytes only in the medium with MMP. At last, the antisense oligodeoxyribonucleotide of GADD45-βcould inhibit the expression of GADD45-βand blocked the terminal differentiation of chondrogenesis induced by TGF-β3. Intervention of antisense oligodeoxyribonucleotide of GADD45-βcould decrease expression of MMP-13, VEGF and COLХefficiently which were the representative cytokines of terminal differentiation in chondrogenesis. Conclusion: Monocloned MSCs came from one marrow cell could transdifferentiate to the mature cells of endoderm and ectoderm. Phosphorylation of Erk1/2 on Thr277 was the key point of MAPK signal pathway which was the other important signal pathway except Smad signal pathway in chondrogenesis, and Smad4 is the junction point of Smad pathway and MAPK pathway. Genetically modified MSCs with TGF-β3 could be used as seed cells in cartilaginous tissue engineering more efficiently. And the engineering TGF-β3 protein could repair the injury of cartilage in all kinds of orthopedic diseases more specifically. The GADD45-βwas the key point of terminal differentiation of chondrogenesis, and the inhibitor of GADD45-βcould block the terminal differentiation more efficiently.更多还原