【作者】 汤旭日；

【导师】 张少成； 王秋根； 侯铁胜； 张秋林； 纪方；

【作者基本信息】 第二军医大学 ， 外科学， 2008， 博士

【摘要】 关节软骨损伤是临床常见疾患,由于其无神经支配、缺乏血液供应、软骨细胞增殖迁移能力低等组织学特点,关节软骨自身修复能力非常有限,损害后易出现不可逆的关节功能障碍,因此关节软骨损伤的治疗是运动医学研究的热点和难点之一。目前实验和临床研究证实应用软骨下骨板钻孔或微骨折等技术,可使骨髓腔内未分化的间充质细胞(BMSCs)迁移到缺损区,并增殖、分化形成类透明软骨组织,尽管远期效果不理想,但也显示了关节软骨组织在特定的条件下具有一定的修复能力。因此提高骨髓间充质干细胞向软骨细胞定向分化能力并进一步改善新生软骨的质量,可能是促进关节软骨损伤修复的有效途径之一。而近年来的研究表明低强度脉冲超声(LowIntensity Pulsed Ultrasound)能促进骨折、骨不连的愈合,其机制是加速了软骨的形成及软骨内骨化。也有动物实验研究报道,低强度脉冲超声波具有促进关节骨-软骨缺损修复的作用。然而目前关于LIPU对BMSCs等生物学效应及作用机制仍缺乏了解,临床应用缺乏足够的理论和实验依据。本课题拟通过构建BMSCs-软骨细胞的藻酸盐三维共培养体系,予以不同强度LIPU刺激,观察BMSCs的增殖能力、细胞表型及合成分泌细胞外特异性基质、细胞调控因子水平的变化,从细胞、分子生物等层面探讨LIPU对BMSCs的生物学效应及可能的作用机制。目的:构建BMSCs、软骨细胞的藻酸盐三维共培养体系,探讨软骨细胞的体外三维培养微环境对BMSCs增殖、成软骨分化的影响;对体外BMSCs-软骨细胞三维共培养体系施加LIPU刺激,观察细胞生物学改变情况,探讨LIPU对BMSCs的生物学效应及作用机制,为LIPU在临床中的进一步合理应用提供理论和实验依据。方法:1.采用机械粉碎结合酶消化法获取兔关节软骨细胞,通过体外平面培养扩增、传代,应用细胞形态学、Ⅱ型胶原免疫组化染色鉴定细胞表型。2.采用密度梯度离心结合贴壁筛选法从兔骨髓组织中分离、纯化BMSCs,进行体外平面培养扩增,应用免疫组化染色、流式细胞仪分析细胞表面标志物等方法对细胞表型、纯度进行鉴定。3.采用藻酸盐凝胶复合软骨细胞、BMSCs构建三维共培养体系,通过细胞计数、绘制生长曲线、H-E染色和Ⅱ型胶原免疫组化染色等观察藻酸盐三维凝胶中细胞生物学性状的变化。4.对BMSCs、软骨细胞的藻酸盐凝胶三维共培养体系予以强度为2mW/cm~2、10mW/cm~2、30mW/cm~2的LIPU刺激,观察细胞形态、细胞增殖及表型的变化;应用Ⅱ型胶原免疫组化染色法、甲苯胺蓝染色法检测细胞基质中Ⅰ型、Ⅱ型、Ⅹ型胶原及蛋白多糖蛋白合成分泌情况;应用RT-PCR法检测细胞aggrecan与Ⅰ型、Ⅱ型、Ⅹ型胶原mRNA的表达情况;应用RT-PCR、Western-bloting法检测细胞分化调控因子TGF-β1、IGF-Ⅰ、bFGF的表达情况。结果:1.原代及第1-3代软骨细胞贴壁后呈多角形,细胞形态、大小相近,融合区细胞呈典型的铺路石状,Ⅱ型胶原免疫组化染色阳性,显示其具有分泌软骨细胞特征性基质Ⅱ型胶原的能力。2.培养的BMSCs贴壁能力很强,细胞形态为短梭形或纺锤形,细胞形态、大小相近;采用免疫细胞化学检查发现培养的第1-3代细胞表达细胞表面抗原CD29、CD44、CD105,不表达CD14、CD34、CD45;同时,Ⅱ型、Ⅹ型胶原免疫组化染色阴性,显示所培养的细胞为未分化的BMSCs。3.藻酸盐凝胶具有一定的弹性和强度及良好的组织相容性,软骨细胞、BMSCs在藻酸盐凝胶中具有良好的生物学活性;免疫组化鉴定发现与软骨细胞共培养的BMSCs的Ⅱ型胶原染色阳性,而对照组为阴性,说明体外软骨细胞三维培养微环境可诱导BMSCs定向分化成软骨细胞。。4.LIPU不影响BMSCs的活性;可促进BMSCs增殖并合成软骨组织特异性细胞外基质,其效应与超声波的强度相关。强度为2mW/cm~2、10mW/cm~2的LIPU促进BMSCs向软骨细胞转化,增加Ⅱ型胶原、蛋白多糖合成分泌,上调TGF-β1、IGF-ⅠmRNA表达。强度为30mW/cm2的LIPU促进BMSCs向肥大软骨细胞转化,增加Ⅱ型、Ⅹ型胶原、蛋白多糖合成分泌增加,上调TGF-β1、IGF-Ⅰ、bFGF mRNA表达。结论:1.通过机械粉碎酶消化法可获取关节软骨细胞,经单层传代培养扩增可在短时间内获得足量、纯化的、具有良好生物学活性兔关节软骨细胞。2.通过密度梯度离心联合贴壁筛选法可分离、纯化骨髓间充质干细胞,经过体外单层培养扩增可获得成分单一、未分化的BMSCs。该方法简便有效,分离、纯化效果较理想,收获的第1至第3代BMSCs可作为后续实验研究的种子细胞。3.软骨细胞-藻酸盐凝胶与BMSCs-藻酸盐凝胶在体外共培养可成功诱导BMSCs分化成软骨细胞,提示除了传统添加生长因子的诱导方法以外,与同类细胞共培养的方法可能是更经济而有效的干细胞诱导分化方法。4.LIPU具有促进BMSCs增殖的作用,强度为30mW/cm~2LIPU的效应最为明显。LIPU刺激可促进BMSCs向软骨细胞分化。2mW/cm~2组、10mW/cm~2组可促进BMSCs增殖,诱导其分化成软骨细胞,以Ⅱ型胶原明显增加为特征;30mW/cm~2组可明显促进BMSCs增殖,诱导其分化成肥大软骨细胞,以Ⅹ型胶原明显增加为特征,长时间刺激可进一步诱导分化为成骨细胞。5.LIPU作用于细胞后产生独特的空泡效应、机械效应和温热效应,通过影响细胞骨架结构、细胞膜的通透性或通过第二信使系统来发挥作用,改变细胞调控因子TGF-β1、IGF-Ⅰ、bFGF等基因表达,再通过细胞自分泌、旁分泌途径诱导BMSCs定向分化成软骨细胞。2mW/cm~2组、10mW/cm~2 LIPU通过上调TGF-β1、IGF-Ⅰ的表达,促进BMSCs定向分化成软骨细胞;30mW/cm~2 LIPU通过同时上调TGF-β1、IGF-Ⅰ、bFGF的表达,促进BMSCs分化成肥大软骨细胞。更多还原

【Abstract】 The avascularity of the articular cartilage and lack of access to the subchondral vascular supply present two of the factors that limit the ability for spontaneous healing of cartilage defects that do not penetrate the subchon -dral bone plate.This has led to the development of surgical techniques such as abrasion,microfractures and drilling of the subchondral bone plan that introduce a vascular-mediated healing response to the injured cartilage by creating access to the subchondral bone marrow vascular system.These socalled marrow stimulation techniques introduce the classic elements of vascular healing by producing a fibrin-rich clot that contains pluripotential blood and bone marrow mesenchymal stem cells(BMSCs),cytokines,and growth factors.Laboratory investigations have shown that microfracture penetration of the subchondral bone results in the formation of a hybrid fibrohyaline repair cartilage tissue.Although unable to produce predictable and durable clinical improvement,this method indicated articular cartilage had the ability for spontaneous healing.Low-intensity pulsed ultrasound accelerates bone healing via upregulation of cartilage formation and maturation phases of endchondral bone formation.In the animal model,daily low-intensity pulsed ultrasound had a significant positive effect on the healing of osteochondral defects.Although a great deal of research has been conducted on the biologic and biochemical regulation of the behavior of these cells,very little is known about the biological effects and cellular mechanisms of low-intensity pulsed ultrasound on BMSCs.Object:To explore the effects of micro-enviroment of chondrocyte culture in alginate bead on BMSCs differentiation.To explore the biologic effects and cellular mechanism of low-intensity pulsed ultrasound(LIPU) on BMSCs viability,proliferation,phenotype,function and gene expression of collagen typeⅡ、aggrecan、TGF-β1、IGF-Ⅰ、bFGF mRNA,which control and/or regulate BMSC to differentiate directionally towards chondrocytes.Method:1.The chondrocytes were isolated from the articular cartilage of rabbits by mechanical smash and enzyme digestion,and proliferated through monolayer culture in vitro.2.The bone marrow mesenchymal stem cells of rabbit were isolated from bone marrow by flowing bone marrow cavity combining with the density gradient centrifugation,purified and proliferated through monolayer culture.3.BMSCs and chondrocytes were co-cultured in alginate beads in vitro, whose morphology,viability and proliferation was observed.4.LIPU was applied to BMSCs and chondrocyte co-culture system which were divided into 3 groups randomly:2mW/cm~2,10mW/cm~2 and 30mW/cm~2.The cell morpho -logy,viability and proliferation was observed periodically.The synthesis and secretion of collagen typeⅡ,collagen typeⅠ,collagen typeⅩand glycosaminoglycans were tested through immunohistochemistry and toluidine blue method,while the gene mRNA and protein expression of aggrecan,Collagen typeⅡ,Collagen typeⅠ,Collagen typeⅩ,TGF-β1,bFGF and IGF-Ⅰwere determined through RT-PCR and Western-bloting.Results:1.A large number of seeding cells with high proliferation ability can be harvested by enzyme digestion and monolayer culture,and cells of 1st-3rd generation chondrocytes have the same morphologic features as the primary source,that can be confirmed by immunocytochemical staining of collagen typeⅡ.2.Purified BMSCs can be obtained through the method of density gradient acentrifugation combing monolayer culture in vitro,whose phenotype can be examined by flow cytometry.3.BMSC and chondrocytes cultured in alginate bead can keep good prolife -ration,and BMSCs which co-cultured with chondrocytes differentiated into chongdrocytes,that was confirmed by positive staining of typeⅡcollagen immunocytochemical stain.4.LIPU had significant effect in accelerating BMSCs proliferation but did not affect the cell viability.The biological effects of LIPU varied with its intensity.LIPU of 2mW/cm~2,10mW/cm~2 can induce BMSC to differentiate into chongdrocytes,that were charactered of improvement of the synthesis and secretion of collagen typeⅡand glycosaminoglycans(GAG),which might be realized by upregulated the gene mRNA expression TGF-β1 and IGF-Ⅰ.LIPU of 30mW/cm~2 can induce BMSC to differentiate into hypertrophic chongdrocytes, that were charactered of improvement of the synthesis and secretion of typeⅩcollagen,which might be realized by upregulated the gene mRNA expression of bFGF.Conclusion:1.The method of mechanical smash combining enzyme digestion and monolayer cultured in vitro can acquire pure and enough isolated rabbits chondrocytes.2.Purified BMSCs can be obtained through the method of density gradient acentrifugation combining monolayer culture.Cells of 1st-3rd generation with high proliferation were fit to the further experiment3.Alginat scaffold appears to provide the satisfactory 3-dimensional support for BMSCs and chondrocytes,and the micro-enviroment of chondrocyte culture in alginate bead can induce BMSCs differentiated into chongdrocytes without adding cytokine in vitro.4.LIPU had significantly positive effects on remaining BMSCs viability, promoting proliferation.The biological effects of LIPU varied with its intensity.LIPU of 2mW/cm~2,10mW/cm~2 can induce BMSC to differentiate direction -ally towards chongdrocytes in vitro,while LIPU of 30mW/cm~2 can induce BMSC to differentiate into hypertrophic chongdrocytes,wbich may differentiate into osteoblasts automatically when cultured for longer time.5.Low-intensity pulsed ultrasound,a form of mechanical energy as high frequency acoustic pressure waves can produce micromechanical stresses on cells,that may plays a role in the modulation of cytokine synthesis and parathyroid hormone response in mesenchymal cells and prechondrocytes,which can influence the synthesis and secretion of collagen typeⅡand collagen typeⅩ.更多还原