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1.
摘要目的:观察自体富血小板纤维蛋白(platelet-rich fibrin, PRF)对体外培养的兔骨髓间充质干细胞(Bone marrow mesenchymal stemcells, BMSCs)成软骨分化的影响。方法:兔心脏采血制备PRF,电镜观察其超微结构;分离培养兔BMSCs,取第3代细胞用于 实验,分为PRF组、阳性对照组、空白对照组。诱导培养21d 后,对三组细胞分别进行形态学观察,成软骨鉴定染色(甲苯胺蓝、II 型胶原免疫组化染色),软骨相关基因表达检测(Ⅱ型胶原、Aggrecan、SOX9)。结果:PRF 组和阳性对照组中BMSCs经诱导后,细 胞由长梭形变为三角形、多角形、圆形;甲苯胺蓝、II 型胶原免疫组化染色均为阳性;Ⅱ型胶原、Aggrecan、SOX9基因表达水平均 较高,两组比较无统计学差异,空白对照组未见相关分化现象。结论:PRF在体外可促进兔BMSCs 成软骨分化,可作为自体生物 材料,在构建组织工程软骨中发挥更好的作用。  相似文献   

2.
童迅  贠喆  张栋  赵新文  曾照辉  于洋  马保安 《生物磁学》2013,(24):4648-4653
摘要目的:研究人正常软骨细胞及骨关节炎软骨细胞的体外分离、培养及鉴定方法,对其生物学特性进行对照并评价其生物学活性。方法:取人创伤性截肢与骨关节炎全膝置换的无菌膝关节软骨,采用两步酶消化法分离培养人关节软骨细胞,并进行传代培养。通过倒置相差显微镜下观察细胞形态,绘制生长曲线,测细胞增殖,甲苯胺蓝染色及Ⅱ型胶原免疫组织化学染色对细胞进行对照研究。结果:骨关节炎软骨细胞形态似成纤维细胞,生长速度明显较正常软骨细胞慢。MTT测细胞增殖显示,第2.4、6代骨关节炎软骨细胞在相同时间点大都比同代正常软骨细胞增殖速度慢(P〈0.05)。甲苯胺蓝及Ⅱ型胶原免疫组化染色显示,骨关节炎软骨细胞染色较正常软骨细胞浅,经多次传代后基本无着色。结论:正常软骨细胞5代以内细胞生长良好,生物学特性明显,5代以后出现去分化现象。骨关节炎软骨细胞增殖慢,生物学特征退变旱,符合软骨细胞退变的表现。这为骨关节炎在软骨细胞水平的研究提供了实验基础。  相似文献   

3.
研究淫羊藿素在GDF-5诱导BMSCs成软骨分化过程中的作用。全骨髓贴壁法分离培养SD大鼠骨髓间充质干细胞(BMSCs),取P3代细胞随机分成4组:对照组,淫羊藿素(Icaritin)组,Growth differentiation factor 5(GDF-5)组,Icaritin+GDF-5联合组。连续诱导培养14 d,倒置相差显微镜观察细胞形态,Alcian Blue染色检测细胞的蛋白聚糖改变,RT-PCR检测软骨分化标记基因Aggrecan、COL2、Sox9及COL1的表达情况,Western Blot检测COL2和COL1蛋白表达水平。结果提示,与对照组及GDF-5组相比,Icaritin+GDF-5联合组蛋白聚糖染色更深;软骨分化标记基因Aggrecan、COL2、Sox9明显增加;Ⅱ型胶原蛋白表达量均明显增加。淫羊藿素能够促进GDF-5诱导BMSCs成软骨分化。  相似文献   

4.
目的:探讨转化生长因子β2(TGF-β2)在低氧条件下诱导骨髓基质干细胞(BMSCs)向软骨细胞分化的作用。方法:无菌条件下分离Wistar大鼠股骨骨髓,采用全贴壁培养法纯化BMSCs。传6代后,将细胞随机分为3组,A组加入25 ng/mL TGF-β2在1%氧浓度条件下培养;B组加入25 ng/mL TGF-β2在21%氧浓度条件下培养;C组仅加入含10%胎牛血清的DMEM-α培养液在1%氧浓度条件下培养。3周后,通过甲苯胺蓝染色检测细胞糖胺多糖,聚合酶链反应检测Ⅱ型胶原和蛋白聚糖(Aggrecan)的表达水平。结果:骨髓细胞经换液后贴壁聚集生长,形态均一,连续传代后形态无明显改变。分组培养第1周,A、C组生长速度低于B组;第2周各组均出现不规则形态细胞,A、C组细胞形态小于B组;第3周各组均可见透明样基质,以A组最明显。3周后行甲苯胺蓝染色,A组细胞内外均可见丰富的蓝染颗粒,B、C组染色较A组略浅。A组Ⅱ型胶原的表达相对量(1.246±0.287)高于B组(0.973±0.365)、C组(0.802±0.196),差异有统计学意义(P〈0.05);B、C组比较无明显差异(P〉0.05)。A组Aggrecan的表达相对量(0.833±0.375)高于B组(0.724±0.173)、C组(0.602±0.091),差异有统计学意义(P〈0.05);B、C组比较无明显差异(P〉0.05)。结论:TGF-β2联合低氧环境可明显促进骨髓基质干细胞分化为软骨细胞。  相似文献   

5.
目的将间充质干细胞诱导分化为软骨细胞,观察分化后的细胞在单层培养和几丁质支架上培养的差别。方法抽取兔股骨骨髓,密度梯度离心分离BMSCs,用TGF-β1诱导第3代的BMSCs向软骨方向分化,2周后用免疫组化检测Ⅱ型胶原表达情况。将分化后的软骨样细胞传代,用无TGF-β1的培养基分别进行单层培养和接种在几丁质支架上培养2周,再用免疫组化检测Ⅱ型胶原表达情况。结果BMSCs经TGF-β1诱导后,具有软骨细胞特点,然而进行无TGF-β1的继续培养后,单层培养的类软骨细胞很快呈去分化表型,而接种在支架上的细胞仍能较好表达Ⅱ型胶原。结论几丁质支架具有延缓软骨样细胞去分化和老化的作用。  相似文献   

6.
目的:比较骨髓间充质干细胞、脂肪间充质干细胞、滑膜间充质干细胞3种间充质干细胞的成软骨分化潜能,为软骨组织工程中种子细胞的选择提供实验依据。方法:采用贴壁法分别分离提取兔骨髓间充质干细胞、脂肪间充质干细胞、滑膜间充质干细胞3种间充质干细胞,并进行传代培养,绘制3种间充质干细胞的生长曲线并比较其倍增时间。将3种间充质干细胞成软骨诱导14 d后,行甲苯胺蓝染色及II型胶原免疫组化染色以观测3种细胞成软骨分化能力。结果:脂肪间充质干细胞的倍增时间短于骨髓间充质干细胞,滑膜间充质干细胞的倍增时间最短;3种细胞成软骨诱导14 d后均产生糖胺聚糖和II型胶原,且组与组之间II型胶原表达水平的差异有统计学意义,骨髓间充质干细胞组高于脂肪间充质干细胞组(P0.01),滑膜间充质干细胞组高于骨髓间充质干细胞组(P0.01)。结论:在一定的培养条件下,3种间充质干细胞均有一定的成软骨细胞分化潜能,滑膜间充质干细胞最快的增殖速度及最强的成软骨分化潜能。  相似文献   

7.
目的:建立人颈椎间盘髓核细胞体外培养体系,并对其细胞表型进行鉴定。方法:采用酶消化法分离人颈椎间盘髓核细胞,进行单层培养,倒置相差显微镜观察细胞生长和形态,流式细胞仪测定细胞周期和凋亡率,并行甲苯胺蓝、Ⅱ型胶原及CK8免疫组化染色对其细胞表型进行鉴定。结果:原代髓核细胞凋亡率6.1±1.4%,S期细胞比例7.3±0.5%。贴壁后形态为多角形或短楔形,传代后生长加速。细胞呈甲苯胺蓝异染性;Ⅱ型胶原免疫组化染色阳性;只有少量椭圆形大细胞CK8免疫组化染色阳性。结论:成功建立人颈椎间盘髓核细胞体外培养模型,并证实成年后髓核内仍有少量细胞保持脊索细胞表型。  相似文献   

8.
目的:探讨低强度周期性静水压力对体外培养的人膝关节软骨细胞增殖、凋亡,以及细胞Ⅱ型胶原分泌表达的影响。方法:体外酶消化法分离培养成人膝关节正常软骨细胞,将培养的第3代软骨细胞分为两组:正常对照组、3.0MPa组压力实验组,应用多功能恒温体外细胞培养中高压静水压力加载装置加载低强度周期性压力,共5d,每天2h。Ⅱ型胶原免疫组织化学染色法和甲苯胺蓝染色法鉴定软骨细胞,流式细胞术检测细胞凋亡,四甲基偶氮唑蓝(MTT)法绘制细胞生长曲线,qRT-PCR、Western-Blot检测Ⅱ型胶原的分泌和表达。结果:软骨细胞Ⅱ型胶原免疫组织化学染色和甲苯胺蓝染色均显示为阳性。与正常对照组相比,3.0MPa组表现出促进软骨细胞增殖,抑制细胞凋亡,且Ⅱ型胶原的合成分泌明显升高(P0.05)。结论:通过体外模拟人生理情况下较低强度(3.0MPa)的周期性静水压力对人软骨细胞增殖、凋亡水平及周围基质分泌合成功能的影响,初步证实了较低强度压力有助于软骨自我修复和自身保护作用的发挥。  相似文献   

9.
目的:探讨转化生长因子β2(TGF-β2)在低氧条件下诱导骨髓基质干细胞(BMSCs)向软骨细胞分化的作用。方法:无菌条件下 分离Wistar 大鼠股骨骨髓,采用全贴壁培养法纯化BMSCs。传6 代后,将细胞随机分为3 组,A组加入25 ng/mL TGF-β2在1%氧 浓度条件下培养;B 组加入25 ng/mL TGF-β2在21%氧浓度条件下培养;C 组仅加入含10%胎牛血清的DMEM-α培养液在1%氧 浓度条件下培养。3 周后,通过甲苯胺蓝染色检测细胞糖胺多糖,聚合酶链反应检测Ⅱ型胶原和蛋白聚糖(Aggrecan)的表达水平。 结果:骨髓细胞经换液后贴壁聚集生长,形态均一,连续传代后形态无明显改变。分组培养第1 周,A、C 组生长速度低于B 组;第 2 周各组均出现不规则形态细胞,A、C 组细胞形态小于B 组;第3 周各组均可见透明样基质,以A组最明显。3 周后行甲苯胺蓝 染色,A 组细胞内外均可见丰富的蓝染颗粒,B、C 组染色较A 组略浅。A 组Ⅱ型胶原的表达相对量(1.246± 0.287) 高于B 组 (0.973± 0.365)、C组(0.802± 0.196),差异有统计学意义(P<0.05);B、C 组比较无明显差异(P>0.05)。A 组Aggrecan 的表达相对量 (0.833± 0.375)高于B组(0.724± 0.173)、C 组(0.602± 0.091),差异有统计学意义(P<0.05);B、C 组比较无明显差异(P>0.05)。结 论:TGF-β2联合低氧环境可明显促进骨髓基质干细胞分化为软骨细胞。  相似文献   

10.
细胞片技术是应用组织工程方法使培养细胞从培养表面分离而形成含有细胞外基质的一层完整片状结构,弥补了传统组织工程技术的不足,是获取种子细胞以及对种子细胞进行转移的一项新技术。为探讨体外生长分化因子-5(GDF5)基因转染修饰的BMSCs细胞片与GDF5转基因BMSCs负载的PLGA支架形成的共聚物修复兔甲状软骨缺损的效果,实验通过腺病毒转染GDF5基因至四代兔BMSCs,温度敏感性培养皿制备GDF5转基因细胞片并与负载有转染GDF5基因BMSCs的PLGA支架复合,移植至同种兔甲状软骨缺损处,分别于术后4、8周行大体观察和组织学检测其修复效果。实验分3组:(A)转基因细胞片包裹负载有转基因BMSCs的PLGA支架组;(B)负载有转基因BMSCs的PLGA支架组;(C)负载BMSCs的PLGA支架组。结果显示,体外成功收获了完整的GDF5转基因细胞片,Real time PCR检测到GDF5 mRNA的表达,行大体组织的II型胶原免疫组化和阿利新蓝染色显示:A组和B组均表达II型胶原和糖胺聚糖(GAG),但A组表达高于B组,有统计学意义(P0.05)。由此可得,转基因细胞片包裹负载转基因BMSCs PLGA支架较传统转基因BMSCs负载PLGA支架方法具有更加优越的成软骨能力,能更有效地促进软骨缺损的修复。  相似文献   

11.
We investigated the influence of the microgravity rotating culture system on the chondrogenic differentiation of bone marrow mesenchymal stem cells (MSCs). During chondrogenic induction, MSCs combined with polyglycolic acid (PGA) were cultured by static culture or microgravity rotating culture and chondrocyte formation was confirmed by toluidine blue staining. Furthermore, the mRNA and protein expressions of a specific cartilage extracellular matrix protein (collagen type II and Aggrecan) were evaluated by real-time RT-PCR and western blot, respectively. Toluidine blue staining indicated the OD values of proteoglycans semi-determination were higher in the microgravity rotating culture group than the static culture group. Following chondrogenic induction, mRNA and proteins of collagen type II and Aggrecan were more significantly expressed in cells of the microgravity rotating culture group compared with the controls. Compared with routine three-dimensional static culture, the microgravity rotating culture system was more effective for the construction of tissue-engineered cartilage in vitro.  相似文献   

12.
Abnormal expression of KDM6A and SOX9 is a key factor in the pathogenesis of osteoarthritis (OA). Cellular treatments of OA with articular cartilage chondrocytes (ACCs) and bone marrow mesenchymal stem cells (BMSCs) are promising, but their underlying mechanisms remain to be explored. The pellet size, weight and sulfated glycosaminoglycan/DNA content of ACCs were measured to evaluate the effect of BMSCs on the chondrogenic differentiation of SCCs. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to analyze the proliferation of ACCs cultured along or cocultured with BMSCs. Quantitative polymerase chain reaction (qPCR) was performed to evaluate the messenger RNA expression of KDM6A, SOX9, type2 collagen, and Aggrecan in ACCs and OA rats. Western blot and immunohistochemistry were performed to analyze the expression of KDM6A and SOX9 proteins. Bisulfite sequencing PCR was performed to assess the DNA methylation level of the SOX9 promoter. Flow cytometry was used to evaluate the apoptotic status of ACCs. The chondrogenic differentiation of ACCs was significantly enhanced by coculturing with BMSCs, especially under a hypoxic condition. The expression of KDM6A, SOX9, type2 collagen, and Aggrecan was remarkably elevated in ACCs cocultured with BMSCs. Also, the DNA methylation of SOX9 promoter was decreased in ACCs cocultured with BMSCs, along with notably reduced apoptosis. Moreover, ACCs cocultured with BMSCs could repair cartilage lesions and prevent the abnormal expression of KDM6A, SOX9, type2 collagen, and Aggrecan in OA rats. In this study, we cocultured ACCs with BMSCs and used them to treat OA rats. Our findings presented a mechanistic basis for explaining the therapeutic effect of BMSCs on OA treatment.  相似文献   

13.
目的 探讨骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)与聚乳酸/羟基乙酸共聚物(poly (lactide-co-glycolide),PLGA)三维生物支架在软骨源性形态发生蛋白1(cartilage-derived morphogenetic protein 1,CDMP1)和转化生长因子-β1(transforming growth factor-β1,TGF-β1)作用下向软骨细胞表型分化及体内修复喉软骨缺损的能力。方法 在体外高密度细胞悬液与PLGA共同构筑的三维立体培养体系下CDMP1和(或)TGF-β1联合诱导BMSCs向软骨细胞分化,观察诱导后细胞表型的表达;将培养体系移植入动物体内,从大体、组织学方面观察其对喉软骨缺损的修复效果。结果 诱导后的培养体系可表达特异性软骨基质Ⅱ型胶原和GAG;将培养体系移植入动物体内,可有效的修复喉软骨缺损。结论 BMSCs与PLGA三维生物支架在CDMP1和TGF-β1作用下所得组织工程化软骨可以有效的修复喉软骨缺损。  相似文献   

14.
目的:探究姜黄素改善慢性低O2高CO2大鼠肺动脉高压肺血管重塑作用途径的研究。方法:建立慢低O2高CO2肺血管重塑模型,以24只雄性大鼠为受试对象,随机分为四组(n=6):I组(常氧空白对照组),Ⅱ(低O2高CO2模型组),Ⅲ组(色甘酸钠对照组),Ⅳ组(姜黄素实验组)。将后3组动物放入常压低O2高CO2舱中,吸入O2浓度8%~11%,CO2浓度3%~5%,每天8h,每周6d,连续4周,Ⅲ组给予色甘酸钠以20mg/kg体重腹注射处理,Ⅳ组给予姜黄素混悬液按150mg/kg体重灌胃处理。光镜下、透射电镜下观察肺动脉血管壁及其周围大细胞超微结构形态学改变,甲苯胺蓝染色法和免疫组织化学法对肺动脉周围的肥大细胞及其脱颗粒状态进行性定量测定。结果:①电镜下,Ⅱ组肺细小动脉中膜平滑肌增生,外膜胶原纤维密集,内弹力板扭曲,内皮细胞起,血管外肥大细胞内颗粒减少,胞膜不完整;光镜下,Ⅱ组相比1组肺细小动脉管腔/管总面积(WA/TA)明显升(P〈0.05)、管腔/管总面积(EA/TA)明显降低(P〈0.05),甲苯胺蓝染色肥大细胞细胞数(NMC)、肥大细胞脱颗率(DR)及免疫组化检测类胰蛋白酶阳性细胞数(TBS)高于I组(P〈0.05);②干预后,电镜下,Ⅲ组、Ⅳ组血管结基本正常,平滑肌增生及胶原增生较Ⅱ组轻,肥大细胞膜完整;光镜下,两干预组相比Ⅱ组WA/TA明显降低(P〈0.05)、ET/TA明显升高(P〈0.05),甲苯胺蓝染色NMC、DR、TBS阳性细胞数分别低于Ⅱ组(P〈0.05)。结论:姜素可通过Mc途径抑制慢性低O2高CO2导致的大鼠肺血管重塑改变。  相似文献   

15.
Human adipose-derived stromal cells (hASCs) possess the potential for chondrogenic differentiation. Recent studies imply that this differentiation process may be enhanced by culturing the cells in low oxygen tension in combination with three-dimensional (3D) scaffolds. We report the evaluation of the chondrogenic potential of hASC pellets in 5 and 21 % O2 and as cell-scaffold constructs using a collagen I/III scaffold with chemical induction using TGF-β3. hASCs from four human donors were cultured both in a micromass pellet system and in 3D collagen I/III scaffolds in either 5 or 21 % O2. Chondrogenesis was evaluated by quantitative gene expression analysis of aggrecan, SOX9, collagen I, II and X and histological evaluation with H&E and toluidine blue staining. Induced pellets cultured in 5 % O2 showed increased peripheral cellularity and matrix deposition compared with 21 % O2. Induced pellets cultured in 5 % O2 had increased control-adjusted gene expression of aggrecan, SOX9 and collagen I and decreased collagen X compared with 21 % O2 cultures. Induced pellets had higher gene expression of aggrecan, SOX9, collagen I, II and X and increased ratios of collagen II/I and collagen II/X compared with controls. As for pellets, scaffold cultures showed cellularity and matrix deposition organized in a zonal manner as a function of the oxygen tension, with a cartilage-like morphology and matrix deposition peripherally in the 5 % O2 group and a more centrally located matrix in the 21 % O2 group. There were no differences in histology and gene expressions between pellet and scaffold cultures. Five percent O2 in combination with chondrogenic culture medium stimulated chondrogenic differentiation of hASCs in vitro. We observed similar patterns of differentiation and matrix disposition in pellet and scaffold cultures.  相似文献   

16.
目的:近年来研究表明,关节软骨细胞凋亡在骨关节炎发病过程中起到了重要的作用,本文旨在探讨microma-15a模拟物对于原代人膝关节软骨细胞增殖与凋亡的影响。方法:取人外伤性截肢后的膝关节软骨,采用双酶消化法分离获得人膝关节软骨细胞,并进行体外培养,通过甲苯胺蓝染色和II型胶原免疫细胞化学染色进行软骨细胞鉴定。将培养的软骨细胞传代后取第l代细胞,分为实验组和对照组,实验组采用mir.15a模拟物(has.mir-15amimics)转染软骨细胞,上调软骨细胞内mir-15a的表达量;对照组分为阴性对照组、空白对照组。采用MTT法测定细胞增殖曲线,流式细胞仪测定细胞凋亡率。结果:原代细胞中细胞呈多角形、圆形与梭型,贴壁生长;甲苯胺蓝染色胞质呈深蓝色,II型胶原染色胞质呈黄褐色,为特异性染色。经统计学分析,实验组与对照组相比增殖速率明显下降(P〈0.05)。实验组凋亡率(7.13%±0.57)与阴性对照组凋亡率(2.66%±0.15)相比明显增高(P〈0.05)。结论:采用双酶消化法成功分离并培养具有生物学特性的原代人膝关节软骨细胞,通过转染mir-15a模拟物外源性增加关节软骨细胞内mir.15a表达量可显著促进其凋亡并抑制其增殖,为阐明骨关节炎发病机制提供了新的理论依据,为’临床治疗提供了新的靶点。  相似文献   

17.
Our study aimed to find out the most effective mode for chondrogenic differentiation based on time, dose and culture method. ADSCs were cultured and identified by CD44, CD49d, and CD106 immumohistochemical staining method, and their differentiation potential to chondrocyte were detected by Alizarin red staining. ADSCs induced by different concentrations of GDF-5 for chondrogenic differentiation were detected by blue and toluidine blue staining and collagen type II and X immumohistochemical staining. The expression of collagen I, II, X and aggrecan gene in GDF-induced ADSCs cultured in 2- and 3-dimension was identified by real-time PCR. Cell microstructure and proliferation in three-dimensional scaffolds at day 7, 14, 21 and 28 were analyzed by scanning electron microscopy and MTS assay. The ADSCs were successfully identified by CD44 and CD49d, and their differentiation potential was detected by Alizarin red staining. Real-time PCR showed that collagen and aggrecan were expressed at high levels in 100 or 200 ng/mL GDF-5 treated cells. The collagen types (I, II) and aggrecan genes were higher expressed in GDF-5 induced scaffold group than that in monolayer group. MTS showed that the cell counts were not significantly different among different treated time. Both collagen type II and aggrecan gene were highly expressed at day 14, while collagen types I and X gene expressions peaked at day 21 and 28. The 100 ng/mL GDF-5 is effective and cost-effective for chondrogenic differentiation when cultured at day 14 in vitro under three-dimensional culture conditions.  相似文献   

18.
TGF-β1 plays a necessary and important role in the induction of chondrogenic differentiation of bone marrow stromal cells (BMSCs). In this study, porcine BMSCs were infected with a replication-deficient adenovirus expression vector carrying the hTGF-β1 gene. The transduced BMSCs were cultured as pelleted micromasses in vitro for 21 days, seeded onto disk-shaped PGA scaffolds for 3 days and subsequently implanted into the subcutaneous tissue of mice. BMSCs transduced with AdhTGF-β1 expressed and secreted more hTGF-β1 protein in vitro than those of the control group. Histological and immunohistological examination of the pellets revealed robust chondrogenic differentiation. Tissues made from cells transduced with AdhTGF-β1 exhibited neocartilage formation after 3 weeks in vivo. The neocartilage occupied 42 ± 5% of the total tissue volume which was significantly greater than that of the control group. Furthermore, there was extensive staining for sulfated proteoglycans and type II collagen in the AdhTGF-β1 group compared to controls, and quantification of GAG content showed significantly greater amounts of GAG in experimental groups. The results demonstrate that transfer of hTGF-β1 into BMSCs via adenoviral transduction can induce chondrogenic differentiation in vitro and enhance chondrogenesis in vivo.  相似文献   

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