绿原酸联合海藻酸钠构建组织工程软骨修复关节损伤的研究
发布时间：2020-06-07 16:01
【摘要】：目的:软骨组织是无血管和无神经的特殊结构,一旦受损,组织再生和修复的能力非常有限。组织工程和再生医学技术的不断成熟,为提高软骨的修复质量带来新的机会。然而,关节腔内的缺氧环境及骨关节炎患者关节腔的炎性环境,使移植细胞活性及存活率明显下降,制约了软骨组织工程的应用和发展。寻找促进软骨细胞增殖、增强软骨细胞分泌基质的有效成分,是研究关注的重点。本项目探讨杜仲叶主要提取物绿原酸(Chlorogenic acid,CGA)交联海藻酸钠支架及原代培养的软骨细胞构成的组织工程学软骨,对雏鸡关节软骨损伤的修复作用,并探讨其可能的分子机制。方法:本实验通过对原代鸡胚软骨细胞的培养从而确定CGA对体外原代培养的软骨细胞的形成作用。然后,我们通过应用雏鸡膝关节软骨损伤模型来评估含有CGA、软骨细胞以及海藻酸钠的三维支架的移植对其功能恢复的影响。通过行为学分析、形态学分析、PCR以及Western blot等实验进一步阐明其基本机制。结果:首先证明在CGA联合海藻酸钠体外能够促进原代软骨细胞的形成。其次通过对步态分析说明含有CGA的复合物加速了由于损伤导致的功能障碍的恢复。然后组织化学的分析表明,CGA复合物的移植物会减少血管的增生,促进软骨细胞的增殖以及软骨基质的合成。接着我们发现CGA可以促进与软骨发生相关因子Sox 9和Col2 a1的表达。此外,这些含有CGA的复合物的应用可通过调节细胞氧化还原稳态来抑制炎性细胞因子IL-1β的和TNF-α以及p-p65的表达。结论:我们的研究表明,将软骨细胞、CGA结合在海藻酸钠支架上构建组织工程软骨能明显促进关节软骨的损伤修复,其作用机理与CGA通过调节氧化应激稳态,抑制损伤部位的血管新生、炎症反应和局部纤维化有关。
【图文】：
22 图3.1 不同CGA浓度对原代软骨细胞增殖影响Fig 3.1. The in vitro assessment of optimal CGA concentration using primary cultureof chondrocytesA: The sketches illustrating the primary culture of chondrocytes isolated from 15-daychick embryonic manubrium and articular cartilage in absence/presence of CGA invitro （see material and method in detail）. B-D: Cell counting kit-8 （CCK8） assaywas implemented to detect the cell viability following in vitro primary culture ofchondrocytes at the 24th(B), 48th(C) and 72th(D) hour in absence/presence of differentconcentrations of CGA. E-F, E1-F1, E2-F2: The pHIS3 immunofluorescent stainingwas implemented on 48h-incubated primary culture of chondrocytes in absence (E) orpresence (F) of 60μM CGA. E1-F1 are DAPI staining while E2-F2 are the mergeimages of pHIS3 immunofluorescent staining and DAPI respectively. H-I, H1-I1, H2-I2: The F-actin fluorescent staining was implemented on 48h-incubated primary cultureof chondrocytes in absence (H) or presence (I) of 60μM CGA. H1-I1 are DAPI stainingwhile H2-I2 are the merge images of F-actin staining and DAPI respectively. G: Thebar chart showing the ratio comparison of pHIS3+cell percentage within totalchondrocytes between control and CGA-treated group
24 图3.2 海藻酸钠三维培养条件下的CGA对软骨再生的影响Fig 3.2. The assessment of chondrogenesis on primary culture of chondrocytesfollowing the combinational application of CGA and alginate.A: The sketches illustrating the primary culture of chondrocytes isolated from 15-daychick embryonic manubrium and articular cartilage in alginate and absence/presence ofCGA in vitro. B-D: The H&E staining was implemented on the 21-day incubatedalginate only (B), chondrocytes + alginate (C) or chondrocytes + alginate + CGA (D).B1-D1: The high magnification images from B-C respectively. E-F: The safranin-ostaining was implemented on the 21-day incubated chondrocytes + alginate (E) orchondrocytes + alginate + CGA (F). E1-F1: The high magnification images from E-Frespectively. G: The bar chart showing the ratio comparison of IOD (integral opticaldensity) of the complexes between cell + alginate group and cell + CGA + alginategroup. Scale bars = 500μm in B-F and 100μm in B1-F1.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2018
【分类号】：R318.08
本文编号：2701659
【图文】：
22 图3.1 不同CGA浓度对原代软骨细胞增殖影响Fig 3.1. The in vitro assessment of optimal CGA concentration using primary cultureof chondrocytesA: The sketches illustrating the primary culture of chondrocytes isolated from 15-daychick embryonic manubrium and articular cartilage in absence/presence of CGA invitro （see material and method in detail）. B-D: Cell counting kit-8 （CCK8） assaywas implemented to detect the cell viability following in vitro primary culture ofchondrocytes at the 24th(B), 48th(C) and 72th(D) hour in absence/presence of differentconcentrations of CGA. E-F, E1-F1, E2-F2: The pHIS3 immunofluorescent stainingwas implemented on 48h-incubated primary culture of chondrocytes in absence (E) orpresence (F) of 60μM CGA. E1-F1 are DAPI staining while E2-F2 are the mergeimages of pHIS3 immunofluorescent staining and DAPI respectively. H-I, H1-I1, H2-I2: The F-actin fluorescent staining was implemented on 48h-incubated primary cultureof chondrocytes in absence (H) or presence (I) of 60μM CGA. H1-I1 are DAPI stainingwhile H2-I2 are the merge images of F-actin staining and DAPI respectively. G: Thebar chart showing the ratio comparison of pHIS3+cell percentage within totalchondrocytes between control and CGA-treated group
24 图3.2 海藻酸钠三维培养条件下的CGA对软骨再生的影响Fig 3.2. The assessment of chondrogenesis on primary culture of chondrocytesfollowing the combinational application of CGA and alginate.A: The sketches illustrating the primary culture of chondrocytes isolated from 15-daychick embryonic manubrium and articular cartilage in alginate and absence/presence ofCGA in vitro. B-D: The H&E staining was implemented on the 21-day incubatedalginate only (B), chondrocytes + alginate (C) or chondrocytes + alginate + CGA (D).B1-D1: The high magnification images from B-C respectively. E-F: The safranin-ostaining was implemented on the 21-day incubated chondrocytes + alginate (E) orchondrocytes + alginate + CGA (F). E1-F1: The high magnification images from E-Frespectively. G: The bar chart showing the ratio comparison of IOD (integral opticaldensity) of the complexes between cell + alginate group and cell + CGA + alginategroup. Scale bars = 500μm in B-F and 100μm in B1-F1.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2018
【分类号】：R318.08
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