【摘要】：目的:軟骨組織是無(wú)血管和無(wú)神經(jīng)的特殊結(jié)構(gòu),一旦受損,組織再生和修復(fù)的能力非常有限。組織工程和再生醫(yī)學(xué)技術(shù)的不斷成熟,為提高軟骨的修復(fù)質(zhì)量帶來(lái)新的機(jī)會(huì)。然而,關(guān)節(jié)腔內(nèi)的缺氧環(huán)境及骨關(guān)節(jié)炎患者關(guān)節(jié)腔的炎性環(huán)境,使移植細(xì)胞活性及存活率明顯下降,制約了軟骨組織工程的應(yīng)用和發(fā)展。尋找促進(jìn)軟骨細(xì)胞增殖、增強(qiáng)軟骨細(xì)胞分泌基質(zhì)的有效成分,是研究關(guān)注的重點(diǎn)。本項(xiàng)目探討杜仲葉主要提取物綠原酸(Chlorogenic acid,CGA)交聯(lián)海藻酸鈉支架及原代培養(yǎng)的軟骨細(xì)胞構(gòu)成的組織工程學(xué)軟骨,對(duì)雛雞關(guān)節(jié)軟骨損傷的修復(fù)作用,并探討其可能的分子機(jī)制。方法:本實(shí)驗(yàn)通過(guò)對(duì)原代雞胚軟骨細(xì)胞的培養(yǎng)從而確定CGA對(duì)體外原代培養(yǎng)的軟骨細(xì)胞的形成作用。然后,我們通過(guò)應(yīng)用雛雞膝關(guān)節(jié)軟骨損傷模型來(lái)評(píng)估含有CGA、軟骨細(xì)胞以及海藻酸鈉的三維支架的移植對(duì)其功能恢復(fù)的影響。通過(guò)行為學(xué)分析、形態(tài)學(xué)分析、PCR以及Western blot等實(shí)驗(yàn)進(jìn)一步闡明其基本機(jī)制。結(jié)果:首先證明在CGA聯(lián)合海藻酸鈉體外能夠促進(jìn)原代軟骨細(xì)胞的形成。其次通過(guò)對(duì)步態(tài)分析說(shuō)明含有CGA的復(fù)合物加速了由于損傷導(dǎo)致的功能障礙的恢復(fù)。然后組織化學(xué)的分析表明,CGA復(fù)合物的移植物會(huì)減少血管的增生,促進(jìn)軟骨細(xì)胞的增殖以及軟骨基質(zhì)的合成。接著我們發(fā)現(xiàn)CGA可以促進(jìn)與軟骨發(fā)生相關(guān)因子Sox 9和Col2 a1的表達(dá)。此外,這些含有CGA的復(fù)合物的應(yīng)用可通過(guò)調(diào)節(jié)細(xì)胞氧化還原穩(wěn)態(tài)來(lái)抑制炎性細(xì)胞因子IL-1β的和TNF-α以及p-p65的表達(dá)。結(jié)論:我們的研究表明,將軟骨細(xì)胞、CGA結(jié)合在海藻酸鈉支架上構(gòu)建組織工程軟骨能明顯促進(jìn)關(guān)節(jié)軟骨的損傷修復(fù),其作用機(jī)理與CGA通過(guò)調(diào)節(jié)氧化應(yīng)激穩(wěn)態(tài),抑制損傷部位的血管新生、炎癥反應(yīng)和局部纖維化有關(guān)。
【圖文】：

22 圖3.1 不同CGA濃度對(duì)原代軟骨細(xì)胞增殖影響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 圖3.2 海藻酸鈉三維培養(yǎng)條件下的CGA對(duì)軟骨再生的影響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.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2018
【分類號(hào)】：R318.08

【參考文獻(xiàn)】

相關(guān)期刊論文 前9條

1 孫凱;年?duì)幒?徐成;李瑞欣;李暉;;絲素蛋白復(fù)合膠原蛋白支架的制備及性能研究[J];中國(guó)修復(fù)重建外科雜志;2014年07期

2 王琳玨;;急性踝關(guān)節(jié)扭傷的治療進(jìn)展[J];中醫(yī)正骨;2014年03期

3 劉哲;李士勇;宋玉林;廖新根;陳偉才;顧玉榮;殷明;;綠原酸對(duì)缺氧環(huán)境下干細(xì)胞來(lái)源軟骨樣細(xì)胞凋亡的影響[J];中國(guó)藥理學(xué)通報(bào);2011年02期

4 王清;朱萱萱;張赤兵;倪文彭;吳旭同;;金銀花提取物抗菌作用的實(shí)驗(yàn)研究[J];中國(guó)醫(yī)藥導(dǎo)刊;2008年09期

5 吳衛(wèi)華;康楨;歐陽(yáng)冬生;周宏灝;;綠原酸的藥理學(xué)研究進(jìn)展[J];天然產(chǎn)物研究與開發(fā);2006年04期

6 李寧華;;中老年人群骨關(guān)節(jié)炎的流行病學(xué)特征[J];中國(guó)臨床康復(fù);2005年38期

7 黃喜茹,劉偉娜,曹冬;金銀花的化學(xué)成分藥理作用研究評(píng)析[J];中醫(yī)藥學(xué)刊;2005年03期

8 曹彬,郭子健,朱元玨,許文兵;The potential role of PDGF, IGF-1, TGF-β expression in idiopathic pulmonary fibrosis[J];Chinese Medical Journal;2000年09期

9 高錦明,張鞍靈,張康健,趙曉明;綠原酸分布、提取與生物活性研究綜述[J];西北林學(xué)院學(xué)報(bào);1999年02期

，

本文編號(hào)：2701659