本文選題：軟骨細(xì)胞　切入點(diǎn)：微孔水凝膠　出處：《華南理工大學(xué)》2014年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】：軟骨內(nèi)部無(wú)血管，無(wú)神經(jīng)，無(wú)免疫反應(yīng)的獨(dú)特特性導(dǎo)致其自愈能力極其有限，軟骨修復(fù)是一大國(guó)際性難題。傳統(tǒng)性修復(fù)技術(shù)尚不能完全滿(mǎn)足臨床需要，目前，軟骨組織工程為軟骨缺損修復(fù)與重建開(kāi)辟了新的途徑。如何保持長(zhǎng)期有效的軟骨組織修復(fù)效果是目前存在的重大挑戰(zhàn)。細(xì)胞所處的微環(huán)境對(duì)其生長(zhǎng)及分化有著重要的影響。因此，針對(duì)軟骨修復(fù)材料的開(kāi)發(fā)，研究材料提供的微環(huán)境對(duì)細(xì)胞行為及功能表達(dá)的影響具有重要意義。 水凝膠以其良好的生物相容性，仿似ECM的3D微環(huán)境，合適的孔隙結(jié)構(gòu)，且能提供短期的機(jī)械穩(wěn)定性以及增加細(xì)胞附著表面積，附著力等特點(diǎn)從而能構(gòu)成細(xì)胞生長(zhǎng)和分化的良好微環(huán)境，，但目前傳統(tǒng)的水凝膠的應(yīng)用瓶頸便是它尚不能提供細(xì)胞足夠的快速生長(zhǎng)空間和適當(dāng)?shù)挠H和位點(diǎn)。正因?yàn)槿绱�，�?jīng)過(guò)一段較長(zhǎng)時(shí)間的體外培養(yǎng)，包埋在水凝膠邊緣軟骨細(xì)胞會(huì)突破水凝膠的阻礙，在邊緣位置會(huì)分泌豐富的軟骨組織基質(zhì)蛋白，水凝膠外表面顯示出真正的軟骨組織特征，與水凝膠內(nèi)部，細(xì)胞的保守生長(zhǎng)形成鮮明對(duì)比。該現(xiàn)象被定義為“邊緣發(fā)展edge flourish（EF）”。這種EF現(xiàn)象只是發(fā)生在傳統(tǒng)水凝膠的邊緣，缺乏可控性。受到EF的啟發(fā)，我們通過(guò)在水凝膠內(nèi)部構(gòu)建微孔，從而人為地引入“凝膠微孔邊緣現(xiàn)象”。這樣，即使在水凝膠的內(nèi)部，也能出現(xiàn)EF現(xiàn)象。本論文的研究目的是利用“微孔”這一結(jié)構(gòu)的特殊EF效應(yīng)來(lái)觀(guān)察其對(duì)軟骨細(xì)胞生長(zhǎng)增殖以及細(xì)胞功能表達(dá)情況以研究材料的結(jié)構(gòu)微環(huán)境與細(xì)胞的相互作用。本論文的研究工作主要開(kāi)展了以下內(nèi)容： 本文首先采用II型膠原酶消化法將豬關(guān)節(jié)軟骨細(xì)胞進(jìn)行體外分離、培養(yǎng)和鑒定。不同代數(shù)的軟骨細(xì)胞生長(zhǎng)剛開(kāi)始較為緩慢，之后進(jìn)入快速增殖期和平臺(tái)期，最后開(kāi)始出現(xiàn)生長(zhǎng)抑制。隨著傳代次數(shù)的增加，細(xì)胞形態(tài)由原代的近似橢圓形逐漸變?yōu)槎嘟切位蛘呒忓N形，原代軟骨細(xì)胞的組織染色與天然軟骨的組織染色相接近。II型膠原免疫組化染色表明其細(xì)胞質(zhì)被染成棕黃色，細(xì)胞核基本不著色。證明該方法能獲得純度高，活性好的原代軟骨細(xì)胞，成功建立了豬關(guān)節(jié)軟骨細(xì)胞分離及培養(yǎng)體系。 本文采用雙乳液法成功制備出形狀規(guī)整，尺寸可控的明膠微球，考察了攪拌速率對(duì)明膠微球粒徑的影響，改變攪拌速率，制備并篩選出具有代表性的三種尺寸分別為小尺寸（80-120μm）、中尺寸（150-200μm）、大尺寸（250-300μm）的明膠微球。以明膠微球?yàn)橹驴讋┏晒?gòu)建了具有以上三種不同尺寸大小結(jié)構(gòu)的新型微孔水凝膠。研究了三種尺寸的“微孔”結(jié)構(gòu)體系對(duì)軟骨細(xì)胞生長(zhǎng)的影響，微孔水凝膠比傳統(tǒng)致密的海藻酸鈉水凝膠具有更好的促細(xì)胞增殖作用。細(xì)胞會(huì)沿著明膠微球造成的“孔洞”結(jié)構(gòu)邊緣富集生長(zhǎng)并最終完全填滿(mǎn)整個(gè)“孔洞”。其中小孔水凝膠體系中軟骨細(xì)胞的增殖效果最顯著。且能顯著表達(dá)軟骨特征基因，微孔海藻酸鈉水凝膠體系是通過(guò)激活MAPK/JNK信號(hào)通路來(lái)促進(jìn)軟骨細(xì)胞的增殖與生長(zhǎng)，小孔尺寸結(jié)構(gòu)的海藻酸鈉水凝膠對(duì)保持軟骨特征效果最優(yōu)。 其次，本文采用微孔水凝膠成功介導(dǎo)了去分化軟骨細(xì)胞的再分化，微孔水凝膠具有使得去分化軟骨細(xì)胞重新恢復(fù)并保持軟骨細(xì)胞特征的能力，表現(xiàn)為軟骨特征基因表達(dá)顯著上調(diào)，能重新合成和分泌細(xì)胞外基質(zhì)II型膠原和糖胺聚糖，基質(zhì)分泌增強(qiáng)，且有大量軟骨陷窩形成。且去分化的軟骨細(xì)胞在微孔水凝膠中重新分化成軟骨細(xì)胞的能力是優(yōu)于傳統(tǒng)致密海藻酸水凝膠。Westernblot結(jié)果進(jìn)一步證明該再分化過(guò)程有p38和Erk1/2信號(hào)通路的參與。 最后，考察了RGD修飾的微孔水凝膠對(duì)介導(dǎo)ATDC5細(xì)胞成軟骨分化早期的影響。RGD的接枝率為1.4%-3.5%，RGD微孔水凝膠的內(nèi)部仍呈多孔結(jié)構(gòu)，孔隙連通性保持完整，且生物相容性提高。ATDC5細(xì)胞在RGD修飾的微孔水凝膠中體外培養(yǎng)4周后，成軟骨相關(guān)基因均有大量上調(diào)表達(dá)。II型膠原和糖胺聚糖的蛋白表達(dá)也得到相應(yīng)增強(qiáng)，會(huì)有序的形成類(lèi)天然軟骨陷窩結(jié)構(gòu)，致密均勻，基質(zhì)分泌明顯。在生長(zhǎng)因子TGF-β3的誘導(dǎo)下，RGD修飾的微孔水凝膠能顯著增強(qiáng)ATDC5細(xì)胞的成軟骨分化。
[Abstract]:No cartilage vessels, no nerve, no immune response leads to the unique characteristics of the self-healing ability is extremely limited, cartilage repair is a major international problem. The traditional repair technology is still not fully meet the clinical needs, at present, has opened a new way for the repair and reconstruction of cartilage tissue engineering cartilage defects. How to maintain the effect of cartilage tissue repair long term is a major challenge at present. The micro environment of the cells have important influence on their growth and differentiation. Therefore, for the development of cartilage repair materials, plays an important role in micro environment to provide research material on the expression and function of cell behavior.
Hydrogels with good biocompatibility, like ECM 3D micro environment, suitable pore structure, and can provide mechanical stability and increase the short-term cell adhesion surface, adhesion etc. thus constitute cell growth and differentiation of good micro environment, but the application bottleneck of traditional hydrogels is that it cannot provide the rapid growth of space enough cells and appropriate affinity sites. Because of this, after a long time of in vitro culture, entrapped in the hydrogel edge of cartilage cells will break through the obstacles in the edge position of the hydrogel, secrete abundant cartilage matrix protein, hydrogel surface showed the cartilage tissue features real, and within the hydrogel cell growth, the conservative stark contrast. This phenomenon is defined as "the edge of edge flourish development (EF)". This phenomenon only occurred in traditional EF hydrogels The edge of the lack of controllability. Inspired by EF, we construct microporous hydrogel in the inside, thereby artificially introduced "gel microporous edge phenomenon." in this way, even in the hydrogel interior, also can appear EF phenomenon. The purpose of this study is to observe the cartilage cell proliferation and cell function expression to the interaction structure of the material micro environment and cells. Using the special structure of the microporous EF effect "of the research work of this thesis mainly includes the following contents:
Firstly, by type II collagenase digestion of porcine articular cartilage cells were isolated, cultured and identified. Chondrocyte growth beginning algebra is relatively slow, and after entering the stage platform period of rapid proliferation, finally began to appear. The growth inhibition with increasing passage number, cell morphology by primary ellipse gradually becomes polygonal or fusiform, tissue staining and natural cartilage chondrocytes by tissue staining is similar to type.II collagen immunohistochemical staining showed that the cytoplasm stained brown, nucleus basic color. The results show that the method can obtain high purity, primary chondrocytes activity, successfully established the isolation and culture system of pig the articular cartilage cells.
Double emulsion was successfully prepared by the regular shape, gelatin microspheres with controllable size, the effects of stirring rate on gelatin microspheres, changing the stirring rate, the preparation and selection of the three dimensions of the representative are of small size (80-120 m), size (150-200 m). Large size (250-300 m) gelatin microspheres. The gelatin microspheres as porogen successfully constructed a novel microporous hydrogel has more than three kinds of different size structure. On three dimensions of "micro" structure of cartilage cell growth, cell proliferation of microporous hydrogel is better than sodium alginate hydrogel the traditional dense cells. Along the gelatin microspheres caused by the "hole" edge enrichment and growth and eventually completely fill the "hole". The cartilage cells of small pore water in gel system and the proliferation of the most significant effect. It can significantly express cartilage characteristic genes. Microporous alginate hydrogel system promotes the proliferation and growth of chondrocytes by activating MAPK/JNK signaling pathway. Alginate hydrogel with small pore size has the best effect on maintaining cartilage characteristics.
Secondly, this paper uses the successful differentiation of microporous hydrogels mediated dedifferentiation of chondrocytes, the microporous hydrogel has the ability to restore and maintain the chondrocyte differentiation characteristics of chondrocytes, expressed in cartilage feature genes were up-regulated to synthesis and secretion of extracellular matrix type II collagen and glycosaminoglycan, matrix secretion increased and there is a lot of chondrocytes and cartilage lacuna formation. The differentiation in the porous hydrogel to chondrogenic differentiation ability is better than the traditional dense alginate hydrogel.Westernblot results further show that the differentiation process of p38 and Erk1/2 signaling pathways.
Finally, the study of RGD modified microporous hydrogel mediated effects of cartilage in early differentiation of the graft ratio of.RGD 1.4%-3.5% ATDC5 cells, RGD porous hydrogel was still inside the porous structure, pore connectivity remain intact, and improve the biocompatibility of.ATDC5 cells cultured in vitro in microporous hydrogels modified with RGD in 4 weeks, a there are a lot of cartilage related genes up-regulated the expression of type.II collagen and glycosaminoglycan protein has also been a corresponding increase, will be ordered form of natural cartilage lacuna structure, compact and uniform, matrix secretion was induced by growth factor TGF- beta 3, RGD modified porous hydrogel can significantly enhance the chondrogenic differentiation of ATDC5 cells.

【學(xué)位授予單位】：華南理工大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R318.17

【參考文獻(xiàn)】

相關(guān)期刊論文 前5條

1 ;MAPK signal pathways in the regulation of cell proliferation in mammalian cells[J];Cell Research;2002年01期

2 李文革;徐莘香;;The expression of N-cadherin,fibronectin during chondrogenic differentiation of MSC induced by TGF-β_1[J];Chinese Journal of Traumatology;2005年06期

3 李兵;吳志宏;劉廣源;肖軍;韓東;邱貴興;;三種軟骨細(xì)胞分離培養(yǎng)方法對(duì)細(xì)胞骨架的影響比較[J];中華實(shí)驗(yàn)外科雜志;2007年09期

4 周強(qiáng),李起鴻,戴剛,石國(guó)華;三步酶消化法高效分離兔原代關(guān)節(jié)軟骨細(xì)胞及體外培養(yǎng)觀(guān)察[J];中華外科雜志;2005年08期

5 張翔;周大利;龍沁;周加貝;譚言飛;柳淑婧;;磷灰石-硅灰石生物活性玻璃陶瓷表面接枝多肽改性研究[J];無(wú)機(jī)材料學(xué)報(bào);2013年10期

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