本文選題：組織工程 + 軟骨細(xì)胞外基質(zhì)��； 參考：《中國(guó)人民解放軍醫(yī)學(xué)院》2014年碩士論文

【摘要】：研究背景：關(guān)節(jié)軟骨無(wú)血液、淋巴供應(yīng)，因此關(guān)節(jié)軟骨的再生修復(fù)能力相當(dāng)有限。目前在臨床上應(yīng)用的外科手術(shù)治療方法在后期均存在一定問(wèn)題，如纖維軟骨形成、修復(fù)邊緣區(qū)域整合性差等。近年來(lái)組織工程技術(shù)作為新的治療手段正逐步興起，為軟骨損傷的治療帶來(lái)了希望。軟骨組織工程三大關(guān)鍵問(wèn)題，即種子細(xì)胞、生物支架、細(xì)胞因子微環(huán)境。自體軟骨細(xì)胞是組織工程的首選種子細(xì)胞，臨床應(yīng)用最為廣泛。但隨著而來(lái)的難題是如何在體外短時(shí)間內(nèi)獲得足夠數(shù)量、高質(zhì)量的種子細(xì)胞，即擴(kuò)增問(wèn)題。微載體培養(yǎng)技術(shù)因其具有擴(kuò)增種子細(xì)胞并維持細(xì)胞表型的特點(diǎn)而廣泛用于動(dòng)物細(xì)胞的大規(guī)模培養(yǎng)。微載體作為種子細(xì)胞的擴(kuò)增載體，細(xì)胞可在微載體表面快速增殖，以滿足后期檢測(cè)分析或應(yīng)用種子細(xì)胞的需求，特別是近年來(lái)以組織細(xì)胞外基質(zhì)為原料制備的微載體。目前在皮膚、脂肪組織再生領(lǐng)域，組織細(xì)胞外基質(zhì)源性微載體已獲得良好結(jié)果，但在軟骨組織再生領(lǐng)域未見相關(guān)報(bào)道。 目的：本研究以天然軟骨組織為基礎(chǔ)，通過(guò)濕法粉碎、分篩過(guò)濾等處理后制備軟骨細(xì)胞外基質(zhì)源性微粒，并對(duì)其與軟骨細(xì)胞復(fù)合后的生物相容性進(jìn)行評(píng)估。研究?jī)?nèi)容主要包括：以天然軟骨組織為材料制備關(guān)節(jié)軟骨細(xì)胞外基質(zhì)源性微粒，并觀察其主要成分；觀察其體外軟骨細(xì)胞相容性；探究關(guān)節(jié)軟骨細(xì)胞外基質(zhì)源性微粒對(duì)兔軟骨細(xì)胞表型的影響。 方法：將豬關(guān)節(jié)軟骨進(jìn)行粉碎處理，通過(guò)分篩過(guò)濾得到直徑220-300μm的軟骨微粒，經(jīng)脫細(xì)胞處理后獲得天然去細(xì)胞關(guān)節(jié)軟骨細(xì)胞外基質(zhì)源性微粒，對(duì)此軟骨微粒進(jìn)行組織學(xué)成分鑒定、DNA殘留量檢測(cè)、GAG含量和總膠原含量檢測(cè)。體外將軟骨細(xì)胞與關(guān)節(jié)軟骨細(xì)胞外基質(zhì)源性微粒共培養(yǎng)，通過(guò)Dead/Live細(xì)胞活性染色和MTT，觀察軟骨細(xì)胞的生長(zhǎng)、增殖情況。以普通平面靜態(tài)培養(yǎng)和Cytodex-3微載體為對(duì)照，在三維微重力培養(yǎng)條件下，通過(guò)Rt-PCCR觀察關(guān)節(jié)軟骨細(xì)胞外基質(zhì)源性微粒對(duì)軟骨細(xì)胞表型的影響，，通過(guò)流式細(xì)胞儀，檢測(cè)軟骨細(xì)胞周期變化。 結(jié)果：掃描電鏡顯示關(guān)節(jié)軟骨細(xì)胞外基質(zhì)源性微粒表面有納米級(jí)別絨毛附著；組織學(xué)染色顯示關(guān)節(jié)軟骨細(xì)胞外基質(zhì)源性微粒無(wú)細(xì)胞殘留，蕃紅花“O”、甲苯胺藍(lán)染色、II型膠原免疫組化染色陽(yáng)性；總膠原、GAG定量檢測(cè)結(jié)果顯示該軟骨微粒保留了軟骨組織的大部分細(xì)胞外基質(zhì)成分。Dead/Live細(xì)胞活性染色顯示關(guān)節(jié)軟骨源性微粒表面的軟骨細(xì)胞成綠色，細(xì)胞形態(tài)呈短梭形或多角形；細(xì)胞數(shù)量逐漸增多，未見紅色細(xì)胞存在；MTT結(jié)果顯示關(guān)節(jié)軟骨細(xì)胞外基質(zhì)源性微粒組的OD值呈持續(xù)上升趨勢(shì)。Rt-PCR結(jié)果顯示關(guān)節(jié)軟骨細(xì)胞外基質(zhì)源性微粒能夠促進(jìn)軟骨細(xì)胞Collagen-II、SOX-9、Aggrecan基因表達(dá)水平，抑制Collagen-II基因表達(dá)水平；細(xì)胞周期檢測(cè)結(jié)果顯示關(guān)節(jié)軟骨細(xì)胞外基質(zhì)源性微粒使多數(shù)細(xì)胞處于S、G2-M期。 結(jié)論：以天然軟骨組織為材料制備的關(guān)節(jié)軟骨細(xì)胞外基質(zhì)源性微粒，脫細(xì)胞處理徹底，軟骨微粒呈類圓形結(jié)構(gòu)，表面附有絨毛狀微絲結(jié)構(gòu)，保留了軟骨組織的大部分細(xì)胞外基質(zhì)成分；無(wú)細(xì)胞毒性，生物相容性良好，能夠促進(jìn)細(xì)胞增殖，是軟骨組織工程種子細(xì)胞的優(yōu)良擴(kuò)增載體；刺激細(xì)胞生長(zhǎng)、增殖的能力優(yōu)于Cytodex-3微載體；可有效維持軟骨細(xì)胞的特有表型其效果與優(yōu)于Cytodex-3。
[Abstract]:Background: articular cartilage has no blood and lymph supply, so the ability of regeneration and repair of articular cartilage is very limited. There are some problems in the clinical application of surgical treatment at the present time, such as fibrous cartilage formation, and poor integration of edge areas. In recent years, tissue engineering technology is being taken as a new treatment method. Bu Xingqi, which has brought hope for the treatment of cartilage injury. The three key problems of cartilage tissue engineering are seed cells, biological scaffolds, and cytokine microenvironments. Autologous chondrocytes are the preferred seed cells for tissue engineering, which are the most widely used in clinical practice. However, the problem is how to obtain sufficient quantity and high quality in short time in vitro. Microcarrier culture is widely used in the large-scale culture of animal cells because it has the characteristics of amplifying seed cells and maintaining the phenotype of the cells. As a carrier of seed cells, microcarriers can rapidly proliferate on the surface of microcarriers to meet the needs of later detection analysis or application of seed cells. In recent years, especially in the tissue of tissue extracellular matrix as a microcarrier, good results have been obtained in the field of skin, adipose tissue regeneration, tissue extracellular matrix derived microcarriers, but no related reports have been reported in the field of cartilage tissue regeneration.
Objective: to prepare cartilage extracellular matrix derived particles by wet crushing, sifting and filtration, and evaluate the biocompatibility of cartilage cells with cartilage cells. The main contents are as follows: the preparation of extracellular matrix derived from articular cartilage by natural cartilage tissue, The main components were observed, the chondrocyte compatibility in vitro was observed, and the effects of extracellular matrix derived particulates of articular cartilage on the phenotype of rabbit chondrocytes were explored.
Methods: the cartilage of porcine articular cartilage was crushed and the cartilage particles with a diameter of 220-300 m were obtained by sifting and filtration. The extracellular matrix derived from the natural acellular articular cartilage was obtained by dehydration. The histological composition of the cartilage particles, the detection of DNA residue, the content of GAG and the total collagen content were detected. The cell and articular cartilage extracellular matrix derived microparticles were co cultured. The growth and proliferation of chondrocytes were observed by Dead/Live cell activity staining and MTT. The normal plane static culture and Cytodex-3 microcarrier were compared. Under the condition of three-dimensional microgravity culture, the extracellular matrix derived from articular cartilage was observed by Rt-PCCR. The effects of cell phenotype were detected by flow cytometry.
Results: the scanning electron microscope showed that the surface of the extracellular matrix derived from articular cartilage was attached to the surface of nanoscale villus; histology staining showed that the extracellular matrix of articular cartilage was no cell residue, crocus "O", toluidine blue staining, II collagen immunohistochemical staining positive; total collagen, GAG quantitative detection results showed the cartilage The microparticles retained most of the extracellular matrix components of the cartilage tissue.Dead/Live cell activity to show that the cartilage cells of the articular cartilage derived particles were green, the cell morphology was short spindle or polygonal, and the number of cells increased gradually, and no red cells were found. MTT results showed the extracellular matrix group of articular cartilage extracellular matrix. The.Rt-PCR results showed that the extracellular matrix derived from articular cartilage cells could promote the expression level of Collagen-II, SOX-9, Aggrecan gene and the expression level of Collagen-II gene, and the results of cell cycle detection showed that the extracellular matrix of articular cartilage made most of the cells in S, G2-M phase.
Conclusion: the extracellular matrix derived from articular cartilage was prepared by natural cartilage tissue. The removal of cells was complete, the cartilage particles were round structure, and the surface was attached with villous microfilament structure, which retained most of the extracellular matrix components of cartilage tissue, and had no cytotoxicity and good biocompatibility, which could promote cell proliferation. An excellent carrier of seed cells for cartilage tissue engineering; the ability to stimulate cell growth and proliferation is superior to Cytodex-3 microcarrier; it can effectively maintain the endemic phenotype of chondrocytes and is superior to Cytodex-3.
【學(xué)位授予單位】：中國(guó)人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R318.08

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