發(fā)布時(shí)間：2018-05-09 08:14

  本文選題：軟骨細(xì)胞 + 應(yīng)力��； 參考：《武漢大學(xué)》2011年博士論文

【摘要】：目的： 隨著我國經(jīng)濟(jì)的高速發(fā)展,人口預(yù)期壽命逐步延長,我國骨關(guān)節(jié)炎的發(fā)病人數(shù)也呈高增長趨勢。在我國75歲以上人群中,骨關(guān)節(jié)炎的發(fā)病率為70%；每年由于骨關(guān)節(jié)炎導(dǎo)致的勞動(dòng)力喪失和藥物治療產(chǎn)生的費(fèi)用龐大,給患者及其家庭帶來了巨大的痛苦。 隨著生活水平的不斷提高,體育愛好者也越來越多,而不恰當(dāng)?shù)捏w育運(yùn)動(dòng)也很容易引起關(guān)節(jié)軟骨的損傷,關(guān)節(jié)軟骨損傷在膝關(guān)節(jié)疾病患者中比較常見。 關(guān)節(jié)軟骨損傷的治療方法目前主要有關(guān)節(jié)清理、鉆孔,自體骨膜或軟骨膜移植,微骨折(Microfracture),自體軟骨細(xì)胞移植(Autologous chondrocyte implantation, ACI)和馬賽克成形術(shù)(Mosaicplasty)等,其中后3種治療方法的療效已得到了公認(rèn)。為了進(jìn)行自體軟骨細(xì)胞移植(ACI)和尋求使軟骨細(xì)胞保持正常表型以便進(jìn)行力學(xué)干預(yù)實(shí)驗(yàn),均需進(jìn)行體外人關(guān)節(jié)軟骨細(xì)胞的分離,培養(yǎng)和鑒定。本課題第一部分選用人關(guān)節(jié)軟骨進(jìn)行體外培養(yǎng),探討了分離及培養(yǎng)人關(guān)節(jié)軟骨細(xì)胞的方法。雖然力學(xué)作用于軟骨細(xì)胞的機(jī)制還未明了,但一定的力學(xué)作用對(duì)關(guān)節(jié)軟骨細(xì)胞有著促進(jìn)其分泌軟骨基質(zhì)并修復(fù)關(guān)節(jié)軟骨的作用已得到大家認(rèn)可。本課題第二部分實(shí)驗(yàn)試圖通過將不同程度的間斷流體剪切力作用于人關(guān)節(jié)軟骨細(xì)胞,觀測軟骨細(xì)胞分泌蛋白多糖和膠原蛋白的能力變化,探討人關(guān)節(jié)軟骨細(xì)胞體外培養(yǎng)適宜的力學(xué)條件,以及間斷流體剪切力對(duì)軟骨基質(zhì)代謝、軟骨細(xì)胞增殖的影響,為以后進(jìn)行關(guān)節(jié)軟骨損傷的修復(fù)和康復(fù)鍛煉提供直接的依據(jù)。 方法： 第一部分實(shí)驗(yàn)以手術(shù)中獲得的人關(guān)節(jié)軟骨為材料,通過改良二步酶消化法進(jìn)行人關(guān)節(jié)軟骨細(xì)胞的提取,采用含小牛血清的高糖型DMEM培養(yǎng)基進(jìn)行培養(yǎng),采用倒置顯微鏡觀察細(xì)胞生長情況,用計(jì)數(shù)板進(jìn)行細(xì)胞計(jì)數(shù),繪制細(xì)胞生長曲線；將獲得的人關(guān)節(jié)軟骨細(xì)胞經(jīng)過原代培養(yǎng)增殖后行甲苯胺藍(lán)染色檢測軟骨細(xì)胞表型,MTT比色法估計(jì)細(xì)胞增殖情況。 第二部分實(shí)驗(yàn)以第一部分實(shí)驗(yàn)獲得的第二代人軟骨細(xì)胞為材料,將第二代軟骨細(xì)胞按搖床作用時(shí)不同的轉(zhuǎn)速分為：控制組(空白對(duì)照組)；低轉(zhuǎn)速組(20轉(zhuǎn)/分)；中等轉(zhuǎn)速組(40轉(zhuǎn)/分)；高轉(zhuǎn)速組(60轉(zhuǎn)/分),共4組；來模擬關(guān)節(jié)軟骨細(xì)胞所受到不同大小的剪切力�？瞻讓�(duì)照組置于5%CO2,37℃培養(yǎng)箱中靜止培養(yǎng)；每天將不同轉(zhuǎn)速組人軟骨細(xì)胞置于搖床上按設(shè)定轉(zhuǎn)速作用2h(相當(dāng)于給予間斷剪切力作用),每天兩次,隔天更換培養(yǎng)液。 通過MTT法檢測細(xì)胞增殖,甲苯胺藍(lán)染色判斷軟骨細(xì)胞表型,軟骨細(xì)胞爬片Ⅱ型膠原免疫組化檢測,Rt-PCR法檢測聚集蛋白聚糖(Aggrecan) mRNA及Ⅱ型膠原(TypeⅡCollagen) mRNA表達(dá)來評(píng)估不同大小間斷剪切力對(duì)人軟骨細(xì)胞增殖和分泌蛋白多糖功能的作用。 實(shí)驗(yàn)結(jié)果顯示： 1.運(yùn)用改良二步酶消化法消化人關(guān)節(jié)軟骨具有高效率的同時(shí)細(xì)胞收獲量也較大,獲得的原代人關(guān)節(jié)軟骨細(xì)胞成活率高。 2.原代人關(guān)節(jié)軟骨細(xì)胞經(jīng)過高糖DMEM培養(yǎng)基培養(yǎng)可以獲得軟骨表型完好的人關(guān)節(jié)軟骨細(xì)胞,能正常分泌蛋白多糖,細(xì)胞附壁良好,可以作為力學(xué)作用的材料細(xì)胞。 3.一定強(qiáng)度間斷剪切力能夠顯著影響人關(guān)節(jié)軟骨細(xì)胞的代謝、增殖活動(dòng),使其合成聚集蛋白聚糖(Aggrecan)及Ⅱ型膠原(TypeⅡCollagen)增多。 4.本實(shí)驗(yàn)的結(jié)果與臨床和基礎(chǔ)研究對(duì)于骨關(guān)節(jié)炎和軟骨損傷的認(rèn)識(shí)有較強(qiáng)的關(guān)聯(lián)性,有必要進(jìn)一步深入研究。研究結(jié)果還提示在軟骨損傷的治療和康復(fù)階段使用非負(fù)重的關(guān)節(jié)鍛煉方式(即間斷剪切力作用)可能得到更好的結(jié)果。
[Abstract]:Objective:
With the rapid development of China's economy, the life expectancy of the population is gradually extended, and the number of osteoarthritis in China is also increasing. The incidence of osteoarthritis is 70% in the population over 75 years of age in our country. The cost of labor loss and drug treatment caused by osteoarthritis is huge and brought to the patients and their families. Great pain.
With the continuous improvement of living standards, sports enthusiasts are becoming more and more, and inappropriate sports can also easily cause joint cartilage damage. Articular cartilage damage is more common in patients with knee joint diseases.
At present, the treatment of articular cartilage injury mainly includes joint cleaning, drilling, autogenous periosteal or periosteal transplantation, micro fracture (Microfracture), autologous chondrocyte transplantation (Autologous chondrocyte implantation, ACI) and mosaic plasty (Mosaicplasty). The effect of the latter 3 methods has been recognized. The body cartilage cell transplantation (ACI) and the search for the cartilage cells to maintain normal phenotype in order to carry out the mechanical intervention experiments, the separation, culture and identification of human articular chondrocytes in vitro are needed. The first part of this topic is to culture human articular cartilage in vitro, and explore the methods of separating and cultivating human articular chondrocytes. The mechanism of chondrocytes is not clear, but the effect of certain mechanical action on articular cartilage cells to promote the secretion of cartilage matrix and repair articular cartilage has been recognized. The second part of the experiment is trying to observe the cartilage cell by using different degrees of intermittent fluid shear force on human articular cartilage cells. The changes in the ability of secreting proteoglycan and collagen to explore the suitable mechanical conditions for the culture of human articular cartilage cells in vitro, and the effect of intermittent fluid shear force on cartilage matrix metabolism and chondrocyte proliferation, provide a direct basis for the repair and rehabilitation of articular cartilage injury.
Method錛，

本文編號(hào)：1865251