發(fā)布時(shí)間：2018-02-21 20:33

  本文關(guān)鍵詞： 骨髓間充質(zhì)干細(xì)胞 裝置 流體應(yīng)力 血管內(nèi)皮細(xì)胞生長因子 誘導(dǎo) 分化 表型　出處：《第三軍醫(yī)大學(xué)》2006年碩士論文　論文類型：學(xué)位論文

【摘要】： 骨移植材料植入體內(nèi)后,必須盡快建立充分的血供,為骨組織修復(fù)或改建提供充足的營養(yǎng),這在完成較大骨缺損修復(fù)時(shí),尤為重要。血管生成是一個集細(xì)胞、細(xì)胞因子、細(xì)胞外基質(zhì)、生物力學(xué)刺激等多種因素的復(fù)雜過程。既往的研究多側(cè)重于應(yīng)用VEGF,bFGF等促血管生成因子,但存在劑量不易調(diào)控,甚至導(dǎo)致血管瘤發(fā)生等缺陷。因此,探討給予外源性的血管內(nèi)皮細(xì)胞來促進(jìn)局部血管生成成為新的研究策略。 獲取自體的血管內(nèi)皮細(xì)胞存在較大的創(chuàng)傷,且細(xì)胞獲取有限,而近年來研究發(fā)現(xiàn)具有多向分化潛能的骨髓間充質(zhì)干細(xì)胞在體內(nèi)外相應(yīng)的誘導(dǎo)條件下,有進(jìn)一步分化為內(nèi)皮細(xì)胞表型,進(jìn)而參與局部的血管生成可能。為此,本課題初步探討流體應(yīng)力對體外培養(yǎng)的兔骨髓間充質(zhì)細(xì)胞向血管內(nèi)皮細(xì)胞表型分化的影響。從而篩選出適宜的力學(xué)條件,為利用自體骨髓MSCs促血管生成奠定理論基礎(chǔ)。 一、目的 模擬體內(nèi)血流動力狀態(tài),建立正壓條件下流體應(yīng)力裝置,并用此裝置單獨(dú)或聯(lián)合血管內(nèi)皮細(xì)胞生長因子來刺激兔BM-MSCs向血管內(nèi)皮細(xì)胞表型的分化,為進(jìn)一步應(yīng)用該細(xì)胞來促血管生成的治療創(chuàng)造條件。 二、方法 1、通過梯度離心分離兔骨髓單核細(xì)胞,再結(jié)合定期換液的體外培養(yǎng)方法,獲取兔BM-MSCs。通過測定細(xì)胞的增殖能力和成骨、成脂肪分化來鑒定細(xì)胞。 2、將目前常規(guī)使用的流體應(yīng)力裝置(平行板流動腔)中流體的負(fù)壓狀態(tài),改良為正壓狀態(tài),通過比較改良前后的平板流動腔內(nèi)的壓強(qiáng)、流量、流動的波形,分析兔骨髓MSCs增殖能力和表型變化來判斷該裝置對細(xì)胞生物學(xué)特性的影響。 以不同的流體應(yīng)力作用于平行板流動腔內(nèi)的兔MSCs,分別于3、6、12和24h后通過胎盤蘭染色檢測活細(xì)胞比例,免疫組織化學(xué)染色檢測細(xì)胞CD31陽性表達(dá)率。將活細(xì)胞比例超過80%并CD31陽性率超過70%的實(shí)驗(yàn)組條件,作為適宜的流體應(yīng)力刺激大小和作用時(shí)間。
[Abstract]:Bone graft after implantation in vivo, must establish adequate blood supply as soon as possible, to provide adequate nutrition for bone tissue repair or reconstruction, which is especially important in the completion of repair of large bone defects,. Angiogenesis is a set of cells, cytokines, extracellular matrix, the complex process of many factors such as past. Biomechanical stimulation the study focuses on the application of VEGF, bFGF of Pro angiogenic factors, but the dose is not easy to control, and even lead to the development of hemangioma and other defects. Therefore, study of vascular endothelial cells to promote angiogenesis for local new strategy.
To obtain autologous vascular endothelial cells have great trauma, and limited access to cells, but recent studies found that the multilineage differentiation potential of bone marrow mesenchymal stem cells induced by the in vivo conditions, further differentiate into endothelial cell phenotype, and then participate in the angiogenesis of local potential. Therefore, this paper discussed the fluid effect of stress on vascular endothelial cells to differentiation of cultured rabbit bone marrow mesenchymal stem cells. In order to find out the suitable mechanical condition for the use of autologous bone marrow MSCs, lay a theoretical foundation for promoting angiogenesis.
First, the purpose
To simulate the in vivo hemodynamic state, establish positive pressure under the condition of fluid stress device, and the device used alone or in combination with differentiation of vascular endothelial growth factor to stimulate the rabbit BM-MSCs to the phenotype of vascular endothelial cells, to create conditions for the further application of the treatment of cells to promote angiogenesis.
Two, method
1, the rabbit bone marrow mononuclear cells were isolated by gradient centrifugation, and then the rabbit BM-MSCs. was obtained by in vitro culture with regular fluid exchange. The cells were identified by measuring cell proliferation and osteogenic differentiation.
2, the routine use of fluid stress device (parallel plate flow chamber) negative pressure fluid, modified for positive pressure, the pressure is modified and the plate flow cavity flow, flow waveform, to determine the effect of the device on the biological characteristics of cells of rabbit bone marrow MSCs proliferation and phenotype change.
With different fluid stress in rabbit MSCs parallel plate flow chamber, respectively at 3,6,12 and 24h after trypan blue staining to detect the proportion of living cells, immunohistochemical staining of CD31 cells. The positive expression rate of viable cells more than the experimental group conditions CD31 positive rate of 80% and more than 70%, as a suitable fluid the size and duration of stimulation.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R329.2

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