發(fā)布時間：2018-02-21 20:33

  本文關鍵詞： 骨髓間充質(zhì)干細胞 裝置 流體應力 血管內(nèi)皮細胞生長因子 誘導 分化 表型　出處：《第三軍醫(yī)大學》2006年碩士論文　論文類型：學位論文

【摘要】： 骨移植材料植入體內(nèi)后,必須盡快建立充分的血供,為骨組織修復或改建提供充足的營養(yǎng),這在完成較大骨缺損修復時,尤為重要。血管生成是一個集細胞、細胞因子、細胞外基質(zhì)、生物力學刺激等多種因素的復雜過程。既往的研究多側(cè)重于應用VEGF,bFGF等促血管生成因子,但存在劑量不易調(diào)控,甚至導致血管瘤發(fā)生等缺陷。因此,探討給予外源性的血管內(nèi)皮細胞來促進局部血管生成成為新的研究策略。 獲取自體的血管內(nèi)皮細胞存在較大的創(chuàng)傷,且細胞獲取有限,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)干細胞在體內(nèi)外相應的誘導條件下,有進一步分化為內(nèi)皮細胞表型,進而參與局部的血管生成可能。為此,本課題初步探討流體應力對體外培養(yǎng)的兔骨髓間充質(zhì)細胞向血管內(nèi)皮細胞表型分化的影響。從而篩選出適宜的力學條件,為利用自體骨髓MSCs促血管生成奠定理論基礎。 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力裝置,并用此裝置單獨或聯(lián)合血管內(nèi)皮細胞生長因子來刺激兔BM-MSCs向血管內(nèi)皮細胞表型的分化,為進一步應用該細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胞,再結合定期換液的體外培養(yǎng)方法,獲取兔BM-MSCs。通過測定細胞的增殖能力和成骨、成脂肪分化來鑒定細胞。 2、將目前常規(guī)使用的流體應力裝置(平行板流動腔)中流體的負壓狀態(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、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胞生物學特性的影響。 以不同的流體應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胞比例,免疫組織化學染色檢測細胞CD31陽性表達率。將活細胞比例超過80%并CD31陽性率超過70%的實驗組條件,作為適宜的流體應力刺激大小和作用時間。
[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.

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

【參考文獻】

相關期刊論文 前10條

1 呂學敏,鄧廉夫,楊慶銘;力學刺激對成骨細胞作用機制的研究進展[J];北京生物醫(yī)學工程;2004年02期

2 白家駟,卞修武,史景泉,鄭江,辛榕;惡性膠質(zhì)瘤細胞誘導內(nèi)皮細胞三維成型的體外實驗[J];第三軍醫(yī)大學學報;2004年22期

3 張烈焚,劉麗;力學信號調(diào)控細胞外基質(zhì)變化的研究進展[J];國外醫(yī)學.口腔醫(yī)學分冊;2005年05期

4 曹金芳,李紓,莊昭霞;不同培養(yǎng)條件下牙周韌帶細胞礦化能力的比較研究[J];臨床口腔醫(yī)學雜志;2003年10期

5 徐良,景在平;人造血管移植物自然內(nèi)皮化的研究[J];上海生物醫(yī)學工程;1997年02期

6 郭學良,王杰軍,高勇,傅麗娜,錢立平,許青,林庚金;多西紫杉醇體外抑制血管生成的作用[J];復旦學報(醫(yī)學版);2003年01期

7 肖海波,梅舉,張寶仁,黃盛東;大鼠骨髓基質(zhì)干細胞體外誘導向內(nèi)皮分化的實驗研究[J];上海醫(yī)學;2005年03期

8 申洪,陸藥丹;免疫組織化學染色的定量方法研究[J];生物醫(yī)學工程學雜志;1993年04期

9 劉肖珩,陳槐卿;流室系統(tǒng)的流體動力學模擬[J];生物醫(yī)學工程學雜志;1999年04期

10 覃開蓉,郭必勝,劉寶玉,柳兆榮;平行平板流動腔中Casson流體脈動流的攝動解[J];生物醫(yī)學工程學雜志;2002年03期

，

本文編號：1522809