【摘要】：目的：利用切應(yīng)力加載裝置,模擬生理狀態(tài)下大動(dòng)脈系統(tǒng)所受的層流切應(yīng)力,作用于大鼠骨髓來(lái)源的內(nèi)皮祖細(xì)胞(EPCs),驗(yàn)證切應(yīng)力是否可以促使EPCs向內(nèi)皮分化,并檢測(cè)在此過(guò)程中局部粘著斑激酶(FAK)、踝蛋白(talin)和樁蛋白(paxillin)三個(gè)蛋白的變化情況,從而為揭示切應(yīng)力促EPCs分化的跨膜信號(hào)轉(zhuǎn)導(dǎo)途徑提供一定的依據(jù),并為完善受損血管內(nèi)皮修復(fù)的具體機(jī)制提供一定的參考。 方法：1、梯度密度離心法從大鼠骨髓中分離提取單個(gè)核細(xì)胞,用加有血管內(nèi)皮生長(zhǎng)因子VEGF、堿性成纖維生長(zhǎng)因子BFGF和15%胎牛血清的M199培養(yǎng)基進(jìn)行培養(yǎng),兩周后用雙吞噬法鑒定是否成功培養(yǎng)出EPC,培養(yǎng)至第三代制成細(xì)胞爬片。 2、將細(xì)胞爬片分為切應(yīng)力加載組和靜止組,利用切應(yīng)力加載裝置,模擬生理狀態(tài)下大動(dòng)脈系統(tǒng)所受的層流切應(yīng)力大小,給予切應(yīng)力加載組細(xì)胞爬片12dyne/cm的力學(xué)負(fù)荷。 3、分別提取切應(yīng)力加載3h、6h、12h組和靜止組細(xì)胞爬片的RNA,用RT-PCR檢測(cè)內(nèi)皮細(xì)胞分化標(biāo)記分子vWF和CD31基因的表達(dá)情況。4、取切應(yīng)力加載組和靜止組的細(xì)胞爬片,用細(xì)胞免疫熒光(IF)觀察FAK、talin和paxillin三個(gè)蛋白的表達(dá)位置的變化；用Western Blot檢測(cè)上述三個(gè)蛋白表達(dá)量的變化。 結(jié)果：1、雙吞噬法雙陽(yáng)性提示成功培養(yǎng)出大鼠骨髓源性?xún)?nèi)皮祖細(xì)胞(EPCs)。 2、切應(yīng)力(12dyne/cm2)作用于EPCs后,顯著增加內(nèi)皮細(xì)胞分化標(biāo)記分子vWF和CD31基因的表達(dá),與靜止組比較差異有統(tǒng)計(jì)學(xué)意義(P0.05)。 3、細(xì)胞免疫熒光(IF)結(jié)果顯示,靜止組中FAK、talin和paxillin三個(gè)蛋白主要位于胞漿中,表達(dá)量豐富,并且都在細(xì)胞的核周比較聚集；切應(yīng)力作用3h以后,talin和paxillin蛋白的熒光強(qiáng)度變?nèi)?而FAK的位置從核周?chē)刂袘?yīng)力作用方向聚集成束狀延伸至胞膜。 4、切應(yīng)力加載3h組和靜止組的Western結(jié)果比較,其FAK蛋白的表達(dá)量上升,差異有統(tǒng)計(jì)學(xué)意義(P0.01)；talin和paxillin蛋白的表達(dá)量均下降,差異均有統(tǒng)計(jì)學(xué)意義(P0.01)。 結(jié)論：切應(yīng)力能促使EPCs向內(nèi)皮分化,而在此過(guò)程中FAK蛋白的表達(dá)位置發(fā)生變化,FAK、talin和paxillin三個(gè)蛋白的表達(dá)量均發(fā)生變化,提示他們有可能參與了其信號(hào)轉(zhuǎn)導(dǎo)過(guò)程。
[Abstract]:Objective: to simulate the laminar shear stress of large artery system under physiological condition, to verify whether shear stress can promote the differentiation of EPCs into endothelial cells by (EPCs), and to detect the changes of focal kinase (FAK), ankle protein (talin) and post protein (paxillin) during this process. Thus, it provides a certain basis for revealing the transmembrane signal transduction pathway of shear stress promoting EPCs differentiation, and provides a reference for perfecting the specific mechanism of damaged vascular endothelial repair. Methods: 1. Mononuclear cells were isolated and extracted from rat bone marrow by gradient density centrifugation and cultured in M199 medium supplemented with vascular endothelial growth factor VEGF, basic fibroblasts growth factor BFGF and 15% fetal bovine serum. two weeks later, the cells were cultured successfully to the third generation by double phagocytosis. 2. The cell crawling was divided into shear stress loading group and static group. The laminar shear stress of great artery system was simulated by shear stress loading device, and the mechanical load of cell climbing slice 12dyne/cm was given to shear stress loading group. 3. The expression of vWF and CD31 genes in endothelial cell differentiation markers was detected by RT-PCR in RNA, of 12 h group and rest group after 3 h of shear stress loading for 3 h and 6 h, respectively. 4. The expression of FAK,talin and paxillin proteins was observed by immunofluorescence (IF), and the expression of FAK,talin and paxillin proteins was detected by Western Blot. Results: 1. Double phagocytosis suggested that rat bone marrow-derived endothelial progenitor cells (EPCs).) were successfully cultured. 2. Shear stress (12dyne/cm2) significantly increased the expression of vWF and CD31 genes in endothelial cell differentiation markers after EPCs, which was significantly different from that in rest group (P 0.05). 3. The results of immunofluorescence (IF) showed that the three proteins of FAK,talin and paxillin in the resting group were mainly located in the cytoplasm, and all of them were aggregated around the nucleus, and the fluorescence intensity of talin and paxillin proteins became weaker after 3 hours of shear stress, while the position of FAK extended into bundles from around the nucleus along the direction of shear stress. 4. The expression of FAK protein in shear stress loading group and rest group was significantly higher than that in static group (P 0.01). The expression of); talin and paxillin protein decreased significantly (P 0.01). Conclusion: shear stress can promote the differentiation of EPCs into endothelial cells, and the expression of FAK protein and the expression of FAK,talin and paxillin proteins change during this process, suggesting that they may be involved in the signal transduction process.
【學(xué)位授予單位】：遵義醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R114

【參考文獻(xiàn)】

相關(guān)期刊論文 前8條

1 吳建國(guó);方國(guó)恩;;內(nèi)皮祖細(xì)胞的研究進(jìn)展[J];解放軍醫(yī)學(xué)雜志;2008年02期

2 楊震;陶軍;王潔梅;涂昌;潘仕榮;唐安麗;董吁剛;馬虹;;流體切應(yīng)力對(duì)人內(nèi)皮祖細(xì)胞組織纖溶酶原激活物基因表達(dá)的影響[J];中華老年心腦血管病雜志;2007年03期

3 陶軍;楊震;;血管損傷與修復(fù)研究進(jìn)展[J];中華老年心腦血管病雜志;2008年02期

4 劉肖珩,陳槐卿;流室系統(tǒng)的流體動(dòng)力學(xué)模擬[J];生物醫(yī)學(xué)工程學(xué)雜志;1999年04期

5 卞琴,王擁軍,施杞;粘附斑激酶信號(hào)轉(zhuǎn)導(dǎo)與串話(huà)研究進(jìn)展[J];中西醫(yī)結(jié)合學(xué)報(bào);2005年06期

6 郝軍生;陳忠;;內(nèi)皮祖細(xì)胞的功能及應(yīng)用研究進(jìn)展[J];心肺血管病雜志;2009年04期

7 成敏;陳槐卿;李毅;吳江;;流體低剪切力上調(diào)內(nèi)皮細(xì)胞IL-8基因表達(dá)的細(xì)胞內(nèi)信號(hào)轉(zhuǎn)導(dǎo)途徑分析[J];醫(yī)用生物力學(xué);2007年01期

8 汪海婭,高平進(jìn),朱鼎良;內(nèi)皮祖細(xì)胞臨床研究進(jìn)展[J];中國(guó)分子心臟病學(xué)雜志;2005年01期

，

本文編號(hào)：2503548