FAK、talin和paxillin在切應力促EPCs分化過程中的作用
發(fā)布時間：2019-06-20 22:13
【摘要】：目的：利用切應力加載裝置,模擬生理狀態(tài)下大動脈系統(tǒng)所受的層流切應力,作用于大鼠骨髓來源的內(nèi)皮祖細胞(EPCs),驗證切應力是否可以促使EPCs向內(nèi)皮分化,并檢測在此過程中局部粘著斑激酶(FAK)、踝蛋白(talin)和樁蛋白(paxillin)三個蛋白的變化情況,從而為揭示切應力促EPCs分化的跨膜信號轉(zhuǎn)導途徑提供一定的依據(jù),并為完善受損血管內(nèi)皮修復的具體機制提供一定的參考。 方法：1、梯度密度離心法從大鼠骨髓中分離提取單個核細胞,用加有血管內(nèi)皮生長因子VEGF、堿性成纖維生長因子BFGF和15%胎牛血清的M199培養(yǎng)基進行培養(yǎng),兩周后用雙吞噬法鑒定是否成功培養(yǎng)出EPC,培養(yǎng)至第三代制成細胞爬片。 2、將細胞爬片分為切應力加載組和靜止組,利用切應力加載裝置,模擬生理狀態(tài)下大動脈系統(tǒng)所受的層流切應力大小,給予切應力加載組細胞爬片12dyne/cm的力學負荷。 3、分別提取切應力加載3h、6h、12h組和靜止組細胞爬片的RNA,用RT-PCR檢測內(nèi)皮細胞分化標記分子vWF和CD31基因的表達情況。4、取切應力加載組和靜止組的細胞爬片,用細胞免疫熒光(IF)觀察FAK、talin和paxillin三個蛋白的表達位置的變化；用Western Blot檢測上述三個蛋白表達量的變化。 結(jié)果：1、雙吞噬法雙陽性提示成功培養(yǎng)出大鼠骨髓源性內(nèi)皮祖細胞(EPCs)。 2、切應力(12dyne/cm2)作用于EPCs后,顯著增加內(nèi)皮細胞分化標記分子vWF和CD31基因的表達,與靜止組比較差異有統(tǒng)計學意義(P0.05)。 3、細胞免疫熒光(IF)結(jié)果顯示,靜止組中FAK、talin和paxillin三個蛋白主要位于胞漿中,表達量豐富,并且都在細胞的核周比較聚集；切應力作用3h以后,talin和paxillin蛋白的熒光強度變?nèi)?而FAK的位置從核周圍沿著切應力作用方向聚集成束狀延伸至胞膜。 4、切應力加載3h組和靜止組的Western結(jié)果比較,其FAK蛋白的表達量上升,差異有統(tǒng)計學意義(P0.01)；talin和paxillin蛋白的表達量均下降,差異均有統(tǒng)計學意義(P0.01)。 結(jié)論：切應力能促使EPCs向內(nèi)皮分化,而在此過程中FAK蛋白的表達位置發(fā)生變化,FAK、talin和paxillin三個蛋白的表達量均發(fā)生變化,提示他們有可能參與了其信號轉(zhuǎn)導過程。
[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.
【學位授予單位】：遵義醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R114
本文編號：2503548
[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.
【學位授予單位】：遵義醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R114
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