FAK、talin和paxillin在切应力促EPCs分化过程中的作用
发布时间：2019-06-20 22:13
【摘要】：目的：利用切应力加载装置,模拟生理状态下大动脉系统所受的层流切应力,作用于大鼠骨髓来源的内皮祖细胞(EPCs),验证切应力是否可以促使EPCs向内皮分化,并检测在此过程中局部粘着斑激酶(FAK)、踝蛋白(talin)和桩蛋白(paxillin)三个蛋白的变化情况,从而为揭示切应力促EPCs分化的跨膜信号转导途径提供一定的依据,并为完善受损血管内皮修复的具体机制提供一定的参考。 方法：1、梯度密度离心法从大鼠骨髓中分离提取单个核细胞,用加有血管内皮生长因子VEGF、碱性成纤维生长因子BFGF和15%胎牛血清的M199培养基进行培养,两周后用双吞噬法鉴定是否成功培养出EPC,培养至第三代制成细胞爬片。 2、将细胞爬片分为切应力加载组和静止组,利用切应力加载装置,模拟生理状态下大动脉系统所受的层流切应力大小,给予切应力加载组细胞爬片12dyne/cm的力学负荷。 3、分别提取切应力加载3h、6h、12h组和静止组细胞爬片的RNA,用RT-PCR检测内皮细胞分化标记分子vWF和CD31基因的表达情况。4、取切应力加载组和静止组的细胞爬片,用细胞免疫荧光(IF)观察FAK、talin和paxillin三个蛋白的表达位置的变化；用Western Blot检测上述三个蛋白表达量的变化。 结果：1、双吞噬法双阳性提示成功培养出大鼠骨髓源性内皮祖细胞(EPCs)。 2、切应力(12dyne/cm2)作用于EPCs后,显著增加内皮细胞分化标记分子vWF和CD31基因的表达,与静止组比较差异有统计学意义(P0.05)。 3、细胞免疫荧光(IF)结果显示,静止组中FAK、talin和paxillin三个蛋白主要位于胞浆中,表达量丰富,并且都在细胞的核周比较聚集；切应力作用3h以后,talin和paxillin蛋白的荧光强度变弱,而FAK的位置从核周围沿着切应力作用方向聚集成束状延伸至胞膜。 4、切应力加载3h组和静止组的Western结果比较,其FAK蛋白的表达量上升,差异有统计学意义(P0.01)；talin和paxillin蛋白的表达量均下降,差异均有统计学意义(P0.01)。 结论：切应力能促使EPCs向内皮分化,而在此过程中FAK蛋白的表达位置发生变化,FAK、talin和paxillin三个蛋白的表达量均发生变化,提示他们有可能参与了其信号转导过程。
[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.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R114
本文编号：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.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R114
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