过表达Axl在高糖缺氧条件下调控HIF-1α表达促进内皮成管的作用及机制
发布时间：2018-12-11 12:40
【摘要】：目的：高糖下调缺氧诱导因子-1α(Hypoxia inducible factor-1α, HIF-1α)的表达,进而抑制缺氧状态下血管新生。Axl能调控内皮细胞功能,促进内皮成管,然而Axl是否参与高糖缺氧条件下内皮成管及HIF-1α的调控尚不清楚。本实验拟观察高糖对缺氧条件下内皮细胞成管作用及Axl表达的影响,检测过表达Axl对高糖缺氧条件下内皮细胞成管作用的影响及HIF-1α表达的调控作用。方法：1.给予人脐静脉内皮细胞(Human umbilical vein endothelial cells, HUVECs)和人脐静脉内皮细胞株(Eahy926)不同浓度葡萄糖(5.5mmol/l、11mmol/l、25 mmol/l和30mmol/l)处理,分别培养24 h、48 h和72 h, MTT检测高糖对细胞活性的影响；2.在高糖条件下给予HUVECs和Eahy926细胞不同时间缺氧处理,MTT检测缺氧时间对细胞活性的影响：3.摸索出合适的高糖缺氧条件后,实验分为高糖缺氧组和对照组(单纯缺氧组),用划痕实验和体外基质胶成管实验检测高糖对缺氧状态下HUVECs和Eahy926细胞迁移和管腔形成能力的影响；4.Western blot方法检测高糖对缺氧状态下HUVECs和Eahy926细胞Axl、HIF-1α蛋白表达的影响；5.构建Axl过表达慢病毒并转染Eahy926细胞,用Western blot检测细胞中Axl的表达情况：6.病毒转染成功后,将实验分为四组：对照组(阴性病毒)、Axl过表达组、R428组(Axl抑制剂R428+阴性病毒)、Axl过表达+R428组。划痕实验和体外基质胶成管实验检测过表达Axl对高糖缺氧条件下内皮细胞迁移和成管作用的影响；7.实时荧光定量PCR和Western blot检测过表达Axl对HIF-1α mRNA及蛋白含量的影响；8.使用蛋白合成抑制剂放线菌酮(Cycloheximide, CHX)、PI3K抑制剂LY294002,MEK抑制剂PD98059和mTOR抑制剂Rapamycin,应用Western blot检测过表达Axl调控HIF-1α表达的分子机制。结果：1.葡萄糖对内皮细胞活性的抑制呈浓度和时间依赖性,30 mmol/I葡萄糖处理HUVECs或Eahy926细胞48小时,能显著抑制细胞活性：2.缺氧时间对高糖状态下内皮细胞活性呈时间依赖性,高糖状态下,缺氧12h和24 h组与单纯高糖组相比,内皮细胞活性均品著降低；3.在HUVECs和Eahy926细胞中,高糖24 h+缺氧6h迁移距离均少于缺氧6 h组,HUVECs两组迁移距离分别为182.9±14.6和303.1±10.8,Eahy926两组迁移距离分别为190.7±24.8和293.4±23.9,差异均有统计学意义(P0.05);高糖24 h+缺氧6h组小管数目少于缺氧6h组,两种细胞成管分别减少了30.8%和52.9%,差异均有统计学意义(P0.05)：4.在HUVECs和Eahy926细胞中,高糖缺氧组的Axl蛋白表达量比单纯缺氧组Axl蛋白表达量少,两种细胞分别减少了48.4%和47.1%,差异均有统计学意义(P0.05)：在Eahy926细胞中,高糖缺氧组的HIF-1α蛋白表达量比单纯缺氧组减少了40.7%(P0.05)；5.转染过表达Axl慢病毒后,Eahy926细胞的Ax1蛋白表达明显上调；6.过表达Axl组(231.3±20.6)细胞迁移距离比对照组(179.5±28.9)长(P0.05);过表达Axl组的小管数目比对照组多66.8%(P0.05)；7.实时荧光定量PCR结果显示,过表达Ax1并不影响HIF-1α的mRNA表达;Western blot结果显示,与对照组相比,过表达Axl组HIF-1 α蛋白表达增加了90.6%(P0.05)；8.Axl过表达+CHX组与Axl过表达组相比,HIF-1 α的蛋白表达减少了95%(P0.05),而CHX组与对照组相比变化不明显,说明蛋白合成途径参与过表达Axl导致的HIF-1α表达增加；过表达Axl能激活Akt、p70S6K磷酸化,对Erk磷酸化无影响,LY294002和Rapamycin能抑制过表达Ax1诱导的HIF-1α表达。结论：1.高糖抑制缺氧条件下脐静脉内皮细胞的成管能力；2.过表达Ax1逆转高糖缺氧条件对脐静脉内皮细胞成管的抑制；3.高糖缺氧条件下,过表达Axl通过调控PI3K/Akt/mTOR/p70S6K通路增加HIF-1α蛋白合成。
[Abstract]:Objective: To reduce the expression of hypoxia-induced factor-1 (HIF-1) and to inhibit the angiogenesis in the condition of hypoxia. Axl can regulate the function of endothelial cells and promote the endothelialization of the endothelial cells. The effect of high-sugar on the tube-forming and Axl expression of endothelial cells under the condition of hypoxia was observed, and the effect of the expression of Axl on the tube-forming function of endothelial cells and the regulation of the expression of HIF-1 were examined. Method: 1. Human umbilical vein endothelial cells (HUVECs) and human umbilical vein endothelial cells (Eay926) were treated with different concentrations of glucose (5.5mmol/ l, 11mmol/ l, 25 mmol/ l and 30mmol/ l), respectively. The effects of high sugar on cell activity were measured by MTT assay. HUVECs and Eay926 cells were treated with hypoxia at different time, and the effect of hypoxia time on cell activity was measured by MTT. The effects of high sugar on the migration of HUVECs and Eay926 cells and the formation of the tube cavity in the hypoxic condition were measured by the experiment of scratch and in vitro matrix gel. 4. The effect of high sugar on the expression of Axl and HIF-1 of HUVECs and Eay926 cells was detected by Western blot. Axl overexpressing lentivirus was constructed and the expression of Axl in cells was detected by Western blot: 6. After the successful transfection of the virus, the experiment was divided into four groups: control group (negative virus), Axl overexpressing group, R428 group (Axl inhibitor R428 + negative virus), Axl overexpression + R428 group. The effect of Axl on the migration of endothelial cells and the effect of tube-forming on the condition of high sugar and hypoxia was detected by the test of scratch and in vitro matrix gel. The effect of Axl on HIF-1 mRNA and protein content was detected by real-time fluorescence quantitative PCR and Western blot. The molecular mechanism of expression of HIF-1 was detected by Western blot using a protein synthesis inhibitor, Cyclohepciide, CHX, PI3K inhibitor LY294002, MEK inhibitor PD98059 and mTOR inhibitor Rapamycin. Results: 1. The inhibition of glucose to endothelial cell activity was in a concentration and time-dependent manner, and the cell activity was significantly inhibited by the treatment of HUVECs or Eay926 cells for 48 hours with 30 mmol/ I glucose. In the condition of high sugar, the activity of endothelial cells was time-dependent, and in the high-sugar state, the activity of endothelial cells was lower than that of the pure high-sugar group. In HUVECs and Eay926 cells, the migration distance of high-sugar 24h + hypoxia was less than that of hypoxia 6h group. The migration distance of HUVECs was 182. 9/ 14. 6 and 303. 1/ 10. 8, and the migration distance between two groups of Eay926 was 190. 7, 24, 8 and 293. 4 and 23. 9, respectively (P0.05). The number of small tubes in the high-sugar 24h + hypoxia group was less than that of the hypoxia group, and the number of the two cells decreased by 30.8% and 52.9%, respectively (P <0.05): 4. In the HUVECs and Eay926 cells, the expression of Axl protein in the high-sugar anoxic group was less than that of the Axl protein in the pure hypoxia group, and the expression of Axl protein in the two cells decreased by 48. 4% and 47.1%, respectively (P0.05): in the Eay926 cells, The expression of HIF-1 in HIF-1 group decreased by 40.7% (P0.05). After transfection of the Axl lentivirus, the expression of the Ax1 protein in the Eay926 cells was up-regulated; The cell migration distance of the overexpressing Axl group (233.1. 3-20.6) was longer than that in the control group (P0.05); the number of the small tubes of the overexpressing Axl group was more than that of the control group by 66. 8% (P0.05); The results of real-time fluorescence quantitative PCR showed that the expression of HIF-1 in the expression of Ax1 did not affect the mRNA expression of HIF-1. The results of Western blot showed that the expression of HIF-1 in the expression Axl group increased by 90.6% (P0.05) in comparison with the control group. The expression of HIF-1 in the expression of HIF-1 was decreased by 95% compared with that of the Axl overexpressing group (P0.05). However, the expression of HIF-1 induced by Axl was not affected by the expression of Axl, and the expression of HIF-1 induced by Ax1 was inhibited by LY294002 and Rapamycin. Conclusion: 1. the ability of high-sugar to inhibit the formation of umbilical vein endothelial cells under the condition of hypoxia; Overexpression of Ax1 reverses the inhibition of umbilical vein endothelial cells in the presence of high-sugar and oxygen-deficient conditions. Under the condition of high sugar and oxygen, the overexpression of Axl increased the synthesis of HIF-1 through the regulation of the PI3K/ Akt/ mTOR/ p70S6K pathway.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R587.2;R54
本文编号：2372573
[Abstract]:Objective: To reduce the expression of hypoxia-induced factor-1 (HIF-1) and to inhibit the angiogenesis in the condition of hypoxia. Axl can regulate the function of endothelial cells and promote the endothelialization of the endothelial cells. The effect of high-sugar on the tube-forming and Axl expression of endothelial cells under the condition of hypoxia was observed, and the effect of the expression of Axl on the tube-forming function of endothelial cells and the regulation of the expression of HIF-1 were examined. Method: 1. Human umbilical vein endothelial cells (HUVECs) and human umbilical vein endothelial cells (Eay926) were treated with different concentrations of glucose (5.5mmol/ l, 11mmol/ l, 25 mmol/ l and 30mmol/ l), respectively. The effects of high sugar on cell activity were measured by MTT assay. HUVECs and Eay926 cells were treated with hypoxia at different time, and the effect of hypoxia time on cell activity was measured by MTT. The effects of high sugar on the migration of HUVECs and Eay926 cells and the formation of the tube cavity in the hypoxic condition were measured by the experiment of scratch and in vitro matrix gel. 4. The effect of high sugar on the expression of Axl and HIF-1 of HUVECs and Eay926 cells was detected by Western blot. Axl overexpressing lentivirus was constructed and the expression of Axl in cells was detected by Western blot: 6. After the successful transfection of the virus, the experiment was divided into four groups: control group (negative virus), Axl overexpressing group, R428 group (Axl inhibitor R428 + negative virus), Axl overexpression + R428 group. The effect of Axl on the migration of endothelial cells and the effect of tube-forming on the condition of high sugar and hypoxia was detected by the test of scratch and in vitro matrix gel. The effect of Axl on HIF-1 mRNA and protein content was detected by real-time fluorescence quantitative PCR and Western blot. The molecular mechanism of expression of HIF-1 was detected by Western blot using a protein synthesis inhibitor, Cyclohepciide, CHX, PI3K inhibitor LY294002, MEK inhibitor PD98059 and mTOR inhibitor Rapamycin. Results: 1. The inhibition of glucose to endothelial cell activity was in a concentration and time-dependent manner, and the cell activity was significantly inhibited by the treatment of HUVECs or Eay926 cells for 48 hours with 30 mmol/ I glucose. In the condition of high sugar, the activity of endothelial cells was time-dependent, and in the high-sugar state, the activity of endothelial cells was lower than that of the pure high-sugar group. In HUVECs and Eay926 cells, the migration distance of high-sugar 24h + hypoxia was less than that of hypoxia 6h group. The migration distance of HUVECs was 182. 9/ 14. 6 and 303. 1/ 10. 8, and the migration distance between two groups of Eay926 was 190. 7, 24, 8 and 293. 4 and 23. 9, respectively (P0.05). The number of small tubes in the high-sugar 24h + hypoxia group was less than that of the hypoxia group, and the number of the two cells decreased by 30.8% and 52.9%, respectively (P <0.05): 4. In the HUVECs and Eay926 cells, the expression of Axl protein in the high-sugar anoxic group was less than that of the Axl protein in the pure hypoxia group, and the expression of Axl protein in the two cells decreased by 48. 4% and 47.1%, respectively (P0.05): in the Eay926 cells, The expression of HIF-1 in HIF-1 group decreased by 40.7% (P0.05). After transfection of the Axl lentivirus, the expression of the Ax1 protein in the Eay926 cells was up-regulated; The cell migration distance of the overexpressing Axl group (233.1. 3-20.6) was longer than that in the control group (P0.05); the number of the small tubes of the overexpressing Axl group was more than that of the control group by 66. 8% (P0.05); The results of real-time fluorescence quantitative PCR showed that the expression of HIF-1 in the expression of Ax1 did not affect the mRNA expression of HIF-1. The results of Western blot showed that the expression of HIF-1 in the expression Axl group increased by 90.6% (P0.05) in comparison with the control group. The expression of HIF-1 in the expression of HIF-1 was decreased by 95% compared with that of the Axl overexpressing group (P0.05). However, the expression of HIF-1 induced by Axl was not affected by the expression of Axl, and the expression of HIF-1 induced by Ax1 was inhibited by LY294002 and Rapamycin. Conclusion: 1. the ability of high-sugar to inhibit the formation of umbilical vein endothelial cells under the condition of hypoxia; Overexpression of Ax1 reverses the inhibition of umbilical vein endothelial cells in the presence of high-sugar and oxygen-deficient conditions. Under the condition of high sugar and oxygen, the overexpression of Axl increased the synthesis of HIF-1 through the regulation of the PI3K/ Akt/ mTOR/ p70S6K pathway.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R587.2;R54
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