【摘要】：目的：高糖下調(diào)缺氧誘導(dǎo)因子-1α(Hypoxia inducible factor-1α, HIF-1α)的表達(dá),進(jìn)而抑制缺氧狀態(tài)下血管新生。Axl能調(diào)控內(nèi)皮細(xì)胞功能,促進(jìn)內(nèi)皮成管,然而Axl是否參與高糖缺氧條件下內(nèi)皮成管及HIF-1α的調(diào)控尚不清楚。本實(shí)驗擬觀察高糖對缺氧條件下內(nèi)皮細(xì)胞成管作用及Axl表達(dá)的影響,檢測過表達(dá)Axl對高糖缺氧條件下內(nèi)皮細(xì)胞成管作用的影響及HIF-1α表達(dá)的調(diào)控作用。方法：1.給予人臍靜脈內(nèi)皮細(xì)胞(Human umbilical vein endothelial cells, HUVECs)和人臍靜脈內(nèi)皮細(xì)胞株(Eahy926)不同濃度葡萄糖(5.5mmol/l、11mmol/l、25 mmol/l和30mmol/l)處理,分別培養(yǎng)24 h、48 h和72 h, MTT檢測高糖對細(xì)胞活性的影響；2.在高糖條件下給予HUVECs和Eahy926細(xì)胞不同時間缺氧處理,MTT檢測缺氧時間對細(xì)胞活性的影響：3.摸索出合適的高糖缺氧條件后,實(shí)驗分為高糖缺氧組和對照組(單純?nèi)毖踅M),用劃痕實(shí)驗和體外基質(zhì)膠成管實(shí)驗檢測高糖對缺氧狀態(tài)下HUVECs和Eahy926細(xì)胞遷移和管腔形成能力的影響；4.Western blot方法檢測高糖對缺氧狀態(tài)下HUVECs和Eahy926細(xì)胞Axl、HIF-1α蛋白表達(dá)的影響；5.構(gòu)建Axl過表達(dá)慢病毒并轉(zhuǎn)染Eahy926細(xì)胞,用Western blot檢測細(xì)胞中Axl的表達(dá)情況：6.病毒轉(zhuǎn)染成功后,將實(shí)驗分為四組：對照組(陰性病毒)、Axl過表達(dá)組、R428組(Axl抑制劑R428+陰性病毒)、Axl過表達(dá)+R428組。劃痕實(shí)驗和體外基質(zhì)膠成管實(shí)驗檢測過表達(dá)Axl對高糖缺氧條件下內(nèi)皮細(xì)胞遷移和成管作用的影響；7.實(shí)時熒光定量PCR和Western blot檢測過表達(dá)Axl對HIF-1α mRNA及蛋白含量的影響；8.使用蛋白合成抑制劑放線菌酮(Cycloheximide, CHX)、PI3K抑制劑LY294002,MEK抑制劑PD98059和mTOR抑制劑Rapamycin,應(yīng)用Western blot檢測過表達(dá)Axl調(diào)控HIF-1α表達(dá)的分子機(jī)制。結(jié)果：1.葡萄糖對內(nèi)皮細(xì)胞活性的抑制呈濃度和時間依賴性,30 mmol/I葡萄糖處理HUVECs或Eahy926細(xì)胞48小時,能顯著抑制細(xì)胞活性：2.缺氧時間對高糖狀態(tài)下內(nèi)皮細(xì)胞活性呈時間依賴性,高糖狀態(tài)下,缺氧12h和24 h組與單純高糖組相比,內(nèi)皮細(xì)胞活性均品著降低；3.在HUVECs和Eahy926細(xì)胞中,高糖24 h+缺氧6h遷移距離均少于缺氧6 h組,HUVECs兩組遷移距離分別為182.9±14.6和303.1±10.8,Eahy926兩組遷移距離分別為190.7±24.8和293.4±23.9,差異均有統(tǒng)計學(xué)意義(P0.05);高糖24 h+缺氧6h組小管數(shù)目少于缺氧6h組,兩種細(xì)胞成管分別減少了30.8%和52.9%,差異均有統(tǒng)計學(xué)意義(P0.05)：4.在HUVECs和Eahy926細(xì)胞中,高糖缺氧組的Axl蛋白表達(dá)量比單純?nèi)毖踅MAxl蛋白表達(dá)量少,兩種細(xì)胞分別減少了48.4%和47.1%,差異均有統(tǒng)計學(xué)意義(P0.05)：在Eahy926細(xì)胞中,高糖缺氧組的HIF-1α蛋白表達(dá)量比單純?nèi)毖踅M減少了40.7%(P0.05)；5.轉(zhuǎn)染過表達(dá)Axl慢病毒后,Eahy926細(xì)胞的Ax1蛋白表達(dá)明顯上調(diào)；6.過表達(dá)Axl組(231.3±20.6)細(xì)胞遷移距離比對照組(179.5±28.9)長(P0.05);過表達(dá)Axl組的小管數(shù)目比對照組多66.8%(P0.05)；7.實(shí)時熒光定量PCR結(jié)果顯示,過表達(dá)Ax1并不影響HIF-1α的mRNA表達(dá);Western blot結(jié)果顯示,與對照組相比,過表達(dá)Axl組HIF-1 α蛋白表達(dá)增加了90.6%(P0.05)；8.Axl過表達(dá)+CHX組與Axl過表達(dá)組相比,HIF-1 α的蛋白表達(dá)減少了95%(P0.05),而CHX組與對照組相比變化不明顯,說明蛋白合成途徑參與過表達(dá)Axl導(dǎo)致的HIF-1α表達(dá)增加；過表達(dá)Axl能激活A(yù)kt、p70S6K磷酸化,對Erk磷酸化無影響,LY294002和Rapamycin能抑制過表達(dá)Ax1誘導(dǎo)的HIF-1α表達(dá)。結(jié)論：1.高糖抑制缺氧條件下臍靜脈內(nèi)皮細(xì)胞的成管能力；2.過表達(dá)Ax1逆轉(zhuǎn)高糖缺氧條件對臍靜脈內(nèi)皮細(xì)胞成管的抑制；3.高糖缺氧條件下,過表達(dá)Axl通過調(diào)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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R587.2;R54

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