【摘要】：背景：血管生成是一個(gè)復(fù)雜的多步驟過程,其參與創(chuàng)面愈合、胚胎發(fā)生和生殖等眾多生理過程以及腫瘤、自身免疫性疾病、年齡相關(guān)黃斑變性和動(dòng)脈粥樣硬化等一系列病理過程。內(nèi)皮細(xì)胞是血管生成過程中必不可少的一員,它出芽、增殖、遷移、圍成管樣結(jié)構(gòu),還與周細(xì)胞共同決定和調(diào)節(jié)新生血管的穩(wěn)定性。近年來,越來越多的人開始關(guān)注交感神經(jīng)在血管新生中充當(dāng)?shù)纳飳W(xué)角色。種種跡象提示我們,交感神經(jīng)(節(jié)后神經(jīng)遞質(zhì)／腎上腺素能受體α-AR/β-AR在血管生成中扮演了重要角色。大量實(shí)驗(yàn)結(jié)果表明,內(nèi)皮細(xì)胞p-AR阻斷后,細(xì)胞體外增殖、遷移及成管皆被抑制,但是a-AR阻斷及α-AR、β-AR同時(shí)阻斷后內(nèi)皮細(xì)胞體外血管生成功能的變化尚不明確。第一部分：普萘洛爾、酚妥拉明抑制人微血管內(nèi)皮細(xì)胞體外血管生成目的研究離體情況下,p-AR阻斷劑普萘洛爾、a-AR阻斷劑酚妥拉明對人微血管內(nèi)皮細(xì)胞增殖、遷移、成管及VEGF、VEGFR-2、Ang1、Ang2、Tie-2表達(dá)的影響。方法1.細(xì)胞免疫熒光鑒定內(nèi)皮細(xì)胞：細(xì)胞采用免疫熒光染色Ⅷ因子進(jìn)行鑒定。將人皮膚微血管內(nèi)皮細(xì)胞和人腦微血管內(nèi)皮細(xì)胞接種于24孔板爬片。待細(xì)胞融合至80%-90%時(shí),4%冰多聚甲醛固定20分鐘,0.2%Triton X-100通透10分鐘,羊血清封閉30分鐘。兔多克�、蜃�(vWF)一抗4℃濕盒內(nèi)過夜。TRITC標(biāo)記的羊抗兔二抗室溫孵育2小時(shí)(避光)。Hochest (1μg/ml)染核15分鐘(避光)后10%甘油封片。正置熒光顯微鏡下觀察、拍片(×200倍)。2. Western blot檢測人微血管內(nèi)皮細(xì)胞a-AR的表達(dá)：未經(jīng)藥物處理的人皮膚微血管內(nèi)皮細(xì)胞和人腦微血管內(nèi)皮細(xì)胞經(jīng)裂解獲得總蛋白。BCA定量,沸水滅活。取總蛋白約40μg進(jìn)行SDS-PAGE凝膠電泳后,轉(zhuǎn)移到PVDF膜上。5%BSA封閉1小時(shí),蛋白樣品分別在4℃冰箱孵育兔多克隆抗α1-AR、α2-AR一抗過夜。次日在37℃孵箱中孵育相應(yīng)的二抗1h。采用超敏ECL化學(xué)發(fā)光試劑盒顯色,于暗室內(nèi)壓片,顯影定影。3.細(xì)胞增殖實(shí)驗(yàn)：將人皮膚微血管內(nèi)皮細(xì)胞種植于96孔板,將不同濃度梯度的普萘洛爾(0,25,50,75,100μM)、酚妥拉明(0,10,30,50,70 μg/ml)分別處理細(xì)胞48h。然后每孔加入10μlCCK-8,孵箱中繼續(xù)培養(yǎng)2h。之后于酶標(biāo)儀測450nm下吸光度值。4.細(xì)胞毒性實(shí)驗(yàn)：將人皮膚微血管內(nèi)皮細(xì)胞種植于96孔板,將不同濃度梯度的普萘洛爾(0,25,50,75,100μM)、酚妥拉明(0,10,30,50,70μg/m1)分別處理細(xì)胞48h。收集細(xì)胞上清液各60μl于96孔板內(nèi),每孔加入60μl乳酸脫氫酶工作液。室溫30分鐘后,于酶標(biāo)儀測490nm下吸光度值。5.細(xì)胞劃痕實(shí)驗(yàn)：將人皮膚微血管內(nèi)皮細(xì)胞種植于24孔板,細(xì)胞融合100%后,低血清培養(yǎng)基饑餓24h,用20μl槍頭在孔板中央劃寬約1mm劃痕,PBS洗去死細(xì)胞,各孔加入含不同濃度普萘洛爾(0,0.1,1,10,20,30μM)、酚妥拉明(0,0.1,1,10,20,40μg/m1)的低血清培養(yǎng)基,繼續(xù)孵箱內(nèi)培養(yǎng)。倒置熒光顯微鏡下觀察0,12,24,48 h劃痕愈合情況并拍照(40倍)。6.細(xì)胞成管實(shí)驗(yàn)：Matrigel基質(zhì)膠(12.5 mg/m1)在4℃溶解,24孔板、槍頭皆預(yù)冷,冰上操作。于24孔板中加入100μl基質(zhì)膠,均勻平鋪。將其放入37℃孵箱,待基質(zhì)膠凝固后,分別均勻加入500μl含1.5×105個(gè)人皮膚微血管內(nèi)皮細(xì)胞和人腦微血管內(nèi)皮細(xì)胞的完全培養(yǎng)基,繼續(xù)在37℃孵箱中孵育。待細(xì)胞貼壁后,去除舊培養(yǎng)基,分別加入含0、50LM普萘洛爾、0、50μg/ml酚妥拉明的完全培養(yǎng)基,倒置熒光顯微鏡下觀察0,4,8,12,24h細(xì)胞在基質(zhì)膠上的管樣形成情況并拍照(40倍)。7. ELISA法檢測細(xì)胞VEGF的變化：將人皮膚微血管內(nèi)皮細(xì)胞種植于6孔板,細(xì)胞融合至70%時(shí),去掉舊培養(yǎng)基,PBS洗去殘留培養(yǎng)基,加入分別含0、50μM普萘洛爾、0、50μg/ml酚妥拉明的高血清無內(nèi)皮細(xì)胞生長添加劑的培養(yǎng)基,孵箱內(nèi)培養(yǎng)48h。提取細(xì)胞總蛋白及細(xì)胞上清液,按VEGF ELISA試劑盒說明方法檢測細(xì)胞內(nèi)和細(xì)胞上清中VEGF的表達(dá)情況。8. ELISA法檢測細(xì)胞VEGFR-2的變化：將人皮膚微血管內(nèi)皮細(xì)胞種植于6孔板,細(xì)胞融合至70%時(shí),去掉舊培養(yǎng)基,PBS洗去殘留培養(yǎng)基,加入分別含0、50μM普萘洛爾、0、50μg/ml酚妥拉明的完全培養(yǎng)基,孵箱內(nèi)培養(yǎng)48h。提取細(xì)胞總蛋白,BCA定量,統(tǒng)一各孔蛋白量,按VEGFR-2 ELISA試劑盒說明方法檢測細(xì)胞內(nèi)VEGFR-2的表達(dá)情況。9. Western blot檢測人皮膚微血管內(nèi)皮細(xì)胞Ang1、Ang2、Tie-2的表達(dá)：將人皮膚微血管內(nèi)皮細(xì)胞分別用含0、50μM普萘洛爾、0、50μg/ml酚妥拉明的完全培養(yǎng)基培養(yǎng)48h后,裂解細(xì)胞獲得總蛋白。BCA定量,沸水滅活。取總蛋白約40μg進(jìn)行SDS-PAGE凝膠電泳后,轉(zhuǎn)移到PVDF膜上。5%BSA封閉1小時(shí),蛋白樣品分別在4℃冰箱孵育兔多克隆抗Ang1、Ang2、鼠單克隆抗Tie-2一抗過夜。次日在37℃孵箱中孵育相應(yīng)的二抗1h。采用超敏ECL化學(xué)發(fā)光試劑盒顯色,于暗室內(nèi)壓片,顯影定影。結(jié)果1.兩種細(xì)胞Ⅷ因子皆陽性,表明這兩種細(xì)胞皆為內(nèi)皮細(xì)胞來源。2.人腦微血管內(nèi)皮細(xì)胞表達(dá)α1-AR(α1A-AR (50kDa)和α1D-AR (60kDa)亞型)和α2-AR (50 kDa)。人皮膚微血管內(nèi)皮細(xì)胞表達(dá)α1-AR(位于34kDato43kDa之間的三個(gè)α1A-AR亞型(35kDa,37kDa和40kDa))和a2-AR(50 kDa),這是第一次報(bào)導(dǎo)人微血管來源內(nèi)皮細(xì)胞表達(dá)α-AR。3. 普萘洛爾和酚妥拉明皆呈劑量依賴性抑制人皮膚微血管內(nèi)皮細(xì)胞增殖,半抑制率分別位于50μM、 50kdg/ml附近。4. 細(xì)胞增殖實(shí)驗(yàn)中采用的濃度梯度內(nèi)的藥物未對細(xì)胞產(chǎn)生明顯毒性作用,間接證明普萘洛爾和酚妥拉明減少細(xì)胞數(shù)量是因?yàn)樗幬锉旧韺?xì)胞增殖的抑制作用,而非藥物對細(xì)胞的毒性作用。5.普萘洛爾和酚妥拉明皆抑制人皮膚微血管內(nèi)皮細(xì)胞遷移。48h后,藥物處理組(普萘洛爾20、30μM;酚妥拉明20、40dg/ml)劃痕寬度明顯大于對照組。低濃度藥物組亦抑制細(xì)胞遷移,但不明顯。6. 普萘洛爾和酚妥拉明皆抑制人皮膚微血管內(nèi)皮細(xì)胞和人腦微血管內(nèi)皮細(xì)胞成管。普萘洛爾抑制人皮膚微血管內(nèi)皮細(xì)胞成管達(dá)40.3%、人腦微血管內(nèi)皮細(xì)胞72.7%,酚妥拉明抑制人皮膚微血管內(nèi)皮細(xì)胞成管達(dá)65.7%、人腦微血管內(nèi)皮細(xì)胞77.7%。7. 普萘洛爾和酚妥拉明皆抑制人皮膚微血管內(nèi)皮細(xì)胞VEGFR-2的表達(dá),但是對胞內(nèi)VEGF和上清VEGF無明顯影響。普萘洛爾對人皮膚微血管內(nèi)皮細(xì)胞VEGFR-2的抑制達(dá)22.4%,酚妥拉明抑制率達(dá)24.4%。對照組細(xì)胞上清中VEGF濃度很低(13.9pg/ml),實(shí)驗(yàn)組上清中基本無VEGF,但二者無明顯差異(p0.05)。8.普萘洛爾和酚妥拉明皆抑制人皮膚微血管內(nèi)皮細(xì)胞Ang1、Ang2的表達(dá),但促進(jìn)Tie-2的表達(dá)。結(jié)論普萘洛爾和酚妥拉明皆抑制內(nèi)皮細(xì)胞增殖、遷移、成管,該作用可能與普萘洛爾和酚妥拉明抑制細(xì)胞VEGFR-2、Ang1、Ang2的表達(dá)有關(guān)。第二部分：同時(shí)阻斷α-AR、β-AR協(xié)同抑制人微血管內(nèi)皮細(xì)胞體外血管生成目的研究離體情況下,同時(shí)阻斷人微血管內(nèi)皮細(xì)胞α-AR、β-AR是否協(xié)同抑制細(xì)胞血管生成。方法1.實(shí)驗(yàn)分組：第一組：酚妥拉明50μg/ml;第二組：普萘洛爾50μM；第三組酚妥拉明50μg/ml+普萘洛爾50μM,分別進(jìn)行增殖、成管實(shí)驗(yàn)及VEGF、 VEGFR-2、Ang1.Ang2、Tie-2的檢測；劃痕實(shí)驗(yàn)中,由于該實(shí)驗(yàn)營養(yǎng)條件降低,藥物濃度降為酚妥拉明20μg/ml/普萘洛爾20μM。2.增殖、劃痕、成管、ELISA.Westem blot具體方法見第一部分。結(jié)果1. 同時(shí)阻斷α-AR、β-AR協(xié)同抑制人皮膚微血管內(nèi)皮細(xì)胞增殖。藥物處理后,酚妥拉明+普萘洛爾組細(xì)胞數(shù)量較酚妥拉明組少45.5%,普萘洛爾組少43.5%。2. 同時(shí)阻斷α-AR、β-AR協(xié)同抑制人皮膚微血管內(nèi)皮細(xì)胞遷移。48h后,酚妥拉明+普萘洛爾組劃痕寬度明顯大于酚妥拉明組和普萘洛爾組。3. 同時(shí)阻斷α-AR、β-AR協(xié)同抑制人皮膚微血管內(nèi)皮細(xì)胞、人腦微血管內(nèi)皮細(xì)胞成管。藥物處理細(xì)胞后4h,酚妥拉明組和普萘洛爾組可見明顯成管,酚妥拉明+普萘洛爾組成管不明顯。藥物處理細(xì)胞后8h,酚妥拉明組和普萘洛爾組成管數(shù)量及長度明顯大于酚妥拉明+普萘洛爾組。4. 同時(shí)阻斷α-AR、β-AR協(xié)同抑制人皮膚微血管內(nèi)皮細(xì)胞VEGFR-2的表達(dá)。藥物作用48h后,酚妥拉明+普萘洛爾組酶標(biāo)儀下OD值較酚妥拉明組少33.3%,較普萘洛爾組少35%。三個(gè)組胞內(nèi)VEGF無明顯差別,上清中基本無VEGF,亦無明顯差別。5. 同時(shí)阻斷α-AR、β-AR協(xié)同抑制人皮膚微血管內(nèi)皮細(xì)胞Ang1、Ang2.Tie-2的表達(dá)。藥物處理細(xì)胞后,酚妥拉明+普萘洛爾組細(xì)胞Ang1表達(dá)輕微下調(diào),Ang2表達(dá)明顯下調(diào),而Tie-2表達(dá)明顯下調(diào),與預(yù)期中Tie-2表達(dá)應(yīng)上升相反。結(jié)論同時(shí)阻斷α-AR、β-AR協(xié)同抑制內(nèi)皮細(xì)胞增殖、遷移、成管,協(xié)同抑制細(xì)胞VEGFR-2、Ang1、Ang2、Tie-2的表達(dá)。后者可能與前者有關(guān)。另外,該結(jié)果提示酚妥拉明和普萘洛爾體外抑制內(nèi)皮細(xì)胞血管生成,可能與VEGF/VEGFR-2、 Ang/Tie-2通路障礙有關(guān)。
[Abstract]:BACKGROUND: Angiogenesis is a complex multi-step process, which involves many physiological processes such as wound healing, embryogenesis and reproduction, as well as a series of pathological processes such as tumors, autoimmune diseases, age-related macular degeneration and atherosclerosis. In recent years, more and more people have begun to pay attention to the biological role of sympathetic nerves in angiogenesis. Various signs suggest that sympathetic nerves (postganglionic neurotransmitter/adrenergic receptor alpha-AR/beta-AR) play an important role in angiogenesis. A large number of experimental results showed that endothelial cell proliferation, migration and tube formation were inhibited in vitro after p-AR blockade, but the changes of angiogenesis in vitro after a-AR blockade and a-AR, beta-AR blockade were not clear. Part I: Propranolol, phentolamine inhibited human microvascular endothelial cells in vitro. Objective To study the effects of propranolol and phentolamine on proliferation, migration, tubulation and expression of VEGF, VEGFR-2, Ang1, Ang2 and Tie-2 in human microvascular endothelial cells in vitro. Endothelial cells and human brain microvascular endothelial cells were inoculated into 24-well climbing plates. When the cells were fused to 80%-90%, 4% ICP was fixed for 20 minutes, 0.2% Triton X-100 was permeated for 10 minutes, and sheep serum was closed for 30 minutes. Rabbit polyclonal factor_ (vWF) 1 antibody stayed overnight in a wet box at 4 C. TRITC labeled sheep anti-rabbit second antibody incubated at room temperature for 2 hours (avoiding light). (1 ug/ml) nucleus stained for 15 minutes (avoiding light) and 10% glycerol sealed. Positive fluorescence microscopy was used to observe the expression of a-AR in human microvascular endothelial cells. Western blot was used to detect the expression of a-AR in human microvascular endothelial cells. After SDS-PAGE gel electrophoresis, about 40 UG White was transferred to PVDF membrane. 5% BSA was blocked for 1 hour. Protein samples were incubated in refrigerator at 4 C for overnight. The next day, the corresponding antibodies were incubated in incubator at 37 C for 1 h. The cells were stained with a hypersensitive ECL chemiluminescent kit, pressed in darkroom, and developed and fixed.3. Experiments: The human skin microvascular endothelial cells were planted on 96-well plates and treated with different concentrations of propranolol (0,25,50,75,100 mu M) and phentolamine (0,10,30,50,70 ug/ml) for 48 hours respectively. Microvascular endothelial cells were planted on 96-well plates and treated with propranolol (0,25,50,75,100 mu M) and phentolamine (0,10,30,50,70 ug/m1) for 48 hours. Cell supernatants were collected and 60 mu L LDH solution was added into 96-well plates. After 30 minutes at room temperature, the absorbance at 490 nm was measured by enzyme labeling apparatus. Scratch test: Human skin microvascular endothelial cells were planted on 24-well plate. After 100% cell fusion, the cells were starved for 24 hours in low serum medium, scratched about 1 mm wide in the center of the plate with a 20-mL gunhead, and the dead cells were washed out with PBS. Low serum medium containing different concentrations of propranolol (0,0.1,1,10,20,30 mu M) and Phentolamine (0,0.1,1,10,20,40 ug/m1) was added to each hole. The scratch healing was observed under an inverted fluorescence microscope for 0,12,24,48 hours and photographed (40 times). 6. Cell tube formation test: Matrigel matrix glue (12.5 mg/m1) dissolved at 4 C, 24 holes plate, gun head were pre-cooled, and operated on ice. 100 ml matrix glue was added into 24 holes plate, evenly spread. It was placed in 37 C incubator until the matrix gel was set. 500 ml complete medium containing 1.5 *105 human skin microvascular endothelial cells and human brain microvascular endothelial cells were added evenly to incubate in an incubator at 37 C. After the cells adhered to the wall, the old medium was removed, and the complete medium containing 0,50LM propranolol and 0,50ug/ml phentolamine was added, respectively. The changes of vascular endothelial growth factor (VEGF) were detected by ELISA. When the cells were fused to 70%, the old culture medium was removed, the residual culture medium was washed out by PBS, and the high serum containing 0,50 micropropranolol and 0,50 microgram/ml phentolamine was added. Total cell protein and supernatant were extracted and the expression of VEGF in cell and supernatant was detected by the method of VEGF ELISA kit. 8. The changes of cell VEGFR-2 were detected by ELISA. The human skin microvascular endothelial cells were planted on 6-well plate and fused to 70% of the cells. In the old medium, PBS was used to wash out the residual medium, and the complete medium containing 0,50 Mu propranolol and 0,50 ug/ml phentolamine was added to incubate for 48 hours. Total cell protein was extracted, BCA was quantified, and the amount of pore protein was unified. The expression of intracellular VEGF R-2 was detected according to the method of VEGF R-2 ELISA kit. 9. Western blot was used to detect the expression of VEGF R-2 in human skin microblood. Expression of Ang1, Ang2 and Tie-2 in endothelial cells: Human skin microvascular endothelial cells were cultured in a complete medium containing 0,50 Mu propranolol and 0,50 ug/ml phentolamine for 48 hours, and then the total protein was obtained. BCA was quantified and inactivated by boiling water. Rabbit polyclonal anti-Ang1, Ang2 and mouse monoclonal anti-Tie-2 monoclonal antibodies were incubated in refrigerator at 4 C for overnight. The next day, the corresponding anti-Tie-2 monoclonal antibodies were incubated in incubator at 37 C for 1 h. The super-sensitive ECL chemiluminescence kit was used for coloration, and the cells were pressed in darkroom for developing and fixing. Source.2. Human brain microvascular endothelial cells express alpha 1-AR (alpha 1A-AR (50 kDa) and alpha 1D-AR (60 kDa) subtypes) and alpha 2-AR (50 kDa). Human skin microvascular endothelial cells express alpha 1-AR (three alpha 1A-AR subtypes (35 kDa, 37 kDa and 40 kDa) and a2-AR (50 kDa). This is the first report that human microvascular endothelial cells express alpha-AR.3. Both propranolol and phentolamine inhibited the proliferation of human skin microvascular endothelial cells in a dose-dependent manner. The semi-inhibitory rate was located at 50 mu M and 50 kdg/ml, respectively. 4. Drugs in the concentration gradient used in cell proliferation experiments did not produce significant cytotoxicity, indirectly demonstrating that propranolol and phentolamine reduced the number of cells because of the decrease in the number of cells. Propranolol and phentolamine both inhibited the migration of human skin microvascular endothelial cells. 48 hours later, the scratch width of the drug treatment group (propranolol 20,30 mu; phentolamine 20,40 dg / ml) was significantly larger than that of the control group. The low concentration drug group also inhibited cell migration, but Both propranolol and phentolamine inhibited the formation of human skin microvascular endothelial cells and human brain microvascular endothelial cells. Propranolol inhibited the formation of human skin microvascular endothelial cells by 40.3%, human brain microvascular endothelial cells by 72.7%, phentolamine inhibited the formation of human skin microvascular endothelial cells by 65.7%, and human brain microvascular endothelial cells by fine tubes. Both propranolol and phentolamine inhibited the expression of vascular endothelial growth factor-2 in human skin microvascular endothelial cells, but had no significant effect on endothelial growth factor and supernatant vascular endothelial growth factor. Propranolol inhibited the expression of vascular endothelial growth factor-2 in human skin microvascular endothelial cells by 22.4% and phentolamine by 24.4%. Propranolol and phentolamine both inhibited the expression of Ang1 and Ang2 in human skin microvascular endothelial cells, but promoted the expression of Tie-2. Conclusion Propranolol and phentolamine both inhibited the proliferation, migration and tube formation of endothelial cells, which may be inhibited by propranolol and phentolamine. The expression of VEGF R-2, Ang-1 and Ang-2 is related. Part 2: To study the effect of blocking alpha-AR and beta-AR on the angiogenesis of human microvascular endothelial cells in vitro. Methods 1. Experimental grouping: Group 1: Phentolamine 50 ug/ml; The second group: propranolol 50 ugm; the third group: phentolamine 50 ug/ml + propranolol 50 ugm, respectively, proliferation, tube test and VEGF, VEGFR-2, Ang1.Ang2, Tie-2 detection; scratch test, because of the experimental nutritional conditions decreased, the drug concentration was reduced to 20 ug/ml of phentolamine / propranolol 20 ugm.2. proliferation, scratch, tube, ELISA.Westem B. Results 1. The number of cells in Phentolamine + Propranolol group was 45.5% less than that in Phentolamine group and 43.5% less in Propranolol group. 2. At the same time, blocking alpha-AR and beta-AR synergistically inhibited the migration of human skin microvascular endothelial cells. The scratch width of phentolamine + propranolol group was significantly wider than that of phentolamine group and propranolol group. Phentolamine group and propranolol group were significantly longer than phentolamine + propranolol group at 8 hours after treatment. 4. At the same time, blocking alpha-AR, beta-AR synergistically inhibited the expression of VEGFR-2 in human skin microvascular endothelial cells. After 48 hours of treatment, the OD value of phentolamine + propranolol group was higher than that of phentolamine group. Vascular endothelial growth factor was 33.3% less in group A and 35% less in group B than that in group B. There was no significant difference in endothelial growth factor between the three groups. Conclusion Blockade of alpha-AR and beta-AR can inhibit the proliferation and migration of endothelial cells in vitro. Tubular formation can inhibit the expression of VEGF R-2, Ang1, Ang2 and Tie-2. The latter may be related to the former. In addition, the results suggest that phentolamine and propranolol can inhibit endothelial fineness in vitro. Cellular angiogenesis may be related to VEGF/VEGFR-2 and Ang/Tie-2 pathway disorders.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R622

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