阻断肾上腺素能受体对人内皮细胞体外血管生成的影响
发布时间：2018-08-10 20:06
【摘要】：背景：血管生成是一个复杂的多步骤过程,其参与创面愈合、胚胎发生和生殖等众多生理过程以及肿瘤、自身免疫性疾病、年龄相关黄斑变性和动脉粥样硬化等一系列病理过程。内皮细胞是血管生成过程中必不可少的一员,它出芽、增殖、迁移、围成管样结构,还与周细胞共同决定和调节新生血管的稳定性。近年来,越来越多的人开始关注交感神经在血管新生中充当的生物学角色。种种迹象提示我们,交感神经(节后神经递质／肾上腺素能受体α-AR/β-AR在血管生成中扮演了重要角色。大量实验结果表明,内皮细胞p-AR阻断后,细胞体外增殖、迁移及成管皆被抑制,但是a-AR阻断及α-AR、β-AR同时阻断后内皮细胞体外血管生成功能的变化尚不明确。第一部分：普萘洛尔、酚妥拉明抑制人微血管内皮细胞体外血管生成目的研究离体情况下,p-AR阻断剂普萘洛尔、a-AR阻断剂酚妥拉明对人微血管内皮细胞增殖、迁移、成管及VEGF、VEGFR-2、Ang1、Ang2、Tie-2表达的影响。方法1.细胞免疫荧光鉴定内皮细胞：细胞采用免疫荧光染色Ⅷ因子进行鉴定。将人皮肤微血管内皮细胞和人脑微血管内皮细胞接种于24孔板爬片。待细胞融合至80%-90%时,4%冰多聚甲醛固定20分钟,0.2%Triton X-100通透10分钟,羊血清封闭30分钟。兔多克隆Ⅷ因子(vWF)一抗4℃湿盒内过夜。TRITC标记的羊抗兔二抗室温孵育2小时(避光)。Hochest (1μg/ml)染核15分钟(避光)后10%甘油封片。正置荧光显微镜下观察、拍片(×200倍)。2. Western blot检测人微血管内皮细胞a-AR的表达：未经药物处理的人皮肤微血管内皮细胞和人脑微血管内皮细胞经裂解获得总蛋白。BCA定量,沸水灭活。取总蛋白约40μg进行SDS-PAGE凝胶电泳后,转移到PVDF膜上。5%BSA封闭1小时,蛋白样品分别在4℃冰箱孵育兔多克隆抗α1-AR、α2-AR一抗过夜。次日在37℃孵箱中孵育相应的二抗1h。采用超敏ECL化学发光试剂盒显色,于暗室内压片,显影定影。3.细胞增殖实验：将人皮肤微血管内皮细胞种植于96孔板,将不同浓度梯度的普萘洛尔(0,25,50,75,100μM)、酚妥拉明(0,10,30,50,70 μg/ml)分别处理细胞48h。然后每孔加入10μlCCK-8,孵箱中继续培养2h。之后于酶标仪测450nm下吸光度值。4.细胞毒性实验：将人皮肤微血管内皮细胞种植于96孔板,将不同浓度梯度的普萘洛尔(0,25,50,75,100μM)、酚妥拉明(0,10,30,50,70μg/m1)分别处理细胞48h。收集细胞上清液各60μl于96孔板内,每孔加入60μl乳酸脱氢酶工作液。室温30分钟后,于酶标仪测490nm下吸光度值。5.细胞划痕实验：将人皮肤微血管内皮细胞种植于24孔板,细胞融合100%后,低血清培养基饥饿24h,用20μl枪头在孔板中央划宽约1mm划痕,PBS洗去死细胞,各孔加入含不同浓度普萘洛尔(0,0.1,1,10,20,30μM)、酚妥拉明(0,0.1,1,10,20,40μg/m1)的低血清培养基,继续孵箱内培养。倒置荧光显微镜下观察0,12,24,48 h划痕愈合情况并拍照(40倍)。6.细胞成管实验：Matrigel基质胶(12.5 mg/m1)在4℃溶解,24孔板、枪头皆预冷,冰上操作。于24孔板中加入100μl基质胶,均匀平铺。将其放入37℃孵箱,待基质胶凝固后,分别均匀加入500μl含1.5×105个人皮肤微血管内皮细胞和人脑微血管内皮细胞的完全培养基,继续在37℃孵箱中孵育。待细胞贴壁后,去除旧培养基,分别加入含0、50LM普萘洛尔、0、50μg/ml酚妥拉明的完全培养基,倒置荧光显微镜下观察0,4,8,12,24h细胞在基质胶上的管样形成情况并拍照(40倍)。7. ELISA法检测细胞VEGF的变化：将人皮肤微血管内皮细胞种植于6孔板,细胞融合至70%时,去掉旧培养基,PBS洗去残留培养基,加入分别含0、50μM普萘洛尔、0、50μg/ml酚妥拉明的高血清无内皮细胞生长添加剂的培养基,孵箱内培养48h。提取细胞总蛋白及细胞上清液,按VEGF ELISA试剂盒说明方法检测细胞内和细胞上清中VEGF的表达情况。8. ELISA法检测细胞VEGFR-2的变化：将人皮肤微血管内皮细胞种植于6孔板,细胞融合至70%时,去掉旧培养基,PBS洗去残留培养基,加入分别含0、50μM普萘洛尔、0、50μg/ml酚妥拉明的完全培养基,孵箱内培养48h。提取细胞总蛋白,BCA定量,统一各孔蛋白量,按VEGFR-2 ELISA试剂盒说明方法检测细胞内VEGFR-2的表达情况。9. Western blot检测人皮肤微血管内皮细胞Ang1、Ang2、Tie-2的表达：将人皮肤微血管内皮细胞分别用含0、50μM普萘洛尔、0、50μg/ml酚妥拉明的完全培养基培养48h后,裂解细胞获得总蛋白。BCA定量,沸水灭活。取总蛋白约40μg进行SDS-PAGE凝胶电泳后,转移到PVDF膜上。5%BSA封闭1小时,蛋白样品分别在4℃冰箱孵育兔多克隆抗Ang1、Ang2、鼠单克隆抗Tie-2一抗过夜。次日在37℃孵箱中孵育相应的二抗1h。采用超敏ECL化学发光试剂盒显色,于暗室内压片,显影定影。结果1.两种细胞Ⅷ因子皆阳性,表明这两种细胞皆为内皮细胞来源。2.人脑微血管内皮细胞表达α1-AR(α1A-AR (50kDa)和α1D-AR (60kDa)亚型)和α2-AR (50 kDa)。人皮肤微血管内皮细胞表达α1-AR(位于34kDato43kDa之间的三个α1A-AR亚型(35kDa,37kDa和40kDa))和a2-AR(50 kDa),这是第一次报导人微血管来源内皮细胞表达α-AR。3. 普萘洛尔和酚妥拉明皆呈剂量依赖性抑制人皮肤微血管内皮细胞增殖,半抑制率分别位于50μM、 50kdg/ml附近。4. 细胞增殖实验中采用的浓度梯度内的药物未对细胞产生明显毒性作用,间接证明普萘洛尔和酚妥拉明减少细胞数量是因为药物本身对细胞增殖的抑制作用,而非药物对细胞的毒性作用。5.普萘洛尔和酚妥拉明皆抑制人皮肤微血管内皮细胞迁移。48h后,药物处理组(普萘洛尔20、30μM;酚妥拉明20、40dg/ml)划痕宽度明显大于对照组。低浓度药物组亦抑制细胞迁移,但不明显。6. 普萘洛尔和酚妥拉明皆抑制人皮肤微血管内皮细胞和人脑微血管内皮细胞成管。普萘洛尔抑制人皮肤微血管内皮细胞成管达40.3%、人脑微血管内皮细胞72.7%,酚妥拉明抑制人皮肤微血管内皮细胞成管达65.7%、人脑微血管内皮细胞77.7%。7. 普萘洛尔和酚妥拉明皆抑制人皮肤微血管内皮细胞VEGFR-2的表达,但是对胞内VEGF和上清VEGF无明显影响。普萘洛尔对人皮肤微血管内皮细胞VEGFR-2的抑制达22.4%,酚妥拉明抑制率达24.4%。对照组细胞上清中VEGF浓度很低(13.9pg/ml),实验组上清中基本无VEGF,但二者无明显差异(p0.05)。8.普萘洛尔和酚妥拉明皆抑制人皮肤微血管内皮细胞Ang1、Ang2的表达,但促进Tie-2的表达。结论普萘洛尔和酚妥拉明皆抑制内皮细胞增殖、迁移、成管,该作用可能与普萘洛尔和酚妥拉明抑制细胞VEGFR-2、Ang1、Ang2的表达有关。第二部分：同时阻断α-AR、β-AR协同抑制人微血管内皮细胞体外血管生成目的研究离体情况下,同时阻断人微血管内皮细胞α-AR、β-AR是否协同抑制细胞血管生成。方法1.实验分组：第一组：酚妥拉明50μg/ml;第二组：普萘洛尔50μM；第三组酚妥拉明50μg/ml+普萘洛尔50μM,分别进行增殖、成管实验及VEGF、 VEGFR-2、Ang1.Ang2、Tie-2的检测；划痕实验中,由于该实验营养条件降低,药物浓度降为酚妥拉明20μg/ml/普萘洛尔20μM。2.增殖、划痕、成管、ELISA.Westem blot具体方法见第一部分。结果1. 同时阻断α-AR、β-AR协同抑制人皮肤微血管内皮细胞增殖。药物处理后,酚妥拉明+普萘洛尔组细胞数量较酚妥拉明组少45.5%,普萘洛尔组少43.5%。2. 同时阻断α-AR、β-AR协同抑制人皮肤微血管内皮细胞迁移。48h后,酚妥拉明+普萘洛尔组划痕宽度明显大于酚妥拉明组和普萘洛尔组。3. 同时阻断α-AR、β-AR协同抑制人皮肤微血管内皮细胞、人脑微血管内皮细胞成管。药物处理细胞后4h,酚妥拉明组和普萘洛尔组可见明显成管,酚妥拉明+普萘洛尔组成管不明显。药物处理细胞后8h,酚妥拉明组和普萘洛尔组成管数量及长度明显大于酚妥拉明+普萘洛尔组。4. 同时阻断α-AR、β-AR协同抑制人皮肤微血管内皮细胞VEGFR-2的表达。药物作用48h后,酚妥拉明+普萘洛尔组酶标仪下OD值较酚妥拉明组少33.3%,较普萘洛尔组少35%。三个组胞内VEGF无明显差别,上清中基本无VEGF,亦无明显差别。5. 同时阻断α-AR、β-AR协同抑制人皮肤微血管内皮细胞Ang1、Ang2.Tie-2的表达。药物处理细胞后,酚妥拉明+普萘洛尔组细胞Ang1表达轻微下调,Ang2表达明显下调,而Tie-2表达明显下调,与预期中Tie-2表达应上升相反。结论同时阻断α-AR、β-AR协同抑制内皮细胞增殖、迁移、成管,协同抑制细胞VEGFR-2、Ang1、Ang2、Tie-2的表达。后者可能与前者有关。另外,该结果提示酚妥拉明和普萘洛尔体外抑制内皮细胞血管生成,可能与VEGF/VEGFR-2、 Ang/Tie-2通路障碍有关。
[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.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R622
本文编号：2176059
[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.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R622
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