毕业论文 同型半胱氨酸对人内皮细胞的损伤机制及丹皮酚保护作用的实验研究.pdf

目 录中文摘要.1英文摘要.6英文缩写.13研究论文同型半胱氨酸对人内皮细胞的损伤机制及丹皮酚保护作用的 实验研究第一部分同型半胱氨酸对人脐静脉内皮细胞的损伤机制研究前言.15材料与方法.15结果.27附图.30附表.40讨论.44小结.47参考文献.48第二部分丹皮酚对同型半胱氨酸损伤的人脐静脉内皮细胞保护作用的研究前言.51材料与方法.51结果.57附图.59附表.68讨论.72小结.74参考文献.75结论.77综 述.78致 谢.89个人简历.90中文摘要同型半胱氨酸对人内皮细胞的损伤机制及丹皮酚 保护作用的实验研究摘 要近年来大量临床和实验研究证明，同型半胱氨酸(homocysteine,Hey)已成为致动脉粥样硬化(atherosclerosis,AS)的一种独立的危险因素7 在心脑血管疾病及外周血管硬化等疾病发病机制中起重要作用。但Hey 致AS的具体机制尚不清楚，因此探讨同型半胱氨酸诱导AS的发病机制 具有十分重要的意义。丹皮酚作为传统的中药有效成分，具有广泛的药理活性，其中在心血 管方面有抗AS,抗血栓，抗心律失常等作用。目前，已有资料表明丹 皮酚在整体水平保护血管内皮细胞，减轻AS的进程，但丹皮酚在细胞水 平保护Hey所致AS的研究尚未见报道。本研究以Hey为损伤因子建立 内皮细胞损伤模型，观察Pae对人脐静脉内皮细胞(Human Umbilical Veins Endothelial Cells,HUVECs)的保护作用，并探讨其分子机制，为临床应 用Pae防治动脉粥样硬化提供有价值的实验和理论依据。第一部分 同型半胱氨酸对人脐静脉内皮细胞的损伤机制研究目的：观察同型半胱氨酸(Hey)对人脐静脉内皮细胞(HUVECs)NFkB通 路、eNOS及NO含量的影响，探讨Hey对内皮细胞损伤的可能的分子机 制，为防治动脉粥样硬化提供新的治疗靶点。方法：L HUVECs的分离培养：用0.1%I型胶原酶与0.25%胰蛋白酶0.02%EDTA等比混合消化液消 化法收集HUVECs,加入含20%胎牛血清、20pg/L bFGF的DMEM培养基 在37、5%C()2培养箱中培养，用0.125%胰蛋白酶0.01%EDTA进行消 化传代培养。2.实验分组：实验用第2、3代细胞，实验分为正常对照组(HO),Hey剂量组。正 中文摘要常对照组细胞加含20%胎牛血清、20pg/L bFGF的DMEM培养基；Hey剂 量组细胞在上述培养基基础上再分为4组，分别加入2.5mmol/LHcy(Hl)、5mmol/LHey(H2)、10mmol/LHey(H3)及 15mmol/LHcy(H4)。各分 组细胞继续培养24h。3.倒置显微镜观察各组HUVECs细胞形态变化。4.MTT法检测各组HUVECs细胞活力。5.RT-PCR检测各组HUVECs NF-kB p65、eNOS mRNA的表达。6.Western blotting 检测各组 HUVECs IkB、ICAM-1 蛋白的表达。7.免疫组化法检测各组HUVECs中NF-kB p65核转移的变化。8.硝酸还原酶法检测各组HUVECs培养液中NO的含量。结果：1.HUVECs的分离培养原代培养的HUVECs呈典型的单层铺路石样或鹅卵石样镶嵌排列。传 代细胞的形态及生长方式与原代培养细胞类似，汇合后细胞也呈单层铺路 石状或鹅卵石样镶嵌排列。2.倒置显微镜观察细胞形态变化如下：与正常对照组相比，H1组细胞形态无明显变化，H2组细胞形态发生 变化，细胞收缩，间隙增宽，变圆。H3、H4组损伤作用明显，细胞呈团 簇状，轮廓不清.有较多细胞脱落。3.MTT法检测各组HUVECs细胞活力与正常对照组比较，H1组细胞活力无明显变化(尸0.05),无统计 学意义；H2、H3、H4组细胞活力明显下降(PV0.01)。4.RTPCR检测各组HUVECs NF*B p65、eNOS mRNA的表达4.1 NF-kB p65 mRNA的表达：与正常对照组比较，Hey各剂量组 NFKBp65mRNA的表达明显增强(PV0.05,P0.01),且呈现一定的剂 量依赖性(P0.05)o H2、H3、H4组eNOS mRNA 的表达明显减少(PV0.01),且呈现一定的剂量依赖性(PV0.05)。5.Western blotting 检测各组 HUVECs kB-a ICAM-1 蛋白的表达5.l kB-a蛋白的表达：与正常对照组比较，Hey各剂量组IkB蛋白 2中文摘要的表达均明显降低(PV0.05,PV0.01),且具有一定的剂量依赖性(P0.05)o 5.2ICAM4蛋白的表达：与正常对照组比较，Hey各剂量组ICAM-1 蛋白的表达均明显增多(PV0.05,P0.01)o6.免疫组化法检测各组HUVECs细胞NF-kB p65核转移变化NF*Bp65阳性表达产物为棕黄色颗粒。正常对照组中，NF-kB p65 在细胞中基本无表达。H1组棕黄色颗粒较少，H2、H3、H4组棕黄色颗 粒明显增加，并且随着Hey浓度的增加，细胞核内棕黄色颗粒明显增多,表明具有明显的核转移倾向。与正常对照组比较,Hey各剂量组NFKBp65 的表达明显增强(尸0.01)。7.硝酸还原酶法检测各组HUVECs培养液中NO的含量与正常对照组比较，Hey各剂量组HUVECs生成的NO逐渐减少(P0.05,P2,倒置显微镜观察各组HUVECs细胞形态学变化。中文摘要3.MTT法检测各组HUVECs细胞活力。4.RT-PCR法检测各组HUVECs NF-kB p65、eNOS mRNA的表达。5.Western blotting 检测各组 HUVECsIkBf、ICAM-l 蛋白的表达。6.免疫组化法检测各组HUVECs中NF-kB p65核转移的变化。7.硝酸还原酶法检测各组HUVECs培养液中NO的含量。结果：1.倒置显微镜观察细胞形态变化如下：与正常对照组比较,Hey损伤模型组的细胞出现片状分离，脱落现象。Pae保护组使细胞间隙变窄，形态趋于正常。2.MTT法检测各组HUVECs细胞活力与正常对照组比较，Hey损伤模型组细胞活力明显降低（PV0.01）。与Hey损伤模型组比较，Pae各剂量组的细胞活力明显升高（PV0.01）,且具有一定的剂量依赖性（PV0.05）。3.RT-PCR 检测各组 HUVECs NF-kB p65、eNOS mRNA 的表达3.1 NF-kB p65 mRNA的表达：与正常对照组比较，Hey损伤模型组 NFKBp65mRNA的表达明显增强（PV0.01）。与Hey损伤模型组比较,Pae翻fi组（低、中、高）的NF-kB p65 mRNA表达明显降低（P0.05,P0,01）,并呈现一定的剂量依赖性（PV0.05）o3.2 eNOS mRNA的表达：与正常对照组比较，Hey损伤模型组eNOS mRNA的表达明显降低（PV0.01）。与Hey损伤模型组比较，Pae各剂 量组（低、中、高）的eNOS mRNA的表达明显增强（PV0.01）,并呈 现一定的剂量依赖性（PV0.05）。4.Western blotting检测各组HUVECs IkB（x、ICAMJ蛋白的表达 蛋白的表达与正常对照组比较，Hey损伤模型组IkB蛋白表达明显降低（P0.01）o与Hey损伤模型组比较，Pae各剂量组（低、中、高）IkBp蛋白表达明显增加（PV0.01）。4.2 ICAMJ蛋白的表达:与正常对照组比较,Hey损伤模型组ICAM 蛋白表达明显增加（尸V0.01）。与Hey损伤模型组比较,Pae各剂量组（低、中、高）ICAM-1的表达逐渐降低，差异具有统计学意义（PV0.05,PV0.01）。5.免疫组化法检测各组HUVECs中NF-kB p65核转移的变化Hey损伤模型组中胞质和胞核中均有棕黄色颗粒。与正常对照组比 4中文摘要较，差异有显著性（PV0.01）。Pae低剂量组中胞核内仍有大量棕黄色颗 粒，Pae中剂量组中胞核内的棕黄色颗粒明显减少，Pae高剂量组中，胞 核中几乎无棕黄色颗粒。与Hey损伤模型组比较，Pae各剂量组在细胞核 内NFKBp65的阳性表达产物明显减少（PV0.01）。6.硝酸还原酶法检测各组HUVECs培养液中NO的含量与正常对照组比较，Hey损伤模型组HUVECs生成的NO明显减少（P0.05).The cell viability in the other groups(H2,H3,H4)decreased obviously compared with nomal control group(PV0.01).4.The expression of NF-kB p65,eNOS mRNA in HUVECs in each group detected by RT-PCR.4.1 The expression ofNF-KB p65 mRNA:Compared with nomal control group,The expression of NF-kB p65 mRNA in Hey dose groups increased significantly(P0.05,P0.01)in a dose-dependent manner(P0.05).The eNOS mRNA expression in the other groups was remarkably lower than that of nomal control group in a dose-dependent manner(P0.05).5.The expression of kB-a,ICAM-1 protein in HUVECs in each group detected by Western blotting.5.1 The expression oflKB-a protein:The expression of IkB-u protein in Hey dose groups reduced significantly compared with nomal control group(P0.05,P0.01)in a dose-dependent 8英文摘要manner(P 0.05).5.2 The expression of IC AM-1 protein:The expression of ICAM-1 protein in Hey dose groups increased significantly compared with nomal control group(P0.05,尸V0.01).6.Immunohistochemistry was used to observe the change of NFkB p65 nuclear translocation.NF-kB p65 positive expression products were buffy particles.In the normal control group,NF-kB p65 almost did not have the positive particles in the cells.The bufly particles in Hl group were few.The buffy particles in the other groups increased obviously.Buffy particles in the nucleus increased significantly with the increasing concentrations of Hey indicating a clear tendency of nuclear translocation.Compared with the normal control group,the positive NF-kB p65 expression particles increased significantly in Hey dose groups(P0.01).7.NO was detected in the cultured supemate of HUVECs in each group by the method of nitric acid reductase.Compared with the normal control group,the content of NO in the cultured supemate of Hey dose groups decreased gradully in a concentration-dependent manner(P0.05,P0.01).Conclusions:1.Hey can increase the expression of NF-kB p65 mRNA obviously,and increase the degradation of IkB-q,allowing the translocation of active NF-kB into the nucleus.Then,NF-kB up-regulates the expression of intercellular adhesion molecule-1.Taken together,Hey can lead to atherosclerosis in the end.2.Hey can inhibit the expression of eNOS mRNA obviously,so it can reduce the synthesis of eNOS and decrease the activation of eNOS.Therefore,NO production was reduced.So Hey can accelerate the pathogenesis and development of atherosclerosis.9英文摘要Part II Explore the protective effect of Pae on HUVECs injured by homocysteine(Hcy)Objective:We used human umbilical vein endothelial cells(HUVECs)as the research objects,and we set up damaged endothelial cells models induced by homocysteine.We investigated the effects of Paeonol on the NF-kB pathway,eNOS and the content of NO in the endothelial cells injured by homocysteine.We explored the protective effects of Paeonol and its anti-atherosclerotic molecular mechanisms.Methods:1.Experimental group:We divided the HUVECs into 5 groups randomly:normal control group,Hey injured model group(lOmmol/L Hey),the low-dose group of Pae(lOmmol/L Hey+0.15mmol/L Pae),the middle-dose group of Pae(lOmmol/L Hey+03mmol/L Pae),the high-dose group of Pae(lOmmol/L Hey+0.6mmol/L Pae).2.The morphological changes of HUVECs were observed in each group respectively with inverted microscope.3.Cell viability was detected in HUVECs in each group by the method of MTT.4.RT-PCR was used to measure the expression of NF-kB p65,eNOS mRNA in HUVECs in each group.5.Western blotting was used to detect the expression of IkB-g,ICAM-1 protein in HUVECs in each group.6.Immunohistochemistry was used to observe the change of NF-kB p65 nuclear translocation.7.NO was detected in the cultured supemate of HUVECs in each group by the method of nitric acid reductase.Results:1.The morphological changes of HUVECs were observed in each group respectively with inverted microscope.Compared with normal control group,Hey injured model group showed 英文摘要flake separation and shedding phenomenon.In Pae dose groups the intercellular space became narrow,and the cell shape tended to be normal.2.Cell viability was detected in HUVECs in each group by the method of MTT.The cell viability in Hey injured model group decreased obviously compared with nomal control group(P0.01).The cell viability in the low-dosethe middle-dose and the high-dose group of Pae was remarkably higher than that of Hey injured model group(P0.05).3.The expression of NF-kB p65,eNOS mRNA in HUVECs in each group detected by RT-PCR.3.1 The expression of NF-kB p65 mRNA:Compared with nomal control group,NF-kB p65 mRNA expression in Hey injured model group increased significantly(P0,01),NF-kB p65 mRNA expression in the low-dose,the middle-dose and the high-dose group of Pae was remaikably lower than that of Hey model group(P0.05,P0,01)and they were in a dose-dependent manner(PV0.05).3.2 The expression of eNOS mRNA:Compared with nomal control group,eNOS mRNA expression in Hey injured model group decreased significantly(P0.01).The eNOS mRNA expression in the low-dose,the middle-dose and the high-dose group of Pae was remarkably increased than that of Hey injured model group(P0.01)and they were in a concentration-dependent manner(P0.05).4.Western blotting was used to detect the expression of IxB-a,ICAM-1 protein in HUVECs in each group.4.1 The expression oflKB-a protein:Compared with nomal control group,IxB-a protein expression in Hey injured model group decreased significantly(P 0.01).kB-a protein expression in the low-dose,the middle-dose and the high-dose group of Pae was remarkably increased than that of Hey model group(PV0.01).4.2 The expression of ICAM-1 protein:Compared with nomal control group,ICAM-1 protein expression in Hey ii英文摘要injured model group increased significantly(P0.01).ICAM-1 protein expression in the low-dose,the middle-dose and the high-dose group of Pae was remarkably lower than that of Hey injured model group(P0,05,P0,01).5.Immunohistochemistry was used to observe the change of NF-kB p65 nuclear translocation.The buffy particles located in nucleus and cytoplasm in Hey injured model group.Compared with the normal control group,the difference was significant in Hey injured model group(P0,01).In Pae low-dose group,there were still many positive particles in the nucleus,and the positive particles in the nucleus in Pae middle-dose group decreased significantly.Furthermore,there were almost no buffy particles in the nucleus in Pae high-dose group.Compared with Hey injured model group,the positive NF-kB p65 expression particles in the nucleus was significantly reduced in Pae dose groups(尸0,01).6.NO was detected in the cultured supemate of HUVECs in each group by the method of nitric acid reductase.Compared with nomal control group,the content of NO in the cultured supemate of Hey injured model group decreased significantly(P0,01).The content of NO in Pae dose groups was remarkably higher than that of model group(P0.01)and they were in a concentration-dependent manner(P 200g/ml 300gg/ml 400|ig/mk 500|ig/ml将待测样品5倍稀释，每孔加200皿工作液，37保温30分钟，用酶标 仪测定56211m的吸光度，绘制标准曲线，据此求出样品总蛋白浓度。2.6.3 SDS-PAGE 电泳把蛋白样品与5xLoading buffer按4:1的体积比混合均匀，95c加热 lOmin使蛋白质变性。根据目的蛋白的分子量，配制5%的浓缩胶和12%的分离胶p-actin）8%的分离胶（ICAM-1）o取30pig蛋白样 品上样，先以80V进行稳压电泳，当样品进入分离胶后，再以120V电压 继续电泳至溟酚蓝到达分离胶边缘。2.6.4 蛋白质的电转移用甲薛激活PVDF膜3min,然后连同滤纸、棉垫一起放入转膜缓冲液 中浸泡15min,将PVDF膜平铺于胶上，覆盖住目的蛋白，在胶和膜的两 侧分别放3张滤纸和2张棉垫组成三明治结构，4mA/cm2,稳流转膜2h；取 出PVDF膜，用5%脱脂奶粉4封闭过夜。265检测目的蛋白加入一抗（kBa、ICAM-1均为1:200,pactin为1:1000）,室温摇床 孵育2h,TBST洗涤（15minxi次，10min 90%、95%乙醇溶液和无水乙醇I及 无水乙醇II中梯度脱水。将玻片放入二甲苯I及二甲苯H以透明，最后用 中性树胶封片。在Mivnt显微图像分析系统下进行半定量分析。显棕黄色颗粒为阳 性。对采集的图片中棕黄色阳性颗粒进行密度扫描，测平均灰度值，观察26研究论文NF-kB p65核转移变化。实验中设置阴性对照，阴性对照片用PBS代替 一抗，其余操作步骤同上。2.8 硝酸还原酶法检测各组HUVECs培养液中NO的含量NO化学性质活泼，在体内很快转为NO?和NO,而NO?一又进一步 转化为NO,利用硝酸还原酶特异性将NCP-还原为N0,通过显色深浅 测定其浓度的高低。吸取6孔培养板中的细胞培养液，按南京建成生物工 程研究所的试剂盒说明书进行测定，结果按下列公式计算：NO(pmol/L)=测定管OD值-空白管OD值 标准管。植二空白管。植X标准品浓度(lOOpmol/L)x样品测试前稀释倍数3统计学方法实验数据均以;土s表示，应用SPSS 1L5软件进行统计学分析，多组 间计量资料均数比较采用单因素方差分析，组间两两比较采用LSD-t检 验。PV0.05为差异有统计学意义。结 果1.HUVECs的分离培养原代培养的HUVECs呈典型的单层铺路石样或鹅卵石样镶嵌排列。传 代细胞的形态及生长方式与原代培养细胞类似，汇合后细胞也呈单层铺路 石状或鹅卵石样镶嵌排列。见Figi、Fig2、Fig3o2,倒置显微镜观察各组HUVECs细胞形态变化与正常对照组比较，H1组细胞形态无明显变化，H2组细胞形态发生 变化，细胞收缩，间隙增宽，变圆。H3、H4组细胞呈团簇状，轮廓不清。有较多细胞脱落。与H3组比较，H4组细胞密度明显降低，细胞数量明 显减少，损伤严重见图Fig 4、Fig 5、Fig 6、Fig7 Fig 8。3.MTT法检测各组HUVECs细胞活力不同浓度Hey作用于HUVECs 24h后,MTT结果显示，与正常对照 组比较，Hey低浓度(2.5mmol/L)对正常细胞生长的抑制作用不明显，H1组细胞活力无明显变化(P0.05),无统计学意义。随着Hey浓度的 增高(515mmol/L),Hey可明显抑制HUVECs的生长，H2、H3、H4 27研究论文组细胞活力明显下降(P0.01)o结果见Table 1,Chart L4.RT-PCR检测各组HUVECs NF-kB p65、eNOS mRNA的表达4.1 在350450 bp区域之间,可见清晰的NF-kB p65条带，在450-550 bp区域之间，可见清晰的pactin条带(Fig 9)。RT-PCR结果显示，不 同浓度Hey作用于HUVECs24h后,与正常对照组比较,Hey剂量组NFkB p65 mRNA的表达明显增强(PV0.05,P005)。随着Hey浓度的升高(515mmol/L),同型半胱氨酸可明显抑制eNOS mRNA的表达,eNOS mRNA的表达明显 降低(PV0.01),且呈现一定的剂量依赖性(PV0.05),结果见Table3,Chart 3 o5.Western blotting检测各组HUVECs IkB、ICAM1蛋白的表达5.1 Western Blotting结果显示，在蛋白Marker 41KD水平处，可见 IkB条带;在蛋白Marker 43KD水平处，可见。actin条带(Fig 11)不同浓度Hey作用于HUVECs 24h后，与正常对照组比较，Hey各剂量 组IkB(x蛋白的表达均明显降低(PV0.05,PV0.01),且具有一定的剂量 依赖性(P0.05,*P0,05,*P0,01.HO:normal control groupH2:5mmol/L Hey groupH4:15mmol/L Hey groupHl:2.5mmol/L Hey groupH3:lOmmol/L Hey group36研究论文Chart 2 NF-kB p65 mRNA expression of HUVECs in each group by RT-PCR.Compared with normal control group,*P0.05,*P0.05,*PV0.0L37研究论文Chart 4 kB-a protein expression of HUVECs in each group by Western blottingCompared with normal control group,*P0.05,*P0,01.Chart 5 ICAM-1 protein expression of HUVECs in each group by Western blotting.Compared with normal control group,*P0,05,*P0,01.38研究论文Chart 6 Expression ofNF-KB p65 in HUVECs by immunohistochemistry assayCompared with normal control group,*P0.01.Chart 7 The content of NO in the cultured supemate of HUVECs in each group by the method of nitric acid reductase.Compared with normal control group,*P0.05,*P0.05,*P0.05,*P0,01.HO:normal control group H1:2.5mmol/L Hey groupH2:5mmol/L Hey group H3:1 Ommol/L Hey groupH4:15mmol/L Hey groupRT-PCR.(xs,n=5)Table 2 NF-kB p65 mRNA expression of HUVECs in each group byGroupconcentrations of Hey(mmol/L)NF-kB p65/p-actinHO0.46330.0308Hl2.50.53940.0102*H250.68380.0348*H3100.7799W0212*H4150912600164*Compared with normal control group,*P0.05,*P0,05,*P0.01.Table 4 IkB protein expression of HUVECs in each group by Western blotting.(xs,n=5)Groupconcentrations of Hey(mmol/L)kB-a/p-actinHO1.10950.01201Hl2.5L03690.02989*H250.91810.04401*H3100.59710.02244*H4150.32970.02430*Compared with normal control group,*P0,05,*P0.01.41研究论文Table 5 ICAM-1 protein expression of HUVECs in each group by Western blotting.(xs,n=5)Groupconcentrations of Hey(mmol/L)ICAM-l/p-actinHO0.83550.0496Hl2.5L1727土0.0279*H251.58060.0416*H3101.77600.0358*H4152.154410,0399*Compared with normal control group,*P0.05,*P0.01.Table 6 Expression of NF-kB p65 in HUVECs by immunohistochemistry assay(xs,n=5)Groupconcentrations of Hey(mniol/L)A valueHOHlH2H3H4 0.02260.01332.5 0.10360.0176*5 0.22780.0201*10 0.59970.0391*15 0.82220.0249*Compared with normal control group,*P0.01.研究论文Table 7 The content of NO in the cultured supemate of HUVECs in each group by the method of nitric acid reductase.Groupconcentrations of Hey(mmol/L)NO(gmol/L)HO179.32582.7060Hl2.517L68543.4994*H25125.61803.8433*H310103.59552.9084*H41588.31463.2371*Compared with normal control group,*P0.05,*P 血管 细胞黏附分子1(VCAM-1)、E-选择素等，有实验证实这些黏附分子都参 与了鼠模型动脉粥样硬化的发展过程。ICAM-1,又名CD54,是最早发 现的免疫蛋白超家族粘附分子之一，是一种细胞表面单链跨膜蛋白抗原，其主要功能是介导细胞与细胞、细胞与细胞外基质之间的黏附作用口，正 常情况下，内皮细胞很少表达或不表达ICAM-L当受到炎性细胞因子等 刺激因素作用后，ICAMJ的表达迅速上调，大量表达于血管内皮细胞表 面，增强单核细胞与血管内皮细胞之间的黏附，促进炎症的发生和发展，进而参与AS的发生、发展过程。目前已有实验证实ICAM-1表达的