局部应用VEGF-165抑制兔颈移植静脉内膜增生的研究

目的 探讨局部应用VEGF-165抑制兔颈移植静脉内膜增生的机制。方法 健康家兔24只,均建立颈外静脉-颈总动脉移植模型并分为对照组及实验组。对照组移植静脉周围仅应用Pluronic-F-127,实验组移植静脉周围应用Pluronic-F-127+VEGF-165。观察静脉移植血管外膜、内膜增生情况,移植静脉VEGF及eNOS的表达量,增殖细胞核抗原PCNA表达情况。分析外膜厚度与静脉桥血管内膜增生的关系。结果 与对照组相比,实验组移植静脉内膜增厚明显减轻、而外膜明显增厚,移植静脉eNOS的表达量明显增加;实验组移植静脉平滑肌细胞向外膜方向迁移;术后免疫组化检查显示,实验组移植静脉血管壁内膜VEGF-165的蛋白含量高;两组移植静脉PCNA的表达量未及明显差异。结论 VEGF-165可以通过增加内皮eNOS表达加速移植静脉血管内皮化,促进移植静脉外膜生长促进平滑肌向外膜迁移减轻移植静脉内膜增厚。     

应用c-myc反义寡核苷酸防治自体静脉移植物内膜增生的实验研究

目的:研究局部应用反义寡核苷酸c-myc对自体静脉移植物内膜增生的防治作用,为反义技术防治血管内膜增生的早期临床应用提供实验依据.方法:30只新西兰兔颈外静脉间置于同侧颈总动脉后随机分成对照组、蛋白胶组、正义组、反义组及错配组,以医用生物蛋白胶分别溶解反义、正义及错配的c-myc基因片段(330μg)后直接涂于静脉移植物外膜.术后28 d取材制片,显微镜进行形态观察,并用计算机图像分析系统测量和计算内膜厚度、中膜厚度及二者之比值,内膜面积、中膜面积及二者之比值,以q检验行统计学分析.结果:反义组增生内膜厚度较对照组降低36.77%,内膜面积降低48.22%,对照组、蛋白胶组、正义组及错配组间内膜厚度及内膜面积均无明显差异(P>0.05);各组间中膜厚度及中膜面积均无明显差异(P>0.05).结论:医用生物蛋白胶携载反义寡核苷酸c-myc局部用于静脉移植物外膜能显著防治其内膜增生,有效防止管腔狭窄.     

静脉移植物巨噬细胞浸润途径的实验研究

目的：探讨外膜是否是静脉移植物巨噬细胞浸润的一条重要途径。方法：应用免疫荧光组织化学技术对30个静脉移植物再塞标本中CD68（巨噬细胞的标记物）、CD31（内皮细胞的标记物）的表达进行了检测,用激光共聚焦显微镜拍片,图片用Silicon Graphics Octane进行处理,并对CD68阳性细胞进行计数。结果：在正常静脉血管,外膜中有少量CD68阳性细胞,中膜和内膜中少见;在病变的血管中,CD68免疫阳性细胞在外膜、中膜和内膜都可观察到,与正常血管相比差异有统计学意义（P<0.05）,且有从外膜向中膜和内膜迁移的趋势;中膜CD68免疫阳性细胞的分布和中膜毛细血管的生成过程密切相关。结论：外膜可能是静脉移植物巨噬细胞浸润的一条重要途径。     

静脉移植物中整合素α5β1的表达及其与再狭窄的关系

目的 检测人静脉移植物中整合素α5β1的表达.方法 应用免疫荧光组织化学技术对30个静脉移植物再狭窄标本中整合素α5β1和α-smooth muscle actin(α-SMA)的表达进行了检测,用激光共聚焦显微镜拍片,图片用SiliconGraphics Octane进行处理.结果 在正常静脉血管中,整合素α5β1在中膜平滑肌细胞和内皮细胞呈微弱表达;α-SMA在中膜平滑肌细胞表达;在病变静脉血管,整合素α5β1在中膜平滑肌细胞、内膜内皮细胞中的表达呈强阳性,在内膜平滑肌细胞中也有弱表达.α-SMA除在中膜平滑肌细胞表达外,在内膜平滑肌细胞也有表达.结论 静脉移植物整合素α5β1在中膜平滑肌细胞、内膜内皮细胞表达显著上调,提示整合素α5β1参与静脉移植物再狭窄的形成.     

联合转染eNOS基因和反义ET核酸对自体移植静脉内膜增生的影响

目的：探讨联合转染eNOS基因和反义ET核酸对自体移植静脉内膜增生的影响。方法：制作20只自体颈静脉腹主动脉移植Wistar大鼠模型，实验组、对照组各10只，实验组移植血管行腺病毒介导的eNOS溶液泡和反义ET核酸凝胶涂布，对照组仅行空载腺病毒溶液浸泡和凝胶涂布。要后2周取出植物血管，利用病理学、免疫组织化学、RT－PCR方法检测移植血管内膜厚度、管腔狭窄度、内膜VSMC数及PCNA阳性表达、血管ETmRNA、eNOS、mRNA表达情况。结果：实验组移植血管内膜厚度、管腔狭窄度及VSMC数均较对照组减小或减少，PCNA阳性表达及ETmRNA表达较对照组减少，而eNOSmRNA表达则明显增加。结论：联合传染eNOS基因和反义ET核酸可有效地抑制移植静脉内膜的增生，是一种有效的防治移植静脉再狭窄的基因疗法。     

自体血管移植后内膜增生与eNOS mRNA表达关系的实验研究

目的探讨自体血管移植后内膜增生的机制以及内皮型一氧化氮合酶（eNOS）和内膜增生的关系。方法建立自体动脉静脉血管搭桥模型,分成静脉组、游离动脉组和非游离动脉组,分别于移植后1-10周切取移植段血管,进行组织形态学检查观察内膜增生情况,以及运用原位杂交分子生物学方法检测血管壁eNOS mRNA的表达。结果静脉组内膜增生最为明显,而非游离动脉组无明显内膜增生。同一时间点静脉组eNOS mRNA表达信号明显低于游离动脉组（6周内）（P〈0.01）,而非游离动脉组eNOS mRNA表达信号与正常血管无差别。静脉组和游离动脉组血管壁eNOS mRNA表达与血管搭桥后内膜增生显著相关。结论静脉桥eNOS mRNA表达比动脉桥弱是静脉桥内膜增生明显的重要机制。     

应用c—mtc反义寡核苷酸防治自体静脉移植物内膜增生的实 …

目的：研究局部应用反义寡核苷酸c-myc对自体静脉移植物内膜增生的防治作用,为反义技 术防治血管人膜增生的早期临床应用提供实验依据。方法：３０只新西兰兔颈外静脉间置于同侧颈总动脉后随机分厉对照组、蛋白胶组、正义组、反义组及错配组,以医用生物蛋白胶分别溶解反义、正义及错配的c-myc基因片段（３３０ug）后直接涂于静脉移植物外膜。术 后２８d取材制片,显微镜进行形态观察,并用刘算机图像分析系统测量和计算内膜厚度及     

血管内皮生长因子在血管外支架预防移植静脉再狭窄中的作用

目的:研究血管内皮生长因子(VEGF)在血管外支架预防移植静脉再狭窄中的作用及不同管径材料外支架的作用效果。方法:通过动物实验获得外支架干预下的移植静脉及其对照标本,用免疫组化及形态学方法对标本中的VEGF、增殖细胞核抗原(PCNA)及滋养血管的表达进行检测、分析。结果:与对照组相比,外支架干预的移植静脉组中膜VEGF含量显著降低(P<0.05),而外膜VEGF含量显著增高(P<0.05),中膜PCNA含量显著降低(P<0.05),外膜滋养血管密度显著增高(P<0.05)。结论:血管外支架可有效促进VEGF向外膜聚集,减少其在中膜、内膜聚集,进而促进滋养血管的生长,减少中膜增生,减轻再狭窄。     

用自体心包行静脉周支持对静脉移植物内膜增生的影响

目的探讨自体心包外周支持对静脉移植物内膜增生的影响。方法取犬一侧颈外静脉，对半剪成两段后分别吻合在双侧股动脉上，其中一侧静脉移植物外包裹自体心包（实验组），另一侧为单纯的静脉移植（对照组）。术后2、4周分别切除移植静脉，计算机图像分析系统测量和计算内膜厚度、内膜面积，进行扫描电镜检查，用免疫组织化学方法检测静脉移植物平滑肌增殖细胞核抗原（PCNA）的表达。结果实验组静脉移植术后2、4周内膜厚度和内膜截面积明显低于对照组（P〈0．01）。实验组静脉移植物术后2、4周的PCNA指数也明显低于对照组（P〈0．01；P〈0．05）。扫描电镜检查显示实验组静脉移植物内皮层破坏程度轻于对照组。结论自体心包外周支持能抑制静脉移植物内膜的增生。     

血管外膜局部应用缬沙坦防治自体移植静脉再狭窄的实验研究

目的 探讨局部应用缬沙坦防治自体移植静脉再狭窄的可行性及其可能机制.方法 建立家兔颈外静脉颈总动脉移植模型,随机分为pluronic-F-127 gel组和缬沙坦组,pluronic-F-127 gel组在静脉移植物外膜涂抹pluronic-F-127 gel凝胶,缬沙坦组在静脉移植物外膜涂抹由pluronic-F-127 gel凝胶携带的缬沙坦.分别于术后7、14、28d用免疫组织化学方法检测移植静脉增殖细胞核抗原(PCNA)的表达情况;HE、Masson染色后应用计算机图像分析系统检测术后14d移植静脉内膜、中膜增生情况及内膜厚度/中膜厚度值(I/M);RT-PCR 法检查术后14d移植静脉血管紧张素Ⅱ2型受体(AT2)mRNA 表达情况.结果 术后14d,缬沙坦组移植静脉内膜厚度、中膜厚度、I/M值均明显低于pluronic-F-127 gel组,差异有统计学意义(P<0.05);术后7、14、28d,在同一时间点,缬沙坦组的血管平滑肌细胞增殖率均低于pluronic-F-127 gel组,差异有统计学意义(P<0.05);术后14d,缬沙坦组AT2 mRNA 表达较pluronic-F-127 gel组显著增高.结论 用携带缬沙坦的pluronic-F-127 gel涂抹自体移植静脉外膜能抑制血管平滑肌细胞增殖,减轻移植静脉内膜、中膜增生,防治移植静脉再狭窄,其机制可能是上调AT2的表达.     

Effect of Troglitazone on Expression of Adhesion Molecules and eNOS in Human Saphenous Vein Gaft

To investigate whether peroxisome proliferators-activated receptor-γ (PPARγ) ligand Troglitazone can reduce endothelial injury and activation during storage of harvested saphenous vein grafts. Segments of human saphenous vein graft were collected from 9 patients undergoing coronary bypass surgery and then divided into two equal parts of control and test specimens, were stored in ei- ther heparinized blood (control group) or heparinized blood containing 20 μmol/L troglitazone (test group) for 1 h at room temperature. Tissue distribution and protein expression of VCAM-1, ICAM-1, and endothelial nitric oxide synthase (eNOS) were compared using immunohistochemistry and West- ern blot analysis. Myeloperoxidase (MPO) activity, a marker of neutrophil sequestration in human saphenous vein grafts, was also measured in each group. The expression of ICAM-1 (753±132 versus 7201±934; P<0.01),VCAM-1 (3731±294 versus 8292±793; P<0.01), and MPO activity (1.52±0.42 U/g,5.04±1.26 U/g P<0.01) were significantly lower in test group. In contract, eNOS expression (7983±834 versus 3989±1008; P<0.01) was significantly higher in test group. PPARγ ligand troglita- zone might reduce endothelial injury during the storage period of human saphenous vein grafts.     

Effect of Troglitazone on Expression of Adhesion Molecules and eNOS in Human Saphenous Vein Graft

To investigate whether peroxisome proliferators-activated receptor-γ （PPARγ） ligand Troglitazone can reduce endothelial injury and activation during storage of harvested saphenous vein grafts. Segments of human saphenous vein graft were collected from 9 patients undergoing coronary bypass surgery and then divided into two equal parts of control and test specimens, were stored in ei- ther heparinized blood （control group） or heparinized blood containing 20 μmol/L troglitazone （test group） for 1 h at room temperature. Tissue distribution and protein expression of VCAM-I, ICAM-I, and endothelial nitric oxide synthase （eNOS） were compared using immunohistochemistry and Western blot analysis. Myeloperoxidase （MPO） activity, a marker of neutrophil sequestration in human saphenous vein grafts, was also measured in each group. The expression of ICAM-1 （753±132 versus 7201±934; P〈0.01） , VCAM-1 （3731±294 versus 8292±793; P〈0.01）, and MPO activity （1.52±0.42 U/g, 5.04±1.26 U/g P〈0.01） were significantly lower in test group. In contract, eNOS expression （7983±834 versus 3989±1008; P〈0.01） was significantly higher in test group. PPARγ ligand troglitazone might reduce endothelial injury during the storage period of human saphenous vein grafts.     

Expression of Platelet Membrane Glycoprotein Ib/Ⅸ/Ⅴ Complex,a Receptor of Thrombin,in Patients with Hemorrhagic Thrombopathy

To investigate the role of platelet membrane glycoprotein （GP） Ib/Ⅸ/Ⅴ complex and its subunit GP Ibα in patients with hemorrhagic thrombopathy （HT）, the expressions of GP Ib/Ⅸ/Ⅴ complex and GP Ibα,defined as mean fluorescence intensity （MFI）, were assessed by flow cytometry. The maximum aggregation of platelet was determined by turbidity method. These indicators were compared among 68 HT patients with the presenting complaint of hemorrhage, 33 well-controlled HT patients and 32 normal healthy subjects. The results showed that the MFI of GP Ib/Ⅸ /Ⅴ complex and GP Ibα was markedly lower in HT patients with current hemorrhage than that in the healthy subjects, with difference being statistically significant （P〈0.05）. There was no significant difference in the expressions of GP Ib/ Ⅸ/ Ⅴ complex and GP Ibα between well-controlled HT patients and normal healthy subjects （P〉0.05）. It was concluded that the expression of GP Ib/Ⅸ /Ⅴ complex, the receptor of thrombin and von Willebrand factor, was down-regulated in HT patients with current hemorrhage, which might result in the dysfunction of platelet aggregation and recurrence of HT.     

妊高征患者血浆致内皮细胞eNOS mRNA表达增强的研究

目的　研究妊高征 (PIH)患者的血浆对体外培养的内皮细胞合成一氧化氮 (NO)及内皮型一氧化氮合成酶 (eNOSmRNA)表达的影响 ,探讨PIH患者血中NO水平的改变及改变的原因。　方法　在体外培养的脐静脉内皮细胞中分别加入PIH患者和正常晚孕妇女的血浆 ,2 4h后应用Northern杂交法比较加入不同血浆的内皮细胞eNOSmRNA表达的差异。　结果　加入PIH组血浆的内皮细胞eNOSmRNA的表达水平较加入正常晚孕组的内皮细胞的eNOSmRNA的表达水平增高 (P <0 .0 1)。　结论　eNOSmRNA表达增强可能是PIH时的一种代偿机制。     

The role of prourokinase gene in protecting vein grafts from intimal hyperplasia

Objective To study the duration of prourokinase gene expression in vein grafts and the role of the prourokinase gene in protecting vein grafts from neointimal hyperplasia.Methods Fifty-four Wistar rats were used in this study. In each rat, the jugular vein was excised and distended for 30 minutes using a solution containing either Adv5-CMV (control group) or Adv5-CMV/ Pro-UK (treatment group). Next, the jugular vein was reversed and interposed into the divided carotid artery of the same rat. On the 14th day after transfection, vein grafts of the control group were collected in order to perform a fibrinolysis test for prourokinase (Pro-UK) activity. On the 2nd, 7th, 14th, 28th, and 60th day, the vein grafts of the treatment group were likewise collected in order to detect prourokinase activity. On the 28th day, the vein grafts of both groups were explanted to evaluate the 3H-TDR incorporation so that pathologic analysis could be performed.Results Pro-UK activity could not be detected in the control group     

高游离脂肪酸致内皮细胞eNOS活性降低与PKC通路的关系

目的 研究游离脂肪酸(FFA)水平升高导致的内皮一氧化氮合酶(eNOS)活性降低是否与蛋白激酶C(PKC)通路激活有关.方法 传代培养的人脐静脉内皮细胞分为4组:对照组,油酸组(10、50、100、150、200 μmol/L),PKC抑制剂(GFX)组,油酸+GFX组.分别检测各组eNOS活性,检测对照组和油酸组PKC的表达和活性,以及各组eNOS磷酸化(p-eNOS)的表达.结果 与对照组相比,油酸组eNOS活性呈剂量依赖性降低,p-eNOS(1177)的表达降低(0.0854±0.017 vs. 0.0134±0.023, P<0.05);PKC的表达出现转位,由胞浆转向胞膜,胞浆表达活性降低,胞膜表达活性增高,总活性增加.加入GFX后对正常内皮细胞eNOS活性无明显影响,但能部分改善油酸所致的内皮细胞eNOS活性的降低.结论 油酸可损害内皮细胞eNOS的表达及磷酸化,其机理与PKC通路的激活有关.     

miRNA-24对血管内皮细胞eNOS表达/活性及其代谢产物NO生成的影响

目的 探讨微小RNA(miRNA)-24对血管内皮细胞内皮型一氧化氮合酶(eNOS)基因表达、活性调节的分子机制及其代谢产物的影响.方法 构建miRNA-24高表达质粒,并转染人脐静脉内皮细胞(HUVEC).MTT法检测细胞的增殖情况,划痕实验检测细胞的迁移能力,RT-PCR和Western印迹法检测细胞eNOS mRNA和蛋白的表达情况,ELISA法检测eNOS的表达活性,硝酸还原法测定细胞培养上清液中NO的含量.结果 与对照组相比,转染miRNA-24后细胞增殖能力下降54.32%、迁移能力下降48.62%;转染miRNA-24后eNOS mRNA降低43.92%,蛋白表达量减少42.71%;转染miRNA-24后eNOS的酶活性降低73.20%,同时NO的合成与释放减少55.29%.结论 miRNA-24高表达抑制eNOS的表达及酶活性;miRNA-24抑制代谢产物NO的合成与释放,这可能成为心血管疾病防治的分子靶点.     

Adenovirus-mediated transfer of β-galactosidase and prourokinase genes into vein grafts

Objective To study the feasibility of adenovirus mediated gene transfer into vein grafts and the role of the prourokinase gene in protecting vein grafts from thrombosis.Methods Fifty-two Wistar rats underwent implantation of reversed autologous jugular vein interposition grafts in the common carotid arteries. Jugular veins were excised and distended with solution containing three different adenovirus vectors (Adv(5)-CMV, group Ⅰ; Adv(5)-CMV/LacZ, group Ⅱ; Adv(5)-CMV/Pro-UK, group Ⅲ) for 30 min, then the jugular veins were reversed and interposed into the divided carotid arteries, and end-to-end anastomoses were performed. The amount of (51)Cr-labeled platelets in vein grafts of group Ⅰ and group Ⅲ was counted 24 hours postoperatively. On the 14th day, the vein grafts were harvested to examine β-galactosidase activity and prourokinase (Pro-UK) activity and observe thrombosis in vein grafts. Results Extensive blue coloration in the area of intima and media of each vein graft in group Ⅱ was observed. No blue coloration was seen in group Ⅰ. Pro-UK activity was not detected in the vein grafts of group Ⅰ. In group Ⅲ, the amount of Pro-UK gene expression was 308 IU/g tissue. The amount of (51)Cr labeled platelets in group Ⅰ and group Ⅱ was (123.7±19.4) ×10(6)/g dry wt, (34.4±5.3) ×10(6)/g dry wt, respectively. The thrombosis rate and occlusion rate of the vein grafts in group Ⅰ were 30% and 10%, respectively. In group Ⅲ, all vein grafts were patent and free of thrombosis. Conclusions Ex vivo gene transfer before vein grafting is feasible using replication deficient recombinant adenovirus and results in a high level of gene expression in vivo. Direct transfer of the Pro-UK gene into vein grafts may prevent thrombosis.     

缺氧条件下RhoA/Rho激酶及eNOS在血管内皮细胞表达的研究

目的：建立稳定的体外细胞缺氧模型,探讨人体脐静脉内皮细胞（HUVEC）在缺氧条件下RhoA蛋白和Rho激酶对其内皮细胞内皮型一氧化氮合酶（eNOS）表达的影响。方法：利用jetPEI-HUVEC、siRNA分别转染SH-SY5Y细胞、HEK293细胞和HUVEC后制备体外细胞缺氧模型,通过细胞裂解对相关蛋白进行免疫印迹分析。结果：常氧条件下RhoA蛋白在HUVEC中表达水平较低,但在缺氧条件下培育5 h后表达增加,同时缺氧3 h后Rho激酶表达增加,5 h后达高峰,而eNOS的表达恰恰相反。缺氧条件下,活化型RhoA蛋白下调eNOS的表达,而siRNA使RhoA蛋白表达减少从而上调eNOS的表达;Rho结合域抑制Rho激酶活性而上调eNOS的表达。结论：RhoA蛋白和Rho激酶的表达及活化抑制内皮细胞中eNOS的表达,因此可以通过某些药物如他汀类或Rho激酶抑制剂,抑制RhoA蛋白和Rho激酶的活性,从而增加eNOS的表达水平,对心脑血管疾病产生保护作用。