过表达p27基因抑制血管损伤后新生内膜的形成

目的　探讨p2 7基因转染对血管损伤后新生内膜形成的影响。方法　以腺病毒为载体转染p2 7基因 ,用MTT法观察其对血管平滑肌细胞增殖的影响 ,用流式细胞仪检测细胞周期 ;在兔颈总动脉球囊损伤模型上转染p2 7基因 ,采用免疫组织化学法检测p2 7基因的表达 ,检测其对新生内膜形成的影响。结果　p2 7可明显抑制培养的血管平滑肌细胞增殖 ,细胞停滞于G1期。兔颈总动脉球囊损伤后转染p2 7基因可以减少新生内膜的形成达 2 7 4 3%。结论　以腺病毒为载体转染p2 7基因可以有效地抑制血管平滑肌细胞增殖及血管损伤后新生内膜的形成。     

甲氨蝶呤抑制兔颈动脉球囊损伤后新生内膜形成

目的　探讨不同剂量甲氨蝶呤 (MTX)对血管损伤后新生内膜形成的影响。方法　制备兔颈动脉球囊损伤模型 ,动物随机分为对照组、MTX 0 5mg/kg、MTX 1 5mg/kg及MTX 5mg/kg组 ,每组 5～ 8只 ,采用肌肉注射方式给药 ,于术后 2周和 4周取材 ,分别进行伊文思染色观察内皮覆盖情况、增殖细胞核抗原 (PCNA)免疫组化染色和定量组织形态学分析。结果　MTX治疗组血管内膜面积、内膜 /中膜面积比值均显著低于对照组 ,内膜面积抑制率达 4 6 %～ 5 6 %,不同剂量MTX之间差异无显著性。内膜及中膜PCNA阳性细胞数目较对照组减少。伊文思蓝染色显示各组间内皮修复程度差异无显著性。结论　MTX能够有效地抑制球囊损伤后血管平滑肌细胞的增殖和新生内膜形成 ,在一定剂量范围内 ,MTX的作用无明显差异。     

血管球囊损伤后血管紧张素Ⅱ受体在内膜增生中的作用

目的探讨血管紧张素Ⅱ受体（ＡＴＲ）在血管球囊损伤后内膜增生中的作用。方法用放射配基结合分析法测定损伤后血管组织ＡＴＲ及其亚型的变化；免疫组化方法测定增殖细胞核抗原（ＰＣＮＡ）阳性细胞以了解血管内膜增殖过程。结果内膜损伤后第３天组织中ＡＴＲ显著增高（Ｐ＜０．０５）；第１４天ＡＴＲ的最大结合容量是对照组的３倍，而受体的平衡解离常数无显著变化；第２８天组织ＡＴＲ降至基础水平。增高的ＡＴＲ为血管紧张素Ⅱ１型受体（ＡＴ１Ｒ）。非肽类ＡＴ１Ｒ拮抗剂Ｉｒｂｅｓａｒｔａｎ显著抑制血管平滑肌细胞增殖，减少新生内膜面积；非肽类血管紧张素Ⅱ２型受体（ＡＴ２Ｒ）拮抗剂ＣＧＰ４２１１２Ａ则无此作用。结论血管内皮损伤后ＡＴＲ上调，ＡＴ１Ｒ参与血管内皮损伤后内膜增殖。     

水飞蓟宾对实验兔球囊血管损伤后内膜增生的影响

探讨水飞蓟宾对兔球囊血管损伤后内膜增生的影响。将新西兰大白兔随机分为对照组 (n =6 )、小剂量水飞蓟宾组 [n =8,2 0mg (kg·d) ]、大剂量水飞蓟宾组 [n =8,4 0mg (kg·d) ]。用球囊导管对实验兔行髂动脉损伤 ,用药组于术前 3d分别用小剂量及大剂量水飞蓟宾 ,术后 2 8d取病变血管染色并免疫组织化学检查 ,以计算机图像分析系统分析血管内膜、中膜厚度和腔面积的变化 ,计算内膜增生细胞核抗原增殖指数。结果发现 ,小剂量水飞蓟宾对血管内膜和中膜厚度、腔面积、内膜增生细胞核抗原阳性细胞百分比无明显影响 (P >0 .0 5 ) ;大剂量水飞蓟宾使腔面积扩大、内膜厚度减少、内膜增生细胞核抗原阳性细胞百分比减少 (P <0 .0 5 )。提示水飞蓟宾能抑制血管内膜增生 ,有可能用于防治再狭窄。     

组织肾素-血管紧张素系统在血管球囊损伤后内膜增生中的作用

目的 :探讨组织肾素 -血管紧张素系统 ( RAS)在血管球囊损伤后内膜增生中的作用。方法 :用放射免疫法测定血管球囊损伤后大鼠的血浆肾素活性 ( RA)、血管紧张素 ( Ang ) ;用生物化学方法检测血清和组织血管紧张素转换酶 ( ACE)活性 ;免疫组化法观察血管壁细胞增殖过程。结果 :损伤后循环中 RAS各成分无明显变化 ,球囊损伤后第 3天组织中 ACE活性增高 ( P <0 .0 1) ,第 14天达最高 ,第 2 8天降至基础水平。ACE活性与 PCNA阳性细胞数呈正相关。ACE抑制剂 Perindopril抑制组织中 ACE活性 ,减少内膜增生。结论 :组织 RAS而非循环 RAS在血管球囊损伤后内膜增生中起重要作用。通过抑制组织 ACE活性可以预防血管成形术后再狭窄。     

大鼠血管紧张素Ⅱ2型受体基因转染对颈动脉球囊损伤后新生内膜形成的影响

目的　探讨腺病毒载体介导的大鼠血管紧张素Ⅱ2型受体(AT2R)基因转染对大鼠颈动脉球囊损伤后新生内膜形成的影响。方法　应用带有绿色荧光蛋白报告基因的重组腺病毒载体将大鼠AT2R基因转移至颈动脉球囊损伤模型中,2 1d后取材,应用免疫组织化学方法检测AT2R在动脉壁中的表达,图像分析观察其对新生内膜形成的影响。结果　在体大鼠颈动脉腺病毒载体的转染率达4 0 % ,AT2R基因在血管壁的新生内膜、中膜、外膜稳定表达,转染AT2R基因的球囊损伤后颈动脉与绿色荧光蛋白基因对照组相比,血管新生内膜 中膜面积比降低了4 8%。结论　AT2R基因转染可抑制颈动脉球囊损伤后新生内膜的形成,对血管成形术后再狭窄可能有一定的预防作用。     

降钙素基因相关肽对颈动脉球囊介入损伤后大鼠新生内膜形成的影响

目的探讨降钙素基因相关肽(CGRP)对颈动脉球囊介入损伤后大鼠新生内膜形成的影响。方法采用雄性SD大鼠建立颈总动脉球囊损伤模型,将60只模型大鼠随机分为对照组,低剂量CGRP(CGRP-L)组和高剂量CGRP(CGRP-H)组。CGRP-L组和CGRP-H组于球囊损伤后每天腹腔注射50、100μg/kg CGRP,而对照组于球囊损伤后每天腹腔注射等体积生理盐水。采用流式细胞术检测内皮祖细胞(EPC)动员情况,酶联免疫吸附法检测外周血中促血管修复因子〔血管内皮生长因子(VEGF)、表皮生长因子(EGF)、基质细胞衍生因子(SDF)-1和碱性成纤维因子(b FGF〕及血清内皮素(ET)-1水平,分光光度法检测血清一氧化氮(NO)水平,HE染色观察颈动脉在球囊损伤后的形态学变化及内膜增生情况。结果CGRP-L组和CGRP-H组外周血中CD34和VEGF受体共同标记的EPC动员比例分别为(15.26±2.70)%和(23.55±3.92)%,均高于对照组的(7.12±1.14)%(P0.05);与对照组相比,CGRP-L处理后的VEGF、SDF-1、b FGF、EGF及NO水平均升高,ET-1水平降低,且内膜面积、中膜面积及两者比值均降低(P0.05);CGRP-H组的以上指标均优于CGRP-L组(P0.05)。结论 CGRP可抑制颈动脉球囊介入损伤后大鼠新生内膜形成,可通过促进EPC动员及升高外周血促血管修复因子辅助内皮损伤修复。     

金属蛋白涂层支架用于小型猪冠状动脉质粒介导下转基因研究

目的 :评价在质粒介导下 ,金属蛋白涂层支架向血管内局部转基因的可行性、效率和选择性。　　方法 :金属支架由 316 L不锈钢丝编织而成 ,其涂层是通过把支架放入有交联剂的明胶溶液中浸泡而成。基因载体为Pc DNA2质粒 ,并携带有β-半乳糖苷酶标记基因 ,该基因编码核特异性β-半乳糖苷酶。首先将蛋白涂层支架分别固定在3.0 mm或 3.5 mm经皮冠状动脉腔内成形术球囊上并在浓度为 8μg/μl的基因原液中浸泡 3分钟 ,然后通过 8F大腔引导导管将支架送入小型猪冠状动脉前降支中段 (转基因组 ,n=3) ,另外把没有浸泡过基因的支架也送入小型猪冠状动脉前降支中段 (对照组 ,n=3)。在支架植入后 7天处死动物。β-半乳糖苷酶表达由 X- Gal染色评估。　　结果 :所有转基因动物均有基因表达。转基因表达出现在内膜、中层和外膜。中层平滑肌细胞转染率为 3.0 %。远处器官和对照组冠状动脉均未显示核特异性β-半乳糖苷酶阳性表达。　　结论 :蛋白涂层支架在质粒介导下向血管内转基因有效、可行 ,因此 ,它有可能成为冠状动脉腔内成形术后再狭窄基因治疗的有效转基因系统。     

Neuronal Nitric Oxide Synthase Gene Transfer into the Rat Prostate Using In Vivo Electroporation

Objectives: It has been reported that nitric oxide (NO) mainly contributes to prostate or urethral smooth muscles relaxation, and that nitrergic innervation and neuronal NO synthase (nNOS) levels are decreased in benign prostatic hyperplasia. The purpose of the present study was to evaluate the feasibility to gene therapy for benign prostatic hyperplasia by transferring nNOS gene into the rat prostate with in vivo electroporation (EP) procedure. Methods: Male Sprague–Dawley rats were divided into four groups (sham, only EP, only nNOS injection, and nNOS gene injection with EP groups). Fifty micrograms of luciferase gene and nNOS expression vectors in 50 µL of K‐PBS (potassium‐phosphate buffered saline) were injected into the prostate. Immediately after the injection of these vectors, the vector injection points were electroporated by the two‐square parallel electrodes. Two days after gene transfer, luciferase analysis and an immunohistochemical staining for nNOS were performed, and NO₂^?/NO₃^? (NO_X) release was measured using high‐performance liquid chromatography coupled with the microdialysis procedure. Results: The optimal electric pulse conditions were 50 V, 1 Hz and 10 msec. In vivo EP with these conditions showed the increase in the luciferase gene expression approximately 300‐fold of the control group. In the nNOS gene injection with EP group, the marked nNOS immunoreactivity was observed, and NO_X release was significantly higher, as compared to other groups. Conclusion: The results suggest that EP is a feasible technique for in vivo gene transfer into the rat prostate, and that the transferred nNOS gene functionally expresses and contributes to NO production.     

Selective Photothermolysis of Blood Vessels Following Flashlamp-Pumped Pulsed Dye Laser Irradiation: In Vivo Results and Mathematical Modelling Are in Agreement

Laser therapy using the pulsed dye laser is the standard treatment for port-wine stains (PWS). But the mechanism of action has not been elucidated completely, yet. The dorsal skin-fold chamber model in hamsters was used to investigate the effects of laser treatment (λ_em=585 nm; pulse duration: 0.45 ms; fluence: 6 J per cm²) on blood vessels. Vessels (n=3394) were marked with FITC dextran (MW 150 kDa) and diameters (2–186 μm) were measured using intravital fluorescence microscopy up to 24 h following irradiation. Histology (H&E, TUNEL, CD31) was taken 1 or 24 h after irradiation. The experimental results were compared with the predictions of a mathematical model based on the finite-element method. Following irradiation treatment the number of unperfused vessels decreases with decreasing vessel diameter in vivo . Histology indicated a restriction of tissue injury to the irradiated area after 1 h. Blood vessels contained aggregated red blood cells. After 24 h tissue damage occurred also outside the irradiated area and thrombus formation was visible. These results were in agreement with the mathematical calculations. In addition to initial physical effects after pulsed dye laser treatment delayed biological processes contribute significantly to the reduction of perfused blood vessels. Because of incomplete photocoagulation of smaller blood vessels (∅ 2–16 μm) a complete bleaching of PWS seems to be unlikely.     

核因子κB的反义和诱骗性寡核苷酸对大鼠球囊损伤后血管狭窄和新生内膜形成的影响

探讨核因子κBp6 5亚基的反义和诱骗性寡核苷酸单独或联合作用对大鼠颈动脉球囊损伤细胞间粘附分子、单核细胞趋化因子的作用。SD大鼠随机分为 7组 ,每组分为 6个时相点 (6h和 1、3、5、7、1 4d) ,每个时相点 3只大鼠。制作血管球囊损伤模型。相应时间点处死动物。模型组、正义组、诱骗对照组血管内膜面积、中膜面积、内膜 中膜在第 5天增加 ,1 4d达到高峰。管腔面积随时相点减小。反义组、诱骗组、反义组 +诱骗组干预后 ,血管上述病理指标明显改善 (P <0 .0 5 ) ,反义组 +诱骗组较反义组、诱骗组治疗效果更明显 (P <0 .0 5 )。内皮细胞间粘附分子、单核细胞趋化因子mRNA表达在血管损伤后 6h即可检测到 ,3、5、7d后持续表达增加 ,1 4d后表达降低。免疫组织化学检测显示 ,内皮细胞间粘附分子 1、单核细胞趋化因子 1蛋白质表达在 6个时相点均为阳性染色 ,1 4d达到高峰 ;反义组、诱骗组、反义组 +诱骗组治疗后 ,与模型组、正义组、诱骗对照组相比 ,内皮细胞间粘附分子、单核细胞趋化因子mRNA表达和蛋白合成在各时相点均降低 (P <0 .0 5 )。免疫印迹法检测核蛋白抽提物显示 ,核因子κBp6 5在血管损伤后 6h有一定蛋白表达 ,1d后蛋白表达明显增加 ,至 7d达高峰 ,1 4d后蛋白表达略降低。反义组、诱骗组、反义组     

A VSV-G Pseudotyped Last Generation Lentiviral Vector Mediates High Level and Persistent Gene Transfer in Models of Airway Epithelium In Vitro and In Vivo

The aim of this work was to evaluate the efficiency and duration of gene expression mediated by a VSV-G pseudotyped last generation lentiviral (LV) vector. We studied LV efficiency in ex-vivo models of respiratory epithelial cells, obtained from bronchial biopsies and nasal polyps, by GFP epifluorescence and cytofluorimetry. In vivo efficiency and persistence of gene expression was investigated by GFP immunohistochemistry and luciferase activity in lung cryosections and homogenates, respectively, upon intranasal and intratracheal administration protocols in C57Bl/6 mice. Both primary bronchial and nasal epithelial cells were transduced up to 70-80% 72 hr after the LV infection. In vivo nasal luciferase expression was increased by lysophosphatidylcholine pre-treatment of the nose. Conversely, the bronchial epithelium was transduced in the absence of any pre-conditioning treatment and luciferase expression lasted for at least 6 months without any decline. We conclude that a last generation LV vector is a promising gene transfer agent in the target organ of genetic and acquired lung diseases, as in the case of cystic fibrosis.     

应用内皮型一氧化氮合酶基因转染冠状动脉旁路移植血管桥的研究进展

冠状动脉旁路移植术后血管桥再狭窄一直困扰着冠状动脉外科的发展,许多研究表明血管桥再狭窄的发生与一氧化氮的功能异常相关,用转基因方法转移重组内皮型一氧化氮合酶基因,重建内源性一氧化氮功能,是冠状动脉旁路移植术后血管桥再狭窄治疗的新策略,本文就血管桥转基因一氧化氮合酶同工酶的选择、靶细胞的选择、转基因载体、就转基因的检测及应用等国内外研究进展作一综述。