自体静脉移植围手术期血浆ET-1测定的护理学价值

随着我国人们生活水平的提高,饮食结构的改变,社会老龄化日趋严重,动脉硬化及其闭塞性疾病的发病率不断升高,周围动脉硬化闭塞症(ASO)的发病率亦越来越高[1],恢复血管通畅性、促进侧枝循环的建立、防止肢体坏死、减少截肢率、尽可能地保全肢体是外科治疗的原则,自体静脉移植是治疗ASO最常见的手术方法,但由于自体静脉移植术后血管活性因子的异常,尤其是以ET-1为代表的血管收缩性活性因子的升高,导致血管内膜增生,平滑肌细胞增殖,引起血管狭窄甚至闭塞,严重影响了移植血管的通畅率,严重影响了手术治疗效果.为探讨动脉硬化闭塞症自体静脉移植术后血浆ET-1水平的变化规律及对护理监测的意义,我们对34例围手术期患者的血浆ET-1水平进行了监测,现将结果报告如下.     

纳米粒子介导Egr-1 DNA酶转染抑制移植静脉内膜增生**☆

背景：应用基因防治移植血管狭窄、闭塞现已被普遍公认,但如何增加其转染效率现已成为焦点及热点问题。 目的：观察以纳米粒子为载体的外源性Egr-1 DNA酶局部转染对移植静脉内膜增生的影响。 方法：构建Egr-1 DNA酶,应用聚乳酸聚乙醇酸共聚物和聚乙烯醇包载Egr-1 DNA酶,制备纳米级粒子混合物。建立自体静脉移植模型210只,随机分成3组,转基因组转染以纳米粒子为载体的Egr-1 DNA酶,空载体组单纯转染纳米粒子包载的空载体,对照组不予特殊处理。 结果与结论：转基因组内膜中Egr-1基因的mRNA及蛋白产物表达较其他两组明显减少(P < 0.05);在术后7,14,28 d,转基因组内膜增生厚度较其他两组明显减少(P < 0.01);转基因组血管平滑肌细胞凋亡百分比较其他两组明显增高(P < 0.05)。结果表明Egr-1 DNA酶的表达能有效抑制自体移植静脉内膜的增生及促进血管平滑肌细胞凋亡。 关键词：纳米粒子;DNA酶;移植静脉;转染;血管平滑肌细胞 doi:10.3969/j.issn.1673-8225.2012.08.006     

腺病毒介导Gax基因对血清刺激后兔血管平滑肌细胞增殖和凋亡的影响

目的:通过腺病毒介导人生长终止特异性同源异形盒基因(Gax)转染体外兔血管平滑肌细胞(VSMCs),观察人Gax基因过表达对血清刺激后兔VSMCs增殖和凋亡的影响,为Gax基因成为理想的静脉桥再狭窄基因治疗的候选基因提供实验基础。方法:Ad5-hGax重组腺病毒载体转染兔VSMCs后,采用Western blot检测不同时间细胞中hGax蛋白的表达;MTT法检测Ad5-hGax对血清刺激后兔VSMCs的体外增殖抑制情况;转染72 h后流式细胞术检测Ad5-hGax诱导血清刺激后VSMCs的凋亡率。结果:转染后1 d、3 d、5 d,Ad5-hGax组细胞中的hGax蛋白均有明显表达;MTT法检测显示hGax基因过表达能明显抑制血清刺激后兔VSMCs的增殖;流式细胞术检测显示转染72 h后,hGax过表达能显著诱导血清刺激后兔VSMCs的凋亡。结论:hGax基因过表达能有效抑制血清刺激后VSMCs的增殖,并促进其凋亡,为腺病毒介导Gax基因治疗静脉桥再狭窄提供重要的实验基础。     

冠状动脉旁路移植术后静脉桥再狭窄的基因治疗研究进展

冠状动脉旁路移植术是治疗缺血性心脏病安全、有效的方法.自体大隐静脉是术中最常用的移植血管材料.但是血栓形成、内膜增厚和粥样硬化导致的静脉桥再狭窄严重影响了其通畅率,而且至今尚无有效的治疗药物.分子生物学技术理论的发展为冠状动脉旁路移植术后静脉桥再狭窄的防治提供了新思路,基因治疗静脉桥再狭窄已成为目前研究的热点.本文主要从静脉桥再狭窄防治的目的基因、基因导入方式、基因载体等方面,对静脉桥再狭窄基因治疗的研究进展进行综述.     

纤维蛋白胶外支架转染PCNA基因反义寡核苷酸预防移植静脉再狭窄

背景：前期研究成果,纤维蛋白胶不仅是一种良好的非限制性、生物可降解血管外支架,能够预防移植静脉内膜和中膜增生,而且是一种良好的药物缓释系统,能够提高血管外膜基因转染效率。 目的：验证应用纤维蛋白胶外支架转染PCNA基因反义寡核苷酸预防移植静脉再狭窄的作用。 方法：建立兔颈外静脉颈总动脉旁路移植模型,随机分为模型组、纤维蛋白胶支架组、纤维蛋白胶联合反义PNCA组。后两组分别给予纤维蛋白胶和纤维蛋白胶与携带PCNA反义寡核苷酸腺病毒的混合物于静脉桥外周均匀喷洒。 结果与结论：移植后第28天模型组静脉移植物内膜和中膜增生明显,纤维蛋白胶外支架组静脉移植血管内膜和中膜增生程度明显减少(P  < 0.01),纤维蛋白胶联合反义PNCA组内膜和中膜增生程度明显少于纤维蛋白胶外支架组(P  < 0.05)。纤维蛋白胶联合反义PNCA组PCNA基因mRNA及蛋白表达明显减少纤维蛋白胶外支架组(P < 0.05),纤维蛋白胶外支架组表达明显弱于模型组(P  < 0.05)。提示血管外膜反义PCNA转染可进一步抑制移植静脉管壁PCNA表达,纤维蛋白胶外支架联合反义PCNA对移植静脉内膜和中膜增生有协同抑制作用。     

转染TFPI基因对人前列腺平滑肌细胞增殖的影响

目的良性前列腺增生（BPH）是严重危害老年男性健康的常见疾病,本研究旨在研究组织因子途径抑制因子（TFPI）基因对前列腺平滑肌细胞生长的影响,为良性前列腺增生的基因治疗提供参考依据。方法取前列腺增生患者手术切除的前列腺组织,采用酶消化法分离前列腺平滑肌细胞;免疫组化方法进行细胞鉴定;pIRES—TFPI基因转染前列腺平滑肌细胞,以pIRES基因作为基因转染阴性对照;用细胞计数法和四氮唑蓝MTT法观察细胞增殖情况;采用RT-PCR检测细胞内TFPI基因的表达情况。结果SMA免疫组化染色和MASSON染色显示：经过5次传代后,前列腺平滑肌细胞的纯度达到95％以上;TFPI基因转染后,前列腺平滑肌细胞内的TFPImRNA水平明显提高,是未转染组的7倍、阴性对照基因转染组的3．5倍;基因转染4d后,TFPI基因转染组的前列腺平滑肌细胞数明显低于阴性对照基因转染组。结论提示TFPI基因对前列腺平滑肌细胞的增殖具有调控作用,有必要对其作用机理进行进一步研究。     

血管性假血友病因子预测静脉留置针致兔耳缘静脉血栓形成的研究

目的观察静脉留置针致兔耳缘静脉血栓形成、局部血管内皮损伤以及血浆血管性假血友病因子（von Willebrand factor,vWF）表达情况,分析vWF改变与血栓形成的相关性。方法60只大耳白兔随机分成3、5、7d三组,取右耳外侧耳缘静脉置20号留置针,每日经留置针输入生理盐水20ml模拟临床输液,检测置针前后血浆vWF水平变化,并各组分别于第3、5、7d取置针侧耳缘静脉做病理切片,光镜下观察血管内血栓形成,透射电镜观察血管内皮超微结构变化。结果3、5、7d组血栓形成率分别为45%、78.9%、89.5%。3d组血管内皮层完整,线粒体、内质网无明显变化;5d组血管内皮层基本完整,局部内皮细胞增生,线粒体、内质网水肿;7d组血管内皮层多破坏缺失,暴露内皮下基质,严重者血管被破坏,结构不清,可见大量脱落坏死细胞。3d组、5d组vWF水平与血栓形成情况呈正相关,7d组内vWF水平与血栓形成情况呈负相关。结论血管内皮损伤为静脉留置针血栓形成机制之一,vWF可以作为预测静脉留置针血栓发生风险指标。     

真核表达载体pCMV-(Kozak)TFPI的构建及其在内皮细胞中的表达

目的构建真核表达载体pCMV-(Kozak)TFPI并检测其在内皮细胞中的表达,为冠状动脉旁路移植和经皮冠状动脉内成形术中转染血管内皮细胞抗凝治疗作了实验和理论的探索。方法用RT-PCR的方法提取人组织因子途径抑制因子(TFPI)基因,并引入Kozak序列,将(Kozak)TFPI亚克隆入pCMV质粒中。在阳离子脂质体介导下,转染人脐静脉内皮细胞(HUVEC)。RT-PCR、免疫荧光和Western blot检测HUVEC中外源TFPI基因mRNA和蛋白的表达。结果(Kozak)TFPI成功克隆入pC-MV中。RT-PCR、免疫荧光和Western blot检测到外源TFPI基因mRNA和蛋白的表达。结论成功构建了pCMV-(Kozak)TFPI真核表达载体,并表达出相应的蛋白。     

缺氧诱导因子1在创伤性深静脉兔模型血栓形成及消退过程中的表达

背景：深静脉血栓形成及消退过程中的分子机制极其复杂,目前已有研究表明,缺氧与损伤因素参与了深静脉血栓形成及消退过程。 目的：观察静脉壁中缺氧诱导因子1在创伤性深静脉血栓模型兔血栓形成及消退过程中的表达变化。 方法：将日本大耳白兔采用钳夹双侧股静脉+石膏固定双下肢建立兔创伤性深静脉血栓模型。PCR及ELISA检测股静脉组织中缺氧诱导因子1 mRNA、蛋白在兔创伤性深静脉血栓建模后表达的变化。 结果与结论：模型兔创伤后24 h,经B超监测,血栓形成率为58%;血栓栓子随造模后时间延长逐渐缩小机化,创伤后第5天开始经B超监测模型组血栓形成的血管腔有断续血流信号。模型兔股静脉组织中缺氧诱导因子1在造模后1,3,5 d表达逐渐升高(P < 0.05或P < 0.01),此后7-14 d表达逐渐下降(P < 0.05或P < 0.01)。结果证实,在创伤性深静脉血栓形成过程,缺氧诱导因子1表达上调,在血栓形成后的溶解及机化再通过程中,缺氧诱导因子1表达下调。     

自体移植静脉模型中环氧化酶2表达及其与移植静脉狭窄的关系

背景：环氧化酶2是与炎症反应密切相关的酶,其表达程度可能与移植静脉的狭窄相关。 目的：观察环氧化酶2在移植静脉中的表达情况,分析环氧化酶2的表达与移植静脉血管狭窄的相关性。 方法：实验分为2组,实验组在建立兔自体颈外静脉-颈总动脉移植模型后应用环氧化酶2抑制剂干预,对照组建模后正常喂养,于静脉移植后2,8,12周观察移植静脉血管壁增生情况及血管中环氧化酶2表达的差异。 结果与结论：术后各移植静脉均保持通畅。正常静脉无明显环氧化酶2表达,移植静脉环氧化酶2表达明显增高。实验组移植静脉中环氧化酶2 mRNA表达较对照组低,移植静脉内膜及中膜增厚程度也低于对照组。结果表明,环氧化酶2在移植静脉中出现明显的表达增高,抑制移植静脉中环氧化酶2的表达可以减缓移植静脉血管壁的增生。     

Processed human umbilical veins as arterial substitutes — evaluation in canine models

Human umbilical cord vein segments have been used as vessel substitutes for damaged or occluded arteries, as aorto-coronary by-passes and as arterio-venous fistulae for dialysis. The Dardik-Biograft® fixed with glutaraldehyde and the Mindich-Bioflow®, fixed with ethanol and dialdehyde starch, are commercially available. They were implanted in dogs as replacements for a segment of the abdominal aorta. Post-implantation status was followed by angiography. They were evaluated after removal from sacrificed animals with the aid of scanning electron microscopy and histological techniques. Attention was focused on vessel patency, dimensional stability, integrity of the anastomosis line, lumen wall microstructure, evidence of suture damage and thrombus deposition pattern. Both types of grafts gave functional by-passes for at least until 6 months post-implantation. The Dardik-Biograft® appeared more prone to thrombus formation near the anastomosis. Sparse cellular development was also noted. The Mindich-Bioflow® gave rise to a prosthesis of superior thromboresistance which was more subject to mechanical damage.     

Prebypass histological and ultrastructural evaluation of the long saphenous vein as a predictor of early graft failure

BACKGROUND: Twenty percent of the long saphenous vein (LSV) grafts that are employed as coronary bypass conduits occlude during the first year after the operation. The aim of this study was to evaluate the morphological parameters of the LSV grafts before implantation as predictors for the early occlusion of the grafts. METHODS: Forty-two samples of LSV grafts were examined via light, transmission electron, and scanning electron microscopy and evaluated clinically and by angiography at 6 months and 2 years after the operation. Morphological parameters were statistically analyzed and examined for their significance on the viability of the vein grafts. RESULTS: Six (14.28%) of the examined grafts occluded within the first 6 months after the operation, and 11 grafts (26.19%) occluded within the first 2 years. The grafts that occluded at 6 months were characterized by thick intima (mean value, 206+/-32.29 vs. 67.44+/-10.17 in the group functioning normally and 98.42+/-34 in the group occluded within 2 years), low endothelial coverage (22.7+/-4.04 vs. 64.61+/-2.89 and 26.06+/-1.78 in the corresponding groups), and narrow lumen (46.73+/-9.69 vs. 527.18+/-45.78 and 204.26+/-16.5 in the corresponding groups). The presence of foam cells, edema, calcification, neovascularization, and thrombus in the lumen of the veins is frequently observed in the wall of the occluded vein grafts, whereas fibrosis does not seem to be related. CONCLUSIONS: LSV grafts with low endothelial cell coverage, stenosis of the lumen, and thick walls are at an increased risk of developing intrawall lesions that lead to early graft failure.     

Salusin-a基因真核表达载体的构建及在HEK293细胞中的表达

目的:研究增强型绿色荧光蛋白真核表达载体Salusin-a(pEG-FP-Salusin-a)的构建及在人胚胎肾细胞系293细胞(HEK293)中的表达。方法:提取人单核细胞系THP-1细胞Salusin-a的mRNA,逆转录多聚酶链反应(RT-PCR)扩增Salusin-a基因,克隆入pEGFP-N3载体,双酶切、菌落PCR及测序鉴定pEGFP-Salusin-a质粒。用脂质体转染pEGFP-Salusin-a入HEK293细胞,分为转染细胞组、质粒对照组和空白对照组,检测分析各组荧光蛋白和Salusin-a基因的表达。结果:成功构建pEGFP-Salusin-a质粒,并转染HEK293,细胞转染效率86%,转染细胞组和质粒对照组绿色荧光蛋白的表达明显高于空白对照组(P0.05);转染细胞组Salusin-amRNA的表达明显高于质粒对照组和空白对照组(P0.05)。结论:重组pEGFP-Salusin-a质粒能转染HEK293细胞并表达Salusin-a,为Salusin-a基因功能的进一步研究提供了实验基础。     

人脐动脉平滑肌细胞培养及转染TFPI基因的实验研究

目的 探讨组织因子途径抑制因子（TFPI）基因对人脐动脉血管平滑肌细胞生长的影响,为TFPI基因用于血管再狭窄的治疗提供理论依据和实验基础.方法 从人脐动脉分离平滑肌细胞,通过免疫组化方法进行细胞鉴定;用不同剂量pIRES-TFPI基因（分别为1,2,3 μg/mL）转染血管平滑肌细胞,采用RT-PCR测定细胞内TFPI表达以优化基因转染条件;通过MTT法测定TFPI基因对人脐动脉血管平滑肌细胞生长的影响.结果 分离得到的血管平滑肌细胞的纯度高于90%;3个剂量的基因转染后,细胞内TFPI基因的表达水平无明显差异.采用2 μg/mL转染剂量时,TFPI基因转染后第5天,脐动脉血管平滑肌的生长受到明显抑制.结论 通过基因转染的方式将TFPI基因导入细胞对人脐动脉平滑肌的增殖具有抑制作用.     

Pretreatment of a Dacron graft with tissue factor pathway inhibitor decreases thrombogenicity and neointimal thickness: a preliminary animal study

The purpose of this study was to evaluate the effects of locally applied TFPI on the reduction of neointimal thickness in Dacron grafts. Seven millimeter internal diameter 5 cm lengths of albumin coated knitted Dacron grafts were interposed in the infrarenal aorta in 14 mongrel dogs. Before implantation, the grafts were immersed in saline solution containing 200 microg/ml of TFPI (TFPI group, n = 7) or 100 IU/ml of heparin (control group, n = 7) for 15 minutes at room temperature. Three months after implantation, neointimal thickness and percentage of graft stenosis were measured by computerized planimetry. All grafts were patent 3 months after implantation. Thrombus was found in one graft in the TFPI group, but observed in three of seven control grafts. Neointimal thickness in the TFPI group was significantly smaller than that in the control group (mean +/- SD, 0.26 +/- 0.1 mm vs. 0.57 +/- 0.15 mm, p < 0.001). Percentage of graft stenosis was significantly lower in the TFPI group than in the control group (13.4 +/- 5.3% vs. 26.9 +/- 7.0%, p < 0.001). Scanning electron micrographs showed that the neointima of TFPI treated grafts were completely covered by endothelial cells. The present results indicate that locally applied TFPI reduces thrombogenicity and neointimal thickness in albumin coated knitted Dacron grafts.     

siRNA沉默AFP基因对肝癌细胞系 EGHC-9901 增殖的影响

 目的 建立稳定表达AFP-siRNA质粒的肝癌细胞系并探讨对其增殖的影响。方法 构建AFP-siRNA,用脂质体法转染肝癌细胞系,G418筛选4～5周,Western blot 及RT-PCR检测靶基因表达,分组：实验组,转染AFP-siRNA组;阳性对照组,转染空载体组;空白对照组,未处理组。MTT绘制生长曲线,平板克隆实验观察集落形成,流式细胞仪分析细胞周期。结果 成功建立稳定表达AFP-siRNA的肝癌细胞系,实验组表达AFP近乎完全抑制;实验组增殖能力显著低于空载体组;实验组＞75μm集落形成数19±2,低于空载体组62±6;实验组G1期细胞数较空载体组提高20％左右。 结论 成功建立稳定表达AFP-siRNA的肝癌细胞系,抑制AFP表达可使肝癌细胞发生G1期阻滞,明显抑制其增殖及集落形成能力。     

人组织因子途径抑制因子重组蛋白表达菌株的建立

组织因子途径抑制因子是机体凝血过程的主要抑制因子，在血栓形成性疾病的防治中具有广阔的应用前景。本研究采用pGEX—2T为表达载体、大肠杆菌细胞为表达宿主进行了人组织因子途径抑制因子重组蛋白的制备。制备的重组蛋白产量较高，纯化过程简单，且具有良好的抑制组织因子的功能。     

Patient-derived endothelial progenitor cells improve vascular graft patency in a rodent model

Late outgrowth endothelial progenitor cells (EPCs) derived from the peripheral blood of patients with significant coronary artery disease were sodded into the lumens of small diameter expanded polytetrafluoroethylene (ePTFE) vascular grafts. Grafts (1 mm inner diameter) were denucleated and sodded either with native EPCs or with EPCs transfected with an adenoviral vector containing the gene for human thrombomodulin (EPC + AdTM). EPC + AdTM was shown to increase the in vitro rate of graft activated protein C (APC) production 4-fold over grafts sodded with untransfected EPCs (p < 0.05). Unsodded control and EPC-sodded and EPC + AdTM-sodded grafts were implanted bilaterally into the femoral arteries of athymic rats for 7 or 28 days. Unsodded control grafts, both with and without denucleation treatment, each exhibited 7 day patency rates of 25%. Unsodded grafts showed extensive thrombosis and were not tested for patency over 28 days. In contrast, grafts sodded with untransfected EPCs or EPC + AdTM both had 7 day patency rates of 88-89% and 28 day patency rates of 75-88%. Intimal hyperplasia was observed near both the proximal and distal anastomoses in all sodded graft conditions but did not appear to be the primary occlusive failure event. This in vivo study suggests autologous EPCs derived from the peripheral blood of patients with coronary artery disease may improve the performance of synthetic vascular grafts, although no differences were observed between untransfected EPCs and TM transfected EPCs.