低能量红激光局部照射对自体移植静脉内膜增生的影响

目的探讨低能量红激光局部照射对自体移植静脉内膜增生的影响.方法取80只大鼠,随机分为单纯自体静脉移植组(对照组,n=40)和低能量红激光局部照射组(激光组,n＝40).两组大鼠分别于术后第3 d、7 d、14 d及28 d取移植静脉段(每时相组各l0只)固定,行HE染色、弹力纤维染色及增殖细胞核抗原(PCNA)免疫组化染色,应用光学显微镜和计算机图像分析系统分析测定其内膜厚度和管腔面积.结果术后第3 d,两组管腔横截面积及内膜平均厚度差异无显著性意义(P＞0.05);激光组术后第7 d、14 d、28 d内膜平均厚度低于对照组(P＜0.05),管腔横截面积明显大于对照组(P＜0.05);PCNA免疫组化染色结果显示,激光组移植静脉平滑肌细胞(VSMC)增殖受到明显抑制(P＜0.01).结论低能量红激光局部照射可以抑制自体移植静脉内膜及VSMC的增生,减少再狭窄的发生.     

反义寡脱氧核苷酸抑制survivin基因表达对移植静脉内膜增生的影响

目的探讨生存素（survivin）的反义寡脱氧核苷酸（ASODNs）抑制survivin基因表达对移植静脉内膜增生的抑制作用。方法Wistar大鼠60只，建立自体静脉移植模型，然后随机均分为对照组、survivin ASODNS 50μg和200μg组、正义寡脱氧核苷酸组（ODNs组）及Lipofectin＋pluronic组共5组，施加不同的处理因素，分别在静脉移植术后7和14d取材。组织形态学方法比较移植静脉内膜增生程度，半定量RT—PCR法检测survivin基因mRNA的表达，Western blot检测survivin基因的蛋白产物表达，免疫组化法检测survivin及增殖细胞核抗原（PCNA）的表达，TUNEL检测血管平滑肌细胞（VSMC）凋亡的变化。结果对照组、ODNs组和Lipofectin＋pluronic组移植术后7d、14d移植静脉内膜增生明显，但3组间差异无统计学意义（P〉0．05），局部转染50μg survivin ASODNs能明显抑制其内膜增生（P〈0．05），200μg组受抑制程度更明显（P〈0．05）。对照组survivin mRNA及蛋白产物表达显著增加，而survivin ASODNs组却显著减少（P〈0．05），VSMC中PCNA表达也同时减少（P〈0．05），而VSMC凋亡明显增加（P〈0．05）。结论survivin ASODNs可显著抑制移植静脉内膜增生，其作用可能是通过抑制survivin的基因及蛋白产物表达，从而减轻VSMC增殖，促进其凋亡而实现的。     

p27kip1基因表达对大鼠移植静脉内膜平滑肌细胞增殖与凋亡的影响

目的观察以聚乳酸聚乙醇酸（PLGA）纳米粒子为载体的人p27^kip1基因局部转染自体移植静脉后，对大鼠静脉内膜平滑肌细胞增殖及凋亡的影响。方法Wistar大鼠120只建立自体静脉移植模型，随机分成3组。转基因组：移植静脉转染以PLGA纳米粒子为载体的p27^kip1基因；空白对照组：转染不含有p27^kip1基因的单纯PLGA纳米粒子；单纯静脉移植组：使用生理盐水。分别于术后3、7、14、28d取材，常规HE、Verhoeff弹力纤维染色，Western blot检测p27^kip1蛋白的表达，免疫组化（SABC）法检测增殖细胞核抗原（PCNA）的表达、TUNEL法观察内膜平滑肌细胞凋亡的动态变化。结果转基因组内膜中p27^kip1蛋白表达水平高于其他组；内膜平均厚度7、14、28d低于其他2组（P〈0．01）；转基因组内膜PCNA的表达7、14d明显受到抑制（P〈0．01），平滑肌细胞的凋亡细胞百分比于7、14d较对照组明显增加（P〈0．01），单纯静脉移植组与空白对照组之间各项监测指标差异无统计学意义。结论p27^kip1基因的过表达能够有效抑制自体静脉移植后的内膜增生（IH），促进平滑肌细胞的凋亡。     

转染survivin反义寡脱氧核苷酸对移植血管内膜增生的影响

目的研究survivin基因的反义寡脱氧核苷酸（ASODN）对移植血管内膜增生的影响。方法Wistar大鼠60只，建立自体静脉移植模型，术后随机分为5组：对照组，survivin ASODN 50μg组，200μg组，正义对照组，lipoectin＋pluronic组。分别施加不同的处理因素，在移植后1，2周取材。用组织形态学方法比较内膜增生程度；用半定量RT-PCR检测survivin基因的mRNA表达；Western blot检测survivin基因的蛋白产物表达；免疫组化方法检测survivin及增殖细胞核抗原（PCNA）的表达，TUNEL法检测血管平滑肌细胞（VSMC）凋亡。结果静脉移植1～2周内膜增生明显，局部转染50μg survivin ASODN后明显抑制内膜增生（P〈0．05），200μg组受抑制程度较50μg组更为显著（P〈0．05）。静脉移植后，对照组survivin的mRNA及蛋白产物表达显著增加，而survivin ASODN组却显著减少（P〈0．05），VSMC中PCNA表达也同时减少，而TUNEL阳性细胞明显增加。结论survivin ASODNs可显著抑制移植静脉的内膜增生；其作用可能是通过抑制survivin的基因及蛋白产物表达，促进VSMC凋亡而实现的。     

反义寡核苷酸抑制Survivin基因表达对移植静脉内膜增生的影响

目的研究反义寡脱氧核苷酸（ASODN）抑制Survivin基因表达对移植静脉内膜增生的抑制作用。方法Wistar大鼠60只，建立自体静脉移植模型，术后随机分为：对照组、Survivin ASODN 50、200μg组、正义对照组、Lipofectin＋pluronic等五个组，施加不同的处理因素，在移植后1、2周取材。组织形态学方法比较内膜增生程度，逆转录．聚合酶链反应（RT-PCR）检测Survivin基因的mRNA表达，Westem blot检测Survivin基因的蛋白产物表达，免疫组织化学方法检测Survivin及增殖细胞核抗原（PCNA）的表达，脱氧核苷酸转移酶末端标记法（TUNEL）检测血管平滑肌细胞（VSMC）凋亡的变化。结果移植后1、2周内膜增生明显，局部转染50μg Survivin ASODN组内膜增生明显受抑制（P〈0．05），200μg组受抑制程度更为明显（P〈0．05）；与对照组相比，Survivln ASODN组Survivin的mRNA及蛋白产物表达显著减少（P〈0．05），PCNA阳性表达同时减少，而TUNEL阳性细胞却明显增加。结论Survivin ASODN可显著抑制移植静脉的内膜增生，其作用可能是通过抑制Survivin基因及其蛋白产物表达，从而抑制VSMC增殖、促进其凋亡而实现的。     

纳米粒子包载反义雷帕霉素靶蛋白基因转染对移植静脉内膜增生的影响

目的观察纳米粒子包载的反义雷帕霉素靶蛋白（mTOR）基因局部转染对移植静脉内膜增生的影响。方法应用聚乳酸聚乙醇酸共聚物（PLGA）和聚乙烯醇（PVA）包载mTOR基因。建立自体静脉移植模型，随机分成转基因组、空载体组、对照组。转基因组移植静脉转染纳米粒子包载的反义mTOR基因，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。分别于移植3、7、14、28d取材，常规苏木素．伊红（HE）、Verhoeff染色，检测mTOR基因的mRNA及蛋白产物表达的变化，TUNEL法观察血管平滑肌细胞（VSMC）凋亡的动态变化。结果转基因组内膜中mTOR基因的mRNA及蛋白产物表达明显减少（P〈0．01）；转基因组内膜增生程度在术后7、14、28d较其他组明显减少（P〈0．01）；转基因组凋亡细胞明显增多（P〈0．01）。结论纳米粒子可以作为基因载体，反义mTOR基因的表达能够抑制移植静脉内膜的增生，促进VSMC凋亡。     

低强度微波辐射对兔髂外动脉损伤后再狭窄的影响

目的 探讨低强度微波（LIM）辐射对血管再狭窄病理过程的影响。以评价LIM辐射防治再狭窄的可能性。方法健康雄性新西兰大白兔48只。随机分为2组。应用Fogarty导管损伤兔髂外动脉（EIA）内膜，建立再狭窄动物模型。辐射组采用2450MHz微波。功率分别为2、5和10mW／cm^2。局部辐射EIA（1h／d）。分别于术后3、7、14及28d取材。采用HE和EF染色。观察EIA形态学改变及内膜面积、中膜面积及管腔狭窄率；TUNEL标记法检测细胞凋亡；TEM观察血管平滑肌细胞（VSMC）表型及超微结构。结果LIM微波辐射后，再狭窄早期炎症反应受到抑制，附壁血栓减少，各时间点VSMC的增殖、迁移被抑制，内膜面积和管腔狭窄率显著低于对照组（P〈0．01）。细胞凋亡率在术后3d各组间差异无统计学意义（P〉0．05）。其余时间点，辐射组显著高于对照组（P〈0．01）,其中5mW／cm^2者细胞凋亡率明显高于其他各组（P〈0．01）。TEM观察到对照组大量合成型VSMC术后14d达（93．50＋3．45）％,辐射组VSMC以收缩型为主，大量成纤维细胞和VSMC呈凋亡的形态学改变。结论LIM辐射能显著抑制再狭窄病理过程中的早期炎症反应和VSMC的增殖、迁移，促进细胞凋亡。有效抑制再狭窄的发生、发展。LIM辐射存在量效关系和功率窗效应。功率强度为5mW／cm^2时对再狭窄的效应最强。     

周围神经缺损神经移植修复对神经元细胞凋亡的影响

目的：应用移植神经的方法修复周围神经损伤,观察其对神经元的保护作用。方法：选用雄性SD大鼠30只。随机分成正常对照组、神经缺损组、神经损伤组、神经移植组,实验组每组9只大鼠,并按手术先后随机分成7、14、28d3个时间组。将大鼠左侧坐骨神经在梨状肌下缘碾挫或切除0．5cm,分别制备神经损伤或缺损动物模型,并采用神经原位移植的方法修复神经缺损。按术后不同时间处死动物。于术后7、14、28d取L4－6脊髓作TUNEL标记检测,标记凋亡的运动神经元数目。切片作HE、甲苯胺兰染色后计算脊髓内运动神经元的数目。结果：在术后28d,神经移植组的凋亡细胞明显少于另2组（U1＝2．10;U2＝2．89 P＜0．05）。各时间组的神经元数目,神经移植组明显多于另2组（t1=6.84;t2=6.95 P＜0．05）。结论：移植神经对周围神经损伤后的相应神经元,有一定的保护作用。     

晚期糖基化终产物受体在糖尿病大鼠自体移植静脉中的表达及氨基胍的干预作用

目的 探讨晚期糖基化终产物受体（RAGE）在糖尿病大鼠自体移植静脉中的表达及氨基胍对其内膜增生的干预作用。方法 雄性SD大鼠60只，随机均分为氨基胍组、蒸馏水组及对照组，前2组行链脲佐菌素腹腔注射建立糖尿病模型，并分别用氨基胍或蒸馏水灌胃，对照组为未作处理的正常大鼠。3组均建立自体静脉移植模型后，于术后第7d及14d测定血清晚期糖基化终产物（AGE）含量，同时取自体静脉移植标本进行组织形态学观察，Westernblot和免疫组织化学染色方法检测RAGE及NF-KBp65的蛋白表达，半定量RT-PCR检测RAGE及NF—xBp65mRNA的表达。结果 术后第7d及14d，相对于对照组大鼠，蒸馏水组糖尿病大鼠自体移植静脉内膜增生加重，血清AGE含量增加，RAGE和NF—kB p65mRNA和蛋白表达增强，差异均有统计学意义（P〈0．05）；氨基胍组血清AGE含量、NF—kB p65蛋白和mRNA表达及内膜增生均较蒸馏水组减少或减轻（P〈0．05），与对照组相比差异无统计学意义（P〈0．05）。结论 糖尿病大鼠自体移植静脉RAGE表达增强，激活NF—kB，与移植静脉内膜增生关系密切；氨基胍抑制AGE的产生，可阻断AGE-RAGE结合，减轻内膜增生。     

联合转染tPA基因和PCNA反义寡核苷酸抑制兔自体移植动脉再狭窄研究

目的研究血管局部联合转染组织型纤溶酶原激活物（tPA）基因和增殖细胞核抗原反义寡核苷酸（PCNA—ASODN）对自体移植动脉血管再狭窄的防治作用。方法120只新西兰雄性白兔，按数字表法随机均分为4组（对照组、PCNA—ASODN组、tPA组、tPA＋PCNA—ASODN组）。将同一只兔的左、右髂外动脉（各1、0cm长）对换移植，移植血管及吻合口缝线分别用pBudCE4．1／tPA和PCNA-ASODN液浸泡。于术后3、7、14、28及56d取移植血管制片，显微镜下观察内膜增生情况，计算机图像分析测量其内膜面积和移植血管狭窄率，扫描电镜观察移植血管壁血栓形成情况，并分别用RT—PCR和免疫组织化学染色法检测tPA基因的mRNA表达率与PCNA阳性细胞百分率。结果术后3、7、14和28d时，tPA组与tPA＋PCNA-ASODN组的tPA基因mRNA表达率明显高于另2组（P〈0．01），PCNA—ASODN组、tPA组和tPA＋PCNA—ASODN组PCNA阳性细胞百分率均明显低于对照组（P〈0．05，P〈0、01）。tPA＋PCNA—ASODN组和PCNA—ASODN组、tPA组各时相血管壁内膜增生比对照组明显减轻，内膜面积和管腔狭窄程度显著低于对照组（P〈0、05．P〈0．01），且tPA＋PCNA—ASODN组又明显低于另2组（P〈0．05）。扫描电镜观察显示，tPA组和tPA＋PCNA-ASODN组血管壁只见少量血小板附着，未见血栓形成，而对照组血管壁可见大量血小板附着，且有血栓形成。结论血管局部联合转染tPA基因和PCNA—ASODN能有效抑制VSMC增殖和血栓形成，阻止内膜增生，防止移植血管狭窄。     

Inhibitory effect of brachytherapy on intimal hyperplasia in arteriovenous fistula

PURPOSE: To determine whether endovascular radiation can inhibit intimal hyperplasia in a swine model of hemodialysis access. MATERIALS AND METHODS: Polytetrafluoroethylene arteriovenous grafts (6 mm in diameter) were placed between the common carotid artery and the external jugular vein bilaterally in 8 pigs. Two days after the surgery, fistulography was performed. Gamma radiation (12 Gy) was delivered endovascularly to one of the grafts at the venous anastomosis by using an iridium(192) source. Thus, the other graft in each pig served as an untreated control. Fistulas were evaluated with fistulography or venography 6 week after radiation. All grafts were then harvested for histological and immunohistochemical examination. RESULTS: Seven grafts on the treated side and 5 grafts on the control side remained patent for at least 6 weeks. Angiography demonstrated that the percentage stenosis at the venous anastomosis was significantly lower for the treated group (15.9 +/- 14.1 versus 32.6 +/- 16.7%, P = 0.045). Histopathologic analyses revealed that the mean intimal area and maximal intimal thickness were significantly lower with reduced smooth muscle cell proliferation at the venous anastomosis on the treated side compared to the control side (0.68 +/- 0.30 versus 1.06 +/- 0.29 mm(2), P = 0.017, and 0.18 +/- 0.08 versus 0.26 +/- 0.07 mm, P = 0.004, respectively). The residual lumen was significantly greater for the treated group (1.59 +/- 0.42 versus 1.06 +/- 0.37 mm(2), P = 0.031). No significant differences were found in the area, nor maximal thickness in the vein either proximal or distal to the anastomosis between the two groups. CONCLUSIONS: In an animal model of hemodialysis access, brachytherapy with iridium(192) delivered 2 days after graft implantation reduces intimal hyperplasia and stenosis at the venous anastomosis. The reduced smooth-muscle cells found in the radiated veins suggests that brachytherapy may exert its effect on neointimal formation by inhibition of smooth muscle cell proliferation.     

Vascular clips have no significant effect on the cellular proliferation, intimal changes, or peak systolic velocity at anastomoses in rabbit vein grafts

OBJECTIVE: This study compares vascular closure staples (VCSs) with conventional sutures in the rabbit carotid vein graft model to determine whether anastomotic technique affects cellular proliferation, blood velocity, or intimal changes when measured over a period of 3 months postoperatively. METHODS: Twenty-six New Zealand White rabbits weighing 3.0-3.2 kg underwent interposition of jugular vein grafts in left carotid arteries. Half of the animals had anastomoses performed with small VCSs (n = 13) and half had anastomoses performed with 8-O interrupted polypropylene suture. Animals were allowed to survive for 1 week (n = 4, VCS; n = 4, suture), 2 weeks (n = 4, VCS; n = 4, suture), and 3 months (n = 5, VCS; n = 5, suture). The peak systolic velocity (PSV) at the distal anastomosis was measured after completion of the graft and again at sacrifice in the 3-month survival groups. At sacrifice, sections were taken from the middle and distal end of the vein graft and the distal carotid artery. Vascular cell proliferation was measured using 5-bromo-2'-deoxyuridine labeling and intimal changes were measured using digitized microscopic images. RESULTS: All 26 grafts were open at the time of sacrifice. PSV at the distal clipped anastomosis was 40.52 cm/s (t = 0) and 34.3 cm/s (t = 3 months, P = 0.31). PSV at the distal sutured anastomosis was 38.30 cm/s (t = 0) and 39.23 cm/s (t = 3 months, P = 0.82). There was no difference between the two techniques at either t = 0 or t = 3 months (P = 0.51 and P = 0.31, respectively). Endothelial cell proliferation and smooth muscle cell proliferation at the anastomosis was highest during the 2 weeks after the procedure, then returned to baseline levels by 3 months. But there was no significant difference between the clipped and sutured groups with respect to vascular cell proliferation postoperatively. The intimal thickness changed significantly in the vein graft at the anastomosis for both the clipped and sutured groups (P = 0.0007 and P = 0.002). But there was no difference when the intimal changes for each technique were compared (P = 0.94). CONCLUSION: No differences were observed when peak systolic velocity, vascular cell proliferation, and intimal changes were compared between sutured and stapled anastomoses in rabbit vein interposition grafts over a period of 3 months after surgery.     

Long-term inhibition of Rho kinase suppresses intimal thickening in autologous vein grafts in rabbits

BACKGROUND: Rho kinase plays an important role in vascular smooth muscle cell (VSMC) contraction and other cellular functions, such as proliferation, migration, and apoptosis. Recent studies have demonstrated that long-term inhibition of Rho kinase suppresses coronary artery spasm and vascular lesion formation after arterial injury. In the cardiovascular surgery field, intimal thickening in vein grafts is the major cause of late graft failure, for which no effective treatment has yet been developed. In this study, we examined whether long-term inhibition of Rho kinase suppresses intimal thickening in autologous vein grafts in rabbits. METHODS: Male rabbits were randomly divided into two groups and received normal chow (control group) or a special chow containing 0.09% fasudil (fasudil group). After oral administration, fasudil is metabolized to a specific Rho kinase inhibitor, hydroxyfasudil. Each group underwent reversed autologous vein graft surgery with the internal jugular vein into the left common carotid artery. At 1, 2, and 4 weeks after the operation, we examined the extent of intimal thickening of the graft and VSMC proliferation and apoptosis. RESULTS: The intimal thickening was significantly suppressed in the fasudil group compared with the control group at 2 and 4 weeks after the operation. In the fasudil group, VSMC proliferation was suppressed at 1 and 2 weeks after the operation, whereas VSMC apoptosis was enhanced at 2 weeks after the procedure. CONCLUSIONS: These results indicate that Rho kinase is substantially involved in the pathogenesis of intimal thickening of vein grafts and that it is an important therapeutic target for the prevention of graft failure.     

纳米粒子介导反义mTOR基因转染抑制移植静脉内膜增生

目的:观察以纳米粒子为载体的外源性反义雷帕霉素靶蛋白(mTOR)基因局部转染对移植静脉内膜增生的影响。方法:应用聚乳酸聚乙醇酸共聚物(PLGA)和聚乙烯醇(PVA)包载mTOR基因，制备纳米级粒子混合物。建立自体静脉移植模型72只，随机分成转基因组(转染以纳米粒子为载体的反义mTOR基因)，空载体组(单纯转染纳米粒子包载的空载体)和对照组(不予特殊处理)。分别于术后3，7，14，28d取材。检测mTOR基因的mRNA及蛋白产物表达，以及血管平滑肌细胞(VSMC)凋亡的动态变化。 结果:转基因组内膜中mTOR基因的mRNA及蛋白产物表达较其他两组明显减少(P<0.05)；转基因组内膜增生厚度于7，14，28d较其他组明显减少(P<0.01)；转基因组凋亡细胞较其他组明显增高(P<0.05)。结论:纳米粒子可以作为转基因载体。反义mTOR基因的表达能有效抑制自体移植静脉内膜的增生及促进VSMC凋亡。     

RNA干扰沉默蛋白激酶B基因表达对移植静脉内膜增生的影响

摘要：目的:观察纳米粒子包载的靶向蛋白激酶B(PKB)基因的shRNA表达载体局部转染对大鼠移植静脉内膜增生的影响。方法:应用聚乳酸聚乙醇酸共聚物(PLGA)和聚乙烯醇(PVA)包载PKB的RNA干扰基因载体，制备纳米级粒子混合物。建立自体颈静脉-颈总动脉移植模型共72只，随机分成转基因组、空载体组和对照组。分别于术后3，7，14，28 d取材；常规HE及Verhoeff 染色,用Northern blot和Western blot检测PKB基因的mRNA及蛋白的变化，HE和Verhoeff 染色观察内膜厚度，TUNEL法观察血管平滑肌细胞(VSMC)凋亡的动态变化。结果:转基因组内膜中PKB基因的mRNA及蛋白产物表达较其他两组明显减少(P＜0.05)；术后7，14，28 d转基因组静脉内膜增生厚度较其他组明显减少(P＜0.01)；转基因组细胞凋亡率较其他组明显增高(P＜0.05)。结论:纳米粒子可以作为转基因载体；沉默PKB基因表达能有效地抑制自体移植静脉内膜的增生，促进VSMC的凋亡。     

自体静脉外支架对移植静脉内膜增生影响的实验研究

目的 探讨自体静脉外支架对动脉缺损静脉移植后内膜增生的影响.方法 选用36只5月龄雄性新西兰大白兔,体质量2.8～3.0 kg,随机分为A组、B组和C组(n=12).切取右侧颈外静脉6 cm,等分为3段,远心段均用作移植静脉;B组以中段作为自体外支架,A组以近心段套叠中段做成双层自体外支架.游离左侧颈总动脉,中间切除长0.5 cm一段,建立动脉缺损模型,将移植静脉远近端倒置后吻合于动脉缺损,通血后行移植静脉外径测量.分别于术后2、4周切取移植静脉,行HE染色、Masson染色观察移植静脉管壁组织学变化,增殖性细胞核抗原免疫组化染色观察移植静脉平滑肌细胞增殖情况.结果 通血后移植静脉直径A组(1.6±0.3)mm,B组(2.2±0.4)mm,C组(2.6±0.6)mm(P<0.05).术后4周移植静脉内膜增生程度和内膜细胞增殖指数分别为:A组1.01±0.07和6.84±1.98,B组1.32 4±0.08和11.01±2.61,C组1.55±0.03和14.96±4.14,均为A组相似文献   

Non-penetrating vascular clips anastomosis inhibited intimal thickening under poor runoff conditions in canine autogenous vein grafts.

OBJECTIVE: Late graft failure is still a significant problem, particularly in cases with poor runoff vessels. The main cause of late graft failure is intimal thickening of the anastomotic region. Vascular closure system (VCS) clips may provide ideal anastomosis, since they do not penetrate the wall. Therefore, we examined whether the VCS clips affect intimal thickening under poor runoff conditions in the canine autogenous vein grafts. METHODS: A canine poor runoff model was prepared at both femoral veins. Four weeks after the first surgical procedure, two groups were established according to the two different methods of anastomosis employed. The right femoral vein graft was performed using polypropylene sutures, conventional surgical anastomosis (control group), while the left femoral vein graft was performed using VCS clips anastomosis (VCS group). Four weeks after grafting, the vein grafts were removed and the intimal thickening of proximal, distal anastomosis and midportion of the vein grafts were examined histologically. RESULTS: In the control group, flow rate and variation were 26+/-8 ml/min and 51+/-10 dynes/cm(2), respectively. In the VCS group, the flow rate and variation were 23+/-11 ml/min and 44+/-14 dynes/cm(2), respectively. There were no significant differences between the two groups. The average value of intimal thickening of both the anastomotic region and the midportion of the vein graft in the VCS group was significantly inhibited compared to that of the control group. The number of positive cells of masson trichrome stain in the VCS group was significantly less than that of the control group. CONCLUSIONS: These experiments indicate that VCS clips significantly inhibit intimal thickening under poor runoff conditions in canine autogenous vein grafts to a greater extent compared to suture-constructed anastomosis. One mechanism that may account for the decreased intimal thickening is the inhibition of the expression of transforming growth factor-beta (TGF-beta), because the number of positive cells of masson trichrome stain in the VCS group was significantly less than that of the control group.     

Egr-1DNA酶抑制自体移植静脉内膜增生的实验研究

目的 探讨早期生长反应基因-1 (Egr-1) DNA酶(Egr-1 DNA enzyme,EDRz)对血管平滑肌细胞(VSMC)增殖和内膜增生的抑制作用,从而证实基因治疗静脉移植后狭窄、闭塞的可行性.方法 构建EDRz,建立自体静脉移植模型,将大鼠右颈总静脉端-端吻合于肾下腹主动脉,EDRz转染移植静脉,分别于移植后1、2、6、24 h及3、7、14、28、42 d切取移植静脉标本,每个时相按随机数字表法随机抽取10只大鼠.荧光显微镜下观察EDRz转染移植静脉情况;应用原位杂交和RT-PCR方法检测Egr-1 mRNA的表达;应用Western蛋白印迹和免疫组织化学方法检测Egr-1蛋白表达情况;HE染色光镜下观察组织学形态.结果 ①EDRz转染移植静脉情况:移植后1h时,EDRz主要位于移植静脉的外膜、中膜和部分内皮细胞;2、6及24 h时,EDRz则主要位于移植静脉的中膜;7d时,EDRz主要位于移植静脉的内膜; 14、28及42 d时未检测到EDRz的表达.②Egr-1 mRNA表达结果.RT-PCR检测结果:转染EDRz后1h时,Egr-1 mRNA表达出现高峰,2、6及24 h表达下降,3d时表达微弱,移植后7、14、28及42 d未见Egr-1 mRNA的表达,转染EDRz后1h时Egr-1 mRNA表达明显高于其余各时相(P＜0.01).原位杂交检测Egr-1 mRNA表达的变化趋势与RT-PCR结果基本一致.③Egr-1蛋白表达结果.Western蛋白印迹结果:正常静脉中未检测到Egr-1蛋白阳性表达.转染EDRz后2h时,出现Egr-1蛋白阳性表达,6h、24 h及3d时其表达逐渐降低,移植后1h时和移植后7、14、28及42 d时未见Egr-1蛋白阳性表达.移植后2h时的Egr-1蛋白表达的吸光度值高于其他时相(P＜0.01).免疫组织化学方法检测的Egr1蛋白阳性表达的变化趋势与Western蛋白印迹结果基本一致.④EDRz转染移植静脉与未转染同期相比VSMC的增殖程度和内膜厚度明显减轻.结论 EDRz通过减少Egr1在自体移植静脉中的表达,可有效地抑制自体移植静脉中VSMC增殖和内膜增生,可用来防治自体静脉移植后所导致的血管狭窄、闭塞.     

Adenovirus-mediated gene transfer of human inducible nitric oxide synthase in porcine vein grafts inhibits intimal hyperplasia.

OBJECTIVE: The aim of this study is to determine whether adenoviral inducible nitric oxide synthase (iNOS) gene transfer could inhibit intimal hyperplasia (IH) in porcine internal jugular veins interposed into the carotid artery circulation. METHODS: Porcine internal jugular veins were transduced passively with 1 x 10(11) particles of an adenoviral vector carrying either the human iNOS (AdiNOS) or beta-galactosidase (AdlacZ) cDNA for 30 minutes and then interposed into the carotid artery circulation. Segments of each vein graft were maintained in an ex vivo organ culture to measure nitrite accumulation, a marker of nitric oxide synthesis. The grafts were analyzed immunohistochemically for the presence of neutrophils, macrophages, and leukocytes by staining for myeloperoxidase, ED1, and CD45, respectively, at 3 (n = 4) and 7 (n = 4) days. Morphometric analyses and cellular proliferation (Ki67 staining) were assessed at 3 (n = 4), 7 (n = 4), and 21 days (n = 8). RESULTS: AdlacZ-treated vein grafts demonstrated high levels of beta-galactosidase expression at 3 days with a gradual decline thereafter. Nitrite production from AdiNOS-treated vein grafts was approximately fivefold greater than AdlacZ-treated grafts (P =.00001). AdiNOS or AdlacZ treatment was associated with minimal graft inflammation. Cellular proliferation rates were significantly reduced in AdiNOS-treated grafts as compared with controls at both 3 (41%, P =.000004) and 7 days (32%, P =.0001) after bypass. This early antiproliferative effect was most pronounced at the distal anastomosis (65%, P =.0005). The iNOS gene transfer reduced the intimal/medial area ratio in vein grafts at 7 (36%, P =.009) and 21 days (30%, P =.007) versus controls. This inhibition of IH was again more prominent in the distal segments of the grafts (P =.01). CONCLUSION: Adenovirus-mediated iNOS gene transfer to porcine internal jugular vein grafts effectively reduced cellular proliferation and IH. Although iNOS gene transfer reduced IH throughout the entire vein graft, the most pronounced effect was measured at the distal anastomosis. These results suggest potential for iNOS-based genetic modification of vein grafts to prolong graft patency.