反义大鼠凝血酶受体基因的构建及其对主动脉血管平滑肌细胞增生的影响

凝血酶激活凝血酶受体引起的一系列细胞事件和内源性PDGF-A,bFGF的产生可能是血管病变发生发展过程中平滑肌细胞增生的重要因素。为进一步阐明血管损伤后平滑肌细胞增生的机理及探索有效治疗途径,针对凝血酶受体基因中一段包括转录起始点,翻译起始点,凝血酶结合和切割位点的序列作为靶序列,构建了凝血酶受体反义RNA重组表达载体pLXSN／ATR,并将其导入凝血酶刺激的动脉平滑肌细胞。结果表明重组基因转染能抑制凝血酶对大鼠平滑肌细胞DNA合成的刺激作用。提示序列特异的凝血酶受体反义重组基因的表达可以抑制凝血酶受体介导的血管平滑肌细胞增生。     

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.     

血管平滑肌细胞增殖与p21基因启动子甲基化的影响

背景:血管平滑肌细胞增殖、迁移及表型改变是动脉粥样硬化发生的中心环节,一系列相关基因的甲基化参与该过程。目的:观察血管平滑肌细胞p21基因启动子甲基化对其增殖活性的影响。方法:采用组织贴块法培养人脐血管平滑肌细胞,给予不同质量浓度氧化低密度脂蛋白(0,10,20,40 mg/L)孵育24 h,甲基化特异PCR检测p21基因启动子区CpG岛甲基化程度,反转录PCR检测p21 mRNA表达,MTT比色法测定血管平滑肌细胞增殖活性。结果与结论:氧化低密度脂蛋白呈浓度依赖性促进p21启动子甲基化和降低p21 mRNA表达,同时平滑细胞增殖活性增高。提示氧化低密度脂蛋白可以通过p21基因甲基化促进血管平滑肌细胞增殖,p21基因甲基化可能在动脉粥样硬化发生中起到一定作用。     

骨桥蛋白反义核苷酸对大鼠血管平滑肌细胞增殖的影响

背景:骨桥蛋白在血管损伤后新生内膜中的表达明显上调,而且能促进血管平滑肌细胞和外膜细胞的增殖、迁移。目的:探索骨桥蛋白反义核苷酸对大鼠血管平滑肌细胞增殖的影响。方法:培养大鼠A10主动脉血管平滑肌细胞,通过MTT比色法测定骨桥蛋白反义核苷酸对平滑肌细胞增殖的影响,流式细胞仪测定骨桥蛋白反义核苷酸对平滑肌细胞周期分布的影响;通过RT-PCR方法检测血管平滑肌细胞转染骨桥蛋白反义核苷酸对增殖细胞核抗原表达的影响。结果与结论:骨桥蛋白反义核苷酸对血管平滑肌细胞的增殖有明显的抑制作用,随时间延长对细胞增殖抑制率下降。骨桥蛋白反义核苷酸主要作用于G1/S限制点,能阻止血管平滑肌细胞由G0/G1期向S期推进,从而将血管平滑肌细胞阻滞于G0/G1期,抑制血管平滑肌细胞的增殖。血管平滑肌细胞转染骨桥蛋白反义核苷酸后,增殖细胞核抗原的表达水平均较对照组降低(P0.05)?因此,得出骨桥蛋白反义核苷酸通过阻止血管平滑肌细胞由G0/G1期向S期推进,从而抑制血管平滑肌细胞的增殖,进而抑制血管内膜增生。     

联合转染p21基因和反义c-fos核酸对兔自体移植静脉内膜增生的影响

目的：探讨联合转染p21基因和反义c-fos核酸对兔自体移植静脉内膜增生的影响，探索一种理想的血管再狭窄的疗法。方法：选择20只新西兰家兔，实验组、对照组各10只，均行自体颈外静脉、颈总动脉移植手术，实验组行腺病毒介导的p21 cDNA溶液浸泡和反义c-fos寡聚核苷酸凝胶涂布；对照组仅行空载腺病毒溶液浸泡和凝胶涂布。术后2周取出移植血管，分别行病理学、免疫组织化学检测移植血管内膜厚度（IT）、内膜平滑肌细胞（VSMC）数及PCNA阳性表达情况。结果：实验组移植血管IT、管腔狭窄度及VSMC数均较对照组减少，增殖细胞核抗原（PCNA）阳性表达情况亦较对照组明显减少。结论：联合转染p21基因和反义c-fos核酸可有效地抑制移植静脉内膜的增生。     

p16和p27表达对前列腺癌细胞系的作用

目的 探讨p16和p27Kip1 基因蛋白对抑制前列腺癌细胞增殖和调控其凋亡的作用.方法 构建携带人P16和p27基因的腺病毒载体,转染体外培养的前列腺癌细胞系PC-3,采用RT-PCR、Western blot检测目的 基因的表达.通过细胞生长试验、流式细胞仪检测PC-3转染前后细胞增殖和凋亡的变化.结果 病毒滴度Ad-p16为115×10^8 pfuPml 、Ad-p27为112×10^9 pfuPml ,RT-PCR 检测可见p16-mRNA（520bp）和p27Kip12mRNA （320bp）表达,Western blot检测有p16 蛋白（65KD）和P27蛋白（27KD） 特异表达,并可明显抑制PC-3细胞的增殖,诱导其凋亡,联合基因治疗组与单基因组相比差异显著（P＜0.01）.结论 p16和p27可明显抑制前列腺癌细胞株PC-3的增殖,增加细胞的凋亡,转染携带p16和p27的重组腺病毒载体有望成为治疗前列腺癌的有效方法.     

慢病毒介导bFGF和BMP-2双基因转染对兔骨髓间充质干细胞增殖的影响

目的构建携带骨形成蛋白-2(BMP-2)与碱性成纤维细胞生长因子(bFGF)双基因过表达的慢病毒载体,观察其对兔骨髓间充质干细胞(BMSCs)增殖的影响。方法体外培养兔BMSCs,构建携带b FGF和BMP-2双基因的重组慢病毒载体,转染至BMSCs诱导两种基因过表达;流式细胞仪检测转染效率;Western blot方法验证双基因转染模型构建成功;MTT方法检测细胞增殖水平。结果慢病毒以最佳感染复数(MOI)100转染BMSCs时转染效率最佳,Western blot检测到转染后细胞bFGF和BMP-2蛋白表达量明显比对照增加(P0.05);MTT结果发现,过表达bFGF和BMP-2的BMSCs细胞的增殖水平增加(P0.05)。结论 bFGF和BMP-2双基因共表达重组腺病毒载体促进BMSCs细胞增殖。     

人组织激肽释放酶基因重组腺病毒转染对血管平滑肌细胞增殖和迁移的影响

目的:探讨重组腺病毒介导的人组织激肽释放酶(hKLK1)基因转移对血小板源性生长因子-BB(PDGF-BB)诱导下的自发性高血压大鼠(SHR)血管平滑肌细胞(VSMCSHR)增殖和迁移的影响。方法:自行构建双顺反子重组腺病毒载体,携带强绿色荧光蛋白(EGFP)标志基因和目的基因hKLK1;用细胞计数法和四甲基偶氮唑盐(MTT)比色法检测细胞增殖,流式细胞仪检测细胞生长周期;蛋白免疫印迹法(Western blotting)测定细胞周期素依赖性激酶抑制蛋白p27Kip1、p21Cip1的表达。采用改良Boyden微孔膜双槽法测定VSMCSHR迁移。结果:(1)hKLK1基因转移呈感染复数依赖性(20-100MOI)抑制PDGF-BB诱导的VSMCSHR生长,100MOI时抑制率为39.3%;呈时间依赖性抑制VSMCSHR生长,第5d时达高峰,抑制率为35.2%。(2)hKLK1基因转移可显著抑制PDGF-BB诱导的VSMCSHR增殖,峰值抑制率为30.2%(P0.01);细胞周期阻滞于G0/G1期的VSMCSHR明显增多,最大阻滞率为36.4%(P0.01),而缓激肽B2受体特异性阻断剂Hoe140逆转了hKLK的抑制作用。(3)hKLK1基因转移明显上调PDGF-BB诱导VSMCSHR的p27Kip1、p21Cip1表达,Hoe140明显降低p27Kip1、p21Cip1表达。(4)hKLK1基因转移可明显抑制PDGF-BB诱导的VSMCSHR细胞迁移,抑制率为34.6%,且Hoe140不影响该抑制作用。结论:hKLK1基因转移可抑制PDGF-BB诱导的VSMCSHR增殖,主要由缓激肽B2受体介导的,通过上调细胞周期素依赖性激酶抑制蛋白p27Kip1、p21Cip1表达的途径。而hKLK1基因转移抑制VSMCSHR迁移效应可能不通过B2受体。     

p21基因启动子甲基化对血管平滑肌细胞向泡沫细胞转化的影响

目的观察氧化低密度脂蛋白(ox-LDL)对血管平滑肌细胞(VSMC)p21基因启动子甲基化及对VSMC向泡沫细胞转化的影响。方法采用组织贴块法培养人脐血管平滑肌细胞,给予不同浓度氧化低密度脂蛋白ox-LDL(0,10mg/L,20mg/L,40mg/L)孵育24h,甲基化特异PCR(MS-PCR)检测p21基因启动子区CpG岛甲基化程度,MTT比色法测定VSMC增殖活性,油红O染色镜下观察计数泡沫细胞比率。结果 ox-LDL呈浓度依赖性促进p21启动子甲基化,同时平滑细胞增殖活性增高,向泡沫细胞转化增加。结论 ox-LDL可以通过p21基因甲基化促进VSMC向泡沫细胞转化,p21基因甲基化可能在动脉粥样硬化发生中起到一定作用。     

降钙素基因相关肽对血管平滑肌细胞增殖的影响

目的:为阐明神经因素与平滑肌细胞增生调控之间的关系提供理论依据。方法:取人胚胎的主动脉,体外原代培养血管平滑肌细胞。实验分为联胺诱导组、联胺 降钙素基因相关肽(CGRP)组、CGRP组;用细胞计数法和MTT法检测细胞增殖,RT-PCR法检测细胞周期蛋白D1、周期蛋白E mRNA的表达,流式细胞仪检测细胞周期的变化。结果:联胺能诱导血管平滑肌细胞的增殖。CGRP对正常血管平滑肌细胞的增殖有抑制作用,但对联胺诱导增生的血管平滑肌细胞抑制作用更明显。对周期蛋白D1 mRNA的表达有明显下调作用。结论:CGRP对血管平滑肌细胞的增殖,特别对联胺诱导增殖的下调作用更明显。     

反义凝血酶受体基因表达抑制大鼠动脉内膜损伤后内膜增生

目的 了解凝血酶及其受体在血管损伤后动脉内膜增生中的作用,以进一步阐明经皮冠状动脉血管腔内成形术（PTCA）后再狭窄的发生机制,为寻找再狭窄的防治途径提供线索。方法 采用球囊导管损伤动脉内膜的方法建立大鼠颈动脉球囊损伤模型。用纳米粒子包装凝血酶受体重组反义基因质凿pLXSN/ATR,用保护灌注的方法局部定位转染损伤后的大鼠颈动脉。结果 PCR检测发现重组基因整合,RNA斑点杂交观察到实验组大鼠颈动脉壁内有重组反义凝血酶受体基因表达,正义凝血酶受体基因的表达受到明显抑制,反义基因转染组新生内膜,中膜比例降低了40．9％。结论 反义凝因酶受体重组基因转基因表达对大鼠颈动脉球囊损伤后动脉内膜的增生具有抑制作用,提示凝血酶及其受体在PTCA后再狭窄过程中有重要作用,为再狭窄的防治提供了新的线索。     

TR3 nuclear orphan receptor prevents cyclic stretch-induced proliferation of venous smooth muscle cells

In coronary artery bypass surgery, the patency of arterial grafts is higher than that of venous grafts because of vein-graft disease, which involves excessive proliferation of venous smooth muscle cells (SMCs) and subsequent accelerated atherosclerosis. We studied the function of TR3 nuclear orphan receptor (TR3) in the early response of SMCs to mechanical strain, a major initiator of vein-graft disease. We demonstrate that TR3 expression is induced in human saphenous vein segments exposed ex vivo to whole-blood perfusion under arterial pressure. Cultured venous SMCs challenged by cyclic stretch displayed TR3 induction and enhanced DNA synthesis, whereas SMCs derived from the internal mammary artery remained quiescent. Small-interfering RNA-mediated knockdown of TR3 and adenovirus-mediated overexpression of TR3 in venous SMCs enhanced and abolished stretch-induced DNA synthesis, respectively. Accordingly, in organ cultures of wild-type murine vessel segments exposed to cyclic stretch, p27(Kip1) was down-regulated, whereas expression of this cell cycle inhibitor was unaffected by cyclic stretch in TR3-transgenic vessels, concordant with a lower proliferative response. Finally, stretch-mediated proliferation was inhibited by 6-mercaptopurine, an agonist of TR3. In conclusion, TR3 represents inhibitory mechanisms to restrict venous SMC proliferation and may contribute to prevention of vein-graft disease.     

Hepatocyte growth factor/scatter factor and MET are involved in arterial repair and atherogenesis

Several studies have shown that in the arterial wall hepatocyte growth factor/scatter factor (HGF/SF) is expressed by smooth muscle cells (SMCs) but acts on endothelial cells, not SMCs. Other studies, however, have indicated that SMCs can respond to HGF/SF. We have reinvestigated expression and activity of HGF/SF and its receptor MET in arterial SMC and endothelial cell cultures and in whole arteries after superficial or deep injury or atherogenesis. High-density cultures of SMCs produced HGF/SF but did not express MET, whereas SMCs, at the leading edge of injured cultures, expressed both ligand and receptor and showed a dramatic motility and growth response to HGF/SF. In line with these results, HGF/SF and MET expression was undetectable in the media of uninjured carotid arteries but was induced after deep arterial injury in areas of SMC migration in the neointima. Strong MET expression was also observed in the SMCs of the atherosclerotic lesions of homozygous apoE^−/− mice, whereas HGF/SF was expressed by macrophage-derived foam cells. These results demonstrate that MET is induced in migrating and proliferating SMCs and that HGF/SF and MET are key mediators of the SMC response in atherogenesis.     

Clusters of proliferating endothelial cells and smooth muscle cells in rabbit carotid arteries

Schwarz and Benditt found clustering of replicating cells in aortic endothelium in 1976 and discussed how homeostasis of the arterial wall is maintained through this nonrandom distribution of replicating cells. However, it is still unclear how cells of vascular walls turnover. In order to address this issue, we evaluated distribution of the cells in mitotic cycle, labeled by Ki67‐immunostaining, in serial histological sections of twelve carotid arteries of six adult male Japanese rabbits. As a result, a total of 1713 Ki67‐positive endothelial cells (ECs) and 1247 Ki67‐positive smooth muscle cells (SMCs) were identified. The Ki67‐positivity rate in ECs and SMCs were about 0.048% and 0.0027%, respectively. Many of the Ki67‐positive cells clustered in two (EC, 37%; SMC, 33%), three to four (EC, 8%; SMC, 28%), and five to eight cells (EC, 5%; SMC, 10%). Clusters having more than eight cells were not found. Thus, it can be speculated that the cell division of proliferating ECs and SMCs occur four times at most. These novel findings offer great insights for better understanding of the mechanism that underlies cell number regulation of the blood vessel.     

Decorin inhibition of PDGF-stimulated vascular smooth muscle cell function: potential mechanism for inhibition of intimal hyperplasia after balloon angioplasty

Decorin is a small proteoglycan that binds to transforming growth factor-beta (TGF-beta) and inhibits its activity. However, its interaction with platelet-derived growth factor (PDGF), involved in arterial repair after injury, is not well characterized. The objectives of this study were to assess decorin-PDGF and decorin-PDGF receptor (PDGFR) interactions, the in vitro effects of decorin on PDGF-stimulated smooth muscle cell (SMC) functions and the in vivo effects of decorin overexpression on arterial repair in a rabbit carotid balloon-injury model. Decorin binding to PDGF was demonstrated by solid-phase binding and affinity cross-linking assays. Decorin potently inhibited PDGF-stimulated PDGFR phosphorylation. Pretreatment of rabbit aortic SMC with decorin significantly inhibited PDGF-stimulated cell migration, proliferation, and collagen synthesis. Decorin overexpression by adenoviral-mediated gene transfection in balloon-injured carotid arteries significantly decreased intimal cross-sectional area and collagen content by approximately 50% at 10 weeks compared to beta-galactosidase-transfected or balloon-injured, non-transfected controls. This study shows that decorin binds to PDGF and inhibits its stimulatory activity on SMCs by preventing PDGFR phosphorylation. Decorin overexpression reduces intimal hyperplasia and collagen content after arterial injury. Decorin may be an effective therapy for the prevention of intimal hyperplasia after balloon angioplasty.     

Subnormal shear stress-induced intimal thickening requires medial smooth muscle cell proliferation and migration

Arterial intimal thickening is consisted of predominately smooth muscle cells (SMC). The source of these SMCs and mechanisms response for their changes have not been well cleared. Using a model of rabbit common carotid artery (CCA) shear induced intimal thickening, we sought to identify and describe the source of SMCs in intima. The enlarged CCA 28 days after arteriovenous fistula (AVF) creation was subjected to subnormal wall shear stress (WSS) for 1, 3, and 7 days by closure of the AVF. To determine SMC proliferation, BrdU pulse labeling of SMCs was performed. BrdU-labeled SMCs were tracked over time to further confirm SMC migration. In response to subnormal WSS intimal thickening developed progressively. BrdU-labeled SMCs localized in the subendothelial area. When the BrdU-labeled medial SMCs were tracked 1 day after AVF closure, progenies of these BrdU-incorporated SMCs increased by 4.8-fold with 75% of them in the intima. They were 12-fold increased with 83% in the intima 7 days after. En face examination showed an accumulation of SMCs in internal elastic lamina gap after AVF closure, which later migrated into subendothelial area. In situ hybridization revealed increased TGF-beta1 mRNA expression in intimal SMCs. This study demonstrates that the medial SMCs are the predominant cells in subnormal WSS-induced intimal thickening. Early expression of TGF-beta1 may play an important role in the process of intimal thickening.     

Factors controlling smooth-muscle cell proliferation.

Injury to the arterial wall normally elicits a rapid and significant increase in smooth-muscle cell (SMC) replication with the subsequent development of intimal lesions. A variety of factors have been proposed to control SMC replication, but recent work has highlighted the role of basic fibroblast growth factor (bFGF) and platelet-derived growth factor in this process. In the carotid artery of the uninjured rat, we have shown that the SMCs express mRNA for bFGF and that bFGF can be readily extracted from these arteries. Following mechanical injury to the artery, ie, after balloon injury, we suggested that bFGF is released from damaged cells and then stimulates adjacent SMCs. In support of this concept, the infusion of a blocking antibody to bFGF was found to significantly inhibit the early SMC replication induced by use of a balloon catheter. The addition of the antibody at the time of injury, however, did not inhibit the development of intimal lesions. In contrast, studies by us and other investigators have shown that platelet-derived growth factor is not directly important for SMC replication after balloon injury, but that it plays a key role in stimulating the migration of SMCs into the intima. Intimal SMC replication was not inhibited with antibodies to either bFGF or platelet-derived growth factor. Therefore, while significant inroads have been made in understanding the initial events, we still do not fully understand all the processes involved in the proliferation of arterial intimal lesions.     

Retinoic acid inhibits airway smooth muscle cell migration

Airway remodeling in chronic asthma is characterized by increased smooth muscle mass that is associated with the reduction of the bronchial lumen as well as airway hyperresponsiveness. The development of agents that inhibit smooth muscle growth is therefore of interest for therapy to prevent asthma-associated airway remodeling. All-trans retinoic acid (ATRA) suppresses growth of vascular smooth muscle cells (SMCs) from the systemic and pulmonary circulation. The present study investigated the effects of ATRA on human bronchial (airway) SMCs. Human bronchial SMCs were found to express mRNAs for retinoic acid receptor (RAR)-alpha, -beta, -gamma, and retinoid X receptor (RXR)-alpha, -beta, but not RXR-gamma. Although ATRA was not effective in inhibiting proliferation or in inducing apoptosis in airway SMCs, we found that ATRA (0.2-2 microM) inhibited the SMC migration in response to platelet-derived growth factor (PDGF), as determined in a modified Boyden chamber assay. Both RAR and RXR agonists also blocked PDGF-induced airway SMC migration. ATRA also inhibited PDGF-induced actin reorganization associated with migration. PDGF-induced actin reorganization and migration were blocked by inhibitors of phosphatidylinositol 3 kinase (PI3K) and Akt. However, migration was blocked by inhibitors of the MEK/ERK pathway, with no effect on cytoskeletal reorganization. ATRA suppressed PDGF-induced Akt activation without influencing ERK activation. RAR was found to form protein-protein interactions with the p85 PI3K subunit. These results suggest that retinoic acid inhibits airway SMC migration through the modulation of the PI3K/Akt pathway.