【作者】 张良；

【导师】 周胜华；

【作者基本信息】 中南大学 ， 内科学， 2010， 博士

【摘要】 第一章紫杉醇对新西兰大白兔髂动脉球囊损伤后新生内膜形成和Cat K表达的影响背景经皮腔内冠状动脉血管成形术(PTCA)是冠心病治疗的一种重要方法,但是大约20%-40%的患者在术后6个月出现血管内再狭窄。新生内膜形成在血管成形术后再狭窄的发生中起着重要作用。组织蛋白酶K能降解细胞外基质,促进VSMC和EC迁移,与内膜增生密切相关。紫杉醇可抑制VSMC和EC的增殖及迁移,能防治支架内再狭窄。紫杉醇是否通过抑制组织蛋白酶K表达来减少新生内膜形成目前不是很清楚。目的观察紫杉醇对兔髂动脉球囊损伤后新生内膜形成和Cat K及NF-KB表达的影响,探讨紫杉醇减轻血管球囊损伤后新生内膜形成的机制。方法40只雄性新西兰大白兔,随机分为正常对照组(n=8)、手术组(n=16)和紫杉醇组(n=16),均予普通饮食。手术组和紫杉醇组行右侧髂动脉球囊损伤,紫杉醇组术后在喂食普通饲料的基础上予腹腔注射紫杉醇2mg/kg,持续7天或14天。对照组和手术组每日均腹腔注射相同体积的生理盐水。各组分别在术前1天,术后1天,术后第7天和第28天处死兔子时采血。用酶联免疫吸附实验(ELISA)测定血清TNF-a变化。动物喂养在术后第7天和第28天处死兔子,取右侧髂动脉行病理形态学检测,用RT-PCR、Western-blotting和免疫组化方法测定血管中Cat K的表达,用Western-blotting和免疫组化方法检测NF-KB表达。结果1.术前三组血清TNF-a水平无明显差异。手术组术后第1天、第7天和第28天血清TNF-a水平分别为17.92±3.71,23.65±4.51和19.73±3.83ng/l,较术前12.97±2.24 ng/l明显升高,P<0.01。但同一时间点紫杉醇组与手术组TNF-a水平差别不是很明显,术后第7天TNF-a浓度最高。2.与正常对照组相比,手术组和紫杉醇组内膜面积和内膜/中膜面积(I/M)明显增加；手术组术后7天,28天内膜面积和内膜与中膜面积比分别为0.19±0.05mm2,0.35±0.11和0.69±0.1lmm2,1.39±0.13,两者相比,P<0.01。随着术后时间的延长,手术组和紫杉醇组内膜面积和内膜/中膜面积(I/M)明显增加,紫杉醇组内膜面积和内膜/中膜面积(I/M)比手术组同一时间点减少,三组中膜面积在这两个时间点无显著性差异,P>0.05。3.正常对照组基本无Cat K mRNA表达,手术组Cat K mRNA和紫杉醇组Cat K mRNA明显增加,紫杉醇组术后7天,28天Cat K mRNA表达分别为0.559±0.055和0.786±0.025,两者相比,P<0.01。且随着术后时间延长,Cat K mRNA表达增加,与紫杉醇组(0.786±0.025)相比,同一时间点手术组Cat K mRNA表达(0.880±0.065)明显增多,P<0.05。4.正常对照组无明显Cat K蛋白的表达,手术组和紫杉醇组均有Cat K表达,手术组术后7天,28天Cat K表达分别为0.421±0.05和0.819±0.10,两者相比,P<0.01。且随着术后时间延长,Cat K表达增加,与手术组(0.819±0.10)相比,同一时间点紫杉醇组Cat K表达(0.632±0.06)低于同期的手术组,P<0.05。5.免疫组化分析,正常对照组无明显Cat K阳性蛋白的表达,手术组Cat K和紫杉醇组Cat K阳性表达明显增加,且随着术后时间延长,Cat K阳性表达增加,与手术组(16.13±1.27)相比,同一时间点紫杉醇组Cat K免疫组化评分(12.46±1.53)明显减少,P<0.05。6.正常对照组基本无明显NF-KB蛋白的表达,手术组NF-KB和紫杉醇组NF-KB明显增加,且随着术后时间延长,NF-KB表达增加,与手术组(0.369±0.04)相比,同一时间点紫杉醇组NF-KB表达(0.241±0.03)明显减少,P<0.05。7.免疫组化分析,正常对照组无明显NF-KB阳性蛋白的表达,手术组NF-KB和紫杉醇组NF-KB阳性表达明显增加,手术组第7天和28天免疫组化评分分别为4.75±0.79和14.29±1.36,两者相比,P<0.01。且随着术后时间延长,NF-KB阳性表达增加,与紫杉醇组免疫组化评分为(11.37±1.45),低于同一时间点手术组NF-KB免疫组化评分(14.29±1.36),P<0.05。结论新西兰大白兔髂动脉球囊损伤后新生内膜明显增厚,内膜Cat K和NF-KB表达均明显增加,且随着术后时间的延长增加越多。紫杉醇可能通过抑制NF-KB活性减少Cat K表达,减轻髂动脉球囊损伤后新生内膜形成。血清TNF-a水平在兔髂动脉球囊损伤后升高,与在兔血管壁中Cat K表达可能无直接关系。第二章TNF-a对人脐静脉内皮细胞中Cat K表达的影响及其信号通道机制背景肿瘤坏死因子-a(TNF-a)是一种重要的促炎症因子,可以调控多种炎症因子和生长因子的表达。TNF-a在血管球囊损伤后的新生内膜中表达增加。血管损伤时导致多种炎症因子和细胞因子被激活,这些因子相互作用加重了血管壁的炎症反应,促进了新生内膜的形成。组织蛋白酶K能降解细胞外基质,促进VSMC和EC迁移,与内膜增生密切相关。TNF-a可以促进血管内皮细胞中Cat K的表达,但是两者之间的时效和量效关系尚不十分清楚。在人的内皮细胞中TNF-a通过哪些信号通道促进Cat K的表达,也不是很清楚。目的观察TNF-a不同浓度和不同作用时间对人脐静脉内皮细胞中CatK表达的影响,以及不同信号通道阻滞剂对TNF-a引起Cat K表达的抑制作用,探讨TNF-a影响Cat K表达的机制。方法采用HUVEC细胞株传代3-6代时处于对数生长期的细胞作为研究对象。细胞中加入不同浓度的TNF-a作用24h或者加入10ng/ml的TNF-a作用不同时间。在HUVEC中加入10ng/ml的TNF-a,并分别加入特异性的P38MAPK信号通道阻滞剂SB203580、PI3K信号通道阻滞剂LY294002和NF-kB通道阻滞剂PDTC作用24h。用RT-PCR方法和western-blotting方法检测细胞中的Cat K mRNA和蛋白的表达。结果1.正常对照组HUVEC未见明显Cat K mRNA表达。随着TNF-a浓度的增加,Cat K mRNA的表达越来越强,0.1ng/ml,1ng/ml和lOng/ml TNF-a组Cat K mRNA表达分别为0.27±0.03,0.39±0.04和0.50±0.04,两两相比,均P<0.01。1Ong/mlTNF-a组Cat K mRNA表达最强,1OOng/mlTNF-a组Cat K mRNA表达为0.44±0.06,较1Ong/mlTNF-a组反而下降,P<0.05。2.随着TNF-a浓度的增加,Cat K的表达越来越强,0.1ng/ml,1ng/ml和lOng/ml TNF-a组Cat K表达分别为1.12±0.13,1.55±0.11和1.94±0.10,两两相比,均P<0.05。1Ong/mlTNF-a组Cat K表达最强,l00ng/mlTNF-a组Cat K表达(1.36±0.56)较1Ong/mlTNF-a组表达减少,P<0.05。3.予lOng/ml TNF-a作用不同时间后,HUVEC可见Cat K mRNA明显表达,TNF-a干预时间6h,12h和24h时Cat K mRNA表达分别为0.37±0.04,0.59±0.06和0.69±0.04,两两相比,均P<0.01。从6h到24h时,随着TNF-a干预时间的增加,Cat K mRNA的表达越来越强。Cat K mRNA表达在TNF-a24h组最强,TNF-a48h组Cat K mRNA表达(0.61±0.07)较TNF-a24h组明显下降,P<0.05。4. TNF-a干预时间6h,12h和24h时Cat K表达分别为0.36±0.04,0.54±0.05和0.84±0.07,两两相比,均P<0.05,予lOng/ml TNF-a作用从6h到24h时,随着TNF-a干预时间的增加,Cat K的表达越来越强。TNF-a48h组Cat K表达为0.60±0.07,较TNF-a24h组明显减少,P<0.05。5. HUVEC在lOng/ml TNF-a刺激基础上分别加入SB203580、LY294002和PDTC作用24h后,细胞中Cat K mRNA表达分别为0.55±0.04,0.57±0.01和0.52±0.04,较lOng/ml TNF-a组(0.65±0.04)时均明显下降,P<0.05。6. HUVEC在10ng/mlTNF-a刺激基础上分别加入SB203580、LY294002和PDTC作用24h后,细胞中Cat K表达为0.70±0.08,0.67±0.11和0.62±0.09,较10ng/mlTNF-a组(0.91±0.15)时均明显下降,P<0.01。结论TNF-a可以呈剂量和时间依赖性刺激HUVEC中Cat K表达：TNF-a可能通过激活P38、PI3K和转录因子NF-KB信号通路促进HUVEC中Cat K表达。第三章紫杉醇对TNF-a诱导人脐静脉内皮细胞中Cat K表达的影响背景完整内膜在防止血栓形成和再狭窄发生中起重要作用,延迟血栓形成和延迟再狭窄发生可能与紫杉醇抑制内皮再生有关。组织蛋白酶K能降解ECM的多种成分,促进VSMC和EC迁移,与内膜增生密切相关。NF-kB是一个重要的转录因子,可以调控多种基因产物的表达。紫杉醇有明显抑制细胞迁移的作用,紫杉醇对组织蛋白酶K表达的影响目前尚无研究。紫杉醇能否通过抑制内皮细胞中NF-kB表达来减少Cat K表达,目前尚不清楚。目的观察紫杉醇对TNF-a诱导人脐静脉内皮细胞中Cat K及NF-kB表达的影响,探讨紫杉醇在防治内膜增生的作用机制。方法采用HUVEC细胞株传代3-6代时处于对数生长期的细胞作为研究对象。用10ng/mlTNF-a刺激人脐静脉内皮细胞中的Cat K的表达,并分别加入不同浓度的紫杉醇干预24小时,用RT-PCR方法检测细胞中Cat K mRNA的表达；用western-blotting方法检测细胞中的CatK和NF-kB蛋白的表达。结果1.1nmol/l,10nmol/l,100nmol/l和1μml/l紫杉醇干预组Cat K mRNA的表达分别为0.85±0.06,0.68±0.04,0.59±0.02和0.49±0.03,两两相比,均P<0.05,均较TNF-a组(1.02±0.07)表达减少,均P<0.05。紫杉醇可以呈剂量依赖性地抑制TNF-a所致的HUVEC中Cat K mRNA的表达,紫杉醇浓度越高,抑制Cat K mRNA的表达越强。2.1nmol/l紫杉醇组和TNF-a组中Cat K表达分别为1.08±0.07和0.87±0.03,两者相比,P<0.05。紫杉醇可以呈剂量依赖性地抑制TNF-a所致的HUVEC中Cat K蛋白的表达,紫杉醇浓度越高,细胞中CatK的表达越少。3.正常对照组未见明显NF-kB的表达,1nmol/l紫杉醇组和TNF-a组中NF-kB表达分别为0.93±0.04和0.87±0.07,两者相比,P<0.05。紫杉醇可以呈剂量依赖性地抑制TNF-a所致的HUVEC中NF-kB蛋白的表达。紫杉醇浓度越高,细胞中NF-kB的表达越少。结论紫杉醇可以呈剂量依赖性地抑制TNF-a所致的HUVEC中Cat K和NF-kB的表达,紫杉醇可能通过抑制NF-kB活性使Cat K表达减少。更多还原

【Abstract】 BackgroundPercutaneous transluminal coronary angioplasty (PTCA) is an important method of treating coronary heart disease, but about 20% to 40% of patients suffer from vascular restenosis within 6 months after angioplasty. Neointimal formation plays an important role in the pathogenesis of restenosis after angioplasty. Cathepsin K can degrade extracellular matrix, promote VSMC and EC migration, and have an closely relation to intimal hyperplasia. Paclitaxel inhibits vascular smooth muscle cell and endothelial cells proliferation and migration and can prevent in-stent restenosis. It is not clear whether or not Paclitaxel can reduce neointimal formation through decrease Cat K expressionObjectives:The aim of this study was to evaluate the effect of paclitaxel on the neointimal formation, Cat K and NF-KB expression in rabbit iliac artery after balloon injury, to explore the potential mechanisms of paclitaxel for diminishing the neointimal formation.Methods:40 male New Zealand white rabbits were randomly divided into normal control group (n= 8), operation group (n= 16) and the paclitaxel group (n= 16), feeding with normal diet. The rabbits of operation group and the paclitaxel group were performed with balloon injury to the right iliac artery. Paclitaxel group were given paclitaxel 2mg/kg.d from intraperitoneal injection until to 7th day or to 14th day after operation. Control group and operation group were injected daily with the same volume of saline.The serum levels of TNF-a were measured before operation,first day,7th day and 28th day after operation using enzyme-link-immunosorbent assay(ELISA).The right iliac artery were excised 7th day and 28th day while they were killed after operation and then were taken to pathological test.We used RT-PCR, Western-blotting and immunohistochemistry to measure the expression of Cat K of the artery; we also used Western-blotting and immunohistochemistry to detect the expression of NF-KB of the artery.Result1. There was no significantly difference in the level of serum TNF-a between three group before operation.The first day,7th day,28th day after operation level of serum TNF-a in operation group were17.92±3.71, 23.65±4.51 and 19.73±3.83ng/l respectively, all of them were significantly higher than preoperative 12.97±2.24 ng/1, P< 0.01.but There was no significantly difference in the level of serum TNF-a between operation group and paclitaxel group at the same time. the level of serum TNF-a is highest 7th day after operation.2. Compared with the control group, operation group and the paclitaxel group intimal area and intima/media area (I/M) significantly increased, The intimal area and intimal and medial area ratio of 7th day and 28th day after operation in operation group were 0.19±0.05 mm2,0.35±0.11 and 0.69±0.11 mm2,1.39±0.13 respectively,and they comparing, P< 0.01.with the postoperative time being longer, the operation group and the paclitaxel group intimal area and intima/media area ratio (I/M) significantly increased.intimal area and intima/media area (I/M) of the paclitaxel group were less than the operation group at the same time.The medial area had no significant difference in the three groups at the two time points.3. There was little Cat K mRNA expression in the control group operation group and the paclitaxel group Cat K mRNA expression significantly increased,compared with the control group. The expression of Cat K mRNA of 7th day and 28th day after operation in paclitaxel group were 0.559±0.055 and 0.786±0.025, comparing with the two, P< 0.01.with the postoperative time prolonged, Cat K mRNA expression significantly increased in the operation group and the paclitaxel group. Cat K mRNA expression in operation group increased comparing with the paclitaxel group at the same time, P< 0.05.4. There was no obvious Cat K expression in the control group, compared with the control group, operation group and the paclitaxel group Cat K expression significantly increased. The 7th day and 28th day after operation in operation group Cat K expression were 0.421±0.05 and 0.819±0.10, Comparing with them, P< 0.01.As postoperative time moved on, Cat K expression significantly increased in the operation group and the paclitaxel group. Cat K expression(0.632±0.06) in paclitaxel group were less than the operation group (0.819±0.10) at the same time.5. Immunohistochemical analysis, there was no obvious Cat K protein expression in normal controls group, operation group and the paclitaxel group Cat K expression significantly increased.With the postoperative time being longer, The Cat K immunohistochemistry scores significantly increased in the operation group and the paclitaxel group. Compared with the operation group(16.13±1.27),the Cat K immunohisto-chemistry scores in paclitaxel group(12.46±1.53)significantly decreased at the same time, P< 0.05.6. There was little NF-KB expression in the normal control group, operation group and the paclitaxel group NF-KB expression significantly increased.As the postoperative time moved on, NF-KB expression significantly increased in the operation group and the paclitaxel group. Compared with the operation group(0.369±0.04),NF-KB expression in paclitaxel group(0.241±0.03) was less than the operation group at the same time, P< 0.01.7. Immunohistochemical analysis, there was no distinct NF-KB protein expression in normal controls group, the Cat K score of 7th day and 28th day after operation in operation group were 4.75±0.79 and 14.29±1.36 respectively,comparing between them, P< 0.01. with the postoperative time prolonged, The NF-KB immunohistochemistry scores significantly increased in the operation group and the paclitaxel group. The NF-KB immunohistochemistry scores in paclitaxel group(11.37±1.45) were significantly less than the operation group (14.29±1.36)at the same time, P< 0.05.Conclusion:Ballon injury obviously induced New Zealand white rabbits iliac artery neointimal formation, Cat K and NF-KB expression growth in intima, and they increased as postoperative time moved on. Paclitaxel maybe reduce Cat K expression through inhibiting NF-KB to decrease iliac artery neointimal hyperplasia after ballon angioplasty.The levels of serum TNF-a increased after ballon angioplasty,and it maybe has not direct relation with Cat K expression in rabbit vessel wall. BackgroundTumor necrosis factor-a (TNF-a) is an important pro-inflammatory factors, can regulate inflammatory cytokine and growth factor expression. The expression of TNF-a in the vascular neointima after balloon injury increased, Vascular injury leading to multiple inflammatory factors and cytokines being activated, the interaction of these factors increase the inflammatory reaction in the vascular wall, promotes the formation of neointima. Cathepsin K can degrade extracellular matrix, promote VSMC and EC migration, and lead to intimal hyperplasia.It is reported that TNF-a can stimulate vascular endothelial cell expression of Cat K, but the time-response and dose-response effect on TNF-a induced Cat K expression is not complete understood. It is not very clear about the potential signaling pathway mechanism for the effect.Objective:This study was aimed to study the dose-response and time-response effect of TNF-a on the expression of Cat K in human umbilical vein endothelial cells and the effects of different signaling pathway inhibitors on Cat K expression induced by TNF-a, elucidating the underlying signaling pathway mechanism for the expression of Cat K expression induced by TNF-a.Methods:HUVECs in the third to sixth generation were used in the present study.The cells were treated with different concentrations of TNF-a for 24 hours or with lOng/ml TNF-a for different times. The cells were also treated with the P38MAPK pathway inhibitor SB203580,the PI3K pathway inhibitor LY294002 and the NF-kB pathway inhibitor PDTC for 24 hours in the presence of 1Ong/ml TNF-a. We used the RT-PCR method to measure Cathepsin K mRNA levels and used the western-blotting method to measure Cathepsin K protein levels of these cells.Result1.Normal human umbilical vein endothelial cell had very little Cat K mRNA expression. With the increasing concentration of TNF-a, Cat K mRNA expression increased in endothelial cell.The Cat K mRNA expression were 0.27±0.03,0.39±0.04 and 0.50±0.04 respectively in the TNF-a concentration of 0.1ng/ml, 1ng/ml and 10ng/ml, comparing with any two of them,P<0.05. The level of lOng/ml TNF-a group Cat K mRNA expression was the highest,100ng/ml TNF-a group Cat K mRNA expression was 0.44±0.06 and it is less than lOng/ml TNF-a group, P <0.05. 2.With the increasing concentration of TNF-a, Cat K expression is growing, The Cat K expression of 0.1ng/ml, 1ng/ml and 10ng/ml TNF-a concentration group were 1.12±0.13,1.55±0.11 and 1.94±0.10 respectively, comparing with any two of them, P<0.05.The level of lOng/ml TNF-a group Cat K protein expression was the highest, 100ng/ml TNF-a group Cat K protein expression(1.36±0.56) is less than lOng/ml TNF-a group, P< 0.05.3.Compared with control group, the cells with lOng/ml TNF-a for different time had higher Cat K mRNA expression. Cat K mRNA expression were 0.37±0.04,0.59±0.06 and 0.69±0.04 respectively in the TNF-a intervention time for 6h,12h and 24h, comparing with any two of them, P<0.01.The time of induced being longer, the expression of Cat K mRNA became higher from 6 hours to 24 hours. Cat K mRNA expression(0.61±0.07) stimulated by TNF-a for 48 hour was significantly less than stimulated by TNF-a for 24 hour, P<0.05.4.The Cat K expression of TNF-a intervention time for 6h,12h and 24h were 0.36±0.04,0.54±0.05和0.84±0.07 respectively, comparing with any two of them, P<0.05.As the time of induced moved on, the expression of Cat K become higher during 6 hours to 24 hours. Cat K expression stimulated by TNF-a for 48 hour was 0.60±0.07, which was significantly less than stimulated by TNF-a for 24 hour, P<0.05.5.Compared with l0ng/ml TNF-a stimulation group(0.65±0.04), the expression of Cat K mRNA significantly decreased to 0.55±0.04, 0.57±0.01 and 0.52±0.04 respectively in the cells that were treated with SB203580、LY294002 and PDTC for 24 hours with lOng/ml TNF-a stimulation,all P<0.05.6.The expression of Cat K were 0.70±0.08,0.67±0.11 and 0.62±0.09 in the cells that were treated with SB203580、LY294002 and PDTC for 24 hours with lOng/ml TNF-a stimulation, they all were obviously less than the lOng/ml TNF-a stimulation group(0.91±0.15), all P<0.01.Conclusion:TNF-a could induce the expression of Cathepsin K in human umbilical vein endothelial cells in a dose-dependent and time-dependent manner. TNF-a may induce Cat K expression through the activation of signaling pathway of P38,PI3K AND NF-Kb. BackgroundIntegrity of intimal play an important role in preventing thrombosis and restenosis, late thrombosis and restenosis maybe have relations to the inhibition of regeneration of endothelial cells by Paclitaxel. Cathepsin K which could degrade various ECM components and promote VSMC and EC migration is closely associated with intimal hyperplasia. NF-kB is an important transcription factor and could regulates the expression of multiple gene products. Paclitaxel inhibited cell migration, but the effect of paclitaxel on the expression of cathepsin K is still unknown. It is not clear whether or not Paclitaxel can reduce Cat K expression through inhibiting NF-kB in endothelial cells.Objective:The present study was aimed to investigate the effect of Paclitaxel on TNF-a induced human umbilical vein endothelial cells expression of Cat K and NF-kB, trying to elucidate the of mechanism of paclitaxel inhibiting intimal hyperplasia.Methods:HUVECs in the third to sixth generation were used in the present study.The cells were treated with 10ng/ml TNF-a and then different concentrations of paclitaxel were added into it for 24hours.We used the RT-PCR method to measure Cathepsin K mRNA levels and used the western-blotting method to measure Cathepsin K and NF-kB levels of these cells.Results:1. The expression of Cat K mRNA were 0.85±0.06,0.68±0.04,0.59±0.02 and 0.49±0.03 respectively in the paclitaxel treated group of 1nmol/l,10nmol/l, 100nmol/l and 1μmol/1, comparing with any two of them,P<0.05. comparing with TNF-a group(1.02±0.07), their expressions decreased, P<0.05. paclitaxel could inhibit TNF-a induced Cathepsin K mRNA expression in a dose-dependent manner. With the higher concentration of paclitaxel, inhibition of the expression of Cat K mRNA became stronger.2. The Cat K expression of 1nmol/l paclitaxel group and TNF-a group were 1.08±0.07 and 0.87±0.03, both comparing, P<0.05。paclitaxel could inhibit TNF-a induced Cathepsin K protein expression in a dose-dependent manner. The higher concentration of paclitaxel, the less Cat K expression of the cells.3. Normal human umbilical vein endothelial cell had very little NF-kB expression.The NF-kB expression of 1nmol/l paclitaxel group and TNF-a group were 0.93±0.04 and 0.87±0.07, the two comparing, P <0.05. paclitaxel could inhibit TNF-a induced NF-kB protein expression in a dose-dependent manner in endothelial cells. The higher concentration of paclitaxel, the less NF-kB expression of endothelial cells.Conclusion:Paclitaxel could decrease the expression of Cathepsin K and NF-kB stimulated with TNF-a in a dose-dependent manner. paclitaxel maybe inhibit the activation of NF-kB to reduced Cat K expression in endothelial cells.更多还原