抑制NF-κB通路激活对防治旁路移植术后移植血管再狭窄的实验研究
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摘要
第一部分:Cuff套管技术建立大鼠自体静脉移植动物模型
目的:探讨利用Cuff套管技术进行大鼠颈外静脉-肾下腹主动脉的自体静脉移植,建立一种与冠脉旁路移植术后静脉桥再狭窄病理过程相似的动物模型。
方法:采用雄性Wistar大鼠20只,体重350-400g,行无菌外科手术操作。显露并切取一段颈外静脉利用Cuff套管技术端端吻合移植于肾下腹主动脉,建立大鼠自体静脉移植动物模型。分别在术后第2、4周各处死5-6只大鼠切取移植静脉,取不同时期的静脉移植物进行组织病理学检查。实验中同时切取大鼠右侧颈外静脉作为自身对照。
结果:正式手术实验大鼠20只,手术平均时间为60min,移植静脉冷缺血时间10-20min,热缺血时间5-10min,手术过程中:无动物死亡。手术成功17只,术后死亡3只,手术成功率85%。组织病理学检查显示术后第2、4周可见内膜不同程度增生,伴炎症细胞浸润及中层平滑肌细胞增殖。
结论:本研究应用Cuff套管吻合技术成功建立大鼠自体静脉移植动物模型,手术操作简便易行,成功率高,能客观真实地反映冠脉旁路手术后移植静脉内膜增生的病理过程,是进一步研究防治血管移植后再狭窄的理想模型。
第二部分:蛋白酶体抑制剂万珂(注射用硼替佐米)在大鼠自体静脉移植模型中抑制移植血管内膜增生的实验研究
目的:通过建立Wistar大鼠自体静脉移植模型,探讨炎症相关因子术后在移植血管中表达的动态变化及炎性反应对血管再狭窄的相关影响;并在此基础上研究应用蛋白酶体抑制剂硼替佐米后对相关炎症介质表达及对移植血管内膜增生的抑制作用。
方法:采用雄性Wistar大鼠88只,体重350-400g,麻醉后行无菌手术操作。显露并切取一段颈外静脉利用Cuff套管技术端端吻合移植于肾下腹主动脉,建立大鼠自体静脉移植动物模型。术后将大鼠随机分为两组,分别静脉给予硼替佐米(硼替佐米组)和安慰剂(安慰剂组)治疗。每组分别在术后第24、72小时各处死8只大鼠切取移植静脉,取不同时期的静脉移植物进行实时定量逆转录聚合酶链反应(real-time reverse transcription-polymerase chain reaction,RT-PCR)检测趋化因子(CINC-2β和MCP-1)和细胞因子(IL-1,6和TNF-α);与此同时取术后24小时静脉移植物标本行酶联免疫吸附测定(enzyme linkedimmunosorbent assay,ELISA)检测趋化因子(CINC-2β和MCP-1)和细胞因子(IL-1,6和TNF-α)(8只/组),并进行中性粒细胞趋化性试验(8只/组)。在接下的实验中,两用药组分别在术后第2、4周各处死6只大鼠,切取不同时期的静脉移植物行组织病理学检查。以上各实验均同时取大鼠右侧颈外静脉作为自身对照。
结果:组织病理学检查显示,术后2-4周移植静脉新生内膜增生明显。与安慰剂组相比,术后应用硼替佐米可明显抑制各时期静脉移植物的内膜增生(P＜0.05);对炎症相关因子的研究显示,静脉移植物中趋化因子(CINC-2β和MCP-1)和细胞因子(IL-1,6和TNF-α)的基因表达在术后24小时均显著增高(P＜0.05),此后至72小时逐渐下降回归至基线水平(除外MCP-1)。与安慰剂组相比,术后应用硼替佐米可明显抑制CINC-2β,MCP-1,IL-6和TNF-α在静脉移植物中的基因表达(除外IL-1)(P＜0.05);酶联免疫吸附测定显示趋化因子(CINC-2β和MCP-1)和细胞因子(IL-1,6和TNF-α)的蛋白合成在术后24小时亦相应出现显著增高(P＜0.05)。与安慰剂组相比,术后应用硼替佐米可明显抑制CINC-2β,MCP-1,IL-6和TNF-α在静脉移植物中的蛋白合成(除外IL-1)(P＜0.05),并显著抑制静脉移植物组织匀浆对中性粒细胞的趋化活性(P＜0.05)。
结论:1.旁路移植手术后,由血管损伤诱发的局部炎症反应是导致移植血管内膜增厚/再狭窄的主要原因;2.应用蛋白酶体抑制剂硼替佐米可明显抑制移植血管术后的炎性变化和内膜增生,其可成为一种新的药物用于防治旁路移植术后的血管病变。
第三部分:短发夹样NF-κB特异性RNA干扰表达载体的构建及体外功能研究
目的:采用RNA干扰(RNA interference,RNAi)技术,构建大鼠NF-κB(RelA,p65)基因特异性小分子干扰RNA(small interfering RNAs,siRNA)表达载体质粒;体外观察其转染对NF-κB基因的沉默作用,及对NF-κB信号通路激活所致细胞增殖调控的抑制作用,为进一步体内实验研究准备条件。
方法:根据GenBank提供的大鼠NF-κB(p65)基因序列,通过基因BLAST,挑选长为21个碱基的特异性寡核苷酸序列,合成其短发夹状RNA(short hairpinRNAs,shRNA)双链。通过DNA重组技术,将p65 siRNA双链与U6启动子指导的线性化真核表达载体pGenesil-1.2连接,构建p65 siRNA的重组表达载体质粒并命名为pGenesil-1.2-p65siRNA。以重组质粒转化大肠杆菌,用碱裂解法少量制备质粒DNA,进行酶切及DNA测序鉴定,用QIAGEN试剂盒大量制备质粒DNA。取重组质粒以脂质体Lipofectamine2000介导转染体外SVAREC细胞。进一步试验采用内毒素LPS(1μg/ml)刺激SVAREC细胞以激活NF-κB信号通路,Western blot检测细胞内NF-κB p65亚单位蛋白水平,流式细胞仪结合MTT检测以观察不同条件下细胞中NF-κB的活化与细胞增殖的关系及转染质粒pGenesil-1.2-p65siRNA后对NF-κB信号通路激活所致细胞增殖的干扰抑制效力。
结果:成功构建大鼠NF-κB p65基因的siRNA表达质粒载体pGenesil-1.2-p65siRNA,经测序鉴定证实插入序列正确无误;体外实验,Western blot检测显示,转染质粒pGenesil-1.2-p65siRNA能显著抑制LPS刺激后SVAREC细胞内NF-κBp65蛋白的增高(P＜0.05);流式细胞仪结合MTT检测结果显示,LPS刺激激活NF-κB信号通路可促使细胞由G_0/G_1期向S期转换,进而促进内皮细胞的增殖,而转染质粒pGenesil-1.2-p65siRNA可显著抑制LPS刺激后引起的内皮细胞S期细胞比例和增殖指数的增高(P＜0.05)。
结论:1.成功构建大鼠NF-κB p65基因的siRNA表达质粒pGenesil-1.2-p65siRNA;2.质粒pGenesil-1.2-p65siRNA转染大鼠内皮细胞后可有效抑制相应基因及蛋白的表达合成,并显著抑制NF-κB信号通路激活引起的细胞增殖性改变,为进一步体内试验准备了条件。
PartⅠEstablish Abdominal External Jugular Vein Transplatation Model With Cuff Technique in Rats
Objective
To establish an rat mode of autologous vein grafts restenosis with cuff technique, the pathological process of which is similar with cononary artery bypass grafting.
Methods
Twenty male Wistar rats(weight,350 to 400 g)were obtained and housed in conformity with international guidelines for the care and use of laboratory animals. After the rat was anesthetized,a segment of the left external jugular vein was excised and a segment of the abdominal aorta was denuded and disconnected.Then the autologous external jugular vein graft was interposed into the aortic nick according to cuff technique.Subsequently,rats were euthanized at 2 and 4 weeks respectively after grafting and vein grafts were processed for morphometric analysis.Meanwile,the right external jugular vein was obtained as own control.
Results
Abdominal external jugular vein transplantation was performed in twenty rats, seventeen survived while three died.The reasons of death were the lumbar artery damage,the delayed bleeding of abdominal aorta stump,and the intestinal obstruction respectively.The achievement ratio of operation is 85%.The mean operative time was 60min.The cold ischemia time and wam isehemia time of the donor was 10-20min and 5-10min,respectively.Histopathology showed that the intima of grafts thickened gradually during 4 weeks after surgery accompanyed with inflammatory cell infiltration and smooth muscle cell proliferation.
Conclusions
The cuff technique is feasible to establish rat model of abdominal external jugular vein transplantation.This technique would be ideal because it is simple and easier to peform with higher achievement ratio.It is also able to reflect the pathological process of intimal hyperplasia of autologous vein grafting objectively and could be an ideal model for further preventing restenosis after cononary artery bypass grafting.
PartⅡProteasome Inhibitor Bortezomib Inhibit Intimal Hyperplasia of Autologous Vein Grafting in Rat Model
Objective
A autologous vein transplantation model was used to detect whether inflammation played an important role in intimal hyperplasia induced by bypass graft. On this basis,to explore the feasibility and efficiency of proteasome inhibitor bortezomib in the inhibition of neointima formation in transplant-induced vasculopathy for its anti-inflammatory effect.
Methods
Eighty-eight male Wistar rats(weight,350 to 400 g)were obtained and housed in conformity with international guidelines for the care and use of laboratory animals. After the rat was anesthetized,a segment of the left external jugular vein was excised and interposed into the abdominal aorta nick according to cuff technique.All post-surgical rats were divided into two groups randomly and treated with bortezomib or placebo.After 24 and 72 hours,rats were humanly killed and vein grafts were processed for real-time RT-PCR(24 and 72 hours)to test CINC-2β,MCP-1,IL-1, IL-6 and TNF-α,for ELISA(24 hours)to test C1NC-2β,MCP-1,IL-1,IL-6 and TNF-α,and for neutrophil chemotaxis assay(24 hours).Subsequently,rats were euthanized at 2 and 4 weeks after grafting and samples were processed for morphometric analysis.Meanwile,the right external jugular vein was obtained as own control.
Results
Morphometric analysis showed that the intima of grafts thickened gradually during 4 weeks after surgery.Bortezomib resulted in significant inhibition of intimal hyperplasia in each time point,compared with untreated controls(P＜0.05).The expression of mRNA for selective chemokine(CINC-2βand MCP-1)and cytokines (IL-1,6 and TNF-α)increased markedly in injured vessels during the first day after surgery and declined gradually in the following three days(except MCP-1).ELISA showed chemokine(C1NC-2βand MCP-1)and cytokines(IL-1,6 and TNF-α)also had significant rise accordingly in 24 hours after surgry(P＜0.05).Compared with control,Bortezomib significantly attenuated gene expression and protein level in most of the inflammatory mediators and simultaneously inhibited neutrophil chemotactic activity of the vessel homogenates(P＜0.05).
Conclusions
Increasing evidence indicated that inflammation played an important role in intimal hyperplasia induced by autologous vein grafting.Bortezomib could inhibit neointima formation significantly by attenuating the inflammatory response in transplant-induced vasculopathy and should be used as a novel vaso-protective remedium in the clinical field.
PartⅢConstruction of Plasmid Vector Expressing Rat NF-KB Small Interfering RNA and In Vitro Function Identification
Objective
To construct one RNA interference(RNAi)expression vector targeting rat NF-κB(Rela,P65)and identify its inhibitory effect to corresponding gene expression in SVAREC cells transfected with the recombinant plasmid,preparing for the next in vivo study.
Methods
Design small interfering RNA(siRNA)sequence targeting rat NF-κB p65,and synthesize two complementary oligodeoxyribonucleotides encoding NF-κB p65 short hairpin RNA(shRNA).Two complementary oligodeoxyribonucleotides were annealed and ligated into linearized pgenesil-1.2 to construct the recombinant plasmid pGenesil-1.2-p65siRNA.The ligation mixtures were transformed into competent E.coli DH5a.The recombinant plasmids were extracted from small-scale bacterial cultures by Alkaline Lysis and then identified by enzyme analysis and DNA sequence analysis.QIAGEN plasmid maxikit was employed tbr large-scale preparation of recombinant plasmids.Lipofectamine2000 was used for the transfection of SVAREC cells by pGenesil-1.2-p65siRNA.After the transfection,LPS(lμg/ml)was used to stimulate SVAREC cells for activating the NF-κB signal pathway,total protein was isolated from cells and NF-κB p65 protein was analyzed by Western blotting.Then,flow cytometry and MTT assay were performed to observe the relationship between cell proliferation and NF-κB pathway activation,and the suppression effect due to the pGenesil-1.2-p65siRNA transfection.
Results.
It was confirmed by enzyme analysis and DNA sequencing that the insert sequence was successfully cloned into the vector.Western blotting showed that protein level of NF-κB p65 in transfected SVAREC cells reduced significantly as stimulated by LPS(P＜0.05).Flow cytometry with MTT assay suggested that the activation of NF-κB signal pathway stimulated by LPS would prompt cell cycle converting from G_0/G_1 phase to S phase,thereby promoting the cell proliferation. Meanwhile,Rreal transfection could significantly inhibit the increased cell proportion in S phase and cell proliferation index induced by LPS stimulation in SVAREC cells respectively(P＜0.05).
Conclusion
The plasmids vector pGenesil-1.2-p65siRNA which express rat NF-κB p65 siRNA were successfully designed and constructed.It was demonstrated in initiatory study that transfected pGenesil-1.2-p65siRNA in vitro rat endothelial cells could effectively suppress the NF-κB p65 expression,and significantly inhibit cells proliferation induced by NF-κB signal pathway activation,which prepare for further in vivo study.
目的:探讨利用Cuff套管技术进行大鼠颈外静脉-肾下腹主动脉的自体静脉移植,建立一种与冠脉旁路移植术后静脉桥再狭窄病理过程相似的动物模型。
方法:采用雄性Wistar大鼠20只,体重350-400g,行无菌外科手术操作。显露并切取一段颈外静脉利用Cuff套管技术端端吻合移植于肾下腹主动脉,建立大鼠自体静脉移植动物模型。分别在术后第2、4周各处死5-6只大鼠切取移植静脉,取不同时期的静脉移植物进行组织病理学检查。实验中同时切取大鼠右侧颈外静脉作为自身对照。
结果:正式手术实验大鼠20只,手术平均时间为60min,移植静脉冷缺血时间10-20min,热缺血时间5-10min,手术过程中:无动物死亡。手术成功17只,术后死亡3只,手术成功率85%。组织病理学检查显示术后第2、4周可见内膜不同程度增生,伴炎症细胞浸润及中层平滑肌细胞增殖。
结论:本研究应用Cuff套管吻合技术成功建立大鼠自体静脉移植动物模型,手术操作简便易行,成功率高,能客观真实地反映冠脉旁路手术后移植静脉内膜增生的病理过程,是进一步研究防治血管移植后再狭窄的理想模型。
第二部分:蛋白酶体抑制剂万珂(注射用硼替佐米)在大鼠自体静脉移植模型中抑制移植血管内膜增生的实验研究
目的:通过建立Wistar大鼠自体静脉移植模型,探讨炎症相关因子术后在移植血管中表达的动态变化及炎性反应对血管再狭窄的相关影响;并在此基础上研究应用蛋白酶体抑制剂硼替佐米后对相关炎症介质表达及对移植血管内膜增生的抑制作用。
方法:采用雄性Wistar大鼠88只,体重350-400g,麻醉后行无菌手术操作。显露并切取一段颈外静脉利用Cuff套管技术端端吻合移植于肾下腹主动脉,建立大鼠自体静脉移植动物模型。术后将大鼠随机分为两组,分别静脉给予硼替佐米(硼替佐米组)和安慰剂(安慰剂组)治疗。每组分别在术后第24、72小时各处死8只大鼠切取移植静脉,取不同时期的静脉移植物进行实时定量逆转录聚合酶链反应(real-time reverse transcription-polymerase chain reaction,RT-PCR)检测趋化因子(CINC-2β和MCP-1)和细胞因子(IL-1,6和TNF-α);与此同时取术后24小时静脉移植物标本行酶联免疫吸附测定(enzyme linkedimmunosorbent assay,ELISA)检测趋化因子(CINC-2β和MCP-1)和细胞因子(IL-1,6和TNF-α)(8只/组),并进行中性粒细胞趋化性试验(8只/组)。在接下的实验中,两用药组分别在术后第2、4周各处死6只大鼠,切取不同时期的静脉移植物行组织病理学检查。以上各实验均同时取大鼠右侧颈外静脉作为自身对照。
结果:组织病理学检查显示,术后2-4周移植静脉新生内膜增生明显。与安慰剂组相比,术后应用硼替佐米可明显抑制各时期静脉移植物的内膜增生(P＜0.05);对炎症相关因子的研究显示,静脉移植物中趋化因子(CINC-2β和MCP-1)和细胞因子(IL-1,6和TNF-α)的基因表达在术后24小时均显著增高(P＜0.05),此后至72小时逐渐下降回归至基线水平(除外MCP-1)。与安慰剂组相比,术后应用硼替佐米可明显抑制CINC-2β,MCP-1,IL-6和TNF-α在静脉移植物中的基因表达(除外IL-1)(P＜0.05);酶联免疫吸附测定显示趋化因子(CINC-2β和MCP-1)和细胞因子(IL-1,6和TNF-α)的蛋白合成在术后24小时亦相应出现显著增高(P＜0.05)。与安慰剂组相比,术后应用硼替佐米可明显抑制CINC-2β,MCP-1,IL-6和TNF-α在静脉移植物中的蛋白合成(除外IL-1)(P＜0.05),并显著抑制静脉移植物组织匀浆对中性粒细胞的趋化活性(P＜0.05)。
结论:1.旁路移植手术后,由血管损伤诱发的局部炎症反应是导致移植血管内膜增厚/再狭窄的主要原因;2.应用蛋白酶体抑制剂硼替佐米可明显抑制移植血管术后的炎性变化和内膜增生,其可成为一种新的药物用于防治旁路移植术后的血管病变。
第三部分:短发夹样NF-κB特异性RNA干扰表达载体的构建及体外功能研究
目的:采用RNA干扰(RNA interference,RNAi)技术,构建大鼠NF-κB(RelA,p65)基因特异性小分子干扰RNA(small interfering RNAs,siRNA)表达载体质粒;体外观察其转染对NF-κB基因的沉默作用,及对NF-κB信号通路激活所致细胞增殖调控的抑制作用,为进一步体内实验研究准备条件。
方法:根据GenBank提供的大鼠NF-κB(p65)基因序列,通过基因BLAST,挑选长为21个碱基的特异性寡核苷酸序列,合成其短发夹状RNA(short hairpinRNAs,shRNA)双链。通过DNA重组技术,将p65 siRNA双链与U6启动子指导的线性化真核表达载体pGenesil-1.2连接,构建p65 siRNA的重组表达载体质粒并命名为pGenesil-1.2-p65siRNA。以重组质粒转化大肠杆菌,用碱裂解法少量制备质粒DNA,进行酶切及DNA测序鉴定,用QIAGEN试剂盒大量制备质粒DNA。取重组质粒以脂质体Lipofectamine2000介导转染体外SVAREC细胞。进一步试验采用内毒素LPS(1μg/ml)刺激SVAREC细胞以激活NF-κB信号通路,Western blot检测细胞内NF-κB p65亚单位蛋白水平,流式细胞仪结合MTT检测以观察不同条件下细胞中NF-κB的活化与细胞增殖的关系及转染质粒pGenesil-1.2-p65siRNA后对NF-κB信号通路激活所致细胞增殖的干扰抑制效力。
结果:成功构建大鼠NF-κB p65基因的siRNA表达质粒载体pGenesil-1.2-p65siRNA,经测序鉴定证实插入序列正确无误;体外实验,Western blot检测显示,转染质粒pGenesil-1.2-p65siRNA能显著抑制LPS刺激后SVAREC细胞内NF-κBp65蛋白的增高(P＜0.05);流式细胞仪结合MTT检测结果显示,LPS刺激激活NF-κB信号通路可促使细胞由G_0/G_1期向S期转换,进而促进内皮细胞的增殖,而转染质粒pGenesil-1.2-p65siRNA可显著抑制LPS刺激后引起的内皮细胞S期细胞比例和增殖指数的增高(P＜0.05)。
结论:1.成功构建大鼠NF-κB p65基因的siRNA表达质粒pGenesil-1.2-p65siRNA;2.质粒pGenesil-1.2-p65siRNA转染大鼠内皮细胞后可有效抑制相应基因及蛋白的表达合成,并显著抑制NF-κB信号通路激活引起的细胞增殖性改变,为进一步体内试验准备了条件。
PartⅠEstablish Abdominal External Jugular Vein Transplatation Model With Cuff Technique in Rats
Objective
To establish an rat mode of autologous vein grafts restenosis with cuff technique, the pathological process of which is similar with cononary artery bypass grafting.
Methods
Twenty male Wistar rats(weight,350 to 400 g)were obtained and housed in conformity with international guidelines for the care and use of laboratory animals. After the rat was anesthetized,a segment of the left external jugular vein was excised and a segment of the abdominal aorta was denuded and disconnected.Then the autologous external jugular vein graft was interposed into the aortic nick according to cuff technique.Subsequently,rats were euthanized at 2 and 4 weeks respectively after grafting and vein grafts were processed for morphometric analysis.Meanwile,the right external jugular vein was obtained as own control.
Results
Abdominal external jugular vein transplantation was performed in twenty rats, seventeen survived while three died.The reasons of death were the lumbar artery damage,the delayed bleeding of abdominal aorta stump,and the intestinal obstruction respectively.The achievement ratio of operation is 85%.The mean operative time was 60min.The cold ischemia time and wam isehemia time of the donor was 10-20min and 5-10min,respectively.Histopathology showed that the intima of grafts thickened gradually during 4 weeks after surgery accompanyed with inflammatory cell infiltration and smooth muscle cell proliferation.
Conclusions
The cuff technique is feasible to establish rat model of abdominal external jugular vein transplantation.This technique would be ideal because it is simple and easier to peform with higher achievement ratio.It is also able to reflect the pathological process of intimal hyperplasia of autologous vein grafting objectively and could be an ideal model for further preventing restenosis after cononary artery bypass grafting.
PartⅡProteasome Inhibitor Bortezomib Inhibit Intimal Hyperplasia of Autologous Vein Grafting in Rat Model
Objective
A autologous vein transplantation model was used to detect whether inflammation played an important role in intimal hyperplasia induced by bypass graft. On this basis,to explore the feasibility and efficiency of proteasome inhibitor bortezomib in the inhibition of neointima formation in transplant-induced vasculopathy for its anti-inflammatory effect.
Methods
Eighty-eight male Wistar rats(weight,350 to 400 g)were obtained and housed in conformity with international guidelines for the care and use of laboratory animals. After the rat was anesthetized,a segment of the left external jugular vein was excised and interposed into the abdominal aorta nick according to cuff technique.All post-surgical rats were divided into two groups randomly and treated with bortezomib or placebo.After 24 and 72 hours,rats were humanly killed and vein grafts were processed for real-time RT-PCR(24 and 72 hours)to test CINC-2β,MCP-1,IL-1, IL-6 and TNF-α,for ELISA(24 hours)to test C1NC-2β,MCP-1,IL-1,IL-6 and TNF-α,and for neutrophil chemotaxis assay(24 hours).Subsequently,rats were euthanized at 2 and 4 weeks after grafting and samples were processed for morphometric analysis.Meanwile,the right external jugular vein was obtained as own control.
Results
Morphometric analysis showed that the intima of grafts thickened gradually during 4 weeks after surgery.Bortezomib resulted in significant inhibition of intimal hyperplasia in each time point,compared with untreated controls(P＜0.05).The expression of mRNA for selective chemokine(CINC-2βand MCP-1)and cytokines (IL-1,6 and TNF-α)increased markedly in injured vessels during the first day after surgery and declined gradually in the following three days(except MCP-1).ELISA showed chemokine(C1NC-2βand MCP-1)and cytokines(IL-1,6 and TNF-α)also had significant rise accordingly in 24 hours after surgry(P＜0.05).Compared with control,Bortezomib significantly attenuated gene expression and protein level in most of the inflammatory mediators and simultaneously inhibited neutrophil chemotactic activity of the vessel homogenates(P＜0.05).
Conclusions
Increasing evidence indicated that inflammation played an important role in intimal hyperplasia induced by autologous vein grafting.Bortezomib could inhibit neointima formation significantly by attenuating the inflammatory response in transplant-induced vasculopathy and should be used as a novel vaso-protective remedium in the clinical field.
PartⅢConstruction of Plasmid Vector Expressing Rat NF-KB Small Interfering RNA and In Vitro Function Identification
Objective
To construct one RNA interference(RNAi)expression vector targeting rat NF-κB(Rela,P65)and identify its inhibitory effect to corresponding gene expression in SVAREC cells transfected with the recombinant plasmid,preparing for the next in vivo study.
Methods
Design small interfering RNA(siRNA)sequence targeting rat NF-κB p65,and synthesize two complementary oligodeoxyribonucleotides encoding NF-κB p65 short hairpin RNA(shRNA).Two complementary oligodeoxyribonucleotides were annealed and ligated into linearized pgenesil-1.2 to construct the recombinant plasmid pGenesil-1.2-p65siRNA.The ligation mixtures were transformed into competent E.coli DH5a.The recombinant plasmids were extracted from small-scale bacterial cultures by Alkaline Lysis and then identified by enzyme analysis and DNA sequence analysis.QIAGEN plasmid maxikit was employed tbr large-scale preparation of recombinant plasmids.Lipofectamine2000 was used for the transfection of SVAREC cells by pGenesil-1.2-p65siRNA.After the transfection,LPS(lμg/ml)was used to stimulate SVAREC cells for activating the NF-κB signal pathway,total protein was isolated from cells and NF-κB p65 protein was analyzed by Western blotting.Then,flow cytometry and MTT assay were performed to observe the relationship between cell proliferation and NF-κB pathway activation,and the suppression effect due to the pGenesil-1.2-p65siRNA transfection.
Results.
It was confirmed by enzyme analysis and DNA sequencing that the insert sequence was successfully cloned into the vector.Western blotting showed that protein level of NF-κB p65 in transfected SVAREC cells reduced significantly as stimulated by LPS(P＜0.05).Flow cytometry with MTT assay suggested that the activation of NF-κB signal pathway stimulated by LPS would prompt cell cycle converting from G_0/G_1 phase to S phase,thereby promoting the cell proliferation. Meanwhile,Rreal transfection could significantly inhibit the increased cell proportion in S phase and cell proliferation index induced by LPS stimulation in SVAREC cells respectively(P＜0.05).
Conclusion
The plasmids vector pGenesil-1.2-p65siRNA which express rat NF-κB p65 siRNA were successfully designed and constructed.It was demonstrated in initiatory study that transfected pGenesil-1.2-p65siRNA in vitro rat endothelial cells could effectively suppress the NF-κB p65 expression,and significantly inhibit cells proliferation induced by NF-κB signal pathway activation,which prepare for further in vivo study.
引文
1. Francis SE, Hunter S, Holt CM,et al. Release of platelet derived growth factor activity from arteriovenous bypass grafts.Thorac Cardiovasc Surg. 1994;108(3): 540-8.
2. Togni M, Windecker S, MeierB,et al. Treatment of restenosis.Curr Interv Cardiol Rep. 2001;3(4):306-10.
3. Hamano M, Kuroda Y, Saitoh Y. Pancreas transplantation using non-suture cuff technique in the neck. Kobe J Med Sci. 1996; 42(2):93-104.
4. Tomita Y, Zhang QW, Yoshikawa M, et al. Improved technique of heterotopic cervical heart transplantation in mice. Transplantation. 1997; 64(11): 1598-601.
5. Tomita Y, Zhang QW, Uchida T, et al. A technique of cervical aortic graft transplantation in mice. J Heart Lung Transplant. 2001; 20(6):699-702.
6. Nakao A, Ogino Y, Tahara K, et al. Orthotopic intestinal transplantation using the cuff method in rats:a histopathological evaluation of the anastomosis. Microsurgery. 2001;21(1):12-5.
7. Xiu D, Uchida H, To H, et al. Simplified method of heterotopic rat heart transplantation using the cuff technique: application to sublethal dose protocol of methotrexate on allograft survival. Microsurgery. 2001; 21(1):16-21.
8. Guo H, Wu YJ, Zheng SS, et al. Application of modified two-cuff technique and multiglycosides tripterygium wilfordii in hamster-to-rat liver xenotransplant model. World J Gastroenterol. 2003 ;9(7): 1550-3.
9. Peng Y, Gong JP, Yan LN, et al. Improved two-cuff technique for orthotopic liver transplantation in rat. Hepatobiliary Pancreat Dis Int. 2004; 3(1): 33-7.
10. Tan F, Chen Z, Zhao Y, et al. Novel technique for suprahepatic vena cava reconstruction in rat orthotopic liver transplantation. Microsurgery.2005;25(7):556-60.
11. Ross R. The pathogenesisof atherosclerosis:a perspective for the 1990s. Nature. 1993;362(6432):801-9.
12. Fitzgibbon G, Kafka HP, Leach AJ, et al. Coronary bypass graft fate and patient outcome: angiographic follow-up of 5065 grafts related to survival and reoperation in 1388 patients during 25 years. J Am Coll Cardiol. 1996; 28(3): 616-26.
13. Kalra M, Miller VM. Early remoldeling of saphenous vein graft: proliferation, migration and apoptosis of adventitial and medial cells occur simultaneously with changes in graft diameter and blood flow. J Vasc Res. 2000; 37(6):576-84.
14. Bauters C, Lamblin N, Amouyel P, et al. Gene polymorphisms and outcome after coronary angioplasty. Curr Interv Cardiol Rep. 2001; 3(4):281-6.
15. Ruygrok PN, Webster MW, de Valk V, et al. Clinical and angiographic factors associated with asymptomatic restenosis after percutaneous coronary intervention. Circulation. 2001; 104(19):2289-94.
16. Grassman ED, Leya F, Fareed J, et al. A randomized trial of the low molecular weight heparin certoparin to prevent restenosis following coronary angioplasty.J Invasive Cardiol. 2001;13(11):723-8.
17. Nakao A, Tahara K, Inoue S, et al. Combined cuff and suture technique for orthotopic whole intestinal transplantation in rats. Microsurgeiy.2002;22(3):85-90.
18. Akpinar S, Hersekli MA, Demirors H, et al. Effects of methylprednisolone and betamethasone injections on the rotator cuff: an experimental study in rats. Adv Ther. 2002; 19(4): 194-201.
19. Tanaka H, Hashizume K, Enosawa S, et al. Successful transplantation of a 20% partial liver graft in rats: a technical innovation. J Surg Res. 2003; 110(2):409-12.
20. Demirhan M, Atalar AC, Kilicoglu O. Primary fixation strength of rotator cuff repair techniques:a comparative study. Arthroscop. 2003; 19(6): 572-6.
21.Gerber C, Meyer DC, Schneeberger AG, et al. Effect of tendon release and delayed repair on the structure of the muscles of the rotator cuff: an experimental study in sheep. J Bone Joint Surg Am. 2004; 86-A(9): 1973-82.
22. Birsner JH, Wan C, Cheng G, et al. Steatotic liver transplantation in the mouse:a model of primary nonfunction. JSurgRes. 2004; 120(1):97-101.
23. Santana Rodriguez N, Martin Barrasa JL, Lopez Garcia A, et al. Lung transplantation in rats: a viable experimental model. Arch Bronco-neumol.2004;40(10): 438-42.
24. Yoshida S, Sekine Y, Saitoh Y, et al. Surgical technique of experimental lung transplantation in rabbits. Ann Thorac Cardiovasc Surg. 2005; 11(1): 7-11.
25. Kashfi A, Mehrabi A, Pahlavan PS, et al. A review of various techniques of orthotopic liver transplantation in the rat. Transplant Proc.2005; 37(1): 185-8.
26. Lehmann TG, Bunzendahl H, Langrehr JM, et al. Arterial reconstruction in rat liver transplantation-development of a new tubing technique of the common hepatic artery. Transpl Int. 2005; 18(1 ):56-64.
1. Francis SE, Hunter S, Holt CM, et al. Release of platelet derived growth factor activity from arteriovenous bypass grafts. Thorac Cardiovasc Surg. 1994;108(3): 540-8
2. Togni M,Windecker S,MeierB,et al.Treatment of restenosis . Curr Interv Cardiol Rep.2001;3(4):306-10.
3. Kornowski R,Hong M,Fermin F,et al. In-stent restenosisxontributions of inflame-matory responses and arterial injury to neointimal hyperplasia. J Am Coll Cardiol. 1998; 31(1):224-30.
4. Farb A, Weber DK, Kolodgie FD, et al. Morphological predictors of restenosis after coronary stenting in humans. Circulation. 2002; 105(25): 2974-80.
5. Gul, Tseng s, Rollins B. Monocyte chemoattractant protein 1. Chem Immunol. 1999; 72(1):7-29.
6. Cipollone F, Marini M, Fazia M, et al. Elevated circulating levels of monocyte chemoattractant protein-1 in patients with restenosis after coronary angioplasty. Arterioscler Thromb Vasc Biol; 2001,21 (3):327-34.
7. Qi X, Li J, Gu J, et al. Plasma levels of IL-8 predict early complications in patients with coronary heart diease after percutaneous coronary intervention. Jpn Heart J. 2003; 44(4):451-61.
8. Abdelmouttaleb I, Danchin N, Ilardo C, et al. C reactive protein and coronary artery disease:additional evidence of the implication of an inflammatory process in acute coronary syndromes. Am Heart J. 1999; 137(2):346-51.
9. Lentsch A B, Ward P A. The NF-kappa B/Ikappa B system in acute inflammation. Arch Immunol Ther Exp(Warsz).2000; 48(2):59-63.
10. Epinat J C, Gilmore T D. Diverse agents act at multiple levels to inhibit the Rel/NF-Kappa B signal transduction pathway. Oncogene. 1999; 18(49):6896 -909.
11. Karin M, Ben Neriah Y. Phosphorylation meets ubiquitination:the control of NF-kappa B activity. Annu Rev Immunol. 2000; 18(1): 621-63.
12. Bakker R A,Schoonus S B,Smit M J,et al.Histamine H(1)-receptor activation of nuclear factor-kappa B:roles for G betagamma- and G alpha(q/11)-subunits in constitutive and agonist-mediated signaling. Mol Pharmacol.2001;60(5):1133-42.
13. Adams J, Kauffman M. Development of the proteasome inhibitor Velcade (Bortezomib). Cancer Invest.2004; 22(2):304-11.
14. Henninger N, Sicard KM, Bouley J, et al. The proteasome inhibitor VELCADE reduces infarction in rat models of focal cerebral ischemia. Neurosci Lett. 2006; 398(3): 300-5.
15. Zhang L, Zhang ZG, Liu X, et al. Treatment of embolic stroke in rats with bortezomib and recombinant human tissue plasminogen activator. Thromb Haemost. 2006; 95(1): 166-73.
16. Sinn DI, Lee ST, Chu K, et al. Proteasomal inhibition in intracerebral hemorrhage: Neuroprotective and anti-inflammatory effects of bortezomib. Neurosci Res. 2007; 58(1):12-8.
17. Ross R. The pathogenesisof atherosclerosis:a perspective for the 1990s. Nature. 1993;362(6432):801-9.
18. Fitzgibbon G, Kafka HP, Leach AJ ,et al. Coronary bypass graft fate and patient outcome:angiographic follow-up of 5065 grafts related to survival and reoperation in 1388 patients during 25 years. J Am Coll Cardiol. 1996; 28(3):616-26.
19. Kalra M, Miller VM. Early remoldeling of saphenous vein grafts. Proliferation ,migration and apoptosis of adventitial and medial cells occur simultaneously with changes in graft diameter and blood flow. J Vasc Res. 2000; 37(6): 576-84.
20. Kataoka M, Shimizu H, Mitsuhashi N, et al. Effect of cold-ischemia time on C-X-C chemokine expression and neutrophil accumulation in the graft liver after orthotopic liver transplantation in rats. Transplantation. 2002; 73(11):1730-5.
21. Libby P. Inflammation in atherosclerosis. Nature. 2002; 420(6917):868-74.
22. Miller AP,Feng W,Xing D,et al.Estrogen modulates inflammatory mediator expression and neutrophil chemotaxis in injured arteries. Circulation.2004; 110(12): 1664-9.
23. Kitamoto S, Egashira K,Kataoka C, et al. Increased activity of nuclear factor-kappa B participates in cardiovascular remodeling induced by chronic inhibition of nitric oxide synthesis in rats. Circulation. 2000; 102(7):806-12.
24. Porreca E, Di Febbo C, Reale M, et al. Monocyte chemotactic protein 1 (MCP-1) is a mitogen for cultured rat vascular smooth muscle cells. J Vasc Res. 1997;34(1): 58-65.
25. Naruko T, Ueda M, Haze K, et al. Neutrophil infiltration of culprit lesions in acute coronary syndromes. Circulation. 2002; 106(23):2894-900.
26. Okamoto E, Couse T, De Leon H, et al. Perivascular inflammation after balloon angioplasty of porcine coronary arteries. Circulation. 2001; 104(18):2228-35.
27. Menger MD, Vollmar B. Pathomechanisms of ischemia- reperfiision injury as the basis for novel preventive strategies: is it time for the introduction of pleiotropic compounds .Transplant Proc. 2007; 39(2): 485-8.
28. Welt FG, Rogers C. Inflammation and restenosis in the stent era. Arterioscler Thromb Vasc Biol. 2002; 22(11): 1769-76.
29. Sen R, Baltimore D. Multiple nuclear factors interact.with the immunoglobulin enhancer sequences. Cell.1986; 46(5):705-16.
30. Parry GC, Mackman N. Activators and target genesof Rel/NF-kappa B transcription factors. Biol Chem. 1999; 269(1): 20823-5.
31. Arenzana-Seisdedos F, Turpin P, Rodriguez M, et al.Nuclear localization of I kappa B alpha promotes active transport of NF-kappa B from the nucleus to the cytoplasm. J Cell Sci. 1997; 110(pt 3):369-78.
32. Stancovski I, Baltimore D. NF-kappa B actritation: the I kappa B kinase revealed. Cell. 1997;91(1):299-302.
33. Jacobs MD, Harriso SC. Structure of an I kappa B alpha/NF-kappa B complex. Cell. 1998; 95(1):749-58.
34. Pomerantz JL, Baltimore D. Two pathways to NF-kappaB. Mol Cell. 2002; 10(4): 693-5.
35. Hinz M, Krappmann D, Eichten A, et al. Nuclear factor-kappa B, cancer ,and apoptosis. Mol Cell Biol. 2004; 64(1):2690-8.
36. Bharti AC, Aggarwal BB. Nuclear factor-kappa B and cancer.Its role in prevention and therapy. Biochem Pharmacol. 2002; 64(5-6):883-8.
37. Ghosh S, Karin M. Missing pieces in the NF-kappaB puzzle.Cell.2002; 109 suppl: s81-96.
38. Prendes M, Zheng Y, BegAA. Regulation of developing B cell survival by RelA-containing NF-kappaB complexes. J Immunol. 2003; 171(8):3963-9.
39. Cercek B, Yamashita M, Dimayuga P, et al. Nuclear factor-kappaB activity and arterial response to balloon injury .Atherosclerosis.1997;131(1): 59-66.
40. Okamoto E, Couse T, De Leon H,et al. Perivascular inflammation after balloon angioplasty of porcine coronary arteries. Circulation. 2001; 104(18):2228-35.
41. Lu C, Gallegos R, Li P, et al. Investigation of drug-drug interaction potential of bortezomib in vivo in female Sprague-Dawley rats and in vitro in human liver microsomes. Drug Metab Dispos. 2006; 34(4):702-8.
1. Lentsch A B,Ward P A. The NF-kappa B/Ikappa B system in acute inflame-mation. Arch Immunol Ther Exp. 2000; 48(2):59-63.
2. Bakker R A,Schoonus S B,Smit M J,et al.Histamine H(1)-receptor activation of nuclear factor-kappa B:roles for G betagamma- and Galpha (q/11)-subunits in constitutive and agonist-mediated signaling. Mol Pharmaco.2001; 60(5):1133-42.
3. Epinat J C,Gilmore T D.Diverse agents act at multiple levels to inhibit the Rel/NF-Kappa B signal transduction pathway.Oncogene.1999; 18(49): 6896-909.
4. Karin M,Ben Neriah Y.Phosphorylation meets ubiquitination:the control of NF-kappa B activity.Ann Rev Immunol. 2000; 18(2):621-63.
5. Gitlin L,Karelsky S,Andino R.Short interfering RNA confers intracellular antiviral immunity in human cells.Nature. 2002; 418(6896): 430-4.
6. Qin XF, An DS,Chen IS,et al.Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5. Proc Natl AcadSci USA. 2003; 100(1): 183-8.
7. Yang YS,Chen GM,Dong WP,et al.Construction and analysis of activity of an HIV-1/bovine immunodeficiency virus chimeric clone cDNA. Zhong hua Shi Yan He Lin Chuang Bing Du Xue Za Zhi.2003; 17(2): 143-5.
8. Carthew R W.RNA interference: the fragile X syndrome connection.Curr Biol. 2002;12(24):852-4.
9. Ishizuka A,Siomi M C,Siomi H.A Drosophila fragile X protein interacts with components of RNAi and ribosomal proteins.Genes Dev. 2002; 16(19): 2497-508
10. Gil J,Esteban M.Induction of apoptosis by the dsRNA dependent portein kinase (PKR): mechanism of action Apoptosis.2000;5(2): 107-14.
11. Elbashir SM,Harborth J,Lendeckel W,et al.Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.Nature .2001; 411(6836): 494-8.
12. Yu JY,DeRuiter SL,Turner DL.RNA interfernce by expression of short-interfering RNAs and hairpin RNAs in mammalian cells.Proc Natl Acad Sci U S A. 2002; 99(9): 6047-52.
13. Brummelkamp TR,Bernards R,Agami R.A system for stable expression of short interfering RNAs in mammalian cells.Science.2002; 296(5567): 550-3.
14. Lee NS,Dohjima T,Bauer G,et al.Expression of small interfering RNAs targeted against HIV21 rev transcripts in human cells.Nat Biotechnol. 2002; 20(5):500-5.
15. Miyagishi M,Taira K.U6 promoter 2 driven siRNAs with four uridine 3'overhangs efficiently suppress targeted gene expression in mammalian cells.Nat Biotechnol. 2002;20(5):497-500.
16. Paddison PJ,Cuady AA,Hannon GJ.Stbale suppression of gene expression by RNAi in mammalian cells.Proc Natl Acad sei USA. 2002; 99(3):1443-8.
17. Myers JW, Jones JT, Meyer T,et al. Recombinant Dicer efficiently converts large dsRNAs into siRNAs suitable for gene silencing.Nat Biotechnol.2003;21(3):324-8.
18. Elbashir SM,Martinez J,Patkaniowska A,et al.Functional anatomy of siRNA for mediating efficient RNAi in Drosophila melanogaster embryo- lysate.EMBO J. 2001; 20(23): 6877-88.
19. Yu JY,Taylor J,DeRuiter SL,et al.Simultaneous inhibition of GSK 3 alpha and GSK 3 beta using hairpin siRNA expression vectors.Mol Ther. 2003; 7(2): 228-36.
20. Czauderna F,Fechtner M,Aygun H,et al.Functional studies of the PI(3)-kinase signalling pathway employing synthetic and expressed siRNA. Nucleic Acids Res. 2003;31(2):670-82.
21. Sen R,Baltimore D.Multiple nuclear factors interact with the immune- globulin enhancer sequences.Cell. 1986; 46(5):705-16.
22. Pahl HL.Activators and target genes of Rel/NF-kappa B transcription factors. Oncogene. 1999;18(49):6853-66.
23. Ghosh S,Karin M.Missing pieces in the NF-kappaB puzzle.Cell. 2002; 109(Supp): S81-96.
24. Prendes M,Zheng Y,BegAA. Regulation of developing B cell survival by RelA-containing NF-kappa B complexes J Immunol. 2003; 171(8):3963-9.
25. Cercek B,Yamashita M, Dimayuga P,et al.Nuclear factor-kappaB activity and arterial response to balloon injury. Atherosclerosis. 1997; 131(1): 59-66.
26. Hinz M,Krappmann D,Eichten A,et al.Nuclear factor-kappa B,cancer,and apoptosis. Mol Cell Biol. 2004; 64(2):2690-8.
27. Bharti AC.Aggarwal BB.Nuclear factor-kappa B and cancer.Its role in prevention and therapy.Biochem Pharmacol. 2002; 64(5-6):883-8.
28. Aggarwal BB.Nuclear factor-kappaB:the enemy within. Cancer Cell.2004; 6(3): 203-8.
29. Biswas DK,Cruz AP,Gansberger E,et al.Epidermal growth factor-induced nuclear factor kappa B activation:A major pathway of cellcycle pro- gression in estrogen-receptor negative breast cancer cells[J].Proc Natl Acad Sci U S A. 2000; 97(15):8542-7.
30. Etienne MC,Formento JL,Xebrun-Frenay C,et al.Epidermal growth factor receptor and labeling index are independent prognosticfactors in glial tumor outcome.Clin Cancer Res.1998;(10):2383-90.
31. Bhat-Nakshatri P,Sweeney CJ,Nakshatri H.Identification of signal transduction pathways involved in constitutive NF-kappaB activation in breast cancer cells. Oncogene. 2002; l(13):2066-78.
32. Duffer DC,Chen Z,Dong G,et al.Expression of a dominant negative mutant inhibitor-kappaB alpha of nuclear factor-kappaB in human head and neck squamous cell carcinoma inhibits survivahproinflammatory cytokine expression, and tumor growth in vivo.Cancer Res.1999; 59(14):3468-74.
33. Dong G,Loukinova E,Chen' Z,et al.Molecular profiling of transformed and metastatic murine squamous carcinoma cells by differential display and cDNA microarray reveals altered expression of multiple genes related to growth, apoptosis,angiogenesis,and the NF-κB signal pathway.Cancer Res. 2001;61 (12) :4797-808.
34. Shishodia S,Aggarwal BB.Nuclear factor-kappaB activation:a question of life or death. J Biochem Mol Biol. 2002;35(1):28-40.
35. Suzuki A,Shiraki K.Tumor cell"dead or alive":caspase and surviving regulate cell death, cell cycle and cell survival. Histol Histopathol. 2001; 16(2):583-93.
36. Mitsiades N,Mitsiades CS,Poulaki V,et al.Biologic sequelae of nuclear factor-KB blockade in multiple myeloma:therapeutic applications.Blood.2002; 99(12):4079-86.
37. You Z,Ouyang H,Lopatin D,et al.Nuclear factor-kappa B-inducible death effector domain-containing protein suppresses tumor necrosis factor-mediated apoptosis by inhibiting caspase-8 activity.J Biol Chem. 2001; 276(28):26398-404.
38. Chen S,Fribey A,Wang CY.Potentiation of tumor necrosis factor- mediated apoptosis of oral squamous cell carcinoma cells by adenovirus- mediated genes transfer of NF-κB inhibitor .J Dent Res. 2002; 81(2):98-102.
39. Bian X,McAllister-Lucas LM,Shao F,et al.NF-kappaB activation mediates doxorubicin-induced cell death in N-type neuroblastoma cells.J Biol Chem. 2001; 276(52):48921-9.
40. Hettmann T,DiDonato J,Karin M,et al.An essential role for nuclear factor kappaB in promoting double positive thymocyte apoptosis. J Exp Med. 1999;189(1): 145-58.
41. Bessho R,Matsubara K,Kubota M,et al.Pyrrolidine dithiocarbamate,a potent inhibitor of nuclear factor kappa B (NF-kappaB) activation, prevents apoptosis in human promyelocytic leukemia HL-60 cells and thymocytes.Biochem Pharmacol. 1994; 48(10):1883-9.
42. Chen X,Kandasamy K,Srivastava RK.Differential roles of RelA (p65) and c-Rel subunits of nuclear factor kappa B in tumor necrosis factor- related apoptosis-inducing ligand signaling.Cancer Res. 2003; 63(5): 1059-66.
43. Ivanov VN,Ronai Z.p38 protects human melanoma cells from UV-induced apoptosis through down-regulation of NF-kappaB activity and Fas expression. Oncogene. 2000; 19(26):3003-12.
44. Campbell KJ,Rocha S,Perkins ND.Active repression of antiapoptotic gene expression by RelA (p65) NF-kappaB.Mol Cell. 2004; 13(6):853-65.
45. Chen ZT,Li SL,Cai EQ,et al.LPS induces pulmonary intravascular macrophages producing inflammatory mediators via activating NF-kappaB. J Cell Biochem. 2003;89(6):1206-14.
46. Nakatani K,Yamakuni T,Kondo N,et al.gamma-Mangostin inhibits inhibitor-kappaB kinase activity and decreases lipopolysaccharide induced cyclo-oxygenase-2 gene expression in C6 rat glioma cells.Mol Pharmacol. 2004; 66 (3): 667-74.
47. Dwarakanath RS,Sahar S,Reddy MA,et al.Regulation of monocyte chemoatt ractant protein-1 by the oxidized lipid,13-hydroperoxyoctadecadenoic acid,in vascular smooth muscle cells via nuclear factor- kappa B (NF- kappaB).Mol Cell Cardiol. 2004; 36(4):585-95.
2. Togni M, Windecker S, MeierB,et al. Treatment of restenosis.Curr Interv Cardiol Rep. 2001;3(4):306-10.
3. Hamano M, Kuroda Y, Saitoh Y. Pancreas transplantation using non-suture cuff technique in the neck. Kobe J Med Sci. 1996; 42(2):93-104.
4. Tomita Y, Zhang QW, Yoshikawa M, et al. Improved technique of heterotopic cervical heart transplantation in mice. Transplantation. 1997; 64(11): 1598-601.
5. Tomita Y, Zhang QW, Uchida T, et al. A technique of cervical aortic graft transplantation in mice. J Heart Lung Transplant. 2001; 20(6):699-702.
6. Nakao A, Ogino Y, Tahara K, et al. Orthotopic intestinal transplantation using the cuff method in rats:a histopathological evaluation of the anastomosis. Microsurgery. 2001;21(1):12-5.
7. Xiu D, Uchida H, To H, et al. Simplified method of heterotopic rat heart transplantation using the cuff technique: application to sublethal dose protocol of methotrexate on allograft survival. Microsurgery. 2001; 21(1):16-21.
8. Guo H, Wu YJ, Zheng SS, et al. Application of modified two-cuff technique and multiglycosides tripterygium wilfordii in hamster-to-rat liver xenotransplant model. World J Gastroenterol. 2003 ;9(7): 1550-3.
9. Peng Y, Gong JP, Yan LN, et al. Improved two-cuff technique for orthotopic liver transplantation in rat. Hepatobiliary Pancreat Dis Int. 2004; 3(1): 33-7.
10. Tan F, Chen Z, Zhao Y, et al. Novel technique for suprahepatic vena cava reconstruction in rat orthotopic liver transplantation. Microsurgery.2005;25(7):556-60.
11. Ross R. The pathogenesisof atherosclerosis:a perspective for the 1990s. Nature. 1993;362(6432):801-9.
12. Fitzgibbon G, Kafka HP, Leach AJ, et al. Coronary bypass graft fate and patient outcome: angiographic follow-up of 5065 grafts related to survival and reoperation in 1388 patients during 25 years. J Am Coll Cardiol. 1996; 28(3): 616-26.
13. Kalra M, Miller VM. Early remoldeling of saphenous vein graft: proliferation, migration and apoptosis of adventitial and medial cells occur simultaneously with changes in graft diameter and blood flow. J Vasc Res. 2000; 37(6):576-84.
14. Bauters C, Lamblin N, Amouyel P, et al. Gene polymorphisms and outcome after coronary angioplasty. Curr Interv Cardiol Rep. 2001; 3(4):281-6.
15. Ruygrok PN, Webster MW, de Valk V, et al. Clinical and angiographic factors associated with asymptomatic restenosis after percutaneous coronary intervention. Circulation. 2001; 104(19):2289-94.
16. Grassman ED, Leya F, Fareed J, et al. A randomized trial of the low molecular weight heparin certoparin to prevent restenosis following coronary angioplasty.J Invasive Cardiol. 2001;13(11):723-8.
17. Nakao A, Tahara K, Inoue S, et al. Combined cuff and suture technique for orthotopic whole intestinal transplantation in rats. Microsurgeiy.2002;22(3):85-90.
18. Akpinar S, Hersekli MA, Demirors H, et al. Effects of methylprednisolone and betamethasone injections on the rotator cuff: an experimental study in rats. Adv Ther. 2002; 19(4): 194-201.
19. Tanaka H, Hashizume K, Enosawa S, et al. Successful transplantation of a 20% partial liver graft in rats: a technical innovation. J Surg Res. 2003; 110(2):409-12.
20. Demirhan M, Atalar AC, Kilicoglu O. Primary fixation strength of rotator cuff repair techniques:a comparative study. Arthroscop. 2003; 19(6): 572-6.
21.Gerber C, Meyer DC, Schneeberger AG, et al. Effect of tendon release and delayed repair on the structure of the muscles of the rotator cuff: an experimental study in sheep. J Bone Joint Surg Am. 2004; 86-A(9): 1973-82.
22. Birsner JH, Wan C, Cheng G, et al. Steatotic liver transplantation in the mouse:a model of primary nonfunction. JSurgRes. 2004; 120(1):97-101.
23. Santana Rodriguez N, Martin Barrasa JL, Lopez Garcia A, et al. Lung transplantation in rats: a viable experimental model. Arch Bronco-neumol.2004;40(10): 438-42.
24. Yoshida S, Sekine Y, Saitoh Y, et al. Surgical technique of experimental lung transplantation in rabbits. Ann Thorac Cardiovasc Surg. 2005; 11(1): 7-11.
25. Kashfi A, Mehrabi A, Pahlavan PS, et al. A review of various techniques of orthotopic liver transplantation in the rat. Transplant Proc.2005; 37(1): 185-8.
26. Lehmann TG, Bunzendahl H, Langrehr JM, et al. Arterial reconstruction in rat liver transplantation-development of a new tubing technique of the common hepatic artery. Transpl Int. 2005; 18(1 ):56-64.
1. Francis SE, Hunter S, Holt CM, et al. Release of platelet derived growth factor activity from arteriovenous bypass grafts. Thorac Cardiovasc Surg. 1994;108(3): 540-8
2. Togni M,Windecker S,MeierB,et al.Treatment of restenosis . Curr Interv Cardiol Rep.2001;3(4):306-10.
3. Kornowski R,Hong M,Fermin F,et al. In-stent restenosisxontributions of inflame-matory responses and arterial injury to neointimal hyperplasia. J Am Coll Cardiol. 1998; 31(1):224-30.
4. Farb A, Weber DK, Kolodgie FD, et al. Morphological predictors of restenosis after coronary stenting in humans. Circulation. 2002; 105(25): 2974-80.
5. Gul, Tseng s, Rollins B. Monocyte chemoattractant protein 1. Chem Immunol. 1999; 72(1):7-29.
6. Cipollone F, Marini M, Fazia M, et al. Elevated circulating levels of monocyte chemoattractant protein-1 in patients with restenosis after coronary angioplasty. Arterioscler Thromb Vasc Biol; 2001,21 (3):327-34.
7. Qi X, Li J, Gu J, et al. Plasma levels of IL-8 predict early complications in patients with coronary heart diease after percutaneous coronary intervention. Jpn Heart J. 2003; 44(4):451-61.
8. Abdelmouttaleb I, Danchin N, Ilardo C, et al. C reactive protein and coronary artery disease:additional evidence of the implication of an inflammatory process in acute coronary syndromes. Am Heart J. 1999; 137(2):346-51.
9. Lentsch A B, Ward P A. The NF-kappa B/Ikappa B system in acute inflammation. Arch Immunol Ther Exp(Warsz).2000; 48(2):59-63.
10. Epinat J C, Gilmore T D. Diverse agents act at multiple levels to inhibit the Rel/NF-Kappa B signal transduction pathway. Oncogene. 1999; 18(49):6896 -909.
11. Karin M, Ben Neriah Y. Phosphorylation meets ubiquitination:the control of NF-kappa B activity. Annu Rev Immunol. 2000; 18(1): 621-63.
12. Bakker R A,Schoonus S B,Smit M J,et al.Histamine H(1)-receptor activation of nuclear factor-kappa B:roles for G betagamma- and G alpha(q/11)-subunits in constitutive and agonist-mediated signaling. Mol Pharmacol.2001;60(5):1133-42.
13. Adams J, Kauffman M. Development of the proteasome inhibitor Velcade (Bortezomib). Cancer Invest.2004; 22(2):304-11.
14. Henninger N, Sicard KM, Bouley J, et al. The proteasome inhibitor VELCADE reduces infarction in rat models of focal cerebral ischemia. Neurosci Lett. 2006; 398(3): 300-5.
15. Zhang L, Zhang ZG, Liu X, et al. Treatment of embolic stroke in rats with bortezomib and recombinant human tissue plasminogen activator. Thromb Haemost. 2006; 95(1): 166-73.
16. Sinn DI, Lee ST, Chu K, et al. Proteasomal inhibition in intracerebral hemorrhage: Neuroprotective and anti-inflammatory effects of bortezomib. Neurosci Res. 2007; 58(1):12-8.
17. Ross R. The pathogenesisof atherosclerosis:a perspective for the 1990s. Nature. 1993;362(6432):801-9.
18. Fitzgibbon G, Kafka HP, Leach AJ ,et al. Coronary bypass graft fate and patient outcome:angiographic follow-up of 5065 grafts related to survival and reoperation in 1388 patients during 25 years. J Am Coll Cardiol. 1996; 28(3):616-26.
19. Kalra M, Miller VM. Early remoldeling of saphenous vein grafts. Proliferation ,migration and apoptosis of adventitial and medial cells occur simultaneously with changes in graft diameter and blood flow. J Vasc Res. 2000; 37(6): 576-84.
20. Kataoka M, Shimizu H, Mitsuhashi N, et al. Effect of cold-ischemia time on C-X-C chemokine expression and neutrophil accumulation in the graft liver after orthotopic liver transplantation in rats. Transplantation. 2002; 73(11):1730-5.
21. Libby P. Inflammation in atherosclerosis. Nature. 2002; 420(6917):868-74.
22. Miller AP,Feng W,Xing D,et al.Estrogen modulates inflammatory mediator expression and neutrophil chemotaxis in injured arteries. Circulation.2004; 110(12): 1664-9.
23. Kitamoto S, Egashira K,Kataoka C, et al. Increased activity of nuclear factor-kappa B participates in cardiovascular remodeling induced by chronic inhibition of nitric oxide synthesis in rats. Circulation. 2000; 102(7):806-12.
24. Porreca E, Di Febbo C, Reale M, et al. Monocyte chemotactic protein 1 (MCP-1) is a mitogen for cultured rat vascular smooth muscle cells. J Vasc Res. 1997;34(1): 58-65.
25. Naruko T, Ueda M, Haze K, et al. Neutrophil infiltration of culprit lesions in acute coronary syndromes. Circulation. 2002; 106(23):2894-900.
26. Okamoto E, Couse T, De Leon H, et al. Perivascular inflammation after balloon angioplasty of porcine coronary arteries. Circulation. 2001; 104(18):2228-35.
27. Menger MD, Vollmar B. Pathomechanisms of ischemia- reperfiision injury as the basis for novel preventive strategies: is it time for the introduction of pleiotropic compounds .Transplant Proc. 2007; 39(2): 485-8.
28. Welt FG, Rogers C. Inflammation and restenosis in the stent era. Arterioscler Thromb Vasc Biol. 2002; 22(11): 1769-76.
29. Sen R, Baltimore D. Multiple nuclear factors interact.with the immunoglobulin enhancer sequences. Cell.1986; 46(5):705-16.
30. Parry GC, Mackman N. Activators and target genesof Rel/NF-kappa B transcription factors. Biol Chem. 1999; 269(1): 20823-5.
31. Arenzana-Seisdedos F, Turpin P, Rodriguez M, et al.Nuclear localization of I kappa B alpha promotes active transport of NF-kappa B from the nucleus to the cytoplasm. J Cell Sci. 1997; 110(pt 3):369-78.
32. Stancovski I, Baltimore D. NF-kappa B actritation: the I kappa B kinase revealed. Cell. 1997;91(1):299-302.
33. Jacobs MD, Harriso SC. Structure of an I kappa B alpha/NF-kappa B complex. Cell. 1998; 95(1):749-58.
34. Pomerantz JL, Baltimore D. Two pathways to NF-kappaB. Mol Cell. 2002; 10(4): 693-5.
35. Hinz M, Krappmann D, Eichten A, et al. Nuclear factor-kappa B, cancer ,and apoptosis. Mol Cell Biol. 2004; 64(1):2690-8.
36. Bharti AC, Aggarwal BB. Nuclear factor-kappa B and cancer.Its role in prevention and therapy. Biochem Pharmacol. 2002; 64(5-6):883-8.
37. Ghosh S, Karin M. Missing pieces in the NF-kappaB puzzle.Cell.2002; 109 suppl: s81-96.
38. Prendes M, Zheng Y, BegAA. Regulation of developing B cell survival by RelA-containing NF-kappaB complexes. J Immunol. 2003; 171(8):3963-9.
39. Cercek B, Yamashita M, Dimayuga P, et al. Nuclear factor-kappaB activity and arterial response to balloon injury .Atherosclerosis.1997;131(1): 59-66.
40. Okamoto E, Couse T, De Leon H,et al. Perivascular inflammation after balloon angioplasty of porcine coronary arteries. Circulation. 2001; 104(18):2228-35.
41. Lu C, Gallegos R, Li P, et al. Investigation of drug-drug interaction potential of bortezomib in vivo in female Sprague-Dawley rats and in vitro in human liver microsomes. Drug Metab Dispos. 2006; 34(4):702-8.
1. Lentsch A B,Ward P A. The NF-kappa B/Ikappa B system in acute inflame-mation. Arch Immunol Ther Exp. 2000; 48(2):59-63.
2. Bakker R A,Schoonus S B,Smit M J,et al.Histamine H(1)-receptor activation of nuclear factor-kappa B:roles for G betagamma- and Galpha (q/11)-subunits in constitutive and agonist-mediated signaling. Mol Pharmaco.2001; 60(5):1133-42.
3. Epinat J C,Gilmore T D.Diverse agents act at multiple levels to inhibit the Rel/NF-Kappa B signal transduction pathway.Oncogene.1999; 18(49): 6896-909.
4. Karin M,Ben Neriah Y.Phosphorylation meets ubiquitination:the control of NF-kappa B activity.Ann Rev Immunol. 2000; 18(2):621-63.
5. Gitlin L,Karelsky S,Andino R.Short interfering RNA confers intracellular antiviral immunity in human cells.Nature. 2002; 418(6896): 430-4.
6. Qin XF, An DS,Chen IS,et al.Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5. Proc Natl AcadSci USA. 2003; 100(1): 183-8.
7. Yang YS,Chen GM,Dong WP,et al.Construction and analysis of activity of an HIV-1/bovine immunodeficiency virus chimeric clone cDNA. Zhong hua Shi Yan He Lin Chuang Bing Du Xue Za Zhi.2003; 17(2): 143-5.
8. Carthew R W.RNA interference: the fragile X syndrome connection.Curr Biol. 2002;12(24):852-4.
9. Ishizuka A,Siomi M C,Siomi H.A Drosophila fragile X protein interacts with components of RNAi and ribosomal proteins.Genes Dev. 2002; 16(19): 2497-508
10. Gil J,Esteban M.Induction of apoptosis by the dsRNA dependent portein kinase (PKR): mechanism of action Apoptosis.2000;5(2): 107-14.
11. Elbashir SM,Harborth J,Lendeckel W,et al.Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.Nature .2001; 411(6836): 494-8.
12. Yu JY,DeRuiter SL,Turner DL.RNA interfernce by expression of short-interfering RNAs and hairpin RNAs in mammalian cells.Proc Natl Acad Sci U S A. 2002; 99(9): 6047-52.
13. Brummelkamp TR,Bernards R,Agami R.A system for stable expression of short interfering RNAs in mammalian cells.Science.2002; 296(5567): 550-3.
14. Lee NS,Dohjima T,Bauer G,et al.Expression of small interfering RNAs targeted against HIV21 rev transcripts in human cells.Nat Biotechnol. 2002; 20(5):500-5.
15. Miyagishi M,Taira K.U6 promoter 2 driven siRNAs with four uridine 3'overhangs efficiently suppress targeted gene expression in mammalian cells.Nat Biotechnol. 2002;20(5):497-500.
16. Paddison PJ,Cuady AA,Hannon GJ.Stbale suppression of gene expression by RNAi in mammalian cells.Proc Natl Acad sei USA. 2002; 99(3):1443-8.
17. Myers JW, Jones JT, Meyer T,et al. Recombinant Dicer efficiently converts large dsRNAs into siRNAs suitable for gene silencing.Nat Biotechnol.2003;21(3):324-8.
18. Elbashir SM,Martinez J,Patkaniowska A,et al.Functional anatomy of siRNA for mediating efficient RNAi in Drosophila melanogaster embryo- lysate.EMBO J. 2001; 20(23): 6877-88.
19. Yu JY,Taylor J,DeRuiter SL,et al.Simultaneous inhibition of GSK 3 alpha and GSK 3 beta using hairpin siRNA expression vectors.Mol Ther. 2003; 7(2): 228-36.
20. Czauderna F,Fechtner M,Aygun H,et al.Functional studies of the PI(3)-kinase signalling pathway employing synthetic and expressed siRNA. Nucleic Acids Res. 2003;31(2):670-82.
21. Sen R,Baltimore D.Multiple nuclear factors interact with the immune- globulin enhancer sequences.Cell. 1986; 46(5):705-16.
22. Pahl HL.Activators and target genes of Rel/NF-kappa B transcription factors. Oncogene. 1999;18(49):6853-66.
23. Ghosh S,Karin M.Missing pieces in the NF-kappaB puzzle.Cell. 2002; 109(Supp): S81-96.
24. Prendes M,Zheng Y,BegAA. Regulation of developing B cell survival by RelA-containing NF-kappa B complexes J Immunol. 2003; 171(8):3963-9.
25. Cercek B,Yamashita M, Dimayuga P,et al.Nuclear factor-kappaB activity and arterial response to balloon injury. Atherosclerosis. 1997; 131(1): 59-66.
26. Hinz M,Krappmann D,Eichten A,et al.Nuclear factor-kappa B,cancer,and apoptosis. Mol Cell Biol. 2004; 64(2):2690-8.
27. Bharti AC.Aggarwal BB.Nuclear factor-kappa B and cancer.Its role in prevention and therapy.Biochem Pharmacol. 2002; 64(5-6):883-8.
28. Aggarwal BB.Nuclear factor-kappaB:the enemy within. Cancer Cell.2004; 6(3): 203-8.
29. Biswas DK,Cruz AP,Gansberger E,et al.Epidermal growth factor-induced nuclear factor kappa B activation:A major pathway of cellcycle pro- gression in estrogen-receptor negative breast cancer cells[J].Proc Natl Acad Sci U S A. 2000; 97(15):8542-7.
30. Etienne MC,Formento JL,Xebrun-Frenay C,et al.Epidermal growth factor receptor and labeling index are independent prognosticfactors in glial tumor outcome.Clin Cancer Res.1998;(10):2383-90.
31. Bhat-Nakshatri P,Sweeney CJ,Nakshatri H.Identification of signal transduction pathways involved in constitutive NF-kappaB activation in breast cancer cells. Oncogene. 2002; l(13):2066-78.
32. Duffer DC,Chen Z,Dong G,et al.Expression of a dominant negative mutant inhibitor-kappaB alpha of nuclear factor-kappaB in human head and neck squamous cell carcinoma inhibits survivahproinflammatory cytokine expression, and tumor growth in vivo.Cancer Res.1999; 59(14):3468-74.
33. Dong G,Loukinova E,Chen' Z,et al.Molecular profiling of transformed and metastatic murine squamous carcinoma cells by differential display and cDNA microarray reveals altered expression of multiple genes related to growth, apoptosis,angiogenesis,and the NF-κB signal pathway.Cancer Res. 2001;61 (12) :4797-808.
34. Shishodia S,Aggarwal BB.Nuclear factor-kappaB activation:a question of life or death. J Biochem Mol Biol. 2002;35(1):28-40.
35. Suzuki A,Shiraki K.Tumor cell"dead or alive":caspase and surviving regulate cell death, cell cycle and cell survival. Histol Histopathol. 2001; 16(2):583-93.
36. Mitsiades N,Mitsiades CS,Poulaki V,et al.Biologic sequelae of nuclear factor-KB blockade in multiple myeloma:therapeutic applications.Blood.2002; 99(12):4079-86.
37. You Z,Ouyang H,Lopatin D,et al.Nuclear factor-kappa B-inducible death effector domain-containing protein suppresses tumor necrosis factor-mediated apoptosis by inhibiting caspase-8 activity.J Biol Chem. 2001; 276(28):26398-404.
38. Chen S,Fribey A,Wang CY.Potentiation of tumor necrosis factor- mediated apoptosis of oral squamous cell carcinoma cells by adenovirus- mediated genes transfer of NF-κB inhibitor .J Dent Res. 2002; 81(2):98-102.
39. Bian X,McAllister-Lucas LM,Shao F,et al.NF-kappaB activation mediates doxorubicin-induced cell death in N-type neuroblastoma cells.J Biol Chem. 2001; 276(52):48921-9.
40. Hettmann T,DiDonato J,Karin M,et al.An essential role for nuclear factor kappaB in promoting double positive thymocyte apoptosis. J Exp Med. 1999;189(1): 145-58.
41. Bessho R,Matsubara K,Kubota M,et al.Pyrrolidine dithiocarbamate,a potent inhibitor of nuclear factor kappa B (NF-kappaB) activation, prevents apoptosis in human promyelocytic leukemia HL-60 cells and thymocytes.Biochem Pharmacol. 1994; 48(10):1883-9.
42. Chen X,Kandasamy K,Srivastava RK.Differential roles of RelA (p65) and c-Rel subunits of nuclear factor kappa B in tumor necrosis factor- related apoptosis-inducing ligand signaling.Cancer Res. 2003; 63(5): 1059-66.
43. Ivanov VN,Ronai Z.p38 protects human melanoma cells from UV-induced apoptosis through down-regulation of NF-kappaB activity and Fas expression. Oncogene. 2000; 19(26):3003-12.
44. Campbell KJ,Rocha S,Perkins ND.Active repression of antiapoptotic gene expression by RelA (p65) NF-kappaB.Mol Cell. 2004; 13(6):853-65.
45. Chen ZT,Li SL,Cai EQ,et al.LPS induces pulmonary intravascular macrophages producing inflammatory mediators via activating NF-kappaB. J Cell Biochem. 2003;89(6):1206-14.
46. Nakatani K,Yamakuni T,Kondo N,et al.gamma-Mangostin inhibits inhibitor-kappaB kinase activity and decreases lipopolysaccharide induced cyclo-oxygenase-2 gene expression in C6 rat glioma cells.Mol Pharmacol. 2004; 66 (3): 667-74.
47. Dwarakanath RS,Sahar S,Reddy MA,et al.Regulation of monocyte chemoatt ractant protein-1 by the oxidized lipid,13-hydroperoxyoctadecadenoic acid,in vascular smooth muscle cells via nuclear factor- kappa B (NF- kappaB).Mol Cell Cardiol. 2004; 36(4):585-95.