A20基因对大鼠颈动脉球囊损伤后内膜增生的影响及其分子机制的研究
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摘要
目的:经皮冠状动脉介入治疗(percutaneous coronary intervention, PCI)是治疗冠心病的有效手段,但是再狭窄(restenosis, RS)仍是制约这一技术应用的主要问题。大量研究证实,再狭窄的机制主要由于球囊扩张和支架置入时导致血管壁损伤,引起炎症反应,进而导致平滑肌细胞的增殖和移行。由于血管壁的机械性损伤所致的炎性和增殖效应是造成再狭窄的主要原因,多种生长因子、细胞因子、粘附分子等参与其病理生理过程,单纯抑制某种特定的因子并不能完全抑制病灶形成。因此,阻断由炎症损伤引起的平滑肌细胞增殖、分化、迁移等最终共同通路的关键基因成为有效防止再狭窄的研究方向。
基于导管将基因准确地送至狭窄血管的局部进行基因治疗是预防血管再狭窄较有效的方法之一,而选择合适的基因和载体是基因治疗再狭窄的关键之一。
近年来在细胞内源性保护机制的研究中发现了一类新的蛋白质,即锌脂蛋白A20(zinc finger protein A20),简称A20。A20是NF-κB信号系统重要的负反馈调节因子,也是防止体内炎症反应失控的重要调节蛋白。A20的表达能够被LPS和TNF等所诱导,是NF-κB信号系统活化的产物,同时A20通过抑制TNF和LPS刺激TNFR-1和TLR4诱导的NF-κB的活化而对细胞损伤起保护作用。已有研究证实NF-κB的激活是球囊损伤动脉后血管狭窄发生的重要机制之一。结构研究显示,A20既有去泛素活性,又有泛素连接酶活性,是一种重要的泛素调节酶,在A20等泛素调节酶的作用下泛素调节参与了NF-κB前体的处理、NF-κB抑制性结合蛋白IκB的降解和IκB激酶IKK的活化等主要环节,反馈抑制了NF-κB的活化。
一方面由于再狭窄的炎症机制及A20调控炎症的机制,另一方面由于NF-κB的激活导致球囊损伤后血管狭窄及A20对NF-κB活化的负反馈抑制作用,由此我们选择A20基因作为再狭窄发生发展过程中的干预靶点,运用基因治疗方法对体内A20的表达进行调控,深入探讨球囊损伤术后血管内膜增生过程中A20在损伤局部组织中的表达变化及局部过表达A20对损伤后内膜增生、平滑肌细胞增殖和表型改变,内皮再生及TLR4/NF-κB信号通路的影响,为进一步明确A20对再狭窄过程中炎症反应的调节作用以及揭示机体内源性保护机制提供必要的理论依据。
方法:
1. pCAGGS-GFP/A20重组质粒的鉴定和大量提取:将获得的pCAGGS-GFP/A20质粒菌保涂Amp抗性的平板后,过夜培养。挑单菌落于3ml Amp抗性的LB培养基中37℃摇床中过夜培养,集菌后,进行小量质粒提取,并使用EcoRⅤ/SmaⅠ进行双酶切鉴定,酶切产物1%琼脂糖凝胶电泳。将鉴定好的质粒菌保进行无内毒素质粒的大量提取,将质粒DNA提取物按适当比例稀释,并测定浓度。
2.动物模型制备和实验分组,大鼠颈总动脉内膜球囊剥脱术用来建立再狭窄模型:健康雄性Sprague-Dawley(SD)大鼠体重350～400g,104只随机分为4组(n=26):假手术组;模型组(单纯损伤组);对照组(pCAGGS-GFP+转染试剂组);治疗组(pCAGGS-GFP/A20+转染试剂组)。10%水合氯醛(3ml/kg)腹腔注射麻醉后,取颈正中切口,钝性分离左颈总动脉,暴露左颈总动脉及其分叉处,用显微血管夹暂时夹闭左颈总动脉近心端及颈内动脉,将2F Fogarty球囊导管从颈外动脉切口插入至左颈总动脉,用50μl生理盐水充盈球囊,来回抽动3次以剥脱颈总动脉内膜。退出球囊,治疗组向损伤血管节段内缓慢注入灌注液(Lipofectamine 2000转染试剂40μl + pCAGGS-GFP/A20质粒20μl),对照组向损伤血管节段内缓慢注入灌注液(Lipofectamine 2000转染试剂40μl+ pCAGGS-GFP质粒20μl)局部作用30分钟。模型组只进行球囊损伤术,假手术组只进行左颈外动脉结扎,不进行球囊损伤。术后不同时间点根据不同要求分别处死动物。转染后24h取大鼠局部血管进行冰冻切片荧光显微镜下观察绿色荧光蛋白的表达籍以评估A20在损伤动脉壁的转染效率;术后14d病理组织学方法进行内膜增生的形态学分析;术后10d溴脱氧尿嘧啶核苷(BrdU)标记技术评估血管平滑肌细胞(VSMC)的增殖指数;术后10d免疫组织化学染色观察A20、核因子-κB p65(NF-κB p65)、Toll样受体4(TLR4)在各组大鼠颈总动脉中的表达情况;术后3d、7d、14d、28d通过逆转录-聚合酶链式反应(RT-PCR)、Western-blotting法分别检测A20、NF-κB p65、TLR4在颈动脉组织的mRNA和蛋白表达。
3.健康雄性Sprague-Dawley(SD)大鼠体重350～400g,36只随机分为3组(n=12):假手术组;对照组(pCAGGS-GFP+转染试剂组);治疗组(pCAGGS-GFP/A20+转染试剂组)。球囊损伤后2周伊文思蓝(Evans blue)染色法评估内皮再生情况;术后3d、7d透射电镜观察血管超微结构改变。
4.体外培养大鼠主动脉平滑肌细胞并鉴定,以LPS(20mg/l)作为刺激因素,以不同浓度大黄素(Emodin, EMD)作为干预因素,通过MTT和BrdU掺入方法评估各组VSMC增殖情况,以RT-PCR检测A20 mRNA表达,以Western-blotting法检测A20、NF-κB p65蛋白的表达。
结果:
1.将提取的重组真核表达质粒pCAGGS-GFP/A20使用EcoRⅤ/SmaⅠ进行双酶切鉴定,酶切产物1%琼脂糖凝胶电泳,质粒大小及酶切结果正确。
2.将pCAGGS-GFP/A20于活体转染至大鼠球囊损伤的颈动脉局部,24h后荧光显微镜下可见治疗组残存血管内膜及中膜有大量绿色荧光分布,证明转染成功。
3.大鼠颈总动脉球囊损伤术后14d模型组和对照组血管内膜显著增生。A20基因转染可显著抑制新生内膜面积的增加(减少47.8%,P<0.05)和内/中膜比值的增加(减少42.9%,P<0.05)。术后10d治疗组VSMC体内增殖指数(BrdU指数)(9.6±2.3%)显著少于对照组(26.7±5.1%,P<0.01)。术后10d治疗组血管壁A20免疫组化染色阳性细胞率显著高于对照组(P<0.05),NF-κB p65、TLR4免疫组化染色阳性细胞率显著低于对照组(P<0.05)。术后3d、7d、14d、28d治疗组血管组织A20的mRNA和蛋白表达量显著高于对照组(P<0.05),治疗组血管组织NF-κB p65、TLR4的mRNA和蛋白表达量显著低于对照组(P<0.05)。
4.球囊损伤后2周,pCAGGS-GFP/A20质粒转染治疗组与对照组比较,显示更大面积的内皮修复,由伊文思蓝染色证明。治疗组再内皮化的面积占最初损伤面积的百分数( 73.1±3.7% )显著大于对照组(55.6±3.3%,P<0.05)。说明A20转染治疗组与对照组比较显示更大面积的内皮修复。
5.假手术组大鼠VSMC电镜下呈典型的“收缩表型”,球囊损伤后7d对照组VSMC表型发生明显改变,呈典型的“合成表型”,损伤后3d对照组可见合成型细胞及过渡型细胞,治疗组术后7d可见接近收缩型的VSMC。
6.体外培养的大鼠主动脉VSMC经LPS刺激后,MTT OD值与BrdU掺入OD值均显著高于空白组。经不同浓度EMD干预结果显示随EMD浓度增大,MTT OD值及BrdU掺入OD值均逐渐变小,其中低剂量组与对照组相比无显著性差异(P>0.05),高剂量组与对照组相比存在显著性差异(P<0.05)。RT-PCR和Western-blot结果显示LPS刺激可使A20mRNA和蛋白以及NF-κB p65蛋白表达均增加,经不同浓度EMD干预后A20mRNA和蛋白表达随EMD浓度递增而进一步增加,NF-κB p65蛋白表达随EMD浓度递增而逐渐减少。
结论:
1.大鼠颈动脉球囊损伤后内源性锌指蛋白A20在局部表达增加,并具有明显的时效性,说明损伤情况下,A20表达增加是机体的一种重要的内源性抗炎保护效应机制,可能对于调控损伤后适度的炎症反应具有重要意义。
2.大鼠颈动脉球囊损伤后局部转染A20基因可获得锌指蛋白A20mRNA和蛋白在局部组织细胞内高表达。
3.大鼠颈动脉球囊损伤后局部转染A20基因可抑制损伤血管内膜增生及平滑肌细胞的增殖,其分子机制可能通过其负反馈抑制了TLR4/NF-κB介导的胞内信号转导途径。
4.大鼠颈动脉球囊损伤后局部转染A20基因可促进损伤血管内皮再生,抑制损伤处VSMC表型转化,其分子机制可能通过其负反馈抑制了TLR4/NF-κB介导的胞内信号转导途径。
5. EMD对LPS诱导的大鼠主动脉VSMC增殖具有抑制作用,其机制可能通过增加体内炎症反应调节蛋白A20的表达并进一步负反馈抑制了NF-κB的激活,进一步证实了A20对VSMC增殖的影响。
Object:Percutaneous coronary intervention (PCI) is now one of the most effective methods to treat coronary artery disease, but restenosis (RS) following PCI is a primary problem to limit its long-term benefits. Many investigates confirmed that restenosis was the arterial wall’s healing response to mechanical injury and comprised two main processes- neointimal hyperplasia (smooth muscle proliferation/migration, extracellular matrix deposition) and vessel remodeling. Many growth factors, cytokine and adhesion molecule participate this pathophysiological procedure, and just inhibiting certain factor can not suppress lesion formation completely. Therefore, it becomes an effective approach of inhibiting restenosis to suppress the critical gene which regulates cell proliferation, migration and differentiation and so on the final common path.
Catheter-based gene therapy has emerged as one of the most promising approaches to prevent coronary artery restenosis, because it appears capable of delivering therapeutic gene specifically to the location of the disease, at a precise site in the arterial wall. The choice of efficacious therapeutic gene and appropriate vector are the crux of the matter for a successful gene therapy.
A kind of new protein molecule, zinc finger protein A20 (ZFPA20) has been found in recent-years’researches of cellular endogenous protective mechanism. It is confirmed that zinc finger protein A20 is not only important negative feedback factor to regulate signaling pathway of NF-κB but also essential regulating proteinum to suppress inflammatory responses in vivo. It is product of activated NF-κB sgnaling system induced by LPS or TNF. A20 has a protective effect on damaged cells through terminating the activation of NF-κB induced by TLR4, which is activated by LPS. Previous researches have shown that the activation of NF-κB initiate lesion formation and inflammatory response after vascular injury. A20 has both ubiquitinating and deubiquitinating activities. As an important ubiquitinating modification enzyme, A20 is involed in dealing with NF-κB prosoma, IκBαdegradation and IKK (IκB-kinase) activation. A20 leads to a negative-feedback response that terminates activation of NF-κB though above main mechanisms.
One side because of inflammation mechanism of restenosis and regulation the inflammation mechanism of A20, on the other hand, bacause the activation of NF-κB initiates restenosis after vascular injury and A20 negative-feedback inhibits activation of NF-κB, A20 was served as target of happening and development process of restenos. A20 expression in vivo was regulated by gene therapy. The present study was designed to evaluate the transfection efficiency of A20 gene in injured arterial wall and to investigate the effect of in vivo local transfection of zinc finger protein A20 gene on intimal hyperplasia, proliferation and the phenotype transformation of vascular smooth muscle cells and reendothelialization of balloon-injured rat carotid arteries and its possible molecular mechanism.
Methods:
1. Identification and extraction of recombinant plasmid DNA in large quantity: Smearing the obtained E.coli carrying pCAGGS-GFP/A20 plasmid on ampicillin resistance plate, overnight cultured. puting single colony from plate into 3ml ampicillin resistance LB medium and overnight cultured on 37℃shaking table. After collecting bacteria, extracting recombinant plasmid DNA in small quantity and identificating by using EcoRⅤ/SmaⅠdouble restriction enzyme digestion. The production 1% agarose gel electrophoresis was performed. Endo-free plasmid DNA was extracted in large quantity from E.coli carrying plasmid. Plasmid DNA extraction was diluted in suitable proportion and detected the concentration.
2. Establishment of rat carotid injury model and grouping. Balloon catheter denudation of the endothelium of rat common carotid artery was routinely used as a model of restenosis. One hundred and four male Sprague-Dawley rats weighing 350-400g were randomly divided into 4 groups (n=26): the sham group (no injury), the model group (simple injury), the control group (vacant vector and transfection regent group) and the therapeutic group (A20 gene and transfection regent group). Rats were anesthetized with 10% chloraldurat (3ml/kg intraperitoneally, i.p.). After a midcervial incision, the left common carotid artery and its bifurcation were exposed. Two vascular clips were placed at the distal end and in the middle of the injured arterial segment. A cannula was introduced into the common carotid artery via the external carotid artery. The left common carotid artery was balloon-injured three times with a 2F balloon catheter (2F Fogarty, Edwards Lifesciences Corporation), then pulling out Balloon catheter. pCAGGS-GFP/A20 20μl or pCAGGS-GFP 20μl with 40μl Lipofectamine 2000 (Invitrogen Corporation) was gradually instilled into the lumen for 30 min. Just balloon-injured operation in the model group. The sham group was only tied with a ligature at the external carotid artery without the balloon- injured operation. Rat were sacrificed according to various requirement on the different day after operation respectively. The transfection efficiency of plasmid in injured arterial wall was evaluated by using fluorescence microscope 24 hours after transfection. Quantification of intimal hyperplasia was determined by histopathologic examination 14 days after operation. Proliferation index of VSMCs in vivo was assessed by thymidine analogue bromodeoxyuridine (BrdU) labeling technique 10 days after operation. The expression of A20, nuclear factor-kappaB p65 (NF-κBp65) and toll like receptor 4 (TLR4) of carotid arteries in different groups was confirmed by immunohistochemical staining, reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis at different time point.
3. Thirty six male Sprague-Dawley(SD) rats were randomly divided into three groups (n=12): the sham group(no injury), the control group (vacant vector and transfection regent group) and the therapeutic group (A20 gene and transfection regent group). Rats were sacrificed two weeks after operation to evaluate reendothelialization by Evans blue dye staining. Ultrastructural change of injured artery was observed with transmission electron microscope at day 3 and 7 after balloon injury.
4. Cultured rat aorta smooth muscle cells (SMCs) were identified by specificα-actin antibody. VSMCs were pretreated by emodin (5～50μg/l) for 30min and then were stimulated by LPS (20mg/l) for 24h. VSMCs proliferation was assessed by MTT and BrdU corporation. The quantification of A20 mRNA expression was measured by RT-PCR. The quantification of A20, NF-κBp65 protein expression was measured by Western blot analysis.
Results:
1. After the pCAGGS-GFP/A20 plasmid was digested by EcoRⅤ/SmaⅠdouble restriction enzyme, the production was identified by 1% agarose gel electrophoresis, plasmid size and enzyme digested result was right.
2. Much green fluorescence distribution of zinc finger protein A20 gene existed in the remnant intima and media In vivo 24 hours after transfection by using fluorescence microscope, which confirmed the transgene efficiency in injured vascular wall.
3. Significant intimal hyperplasia was observed in the model and control group at day 14 after injury. A20 gene transfection markedly reduced the neointimal area (47.8% reduction; P< 0.05) and intimal to media area ratio (42.9% reduction; P< 0.05) in the therapeutic group. Proliferation index of VSMCs (BrdU index) at day 10 after operation decreased more significantly in the therapeutic group (9.6±2.3%) than in the control group (26.7±5.1%,P<0.01). The A20 positive cells ratio of the therapeutic group was much more than that of the control group (P<0.05), whereas, the NF-κBp65 and TLR4 positive cells ratio of the therapeutic group was much less than that of the control group at day 10 after injury (P<0.05). A significantly higher level of A20 mRNA, protein expression was measured in the therapeutic group than in control group at every time point after operation (P<0.05). A significantly lower level of NF-κBp65 and TLR4 mRNA, protein expression was observed in the therapeutic group than in control group at every time point after operation (P<0.05).
4. At 2 weeks after balloon injury, compared with control group ,the arteries of animals treated with A20 gene transfection showed a greater area of recovered endothelium, as evidenced by Evans blue staining. Quantification of Re-endothelialization, based on the percentage of re-endothelialization area in the originally injured area re-endothelialized (control 55.6±3.3% vs. therapeutic 79.1±3.7%, P<0.05) revealed a significant acceleration of endothelial recovery in the A20 gene therapeutic group compared with the control group.
5. Representative contractile phenotype of VSMC was shown in the sham group and synthetic phenotype was shown at day 7 after balloon injury in the control group. Synthetic phenotype and transitional phenotype was shown at day 3 after balloon injury in the control group. At day 7 VSMC showed relatively more myofilament and less organelles and looked like contractile phenotype in the therapeutic group.
6. Compered with the vacant group, the control group showed significantly higher MTT and BrdU corporating OD value. We found that emodin induced a concentration-dependent decrease in the level of MTT and BrdU corporating OD value. Compered with the control group, low-dose groups have not significant difference, high-dose group showed significant difference. The expression of A20 mRNA and protein,NF-κBp65 protein were up-reglulated after VSMCs were stimulated by LPS. The expression of A20 mRNA and protein were more increased in a concentration- dependent manner after VSMCs were pretreated by emodin on different concentration. While the expression of NF-κBp65 protein were down-regulated.
Conclusion:
1. Local endogenous zinc finger protein A20 expression was increased in balloon-injured rat carotid artery, and possesed evident chronergy. It is confirmed that zinc finger protein A20 is critical for the regulation of inflammatory responses. It is the important regulation protein for inhibiting inflammatory responses in vivo.
2. Local transfection of A20 gene resulted in the high expression of zinc finger protein A20 mRNA and protein overexpression in balloon- injured rat carotid artery,
3. Local transfection of A20 gene suppressed intimal hyperplasia and VSMC proliferation of balloon-injured rat carotid artery. Its possible molecular mechanism is that A20 is critical for the regulation of inflammatory responses through terminating the activation of NF-κB induced by injury.
4. Local transfection of A20 gene accelerated reendothelialization and inhibited the phenotype transformation of VSMC of balloon-injured rat carotid artery. Its possible molecular mechanism is that A20 is critical for the regulation of inflammatory responses through terminating the activation of NF-κB induced by injury.
5. EMD can inhibit LPS-mediated rat aorta VSMC proliferation..the possible mechanism is that EMD can suppress activation of NF-kB via increasing the expression of inflammatory reaction regulatory protein A20 in vivo ,which has confirmed further the effect of A20 on the VSMC proliferation.
基于导管将基因准确地送至狭窄血管的局部进行基因治疗是预防血管再狭窄较有效的方法之一,而选择合适的基因和载体是基因治疗再狭窄的关键之一。
近年来在细胞内源性保护机制的研究中发现了一类新的蛋白质,即锌脂蛋白A20(zinc finger protein A20),简称A20。A20是NF-κB信号系统重要的负反馈调节因子,也是防止体内炎症反应失控的重要调节蛋白。A20的表达能够被LPS和TNF等所诱导,是NF-κB信号系统活化的产物,同时A20通过抑制TNF和LPS刺激TNFR-1和TLR4诱导的NF-κB的活化而对细胞损伤起保护作用。已有研究证实NF-κB的激活是球囊损伤动脉后血管狭窄发生的重要机制之一。结构研究显示,A20既有去泛素活性,又有泛素连接酶活性,是一种重要的泛素调节酶,在A20等泛素调节酶的作用下泛素调节参与了NF-κB前体的处理、NF-κB抑制性结合蛋白IκB的降解和IκB激酶IKK的活化等主要环节,反馈抑制了NF-κB的活化。
一方面由于再狭窄的炎症机制及A20调控炎症的机制,另一方面由于NF-κB的激活导致球囊损伤后血管狭窄及A20对NF-κB活化的负反馈抑制作用,由此我们选择A20基因作为再狭窄发生发展过程中的干预靶点,运用基因治疗方法对体内A20的表达进行调控,深入探讨球囊损伤术后血管内膜增生过程中A20在损伤局部组织中的表达变化及局部过表达A20对损伤后内膜增生、平滑肌细胞增殖和表型改变,内皮再生及TLR4/NF-κB信号通路的影响,为进一步明确A20对再狭窄过程中炎症反应的调节作用以及揭示机体内源性保护机制提供必要的理论依据。
方法:
1. pCAGGS-GFP/A20重组质粒的鉴定和大量提取:将获得的pCAGGS-GFP/A20质粒菌保涂Amp抗性的平板后,过夜培养。挑单菌落于3ml Amp抗性的LB培养基中37℃摇床中过夜培养,集菌后,进行小量质粒提取,并使用EcoRⅤ/SmaⅠ进行双酶切鉴定,酶切产物1%琼脂糖凝胶电泳。将鉴定好的质粒菌保进行无内毒素质粒的大量提取,将质粒DNA提取物按适当比例稀释,并测定浓度。
2.动物模型制备和实验分组,大鼠颈总动脉内膜球囊剥脱术用来建立再狭窄模型:健康雄性Sprague-Dawley(SD)大鼠体重350～400g,104只随机分为4组(n=26):假手术组;模型组(单纯损伤组);对照组(pCAGGS-GFP+转染试剂组);治疗组(pCAGGS-GFP/A20+转染试剂组)。10%水合氯醛(3ml/kg)腹腔注射麻醉后,取颈正中切口,钝性分离左颈总动脉,暴露左颈总动脉及其分叉处,用显微血管夹暂时夹闭左颈总动脉近心端及颈内动脉,将2F Fogarty球囊导管从颈外动脉切口插入至左颈总动脉,用50μl生理盐水充盈球囊,来回抽动3次以剥脱颈总动脉内膜。退出球囊,治疗组向损伤血管节段内缓慢注入灌注液(Lipofectamine 2000转染试剂40μl + pCAGGS-GFP/A20质粒20μl),对照组向损伤血管节段内缓慢注入灌注液(Lipofectamine 2000转染试剂40μl+ pCAGGS-GFP质粒20μl)局部作用30分钟。模型组只进行球囊损伤术,假手术组只进行左颈外动脉结扎,不进行球囊损伤。术后不同时间点根据不同要求分别处死动物。转染后24h取大鼠局部血管进行冰冻切片荧光显微镜下观察绿色荧光蛋白的表达籍以评估A20在损伤动脉壁的转染效率;术后14d病理组织学方法进行内膜增生的形态学分析;术后10d溴脱氧尿嘧啶核苷(BrdU)标记技术评估血管平滑肌细胞(VSMC)的增殖指数;术后10d免疫组织化学染色观察A20、核因子-κB p65(NF-κB p65)、Toll样受体4(TLR4)在各组大鼠颈总动脉中的表达情况;术后3d、7d、14d、28d通过逆转录-聚合酶链式反应(RT-PCR)、Western-blotting法分别检测A20、NF-κB p65、TLR4在颈动脉组织的mRNA和蛋白表达。
3.健康雄性Sprague-Dawley(SD)大鼠体重350～400g,36只随机分为3组(n=12):假手术组;对照组(pCAGGS-GFP+转染试剂组);治疗组(pCAGGS-GFP/A20+转染试剂组)。球囊损伤后2周伊文思蓝(Evans blue)染色法评估内皮再生情况;术后3d、7d透射电镜观察血管超微结构改变。
4.体外培养大鼠主动脉平滑肌细胞并鉴定,以LPS(20mg/l)作为刺激因素,以不同浓度大黄素(Emodin, EMD)作为干预因素,通过MTT和BrdU掺入方法评估各组VSMC增殖情况,以RT-PCR检测A20 mRNA表达,以Western-blotting法检测A20、NF-κB p65蛋白的表达。
结果:
1.将提取的重组真核表达质粒pCAGGS-GFP/A20使用EcoRⅤ/SmaⅠ进行双酶切鉴定,酶切产物1%琼脂糖凝胶电泳,质粒大小及酶切结果正确。
2.将pCAGGS-GFP/A20于活体转染至大鼠球囊损伤的颈动脉局部,24h后荧光显微镜下可见治疗组残存血管内膜及中膜有大量绿色荧光分布,证明转染成功。
3.大鼠颈总动脉球囊损伤术后14d模型组和对照组血管内膜显著增生。A20基因转染可显著抑制新生内膜面积的增加(减少47.8%,P<0.05)和内/中膜比值的增加(减少42.9%,P<0.05)。术后10d治疗组VSMC体内增殖指数(BrdU指数)(9.6±2.3%)显著少于对照组(26.7±5.1%,P<0.01)。术后10d治疗组血管壁A20免疫组化染色阳性细胞率显著高于对照组(P<0.05),NF-κB p65、TLR4免疫组化染色阳性细胞率显著低于对照组(P<0.05)。术后3d、7d、14d、28d治疗组血管组织A20的mRNA和蛋白表达量显著高于对照组(P<0.05),治疗组血管组织NF-κB p65、TLR4的mRNA和蛋白表达量显著低于对照组(P<0.05)。
4.球囊损伤后2周,pCAGGS-GFP/A20质粒转染治疗组与对照组比较,显示更大面积的内皮修复,由伊文思蓝染色证明。治疗组再内皮化的面积占最初损伤面积的百分数( 73.1±3.7% )显著大于对照组(55.6±3.3%,P<0.05)。说明A20转染治疗组与对照组比较显示更大面积的内皮修复。
5.假手术组大鼠VSMC电镜下呈典型的“收缩表型”,球囊损伤后7d对照组VSMC表型发生明显改变,呈典型的“合成表型”,损伤后3d对照组可见合成型细胞及过渡型细胞,治疗组术后7d可见接近收缩型的VSMC。
6.体外培养的大鼠主动脉VSMC经LPS刺激后,MTT OD值与BrdU掺入OD值均显著高于空白组。经不同浓度EMD干预结果显示随EMD浓度增大,MTT OD值及BrdU掺入OD值均逐渐变小,其中低剂量组与对照组相比无显著性差异(P>0.05),高剂量组与对照组相比存在显著性差异(P<0.05)。RT-PCR和Western-blot结果显示LPS刺激可使A20mRNA和蛋白以及NF-κB p65蛋白表达均增加,经不同浓度EMD干预后A20mRNA和蛋白表达随EMD浓度递增而进一步增加,NF-κB p65蛋白表达随EMD浓度递增而逐渐减少。
结论:
1.大鼠颈动脉球囊损伤后内源性锌指蛋白A20在局部表达增加,并具有明显的时效性,说明损伤情况下,A20表达增加是机体的一种重要的内源性抗炎保护效应机制,可能对于调控损伤后适度的炎症反应具有重要意义。
2.大鼠颈动脉球囊损伤后局部转染A20基因可获得锌指蛋白A20mRNA和蛋白在局部组织细胞内高表达。
3.大鼠颈动脉球囊损伤后局部转染A20基因可抑制损伤血管内膜增生及平滑肌细胞的增殖,其分子机制可能通过其负反馈抑制了TLR4/NF-κB介导的胞内信号转导途径。
4.大鼠颈动脉球囊损伤后局部转染A20基因可促进损伤血管内皮再生,抑制损伤处VSMC表型转化,其分子机制可能通过其负反馈抑制了TLR4/NF-κB介导的胞内信号转导途径。
5. EMD对LPS诱导的大鼠主动脉VSMC增殖具有抑制作用,其机制可能通过增加体内炎症反应调节蛋白A20的表达并进一步负反馈抑制了NF-κB的激活,进一步证实了A20对VSMC增殖的影响。
Object:Percutaneous coronary intervention (PCI) is now one of the most effective methods to treat coronary artery disease, but restenosis (RS) following PCI is a primary problem to limit its long-term benefits. Many investigates confirmed that restenosis was the arterial wall’s healing response to mechanical injury and comprised two main processes- neointimal hyperplasia (smooth muscle proliferation/migration, extracellular matrix deposition) and vessel remodeling. Many growth factors, cytokine and adhesion molecule participate this pathophysiological procedure, and just inhibiting certain factor can not suppress lesion formation completely. Therefore, it becomes an effective approach of inhibiting restenosis to suppress the critical gene which regulates cell proliferation, migration and differentiation and so on the final common path.
Catheter-based gene therapy has emerged as one of the most promising approaches to prevent coronary artery restenosis, because it appears capable of delivering therapeutic gene specifically to the location of the disease, at a precise site in the arterial wall. The choice of efficacious therapeutic gene and appropriate vector are the crux of the matter for a successful gene therapy.
A kind of new protein molecule, zinc finger protein A20 (ZFPA20) has been found in recent-years’researches of cellular endogenous protective mechanism. It is confirmed that zinc finger protein A20 is not only important negative feedback factor to regulate signaling pathway of NF-κB but also essential regulating proteinum to suppress inflammatory responses in vivo. It is product of activated NF-κB sgnaling system induced by LPS or TNF. A20 has a protective effect on damaged cells through terminating the activation of NF-κB induced by TLR4, which is activated by LPS. Previous researches have shown that the activation of NF-κB initiate lesion formation and inflammatory response after vascular injury. A20 has both ubiquitinating and deubiquitinating activities. As an important ubiquitinating modification enzyme, A20 is involed in dealing with NF-κB prosoma, IκBαdegradation and IKK (IκB-kinase) activation. A20 leads to a negative-feedback response that terminates activation of NF-κB though above main mechanisms.
One side because of inflammation mechanism of restenosis and regulation the inflammation mechanism of A20, on the other hand, bacause the activation of NF-κB initiates restenosis after vascular injury and A20 negative-feedback inhibits activation of NF-κB, A20 was served as target of happening and development process of restenos. A20 expression in vivo was regulated by gene therapy. The present study was designed to evaluate the transfection efficiency of A20 gene in injured arterial wall and to investigate the effect of in vivo local transfection of zinc finger protein A20 gene on intimal hyperplasia, proliferation and the phenotype transformation of vascular smooth muscle cells and reendothelialization of balloon-injured rat carotid arteries and its possible molecular mechanism.
Methods:
1. Identification and extraction of recombinant plasmid DNA in large quantity: Smearing the obtained E.coli carrying pCAGGS-GFP/A20 plasmid on ampicillin resistance plate, overnight cultured. puting single colony from plate into 3ml ampicillin resistance LB medium and overnight cultured on 37℃shaking table. After collecting bacteria, extracting recombinant plasmid DNA in small quantity and identificating by using EcoRⅤ/SmaⅠdouble restriction enzyme digestion. The production 1% agarose gel electrophoresis was performed. Endo-free plasmid DNA was extracted in large quantity from E.coli carrying plasmid. Plasmid DNA extraction was diluted in suitable proportion and detected the concentration.
2. Establishment of rat carotid injury model and grouping. Balloon catheter denudation of the endothelium of rat common carotid artery was routinely used as a model of restenosis. One hundred and four male Sprague-Dawley rats weighing 350-400g were randomly divided into 4 groups (n=26): the sham group (no injury), the model group (simple injury), the control group (vacant vector and transfection regent group) and the therapeutic group (A20 gene and transfection regent group). Rats were anesthetized with 10% chloraldurat (3ml/kg intraperitoneally, i.p.). After a midcervial incision, the left common carotid artery and its bifurcation were exposed. Two vascular clips were placed at the distal end and in the middle of the injured arterial segment. A cannula was introduced into the common carotid artery via the external carotid artery. The left common carotid artery was balloon-injured three times with a 2F balloon catheter (2F Fogarty, Edwards Lifesciences Corporation), then pulling out Balloon catheter. pCAGGS-GFP/A20 20μl or pCAGGS-GFP 20μl with 40μl Lipofectamine 2000 (Invitrogen Corporation) was gradually instilled into the lumen for 30 min. Just balloon-injured operation in the model group. The sham group was only tied with a ligature at the external carotid artery without the balloon- injured operation. Rat were sacrificed according to various requirement on the different day after operation respectively. The transfection efficiency of plasmid in injured arterial wall was evaluated by using fluorescence microscope 24 hours after transfection. Quantification of intimal hyperplasia was determined by histopathologic examination 14 days after operation. Proliferation index of VSMCs in vivo was assessed by thymidine analogue bromodeoxyuridine (BrdU) labeling technique 10 days after operation. The expression of A20, nuclear factor-kappaB p65 (NF-κBp65) and toll like receptor 4 (TLR4) of carotid arteries in different groups was confirmed by immunohistochemical staining, reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis at different time point.
3. Thirty six male Sprague-Dawley(SD) rats were randomly divided into three groups (n=12): the sham group(no injury), the control group (vacant vector and transfection regent group) and the therapeutic group (A20 gene and transfection regent group). Rats were sacrificed two weeks after operation to evaluate reendothelialization by Evans blue dye staining. Ultrastructural change of injured artery was observed with transmission electron microscope at day 3 and 7 after balloon injury.
4. Cultured rat aorta smooth muscle cells (SMCs) were identified by specificα-actin antibody. VSMCs were pretreated by emodin (5～50μg/l) for 30min and then were stimulated by LPS (20mg/l) for 24h. VSMCs proliferation was assessed by MTT and BrdU corporation. The quantification of A20 mRNA expression was measured by RT-PCR. The quantification of A20, NF-κBp65 protein expression was measured by Western blot analysis.
Results:
1. After the pCAGGS-GFP/A20 plasmid was digested by EcoRⅤ/SmaⅠdouble restriction enzyme, the production was identified by 1% agarose gel electrophoresis, plasmid size and enzyme digested result was right.
2. Much green fluorescence distribution of zinc finger protein A20 gene existed in the remnant intima and media In vivo 24 hours after transfection by using fluorescence microscope, which confirmed the transgene efficiency in injured vascular wall.
3. Significant intimal hyperplasia was observed in the model and control group at day 14 after injury. A20 gene transfection markedly reduced the neointimal area (47.8% reduction; P< 0.05) and intimal to media area ratio (42.9% reduction; P< 0.05) in the therapeutic group. Proliferation index of VSMCs (BrdU index) at day 10 after operation decreased more significantly in the therapeutic group (9.6±2.3%) than in the control group (26.7±5.1%,P<0.01). The A20 positive cells ratio of the therapeutic group was much more than that of the control group (P<0.05), whereas, the NF-κBp65 and TLR4 positive cells ratio of the therapeutic group was much less than that of the control group at day 10 after injury (P<0.05). A significantly higher level of A20 mRNA, protein expression was measured in the therapeutic group than in control group at every time point after operation (P<0.05). A significantly lower level of NF-κBp65 and TLR4 mRNA, protein expression was observed in the therapeutic group than in control group at every time point after operation (P<0.05).
4. At 2 weeks after balloon injury, compared with control group ,the arteries of animals treated with A20 gene transfection showed a greater area of recovered endothelium, as evidenced by Evans blue staining. Quantification of Re-endothelialization, based on the percentage of re-endothelialization area in the originally injured area re-endothelialized (control 55.6±3.3% vs. therapeutic 79.1±3.7%, P<0.05) revealed a significant acceleration of endothelial recovery in the A20 gene therapeutic group compared with the control group.
5. Representative contractile phenotype of VSMC was shown in the sham group and synthetic phenotype was shown at day 7 after balloon injury in the control group. Synthetic phenotype and transitional phenotype was shown at day 3 after balloon injury in the control group. At day 7 VSMC showed relatively more myofilament and less organelles and looked like contractile phenotype in the therapeutic group.
6. Compered with the vacant group, the control group showed significantly higher MTT and BrdU corporating OD value. We found that emodin induced a concentration-dependent decrease in the level of MTT and BrdU corporating OD value. Compered with the control group, low-dose groups have not significant difference, high-dose group showed significant difference. The expression of A20 mRNA and protein,NF-κBp65 protein were up-reglulated after VSMCs were stimulated by LPS. The expression of A20 mRNA and protein were more increased in a concentration- dependent manner after VSMCs were pretreated by emodin on different concentration. While the expression of NF-κBp65 protein were down-regulated.
Conclusion:
1. Local endogenous zinc finger protein A20 expression was increased in balloon-injured rat carotid artery, and possesed evident chronergy. It is confirmed that zinc finger protein A20 is critical for the regulation of inflammatory responses. It is the important regulation protein for inhibiting inflammatory responses in vivo.
2. Local transfection of A20 gene resulted in the high expression of zinc finger protein A20 mRNA and protein overexpression in balloon- injured rat carotid artery,
3. Local transfection of A20 gene suppressed intimal hyperplasia and VSMC proliferation of balloon-injured rat carotid artery. Its possible molecular mechanism is that A20 is critical for the regulation of inflammatory responses through terminating the activation of NF-κB induced by injury.
4. Local transfection of A20 gene accelerated reendothelialization and inhibited the phenotype transformation of VSMC of balloon-injured rat carotid artery. Its possible molecular mechanism is that A20 is critical for the regulation of inflammatory responses through terminating the activation of NF-κB induced by injury.
5. EMD can inhibit LPS-mediated rat aorta VSMC proliferation..the possible mechanism is that EMD can suppress activation of NF-kB via increasing the expression of inflammatory reaction regulatory protein A20 in vivo ,which has confirmed further the effect of A20 on the VSMC proliferation.
引文
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