针对TGF-β1启动子的ASODNs对新西兰兔腹主动脉支架植入后再狭窄及再内皮化的影响
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摘要
虽然药物洗脱支架(DES)的应用在较大程度上解决了支架术后再狭窄(ISR)的问题，但是许多经皮冠脉介入治疗(PCI)的病人仍然出现ISR。此外，尽管DES可较为有效降低地ISR，但也引入新的问题，DES所携带的药物或/和涂层载体对局部血管再内皮化的不良影响可增加支架内血栓形成的风险。ISR高度局部化的特征使局部的基因治疗成为可能。基因治疗即将核苷酸,或者是功能基因或者是反义寡核苷酸(ASODNs)转移至个体的体细胞内发挥治疗作用。与其它基因技术相比，ASODNs具有诸多优点且应用成熟，将ASODNs与在疾病过程中起关键作用的特异性靶基因结合，具有靶向治疗高效及毒副作用少的优点。将ASODNs涂在药物支架系统，开发出新一代药物涂层支架，也是减少再狭窄的发生率及预防并发症发生的希望所在。
ISR是一个涉及到血管壁与植入物(包括支架本身，涂层药物及载体药物)相互作用的复杂过程，其主要的发病机制包括血管平滑肌细(VSMCs)的增殖与迁移、内皮细胞的损伤、局部血小板的集聚及炎症细胞的激活，而这其中最重要的机制是VSMCs的增殖与迁移。如何抑制VSMCs的增殖与迁移是防止PCI术后ISR的关键。生长转移因子-β1(TGF-β1)是蛋白质超家族中的成员，对VSMCs生长的调控、分化、迁移和凋亡起重要作用。在血管损伤后，TGF-β1被证实能刺激VSMCs的增殖和迁移，最终导致管腔狭窄。因此，我们设想合成针对TGF-β1基因启动子的ASODNs，通过特异性抑制TGF-β1的功能或持续表达，减少VSMCs的增殖和迁移，从而为防止ISR提供新的思路。
研究由两个部分组成：①合成能特异性沉默TGF-β1基因启动子的ASODNs化合物。体外培养VSMCs，观察针对TGF-β1基因启动子的ASODNs对VSMCs的增殖和迁移的影响；②制作特异性沉默TGF-β1基因启动子的ASODNs药物涂层支架，并将其植入新西兰兔的腹主动脉内，观察其对靶血管局部支架内再狭窄及再内皮化产生的影响。
一、针对TGF-β1启动子的ASODNs化合物对VSMCs增殖和迁移的影响
目的合成能特异性沉默TGF-β1基因启动子的ASODNs化合物，通过体外培养VSMCs，观察其对VSMCs的增殖和迁移的影响
方法构建新西兰兔TGF-β1基因启动子的报告基因质粒并将其转染入HEK293细胞，确定TGF-β1基因AP-1增强子位点，根据核苷酸碱基配对的原则确定能特异性沉默TGF-β1基因启动子的ASODNs结构式并合成ASODNs化合物。体外培养新西兰兔腹主动脉VSMCs并行α-肌动蛋白(α-SMA)特异性免疫细胞化学染色鉴定。通过血小板衍生因子(PDGF)10ng/ml诱导VSMCs生长后，将细胞实验共分为3组：分别为control(单纯加入PDGF)组, PDGF+ASODNs (10ug/ml)组，PDGF+mismatchASODNs(10ug/ml)组。通过Westblot法测定各组细胞TGF-β1表达的情况，通过5-溴脱氧尿嘧啶核苷(BrdU)渗入法和水溶性四唑盐-1(WST-1)法测定各组细胞的增殖及代谢情况，通过Transwell细胞迁移实验观察各组细胞的迁移情况。
结果采用胶原酶消化法培养原代VSMCs，细胞生长呈典型的“峰-谷”状，经α-SMA特异性免疫细胞化学染色后，细胞胞浆呈阳性反应，证实为VMSCs。加入PDGF (10ng/ml)，能诱导VSMCs的代谢率增高，促进VSMCs的迁移与增殖。ASODNs能够显著抑制VSMCs的代谢率、VSMCs的迁移及VSMCs的增殖(P<0.01)，而mismatch ASODNs则不能达到此效果(P>0.05)。
结论针对TGF-β1基因AP-1启动子位点的ASODNs可特异性沉默TGF-β1基因，并能显著降低VSMCs的增殖和迁移。能特异性沉默TGF-β1基因启动子的ASODNs化合物应用于药物支架系统预防支架植入后再狭窄的作用值得进一步探讨。
二、针对TGF-β1启动子的ASODNs对新西兰兔腹主动脉支架植入后再狭窄及再内皮化的影响
目的制作特异性沉默TGF-β1基因启动子的ASODNs药物涂层支架，并将其植入新西兰兔的腹主动脉内，观察其对损伤血管局部支架内再狭窄及再内皮化的影响，并探讨其机制。
方法按照1.0μg/mm2的载药量构建2.0×12mm的ASODNs药物涂层支架。使用高效液相色谱法(HPLC)测定支架的体内外药物释放动力学。建立动物模型，在新西兰兔的腹主动脉内植入ASODNs药物涂层支架。实验共分为3组：裸支架组(n=10)，ASODNs药物涂层支架组(n=10)，mismatchASODNs药物涂层支架组(n=10)。每组各3只动物于支架植入4周后处死，留取少量标本行HPLC检测后，将含支架的血管行扫描电镜检查观察各组的支架表面内皮化情况。另外每组各6只动物与支架植入8周后处死，留取少量标本行HPLC检测后，将标本分为两部分。一部分-80°C保存，使用实时聚合酶链反应(Real-time PCR)及蛋白质印迹法(Western Blotting)法检测各组的TGF-β1、结缔组织生长因子(CTGF)的mRNA及蛋白表达情况，另一部分组织包埋入甲基丙烯酸甲酯中，分别从支架的近、中、远三处切取5μm的切片，然后行苏木精-伊红(HE)染色及弹力纤维(ETVG)染色，然后置于显微镜下观察并计算新生内膜厚度、新生内膜面积及血管狭窄面积的平均值。
结果在体外试验中，ASODN药物涂层支架24h内ASODNs释放超过90%；但在体内试验中，支架植入4周和8周后，局部血管组织中分别仍有一定量和微量ASODNs留存。扫描电镜显示，裸支架、ASODNs药物涂层支架、mismatchASODNs药物涂层支架表面均具有较高的内皮化程度。与对照组相比，裸支架组损伤血管局部的TGF-β1和CTGF的mRNA及蛋白表达均明显升高(P<0.01)。ASODN药物涂层支架能够降低损伤血管局部的TGF-β1和CTGF的mRNA及蛋白表达(P<0.01)。与裸支架组相比，ASODNs组支架内新生内膜面积明显降低(P<0.05)，再狭窄程度明显降低(P<0.05)；而mismatch ASODNs药物涂层支架的上述指标与裸支架相比均无明显差异(P>0.05)。
结论针对TGF-β1基因启动子的ASODNs药物涂层支架尽管体外释放药物动力学不理想，但植入体内仍能够显著抑制血管局部的TGF-β1和CTGF的mRNA及蛋白表达，从而能显著减少支架内再狭窄，而且不影响支架表面的再内皮化。
Clinical application of drug eluting stent (DES) has significantly reduced but notcompleptely sovled in-stent restenosis (ISR) for patients undergoing percutaneouscoronary intervention (PCI). Despite the benefits of DES, it also induces anotherunexpected issue, the in-stent thrombosis, which is associated with the delayedvascular healing or re-endotheliazation due to toxicities of the drugs and/or polymersused for manufacturing DES. The focal nature of restenosis makes it a highlyattractive target for gene therapy. Forecasting possibility of restenosis according togenes is conducive to enhancing values of risk stratification and providing morereasonable therapeutic options for patients. Gene therapies include transferringnucleotide, functional genes, or antisense oligonucleotides (ASODNs) and so on tosomatic cells of individuals to generate therapeutic effects. Many studies show thatASODNs has advantages over the traditional or other genic therapies due to thecombining with special target genes playing pivotal role in the disease process,thereby probably preserving more therapeutic efficacy and fewer side-effects. Hence,ASODNs as a candidate may be promising and particularily suitable for thedevelopment of next generation of DES.
ISR is a complicated interaction process between the vessel wall and implantedstent (stent itself, polymers and drugs). The pathophysiological mechanisms of ISRmainly include damage of endothelial cells, aggregation of platelet and activation of inflammatory cells and subsequently over-expression of various growth factors.Despite other probable mechanisms, proliferation and transfer of vascular smoothmuscle cells (VSMCs) has been considered as the most important mechanisminvolved in ISR process. Accordingly, inhibiting the proliferation and transfer ofVSMCs is the key mechanism to prevent ISR. Growth transfer factor-β1(TGF-β1) isa member in the protein super-family, which plays an important role in regulating thedifferentiation, transfer, and apoptosis of VSMCs. Once vessel wall is injured,TGF-β1might serve as one of the dominant mediators stimulating proliferation andtransfer of VSMCs, leading to neoimtimal formation and thickening, vascular lumenloss and re-stenosis. Therefore, we hypothesized that ASODNs targeting TGF-β1could efficiently inhibit the expression of TGF-β1, thus reducing the proliferation andtransfer of VSMCs. The ASODNs targeting TGF-β1represents a promising feasibleagent to be used in drug eluting stent system for preventing ISR.
The study consisted of two parts:(1) To investigate the effects of ASODNstargeting TGF-β1gene on inhibiting the proliferation and transfer of VSMCs in vitro;(2) To investigate the effects of ASODNs-drug eluting stent on preventing in-stentrestenosis and promoting reendothelialization in vivo.
I. Effects of ASODNs targeting TGF-β1promoter gene on proliferation andtransfer of VSMCs in vitro
Objectives: To synthesize ASODNs specifically silencing TGF-β1promoter gene andto observe its effectes on inhibiting the proliferation and transfer of VSMCs in vitro.
Methods: HEK293cells were transfected with recombinant rabbit TGF-β1promoterplasmids to determine AP-1binding sites. ASODNs targeting rabbit TGF-β1was thensynthesized according to base pairing of nucleotide. VSMCs were cultured in vitroand identified by α-SMA immunoflurescence staining. After incubating with PDGF(10ng/ml), the in vitro study consisted of three groups: control group (only PDGF),PDGF+ASODNs (10ug/ml) group, PDGF+mismatch ASODNs (10ug/ml) group.TGF-β1expression was detected by west-blotting, cell proliferation and metabolismby5-BrdU infiltration method and WST-1method, cell transfer by Transwelltechnique.
Results: Enzyme digestion of collagenase I was applied for the culture of normalVSMCs, which showed the typical pattern “Valley-peak”. α-SMA was abundant in theVSMCs by immunoflurescence staining.. PDGF at lower concentrations of10ng/mlrised the metabolic rate of VSMCs and promoted transfer and proliferation of VSMCs.ASODNs but not mismatch ASODNs significantly inhibted metabolic rate, transferand proliferation of VSMCs(P<0.01).
Conclusions: ASODNs targeting AP-1enhancer locus of TGF-β1gene can efficiently silence TGF-β1gene and remarkably reduce the proliferation and transfer of VSMCs.
II. Effects of ASODNs eluting stent targeting TGF-β1promotrer gene on in-stentrestenosis and reendothelialization in vivo
Objectives: To produce ASODNs eluting stent targeting TGF-β1promotrer gene andto evaluate the potential effects of them on in-stent restenosis andre-endothelialization and to investigating the underlying mechanisms.
Methods: ASODNs-drug eluting stent of2.0×12mm were developed with aconcentration of1.0μg/mm2ASODNs. Drug release dynamics of stent was measuredwith the use of HPLC in vitro. The ASODNs-drug eluting stent was then implantedinto aorta abdominalis of New Zealand rabbits. Rabbits were assigned to three groups:naked stent group (n=10), ASODNs-drug eluting stent group (n=10), and mismatchASODNs-drug eluting stent group (n=10). Three animals were sacrificed four weeksafter stent implantation; some tissue samples were preserved for HPLC detection, andsome for scanning electron microscope to observe stent surface endothelialization.Additionally, six animals were sacrificed eight weeks after stent implantation, and thestented segments of abdominal aorta were harvested with some samples preserved forHPLC detection and the renant samples for subsequent molecular and histologicalstudies. For molecular study, the samples were kept under the temperature of-80°C,mRNA and protein expression of TGF-β1and connective tissue growth factor (CTGF)were detected by real-time PCR and western blotting. For histological study, thesamples were embedded with methyl methacrylate, cutted into sections of5μm respectively from near, middle, and distant segment of stents, and then stained withHE and ETVG, the neointimal area and mean thickness was measured under themicroscope.
Results: ASODNs eluting stent released more than90%of ASODNs within24h invitro. There remained some and trace ASODNs detected in local vascular tissuesrespectively after4and8weeks after stent implamtation. Scanning electronmicroscope showed re-endothelialization was nearly complete in all the three stentsgroups (n=3). Compared with the control group, mRNA and protein expressions ofTGF-β1and CTGF in naked stent group were upregulated obviously (P<0.01for all),ASODNs-drug eluting stent significantly reduced mRNA and protein expressions(P<0.01for all) of TGF-β1and CTGF. Compared with naked stent, there wassignificant reduction of neointima area (P<0.05) and restenosis degree (P<0.05for all)in ASODNs-drug eluting stent, and there were no differences of these indexesbetween the naked stent and mismatch ASODNs-drug eluting groups (P>0.05for all).
Conclusions: Despite poorly controlled releasing of ASODNs with ASODNs-drugeluting stent in vitro, ASODNs-drug eluting stent targeting TGF-β1can inhibit mRNAand protein expression of TGF-β1, reduced in-stent restenosis without imparingre-endothelialization.
ISR是一个涉及到血管壁与植入物(包括支架本身，涂层药物及载体药物)相互作用的复杂过程，其主要的发病机制包括血管平滑肌细(VSMCs)的增殖与迁移、内皮细胞的损伤、局部血小板的集聚及炎症细胞的激活，而这其中最重要的机制是VSMCs的增殖与迁移。如何抑制VSMCs的增殖与迁移是防止PCI术后ISR的关键。生长转移因子-β1(TGF-β1)是蛋白质超家族中的成员，对VSMCs生长的调控、分化、迁移和凋亡起重要作用。在血管损伤后，TGF-β1被证实能刺激VSMCs的增殖和迁移，最终导致管腔狭窄。因此，我们设想合成针对TGF-β1基因启动子的ASODNs，通过特异性抑制TGF-β1的功能或持续表达，减少VSMCs的增殖和迁移，从而为防止ISR提供新的思路。
研究由两个部分组成：①合成能特异性沉默TGF-β1基因启动子的ASODNs化合物。体外培养VSMCs，观察针对TGF-β1基因启动子的ASODNs对VSMCs的增殖和迁移的影响；②制作特异性沉默TGF-β1基因启动子的ASODNs药物涂层支架，并将其植入新西兰兔的腹主动脉内，观察其对靶血管局部支架内再狭窄及再内皮化产生的影响。
一、针对TGF-β1启动子的ASODNs化合物对VSMCs增殖和迁移的影响
目的合成能特异性沉默TGF-β1基因启动子的ASODNs化合物，通过体外培养VSMCs，观察其对VSMCs的增殖和迁移的影响
方法构建新西兰兔TGF-β1基因启动子的报告基因质粒并将其转染入HEK293细胞，确定TGF-β1基因AP-1增强子位点，根据核苷酸碱基配对的原则确定能特异性沉默TGF-β1基因启动子的ASODNs结构式并合成ASODNs化合物。体外培养新西兰兔腹主动脉VSMCs并行α-肌动蛋白(α-SMA)特异性免疫细胞化学染色鉴定。通过血小板衍生因子(PDGF)10ng/ml诱导VSMCs生长后，将细胞实验共分为3组：分别为control(单纯加入PDGF)组, PDGF+ASODNs (10ug/ml)组，PDGF+mismatchASODNs(10ug/ml)组。通过Westblot法测定各组细胞TGF-β1表达的情况，通过5-溴脱氧尿嘧啶核苷(BrdU)渗入法和水溶性四唑盐-1(WST-1)法测定各组细胞的增殖及代谢情况，通过Transwell细胞迁移实验观察各组细胞的迁移情况。
结果采用胶原酶消化法培养原代VSMCs，细胞生长呈典型的“峰-谷”状，经α-SMA特异性免疫细胞化学染色后，细胞胞浆呈阳性反应，证实为VMSCs。加入PDGF (10ng/ml)，能诱导VSMCs的代谢率增高，促进VSMCs的迁移与增殖。ASODNs能够显著抑制VSMCs的代谢率、VSMCs的迁移及VSMCs的增殖(P<0.01)，而mismatch ASODNs则不能达到此效果(P>0.05)。
结论针对TGF-β1基因AP-1启动子位点的ASODNs可特异性沉默TGF-β1基因，并能显著降低VSMCs的增殖和迁移。能特异性沉默TGF-β1基因启动子的ASODNs化合物应用于药物支架系统预防支架植入后再狭窄的作用值得进一步探讨。
二、针对TGF-β1启动子的ASODNs对新西兰兔腹主动脉支架植入后再狭窄及再内皮化的影响
目的制作特异性沉默TGF-β1基因启动子的ASODNs药物涂层支架，并将其植入新西兰兔的腹主动脉内，观察其对损伤血管局部支架内再狭窄及再内皮化的影响，并探讨其机制。
方法按照1.0μg/mm2的载药量构建2.0×12mm的ASODNs药物涂层支架。使用高效液相色谱法(HPLC)测定支架的体内外药物释放动力学。建立动物模型，在新西兰兔的腹主动脉内植入ASODNs药物涂层支架。实验共分为3组：裸支架组(n=10)，ASODNs药物涂层支架组(n=10)，mismatchASODNs药物涂层支架组(n=10)。每组各3只动物于支架植入4周后处死，留取少量标本行HPLC检测后，将含支架的血管行扫描电镜检查观察各组的支架表面内皮化情况。另外每组各6只动物与支架植入8周后处死，留取少量标本行HPLC检测后，将标本分为两部分。一部分-80°C保存，使用实时聚合酶链反应(Real-time PCR)及蛋白质印迹法(Western Blotting)法检测各组的TGF-β1、结缔组织生长因子(CTGF)的mRNA及蛋白表达情况，另一部分组织包埋入甲基丙烯酸甲酯中，分别从支架的近、中、远三处切取5μm的切片，然后行苏木精-伊红(HE)染色及弹力纤维(ETVG)染色，然后置于显微镜下观察并计算新生内膜厚度、新生内膜面积及血管狭窄面积的平均值。
结果在体外试验中，ASODN药物涂层支架24h内ASODNs释放超过90%；但在体内试验中，支架植入4周和8周后，局部血管组织中分别仍有一定量和微量ASODNs留存。扫描电镜显示，裸支架、ASODNs药物涂层支架、mismatchASODNs药物涂层支架表面均具有较高的内皮化程度。与对照组相比，裸支架组损伤血管局部的TGF-β1和CTGF的mRNA及蛋白表达均明显升高(P<0.01)。ASODN药物涂层支架能够降低损伤血管局部的TGF-β1和CTGF的mRNA及蛋白表达(P<0.01)。与裸支架组相比，ASODNs组支架内新生内膜面积明显降低(P<0.05)，再狭窄程度明显降低(P<0.05)；而mismatch ASODNs药物涂层支架的上述指标与裸支架相比均无明显差异(P>0.05)。
结论针对TGF-β1基因启动子的ASODNs药物涂层支架尽管体外释放药物动力学不理想，但植入体内仍能够显著抑制血管局部的TGF-β1和CTGF的mRNA及蛋白表达，从而能显著减少支架内再狭窄，而且不影响支架表面的再内皮化。
Clinical application of drug eluting stent (DES) has significantly reduced but notcompleptely sovled in-stent restenosis (ISR) for patients undergoing percutaneouscoronary intervention (PCI). Despite the benefits of DES, it also induces anotherunexpected issue, the in-stent thrombosis, which is associated with the delayedvascular healing or re-endotheliazation due to toxicities of the drugs and/or polymersused for manufacturing DES. The focal nature of restenosis makes it a highlyattractive target for gene therapy. Forecasting possibility of restenosis according togenes is conducive to enhancing values of risk stratification and providing morereasonable therapeutic options for patients. Gene therapies include transferringnucleotide, functional genes, or antisense oligonucleotides (ASODNs) and so on tosomatic cells of individuals to generate therapeutic effects. Many studies show thatASODNs has advantages over the traditional or other genic therapies due to thecombining with special target genes playing pivotal role in the disease process,thereby probably preserving more therapeutic efficacy and fewer side-effects. Hence,ASODNs as a candidate may be promising and particularily suitable for thedevelopment of next generation of DES.
ISR is a complicated interaction process between the vessel wall and implantedstent (stent itself, polymers and drugs). The pathophysiological mechanisms of ISRmainly include damage of endothelial cells, aggregation of platelet and activation of inflammatory cells and subsequently over-expression of various growth factors.Despite other probable mechanisms, proliferation and transfer of vascular smoothmuscle cells (VSMCs) has been considered as the most important mechanisminvolved in ISR process. Accordingly, inhibiting the proliferation and transfer ofVSMCs is the key mechanism to prevent ISR. Growth transfer factor-β1(TGF-β1) isa member in the protein super-family, which plays an important role in regulating thedifferentiation, transfer, and apoptosis of VSMCs. Once vessel wall is injured,TGF-β1might serve as one of the dominant mediators stimulating proliferation andtransfer of VSMCs, leading to neoimtimal formation and thickening, vascular lumenloss and re-stenosis. Therefore, we hypothesized that ASODNs targeting TGF-β1could efficiently inhibit the expression of TGF-β1, thus reducing the proliferation andtransfer of VSMCs. The ASODNs targeting TGF-β1represents a promising feasibleagent to be used in drug eluting stent system for preventing ISR.
The study consisted of two parts:(1) To investigate the effects of ASODNstargeting TGF-β1gene on inhibiting the proliferation and transfer of VSMCs in vitro;(2) To investigate the effects of ASODNs-drug eluting stent on preventing in-stentrestenosis and promoting reendothelialization in vivo.
I. Effects of ASODNs targeting TGF-β1promoter gene on proliferation andtransfer of VSMCs in vitro
Objectives: To synthesize ASODNs specifically silencing TGF-β1promoter gene andto observe its effectes on inhibiting the proliferation and transfer of VSMCs in vitro.
Methods: HEK293cells were transfected with recombinant rabbit TGF-β1promoterplasmids to determine AP-1binding sites. ASODNs targeting rabbit TGF-β1was thensynthesized according to base pairing of nucleotide. VSMCs were cultured in vitroand identified by α-SMA immunoflurescence staining. After incubating with PDGF(10ng/ml), the in vitro study consisted of three groups: control group (only PDGF),PDGF+ASODNs (10ug/ml) group, PDGF+mismatch ASODNs (10ug/ml) group.TGF-β1expression was detected by west-blotting, cell proliferation and metabolismby5-BrdU infiltration method and WST-1method, cell transfer by Transwelltechnique.
Results: Enzyme digestion of collagenase I was applied for the culture of normalVSMCs, which showed the typical pattern “Valley-peak”. α-SMA was abundant in theVSMCs by immunoflurescence staining.. PDGF at lower concentrations of10ng/mlrised the metabolic rate of VSMCs and promoted transfer and proliferation of VSMCs.ASODNs but not mismatch ASODNs significantly inhibted metabolic rate, transferand proliferation of VSMCs(P<0.01).
Conclusions: ASODNs targeting AP-1enhancer locus of TGF-β1gene can efficiently silence TGF-β1gene and remarkably reduce the proliferation and transfer of VSMCs.
II. Effects of ASODNs eluting stent targeting TGF-β1promotrer gene on in-stentrestenosis and reendothelialization in vivo
Objectives: To produce ASODNs eluting stent targeting TGF-β1promotrer gene andto evaluate the potential effects of them on in-stent restenosis andre-endothelialization and to investigating the underlying mechanisms.
Methods: ASODNs-drug eluting stent of2.0×12mm were developed with aconcentration of1.0μg/mm2ASODNs. Drug release dynamics of stent was measuredwith the use of HPLC in vitro. The ASODNs-drug eluting stent was then implantedinto aorta abdominalis of New Zealand rabbits. Rabbits were assigned to three groups:naked stent group (n=10), ASODNs-drug eluting stent group (n=10), and mismatchASODNs-drug eluting stent group (n=10). Three animals were sacrificed four weeksafter stent implantation; some tissue samples were preserved for HPLC detection, andsome for scanning electron microscope to observe stent surface endothelialization.Additionally, six animals were sacrificed eight weeks after stent implantation, and thestented segments of abdominal aorta were harvested with some samples preserved forHPLC detection and the renant samples for subsequent molecular and histologicalstudies. For molecular study, the samples were kept under the temperature of-80°C,mRNA and protein expression of TGF-β1and connective tissue growth factor (CTGF)were detected by real-time PCR and western blotting. For histological study, thesamples were embedded with methyl methacrylate, cutted into sections of5μm respectively from near, middle, and distant segment of stents, and then stained withHE and ETVG, the neointimal area and mean thickness was measured under themicroscope.
Results: ASODNs eluting stent released more than90%of ASODNs within24h invitro. There remained some and trace ASODNs detected in local vascular tissuesrespectively after4and8weeks after stent implamtation. Scanning electronmicroscope showed re-endothelialization was nearly complete in all the three stentsgroups (n=3). Compared with the control group, mRNA and protein expressions ofTGF-β1and CTGF in naked stent group were upregulated obviously (P<0.01for all),ASODNs-drug eluting stent significantly reduced mRNA and protein expressions(P<0.01for all) of TGF-β1and CTGF. Compared with naked stent, there wassignificant reduction of neointima area (P<0.05) and restenosis degree (P<0.05for all)in ASODNs-drug eluting stent, and there were no differences of these indexesbetween the naked stent and mismatch ASODNs-drug eluting groups (P>0.05for all).
Conclusions: Despite poorly controlled releasing of ASODNs with ASODNs-drugeluting stent in vitro, ASODNs-drug eluting stent targeting TGF-β1can inhibit mRNAand protein expression of TGF-β1, reduced in-stent restenosis without imparingre-endothelialization.
引文
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