c-myc反义寡核苷酸对血管成形术后内膜增殖和重逆影响的实验研究(摘要)
作者：刘利 曾定尹 齐国先 王恩华
单位：辽宁省沈阳市，中国医科大学第一临床学院 心内科(11001)
关键词：
目的 目的：本实验旨在研究经导管血管内局部转运c-myc反义寡核苷酸对兔腹主动脉粥样硬化狭窄球囊成形术后内膜增殖和重构影响及其作用机制。
方法：采用高胆固醇饮食加血管内皮剥脱的方法建立腹主动脉粥样硬化狭窄模型(狭窄≥50%)。并随机分为4组：反义治疗组(Ⅰ组)、正义治疗组(Ⅱ组)、盐水对照组(Ⅲ组)及单纯扩张组(Ⅳ组)。分别对各组狭窄段血管进行球囊扩张并进行血管内局部药物转运，分别于局部药物转运后24小时及4周时取局部药物转运段血管，进行反转录PCR(RT-PCR)半定量分析，c-myc蛋白免疫组化分析、组织形态学及平滑肌细胞(SMC)超微结构观察。
, 百拇医药
结果：局部药物转运后24小时，Ⅰ组c-myc mRNA水平明显低于Ⅱ、Ⅲ和Ⅳ组(P<0.01)；局部药物转运后4周时，Ⅰ组新生内膜面积(NIA)与中膜面积(MA)比率(NIA/MA)均明显小于Ⅱ、Ⅲ和Ⅳ组(P均<0.05)，而血管外膜重塑指数则明显大于Ⅱ、Ⅲ和Ⅳ组(P均<0.05)。Ⅰ组c-myc蛋白阳性细胞百分比明显低于Ⅱ、Ⅲ和Ⅳ组(P均<0.01)。电镜观察显示：Ⅰ组SMC排列尚规则，细胞内粗面内质网及高尔基器较少，而肌丝相对丰富；Ⅱ、Ⅲ和Ⅳ组SMC排列紊乱，细胞内粗面内质网及高尔基器丰富，而肌丝相对较少。
结论：①经微孔球囊导管血管内局部应用c-myc反义寡核苷酸可抑制实验性兔腹主动脉粥样硬化狭窄球囊成形术后内膜增厚。②经微孔球囊导管血管内局部用药对血管壁可造成额外损伤，但不影响c-myc反义寡核苷酸对内膜增殖的抑制作用。③c-myc反义寡核苷酸抑制内膜增殖的机制与其抑制c-myc原癌基因转录和产物的表达，抑制SMC表现型的转变有关。④血管内局部应用c-myc反义寡核苷酸可以抑制血管重塑指数的降低，可能是其降低再狭窄的机制之一。
, 百拇医药
Experimental Study of the Effect of Locally Delivered c-myc Antisense Oligonucleotides on Neointimal Thickening and Vascular Remodeling (Abstract)
Department of Cardiology, The First Affiliated Hospital of China Medical University, Shenyang (110001), Liaoning
Liu Li, Zeng Dingyin, Qi Guoxian, et al.
Objective: The vascular smooth muscle cells (SMC) proferate, migrate from media to intima and synthesis extracellular matrix following vascular injury, and all these have been thought to be the major reasons for restenosis after artery intervention. Several proto-oncogenes, which became transiently activated in the vessel wall after balloon dialation play a very important role in modulating SMC proliferation and phenotypic conversion. Although antisense c-myc oligonucleotides have been demonstrated to be able to suppress SMC proliferation in vitro and inhibit intimal hyperplasia in vivo, these studies were done on an underlying nonatherosclerotic vessel. This was different from the human coronary lesions, and the effect of antisense c-myc oligonucleotides on SMC phenotype and vascular remodeling after vascular injury is unknown. The present study was designed to investigate the effect of antisense oligonucleotides targeting c-myc proto-oncogene, when delivered locally using a porous balloon catheter, on neointimal thickening and vascular remodeling after angioplasty and to explore the therapeutic potential of antisense c-myc oligonucleotides for prevention of restenosis.
, 百拇医药
Methods: The celiac arterial endothelium of 75 male Japanese White Rabbits (2～3 kg) were denuded after being fed with cholesterol-supplemented diet [1.5 g/(kg*d)] for 4 weeks, and angioplasty to atherosclerotic stenosis of celiac arteries was performed after 4 additional weeks of high-cholesterol diet. Animals with significantly atherosclerotic stenosis of celiac arteries demonstrated by angiography were randomized to the antisense group (group Ⅰ) receiving c-myc antisense oligonucleotides (1 mg per vessel, total volume 3 ml), sense group (group Ⅱ) receiving c-myc sense oligonucleotides (1 mg per vessel, total volume 3 ml), saline control group (group Ⅲ) receiving saline 3 ml and single angioplasty only group (group Ⅳ). The drugs were intramurally injected using a porous balloon. At 24 hours after local delivery of drugs, 5 animals of each group were killed and vessels were harvested to assess the c-myc mRNA levels by reverse transcription polymerase chain reaction (RT-PCR). At 4 weeks after local delivery of drugs, the remaining animals were killed and celiac arteries were fixed in situ by perfusion with 10% buffered formalin at 120 mmHg perfusion pressure. Vessels, including the treated segment and proximal reference segment, were collected and cross sectioned for immunohistochemistric and morphometric analysis. Neointimal area (NIA), medial area (MA), ratio of NIA to MA (NIA/MA) were measured, and arterial remodeling was assessed. Also, ultrathin sections were prepared for transmission electron-microscopy (TEM) to observe the changes of intracellular ultrastructure.
, 百拇医药
Results: 34 rabbits were excluded due to death or insignificantly atherosclerotic stenosis created (<50% occlusion). The causes of death included diarrhea, infection, anesthetic accident, vessel rupture, hemorrhage during or after operation, or unknown reason. Morphormetric results showed that the ratio of neointimal area (NEA) to medial area (MA) (NEA/MA) in group Ⅰ (1.03±0.24) were significantly less than those of control segments in group Ⅱ (1.58±0.43, p<0.05), group Ⅲ (1.54±0.35, p<0.05) and group Ⅳ (1.52±0.61, p<0.05). The geometric remodeling index at angioplastied site in group Ⅰ [(32.7±14.2)%, p<0.05] was significantly larger than that in group Ⅱ [(24.0±8.9)%, p<0.05], group Ⅲ [(24.0±11.3)%, p<0.05] and group Ⅳ [(23.2±11.3)%,p<0.05]. RT-PCR results showed that c-myc mRNA level in group Ⅰ decreased comparing with group Ⅱ, group Ⅲ and group Ⅳ. Immunohistochemistric analysis also showed that the percentage of c-myc protein positive cells was less in group Ⅰ (4.5±1.1%) than that in group Ⅱ [(11.9±1.6)%, p<0.01], group Ⅲ [(12.7±1.9)%, p<0.01] and group Ⅳ [(9.8±0.8)%, p<0.01]. Furthermore, a slight difference in NEA/MA and the percentage of positive cells was observed when comparing group Ⅳ with group Ⅱ and group Ⅲ. TEM results showed that the organelles related with biosynthesis, ie. rough endoplasmic reticulum, free ribosome, Golgi complex, mitochondria, glycogen granules were decreased, but myofilaments were more abundant in group Ⅰ than those in group Ⅱ, group Ⅲ and group Ⅳ.
, 百拇医药
Conclusion: ① Transcatheter delivery of c-myc antisense oligonucleotides to the site of balloon angioplasty reduced the neointimal hyperplasia after vascular injury. ② The mechanism of c-myc antisense oligonucleotides inhibiting neointimal thickening involved the inhibition of transcripation of c-myc mRNA and expression of c-myc protein. ③ Transcatheter delivery technique with porous balloon increased extra trauma to arterial wall, but did not affect the inhibitory effect of c-myc antisense oligonucleotides on neointimal formation. ④ Transcatheter delivery of c-myc antisense oligonucleotides suppressed the conversion of SMC from contractile to synthetic phenotype. ⑤ Transcatheter delivery of c-myc antisense oligonucleotides inhibited the unfavourable vascular remodeling., 百拇医药
单位：辽宁省沈阳市，中国医科大学第一临床学院 心内科(11001)
关键词：
目的 目的：本实验旨在研究经导管血管内局部转运c-myc反义寡核苷酸对兔腹主动脉粥样硬化狭窄球囊成形术后内膜增殖和重构影响及其作用机制。
方法：采用高胆固醇饮食加血管内皮剥脱的方法建立腹主动脉粥样硬化狭窄模型(狭窄≥50%)。并随机分为4组：反义治疗组(Ⅰ组)、正义治疗组(Ⅱ组)、盐水对照组(Ⅲ组)及单纯扩张组(Ⅳ组)。分别对各组狭窄段血管进行球囊扩张并进行血管内局部药物转运，分别于局部药物转运后24小时及4周时取局部药物转运段血管，进行反转录PCR(RT-PCR)半定量分析，c-myc蛋白免疫组化分析、组织形态学及平滑肌细胞(SMC)超微结构观察。
, 百拇医药
结果：局部药物转运后24小时，Ⅰ组c-myc mRNA水平明显低于Ⅱ、Ⅲ和Ⅳ组(P<0.01)；局部药物转运后4周时，Ⅰ组新生内膜面积(NIA)与中膜面积(MA)比率(NIA/MA)均明显小于Ⅱ、Ⅲ和Ⅳ组(P均<0.05)，而血管外膜重塑指数则明显大于Ⅱ、Ⅲ和Ⅳ组(P均<0.05)。Ⅰ组c-myc蛋白阳性细胞百分比明显低于Ⅱ、Ⅲ和Ⅳ组(P均<0.01)。电镜观察显示：Ⅰ组SMC排列尚规则，细胞内粗面内质网及高尔基器较少，而肌丝相对丰富；Ⅱ、Ⅲ和Ⅳ组SMC排列紊乱，细胞内粗面内质网及高尔基器丰富，而肌丝相对较少。
结论：①经微孔球囊导管血管内局部应用c-myc反义寡核苷酸可抑制实验性兔腹主动脉粥样硬化狭窄球囊成形术后内膜增厚。②经微孔球囊导管血管内局部用药对血管壁可造成额外损伤，但不影响c-myc反义寡核苷酸对内膜增殖的抑制作用。③c-myc反义寡核苷酸抑制内膜增殖的机制与其抑制c-myc原癌基因转录和产物的表达，抑制SMC表现型的转变有关。④血管内局部应用c-myc反义寡核苷酸可以抑制血管重塑指数的降低，可能是其降低再狭窄的机制之一。
, 百拇医药
Experimental Study of the Effect of Locally Delivered c-myc Antisense Oligonucleotides on Neointimal Thickening and Vascular Remodeling (Abstract)
Department of Cardiology, The First Affiliated Hospital of China Medical University, Shenyang (110001), Liaoning
Liu Li, Zeng Dingyin, Qi Guoxian, et al.
Objective: The vascular smooth muscle cells (SMC) proferate, migrate from media to intima and synthesis extracellular matrix following vascular injury, and all these have been thought to be the major reasons for restenosis after artery intervention. Several proto-oncogenes, which became transiently activated in the vessel wall after balloon dialation play a very important role in modulating SMC proliferation and phenotypic conversion. Although antisense c-myc oligonucleotides have been demonstrated to be able to suppress SMC proliferation in vitro and inhibit intimal hyperplasia in vivo, these studies were done on an underlying nonatherosclerotic vessel. This was different from the human coronary lesions, and the effect of antisense c-myc oligonucleotides on SMC phenotype and vascular remodeling after vascular injury is unknown. The present study was designed to investigate the effect of antisense oligonucleotides targeting c-myc proto-oncogene, when delivered locally using a porous balloon catheter, on neointimal thickening and vascular remodeling after angioplasty and to explore the therapeutic potential of antisense c-myc oligonucleotides for prevention of restenosis.
, 百拇医药
Methods: The celiac arterial endothelium of 75 male Japanese White Rabbits (2～3 kg) were denuded after being fed with cholesterol-supplemented diet [1.5 g/(kg*d)] for 4 weeks, and angioplasty to atherosclerotic stenosis of celiac arteries was performed after 4 additional weeks of high-cholesterol diet. Animals with significantly atherosclerotic stenosis of celiac arteries demonstrated by angiography were randomized to the antisense group (group Ⅰ) receiving c-myc antisense oligonucleotides (1 mg per vessel, total volume 3 ml), sense group (group Ⅱ) receiving c-myc sense oligonucleotides (1 mg per vessel, total volume 3 ml), saline control group (group Ⅲ) receiving saline 3 ml and single angioplasty only group (group Ⅳ). The drugs were intramurally injected using a porous balloon. At 24 hours after local delivery of drugs, 5 animals of each group were killed and vessels were harvested to assess the c-myc mRNA levels by reverse transcription polymerase chain reaction (RT-PCR). At 4 weeks after local delivery of drugs, the remaining animals were killed and celiac arteries were fixed in situ by perfusion with 10% buffered formalin at 120 mmHg perfusion pressure. Vessels, including the treated segment and proximal reference segment, were collected and cross sectioned for immunohistochemistric and morphometric analysis. Neointimal area (NIA), medial area (MA), ratio of NIA to MA (NIA/MA) were measured, and arterial remodeling was assessed. Also, ultrathin sections were prepared for transmission electron-microscopy (TEM) to observe the changes of intracellular ultrastructure.
, 百拇医药
Results: 34 rabbits were excluded due to death or insignificantly atherosclerotic stenosis created (<50% occlusion). The causes of death included diarrhea, infection, anesthetic accident, vessel rupture, hemorrhage during or after operation, or unknown reason. Morphormetric results showed that the ratio of neointimal area (NEA) to medial area (MA) (NEA/MA) in group Ⅰ (1.03±0.24) were significantly less than those of control segments in group Ⅱ (1.58±0.43, p<0.05), group Ⅲ (1.54±0.35, p<0.05) and group Ⅳ (1.52±0.61, p<0.05). The geometric remodeling index at angioplastied site in group Ⅰ [(32.7±14.2)%, p<0.05] was significantly larger than that in group Ⅱ [(24.0±8.9)%, p<0.05], group Ⅲ [(24.0±11.3)%, p<0.05] and group Ⅳ [(23.2±11.3)%,p<0.05]. RT-PCR results showed that c-myc mRNA level in group Ⅰ decreased comparing with group Ⅱ, group Ⅲ and group Ⅳ. Immunohistochemistric analysis also showed that the percentage of c-myc protein positive cells was less in group Ⅰ (4.5±1.1%) than that in group Ⅱ [(11.9±1.6)%, p<0.01], group Ⅲ [(12.7±1.9)%, p<0.01] and group Ⅳ [(9.8±0.8)%, p<0.01]. Furthermore, a slight difference in NEA/MA and the percentage of positive cells was observed when comparing group Ⅳ with group Ⅱ and group Ⅲ. TEM results showed that the organelles related with biosynthesis, ie. rough endoplasmic reticulum, free ribosome, Golgi complex, mitochondria, glycogen granules were decreased, but myofilaments were more abundant in group Ⅰ than those in group Ⅱ, group Ⅲ and group Ⅳ.
, 百拇医药
Conclusion: ① Transcatheter delivery of c-myc antisense oligonucleotides to the site of balloon angioplasty reduced the neointimal hyperplasia after vascular injury. ② The mechanism of c-myc antisense oligonucleotides inhibiting neointimal thickening involved the inhibition of transcripation of c-myc mRNA and expression of c-myc protein. ③ Transcatheter delivery technique with porous balloon increased extra trauma to arterial wall, but did not affect the inhibitory effect of c-myc antisense oligonucleotides on neointimal formation. ④ Transcatheter delivery of c-myc antisense oligonucleotides suppressed the conversion of SMC from contractile to synthetic phenotype. ⑤ Transcatheter delivery of c-myc antisense oligonucleotides inhibited the unfavourable vascular remodeling., 百拇医药