C-sis反义寡脱氧核苷酸抑制兔主动脉平滑肌细胞增殖的时效关系

为观察局部处理C-sis反义寡脱氧核苷酸对平滑肌细胞增殖及新生内膜肥厚的抑制作用、球囊导管损伤60只新西兰白兔胸主动脉后。经导管局部处理每只兔1mg（2mL）C-sis反义或正义寡脱氧核苷酸或2mL生理盐水,于损伤后第3、7、14天和第28天，用免疫组织化学方法结合图象分析，评价损伤动脉内膜平滑肌细胞增殖及新生内膜改变。结果发现．C-sis反义寡脱氧核苷酸明显抑制内膜平滑肌细胞增殖，最高抑制作用出现于损伤后第3天；抑制百分率达69．8％，第7、14天和第28天的抑制率依次降低，分别为59％、51.9％和47.8％；而新生内膜面积的抑制百分率分别是48．9％、77．8％、72．6％和70．8％，与生理盐水处理组相比，平滑肌细胞数及新生内膜面积均有明显降低。研究结果表明，局部处理C-sis反义寡脱氧核苷酸，通过反义途径，可明显抑制球囊损伤主动脉内膜平滑肌细胞增殖，其抑制作用随时间而降低，同时，对新生内膜面积肥厚的最大抑制出现于第7天，随后降低，提示局部处理C-sis反义寡脱氧核苷醚，有可能降低再狭窄的发生。     

Infusa Sleeve导管血管壁内局部导入基因预防血管成形术后再狭窄的实验研究(摘要)

我们于１９９７年７月～１２月进行了用Ｉｎｆｕｓａ Ｓｌｅｅｖｅ（ＩＳ）导管导入ｃ－ｍｙｃ反义寡核着酸预防再狭窄的实验研究。探讨ＩＳ导管局部在体导入ｃ－ｍｙｃ反义寡核着酸的可行性及其对血管平滑肌细胞（ＶＳＭＣｓ）和新生内膜的抑制作用。１材料和方法 ｃ－ｍｙｃ寡核苷酸（加拿大ＳＡＮＧＯＮ上海分公司合成）：用硫代磷酸盐修饰,纯度９９％。ｃ－ｍｙｃ正义核昔酸：５’ＡＴＧＣ－ＣＣＣＴＣＡＡＣＧＴＴ。ｃ－ｍｙｃ反义核苷酸：３ＴＡＣＧＧＧＧＡＧＴＴＧＣＡＡ。 家兔２１只,体重２．５～３．５ ｋｇ,雌雄各半。其中反…     

C—sis反义寡核苷酸局干预抑制主动脉损伤后内膜增生

研究主动脉球囊损伤后,Ｃ－ｓｉｓ原癌基因反义寡核酸局部干预对血管内膜增生的影响。方法３６只新西兰白兔随机分为Ｃ－ｓｉｓ原癌基因ＡＯＤＮ组、正义寡核苷酸（ＳＯＤＮ）组及生理盐水组,每组１２只。球囊损伤胸主动脉后,１ｍｇＡＯＤＮ、ＳＯＤＮ用于损伤局部干预,高胆固醇饲养,用ＲＴ－ＰＣＲ技术及这方法结合图像分析,观察ＡＯＤＮ对损伤血管Ｃ－ｓｉｓ表达及内膜增生的影响。结果ＡＯＤＮ明显抑制损伤管Ｃ－ｓｉｓ表达     

C－sis、C－myc癌基因反义寡脱氧核苷酸抑制动脉平滑肌细胞增殖

用合成Ｃ－ｓｉｓ、Ｃ－ｍｙｃ癌基因反义寡脱氧核苷酸（ＡＯＤＮ）与兔动脉平滑肌细胞（ＡＳＭＣｓ）共同培养，旨在通过ＡＯＤＮ对癌基因的封闭，动态观察ＡＯＤＮ对ＡＳＭＣｓ增殖的影响，探讨ＡＯＤＮ对动脉粥样硬化（ＡＳ）尤其是冠脉成形术后再狭窄（ＲＡＣＡ）的防治作用。结果表明：①Ｃ－ｓｉｓＡＯＤＮ、Ｃ－ｍｙｃＡＯＤＮ具有明显抑制ＡＳＭＣｓ增殖作用，其抑制作用随浓度增加而加强；②Ｃ－ｓｉｓＡＯＤＮ＋Ｃ－ｍｙｃＡＯＤＮ具有协同抑制ＡＳＭＣｓ增殖作用；③Ｃ－ｓｉｓＡＯＤＮ、Ｃ－ｍｙｃＡＯＤＮ明显抑制ＤＮＡ合成，二者联合应用，具有加强抑制ＤＮＡ合成作用。研究结果提示：Ｃ－ｓｉｓ、Ｃ－ｍｙｃ癌基因ＡＯＤＮ抑制ＡＳＭＣｓ增殖，尤其是二者联合应用，有可能为ＡＳ，特别是ＲＡＣＡ防治提供一个新的途径。     

碱性成纤维细胞生长因子反义寡脱氧核苷酸抑制胰岛素诱导动脉平滑肌细胞增殖

为研究胰岛素诱导的动脉平滑肌细胞增殖作用和碱性成纤维细胞生长因子反义寡脱氧核苷对培养的Ｗｉｓｔａｒ大鼠主动脉平滑肌细胞生长的影响。采用胰岛素和碱性成纤维细胞生长因子反义寡脱氧核苷酸处理培养的Ｗｉｓｔａｒ大鼠主动脉平滑肌细胞,Ｎｏｒｔｈｅｎｂｌｏｔ检测碱性成纤维细胞生长因子基因ｍＲＮＡ表达,并测定氚标胸腺嘧啶脱氧核苷掺入和细胞计数。结果发现胰岛素能明显诱导平滑肌细胞碱性成纤维细胞生长因子ｍＲＮＡ表达和增殖,且呈浓度依赖性。胰岛素浓度由０．００１ｍｇ／Ｌ增加到１．０ｍｇ／Ｌ,平滑肌细胞增殖率由１０％增加到５３％。碱性成纤维细胞生长因子反义寡脱氧核苷酸（５μｍｏｌ／Ｌ）能明显抑制胰岛素诱导的平滑肌细胞碱性成纤维细胞生长因子ｍＲＮＡ表达和增殖,氚标胸腺嘧啶脱氧核苷掺入被抑制４１．１％（与对照组比较,Ｐ＜０．０１）,细胞计数被抑制３０．２％（与对照组比较,Ｐ＜０．０１）。提示胰岛素可通过诱导平滑肌细胞碱性成纤维细胞生长因子的表达而促进动脉平滑肌细胞增殖,碱性成纤维细胞生长因子反义寡脱氧核苷酸能有效抑制胰岛素诱导的平滑肌细胞的ＤＮＡ合成及其增殖。     

碱性成纤维细胞生长因子反义寡脱氧核苷酸抑制胰岛素诱导动 …

为研究胰岛素诱导的动脉平滑肌细胞增殖作用和碱性成纤维细胞生长因子反义寡脱氧核苷对培养的Ｗｉｓｔａｒ大鼠主动脉平滑肌细胞生长的影响。采用胰岛素和碱性成纤维细胞生长因子反义寡脱氧核苷酸处理培养的Ｗｉｓｔａｒ大鼠主动脉平滑肌细胞,Ｎｏｒｔｈｅｎｂｌｏｔ检测碱性成纤维细胞生长因子基因ｍＲＮＡ表达,并测定氚标胸腺嘧啶脱氧核苷掺入和细胞计数。结果发现胰岛素能明显诱导平滑肌细胞碱性成纤维细胞生长因子ｍＲＮＡ表     

抗基因及反义寡脱氧核苷酸抑制N—ras表达及对肝癌细胞增殖的影响

目的 探讨抗基因及反义寡核苷酸对肝癌的潜在治疗作用。方法 合成了针对Ｎ－ｒａｓ转录，翻译起始区的１２ｍｅｒ硫代磷酸抗基因寡脱氧核苷酸（Ｓ－ＯＤＮａｎ），１９ｍｅｒ硫代磷酸反义寡脱氧核苷酸（Ｓ－ＯＤＮＡａｓ）采用ＥＬＩＳＡ，斑点杂交法分别检测寡脱氧核苷酸处理的肝癌ＢＥＬ７４０２细胞内ｐ２１，Ｎ－ｒａｓｍＲＮＡ水平，观察了ＢＥＬ７４０２肝癌细胞生长的影响。结果 处理细胞内Ｎ－ｒａｓｍＲＮＡ水平低于对照     

抗基因及反义寡脱氧核苷酸抑制N－ras表达及对肝癌细胞增殖的影响

探讨抗基因及反义寡核苷酸对肝癌的潜在治疗作用。方法合成了针对Ｎ－ｒａｓ转录、翻译起始区的１２ｍｅｒ硫代磷酸抗基因寡脱氧核苷酸（Ｓ－ＯＤＨａｎ）、１９ｍｅｒ硫代磷酸反义寡脱氧核苷酸（Ｓ－ＯＤＮＡａｓ）。采用ＥＬＩＳＡ、斑点杂交法分别检测了寡脱氧核苷酸处理的肝癌ＢＥＬ７４０２细胞内ｐ２１、Ｎ－ｒａｓｍＲＮＡ水平，观察了对ＢＥＬ７４０２肝癌细胞生长的影响。结果处理细胞内Ｎ－ｒａｓｍＲＮＡ水平低于对照组，以Ｓ－ＯＤＮａｎ组更明显。两者对ｐ２１合成的抑制率达８０％，６７．５％。两者的细胞生长抑制率达８８％、７７％，并呈剂量依赖性。两者处理的细胞３Ｈ－ＴｄＲ掺入降低为对照组的２１．８％、３０．５５％；ＡＦＰ水平明显低于对照组（Ｐ直＜０．００１）。结论Ｓ－ＯＤＮａｎ．Ｓ－ＤＤＮａｓ能有效抑制Ｎ－ｒａｓ表达及相关肝癌细胞增殖；也进一步说明Ｎ－ｒａｓ在肝癌发生中起作重要作用。     

反义寡核苷酸对培养内皮细胞c—sis表达的抑制作用

用合成的与人ｃ ｓｉｓ ｍＲＮＡ起始码有其后五个密码子互补的硫代型寡聚核苷酸处理培养的人脐带静脉内皮细胞，观察其对细胞ｃ－ｓｉｓ基因表达的抑制作用。实验结果，ｃ－ｓｉｓ反义寡核苷酸可抑制内皮细胞ｃ－ｓｉｓ ｍＲＮＡ量（Ｐ〈０．０１）和其表达产物血反衍化生长因子量，以７２小时作用最强。而用正义寡核苷酸片段及空白做对照。未出现上述结果。实验提示，ｃ－ｓｉｓ反义寡核苷酸可特异地抑制人内皮细胞ｃ－ｓｉｓ基     

反义寡脱氧核苷酸对丙型肝炎病毒基因表达的抑制作用

为了解丙型肝炎病毒（ＨＣＶ）基因调控方式、探索反义寡脱氧核苷酸对丙型肝炎病毒基因表达的体外抑制作用。构建了由ＨＣＶ５′非翻译区（５′ＵＴＲ）翻译启动报道基因－虫荧光素酶（ｌｕｃ）基因的真核表达载体；人工合成了针对ＨＣＶ５′ＵＴＲ及非结构蛋白５（ＮＳ５）区的反义寡脱氧核苷酸（ＯＤＮ）。借助脂质体将ＯＤＮ分别与所构建的载体一同转染人癌细胞系，短期培养后制备细胞提取液，以荧光发光法检测ｌｕｃ基因的表达．     

大鼠胸主动脉球囊损伤对细胞凋亡及凋亡相关基因Fas、Bcl-2表达的影响

目的探讨大鼠胸主动脉球囊损伤后细胞凋亡和凋亡相关基因表达的变化规律。方法将30只400~500 g的雄性SD大鼠随机分为2组,手术组(n=24)行球囊扩张损伤大鼠胸主动脉术;对照组(n=6)不行球囊损伤,作为正常对照。分别于术后2、7、14、28 d取胸主动脉应用HE染色、TUNEL法、免疫组化和计算机图像分析仪进行形态学、细胞凋亡、增殖细胞核抗原(PCNA)、凋亡基因Fas;抗凋亡基因Bcl-2表达水平检测。结果对照组管壁处于非增殖状态;手术组球囊损伤后7 d形成新生内膜,血管平滑肌细胞(VSMC)增殖活跃;14 d内膜明显增厚,但VSMC增殖已减弱;28 d内膜继续缓慢增厚,管腔明显狭窄。动脉损伤后Fas表达和TUNEL法测定的凋亡规律一致,两周内凋亡较明显,但细胞凋亡高峰时间(中膜7 d、内膜14 d)迟于增殖高峰(中膜2 d、内膜7 d),两周后凋亡与增殖均明显下降。动脉损伤后抗凋亡基因Bcl-2表达下调,在中膜和内膜分别在7 d1、4 d达最低水平,后回升,与凋亡基因Fas表达呈明显负相关(r=-0.878,P<0.001)。结论动脉球囊损伤后,平滑肌细胞的凋亡呈现规律性变化,可能在管腔狭窄的病理过程中具有重要作用。     

Antisense Bcl-x oligonucleotide induces apoptosis and prevents arterial neointimal formation in murine cardiac allografts

OBJECTIVE: Cardiac allograft arteriosclerosis, which limits long-term survival of recipients, cannot be prevented by conservative therapies. The arteriopathy is characterized by diffuse intimal thickening comprised of proliferative smooth muscle cells (SMCs). Cell death is a prominent feature of atherosclerosis; Bcl-x is one of the anti-apoptotic mediators. METHODS: To test the hypothesis that antisense bcl-x oligodeoxynucleotide (ODN) is effective in preventing intimal hyperplasia through enhancing apoptosis after cardiac transplantation, we performed single intraluminal delivery of antisense bcl-x ODN into murine cardiac allografts (n = 9). DBA/2 (H-2d) hearts were transplanted into B10.D2 (H-2d) mice. Sense bcl-x ODN (n = 8) and no treatment (n = 8) studies were also performed. RESULTS: Allografts were harvested at 4 weeks after transplantation; all allografts kept beating throughout the period. Coronary intimal thickening had developed in nontreated and sense ODN transfected allografts at 4 weeks after transplantation with enhanced expression of Bcl-x and cell adhesion molecules, and suppressed apoptosis. However, antisense bcl-x ODN prevented neointimal formation through enhanced apoptosis. CONCLUSION: These results indicate that apoptosis of vascular SMCs induced by Bcl-x is associated with initial hyperplasia after heart transplantation. Antisense bcl-x ODN inhibits SMC proliferation by inducing apoptosis in graft coronary arteries.     

球囊损伤大鼠主动脉内皮后血小板活化及凝血酶受体表达的变化

目的 探讨经皮冠状动脉腔内成形术后再狭窄的发生机制。方法建立大鼠主动脉内皮球囊损伤模型，分别于术后3天、7天、14天和28天，通过组织学检查、放射免疫法和逆转录一聚合酶链反应技术检测主动脉球囊损伤后内膜增生的情况、血小板表面GMP-140数目和凝血酶受体mRNA表达的变化。结果凝血酶受体mRNA在正常血管组织的表达较弱，球囊损伤术后第3天已显著增加，术后第14天达峰值，术后第28天开始下降。GMP-140于术后第3天明显升高，术后第7天开始下降。内皮损伤术后第3天已有增殖的血管平滑肌细胞移行至内膜层；术后第7天内膜开始增生；术后第14天血管平滑肌细胞的增殖及内膜增生更为明显；术后第28天血管平滑肌细胞的增殖明显减弱，细胞外基质增加，内膜继续增生。结论血管内皮损伤内膜增生的过程中血小板活化．凝血酶受体mRNA表达增加。     

罗格列酮对大鼠胸主动脉球囊损伤后内皮再生和内膜增生的影响

目的探讨罗格列酮(rosiglitazone,RSG)对血管损伤后内皮再生和内膜增生的影响。方法制备大鼠胸主动脉球囊损伤模型,将SD大鼠随机分为RSG组、对照组和假手术组,于术后第7天和第14天处死动物。分别进行伊文思蓝染色观察内皮覆盖情况,细胞核增殖抗原(PCNA)免疫组化染色和组织形态学定量分析,并测量损伤后14 d时各组血清一氧化氮(NO)含量。结果RSG 7 d组和14 d组的再生内皮覆盖率分别为38.2%和75.2%,均较对照组(32.4%和60.4%)显著增加(P<0.05和P<0.01),且RSG 14 d组血清中的NO含量较对照组升高。球囊损伤后7 d内膜开始有少量增生,14 d时形成明显的新生内膜。RSG使损伤后14 d形成的新生内膜显著减少,内膜面积与中膜面积的比值(IA/MA)较对照组降低60.9%。与对照组比较,RSG 7 d组和14 d组新生内膜内的PCNA阳性表达指数均显著减少。结论RSG可以促进大鼠胸主动脉球囊损伤处的内皮再生,并减少新生内膜的形成。     

Transient down-regulation of L-type Ca(2+) channel and dystrophin expression after balloon injury in rat aortic cells

OBJECTIVE: Migration and proliferation of arterial smooth muscle cells are critical responses during restenosis after balloon angioplasty. We investigated the changes in the expression of Ca(2+) channels and dystrophin, two determinants of contraction, after balloon injury of rat aortas. METHODS: Proliferation and migration of aortic myocytes were triggered in vivo by the passage of an inflated balloon catheter in the aortas of 12-week-old male Wistar rats. We used the whole-cell patch clamp technique to investigate Ba(2+) currents (I(Ba)) through Ca(2+) channels in single cells freshly isolated from media and neointima at various times after injury (days 2, 7, 15, 30 and 45). RESULTS: No T-type Ca(2+) channel current was recorded in any cell at any time. In contrast, a dihydropyridine (DHP)-sensitive L-type I(Ba)was recorded consistently in the media of intact aorta. After aortic injury, I(Ba) decreased dramatically (at days 2 and 7) but recovered over time to reach normal amplitude on days 30 and 45. In the neointima, I(Ba) was absent on day 15 but also increased gradually over time as observed at days 30 and 45. The use of a specific antibody directed against the L-type Ca(2+) channel alpha(1C) subunit showed, both by immunostaining and by Western blotting, no expression of the Ca(2+) channel protein on day 15. Parallel immunodetection of dystrophin showed that this marker of the contractile phenotype of SMCs was also not detectable at this stage in neointimal cells. Both proteins were re-expressed at days 45 and 63. Balloon injury induces a transient down-regulation of I(Ba) in arterial cells. CONCLUSIONS: Cell dedifferentiation and proliferation in vivo abolish the expression of L-type Ca(2+) channels and dystrophin in neointimal cells. These changes may be critical in the regulation of Ca(2+) homeostasis and, thereby, contraction of the arterial SMCs during restenosis following angioplasty.     

Selective Tyrosine Kinase Inhibitor for the Platelet-Derived Growth Factor Receptor In Vitro Inhibits Smooth Muscle Cell Proliferation After Reinjury of Arterial Intima In Vivo

Summary. The long-term success of coronary angioplasty is limited by restonosis. This study was undertaken to investigate whether and to what extent the enhanced proliferative response observed in a balloon reinjury model of rat aorta is regulated by the PDGF receptor (PDGF-R). Balloon injury was performed to 14-day-old pre-existing neointimal lesion in rat aorta. PDGF receptor and ligand immunoreactivity were measured at several time points after the first and second injury, and PDGF-R signaling was blocked with a selective inhibitor of PDGF-R tyrosine kinase. In the neointima, after repeated injury, upregulation of PDGF-AA was seen to coincide with a prompt proliferative response of smooth muscle cells (SMC). Administration of the PDGF-R tyrosine kinase inhibitor in vivo, tested and found to inhibit the proliferation of SMC induced by PDGF-AA and PDGF-BB, but not by IGF-1, EGF, or bFGF, resulted in a 60% reduction in the absolute number and percentage of BrdU+ cells after the second balloon injury to pre-existing neointima, but had no significant effect on proliferation after the first injury. Endpoint lesion are was reduced by 50% in the treated group at 14 days after the second injury. The results suggest that systemic administration of a tyrosine kinase inhibitor specific for the PDGF-R can be useful in the prevention of restenosis.     

卡维地洛对大鼠颈动脉球囊损伤后血管内膜增生的影响

目的探讨卡维地洛(CAR)对大鼠颈动脉球囊损伤后内膜增生的影响。方法雄性Wistar大鼠36只,随机分为假手术组、损伤组和CAR组,每组12只,后2组建立大鼠颈动脉球囊损伤模型。3组均于术后7、14天分别处死6只大鼠。观察颈动脉形态学变化,计算新生内膜面积,免疫组织化学检测增殖细胞核抗原(PCNA)阳性细胞的表达。结果假手术组无新生内膜发生。与假手术组比较,损伤组大鼠术后7天,新生内膜形成并增厚,14天内膜增厚更明显(P0.01)。与损伤组比较,CAR组术后14天,新生内膜面积减少44%(P0.01),管腔面积增加82%(P0.01),内膜PCNA表达显著降低(P0.01)。结论 CAR可有效抑制颈动脉球囊损伤后内膜增生。     

Neutrophil, not macrophage, infiltration precedes neointimal thickening in balloon-injured arteries

Macrophages are abundant after stent-induced arterial injury. Inhibition of macrophage recruitment blocks neointimal growth in this model. In contrast, after superficial injury from balloon endothelial denudation, macrophages are sparse. However, many anti-inflammatory therapies remain effective against neointimal growth after balloon injury. To investigate further the role of leukocytes after injury, 41 New Zealand White rabbits underwent iliac artery balloon denudation. In 18, subcutaneous pumps were placed to deliver intravenous heparin (0.3 mg/kg per hour). Arteries were harvested at 6 hours and at 3, 7, and 14 days. In 8 animals, either M1/70 (a monoclonal antibody [mAb] against adhesion molecule Mac-1) or a nonspecific IgG was given (5 mg/kg IV bolus and then 1 mg/kg SC QOD), and arteries were harvested at 6 hours and 3 days. Computer-aided morphometry was performed as was immunohistochemistry to assess smooth muscle cell (SMC) proliferation (bromodeoxyuridine-positive cells), neutrophil content (RPN357, mAb against rabbit neutrophil/thymocyte), and macrophage content (RAM-11, mAb against rabbit macrophage). Heparin inhibited neointimal growth at 7 and 14 days (64% and 32.5% reduction, respectively; P:<0.05). Neutrophils were observed in the media early after balloon injury, and heparin and M1/70 inhibited this infiltration (82% and 83% reduction, respectively; P:<0.05 each) with a coincident inhibition of medial SMC proliferation at 3 days (49% and 84% reduction, respectively; P:<0.05 each). Macrophages were absent at all time points. Neutrophil, but not macrophage, infiltration occurs early after endothelial denudation. Inhibition of this process is associated with a reduction in medial SMC proliferation. These data suggest a central role for neutrophils in restenosis and help to explain prior reports of an inhibitory effect of anti-inflammatory therapies on neointimal growth after balloon injury.