实验兔血管内膜损伤后不同阶段PDGF-B链、C-myc基因表达及中药对其拮抗作用机制研究

【作者】 昌艳艳；

【导师】 郑洪新；

【作者基本信息】 辽宁中医学院 ， 中医基础， 2003， 博士

【摘要】 目的：依据中医中药理论在继承的基础上，借鉴分子生物学方法，探讨实验性兔胸主动脉内膜损伤后，血管内膜增殖早期主要调控因子-血小板源生长因子(PDGF-B)及相关原癌基因C-myc的病理机制。通过芪麦通对PDGF-B以及C-myc拮抗作用，从不同层次阐明中药益气活血化瘀能在细胞和基因水平发挥干预作用，最终为达到在内膜损伤早期防治血管再狭窄提供新的实验依据。 方法：模拟PTCA术后再狭窄的模型，采用球囊导管剥脱术，造成实验兔胸主动脉内膜损伤，分别于内膜损伤后第1周、第2周、第4周处死，取兔胸主动脉组织，通过HE染色观察血管大体形态学变化；用原位杂交法观察测定PDGF-B及C-myc在血管损伤部位不同阶段的动态表达；采用逆转录PCR(RT-PCR)检测PDGF-BmRNA转录；Westem免疫印迹方法检测PDGF-B蛋白质表达水平。 结果：显微镜下观察组织形态学变化：HE染色可见损伤后造成内皮及部分内膜坏死脱落、内弹力板断裂、血栓形成。损伤后1周，内膜增厚并主要由细胞构成，大多为SMCa-actin阳性，偶见泡沫细胞、淋巴细胞、少许中性细胞。数周后靠近腔内的细胞仍保持增生肥大，但用药组损伤后一周内膜阳性细胞分布明显减少，中层可见少数散在的弱阳性细胞，数周后仍保持稳定的水平。 原位杂交镜下观察测定：PDGF-B链在血管损伤部位不同阶段的动态表达，以及中药对它们的影响。正常的血管内膜可见少量阳性细胞表达，损伤后1周内膜阳性细胞分布明显增多，中膜亦可见少量的散在的阳性细胞分布，4周后呈稳定的水平下降。但用药组损伤1周后，内膜阳性细胞分布明显减少，中层可见少数散在的弱阳性细胞，数周后仍保持稳定的水平。并且中药组与西药组比较阳性表达减弱更为明显。P＜0.001 逆转录(RT-PCR)方法检测：显示胸主动脉血管内膜损伤后、第一周对照组明显高于正常组、P＜0.001；第四周与前两周比逐渐恢复。结果显示胸主动脉内膜损伤后早期PDGF-BmRNA呈增高趋势。而中药芪麦通对胸主动脉血管内膜损伤早期PDGF-BmRNA转录有明显拮抗作用。 Westem免疫印迹方法检测：显示胸主动脉血管内膜损伤后、第一周对照组明显高于正常组、P＜0.001；第四周与前两周比蛋白表达逐渐减少。结果显示胸主动脉内膜损伤后早期PDGF-B蛋白呈增高趋势，而中药芪麦通对胸主动脉血管内膜损伤早期PDGF-B蛋白的表达有明显拮抗作用。 结论：在损伤早期被激活后血小板产生，血小板源生长因子PDGF，被趋势化了的内皮细胞、巨噬细胞，在损伤部位聚集，诱生PDGF－B在早期出现一个高峰，而这些生长因子通过自分泌和旁分泌途径启动血管平滑肌的增殖，这同中医的气虚血瘀状态与病理产物积聚有关。由于病理代谢产物刺激基因异常表达，诱导原癌基因表达并进而分泌某些细胞或体液因子，引起组织异常增生导致再狭窄的发生而在整个病变的发展过程中由于气虚血瘀贯穿本病始终。而芪麦通对其有明显的拮抗作用。说明益气活血化瘀药能在细胞和基因水平发挥干预作用。即使受创血管内皮细胞修复再生同时，芪麦通对其有明显的拮抗作用。又可以迅速抑制受创血管周围血小板的聚集及炎性渗出行血中之气功。气血同治，气阴双补而不滋腻恋邪，既可补虚固本亦可以活血化瘀，从药物组成配伍合用可体现气虚瘀互补功效。更多还原

【Abstract】 PurposerThe experiment was based on the Traditional Chinese Medicine theory and used the method of molecular biology.The purpose of this experiment is to study the mechanism of PDGF-B which is the major regulation and control factor during earlier period of proliferation of post-injuried chest thoracic aorta of the rabbit. Traditional Chinese Medicine QMT can prevent: and intervene in the role of PDGF-B both in cell and gene levels.This experiment also offer the evidence on QMThow to prevent and cure the blood vessel restosis after earlier period of tunica intima damaged.Method :Foley’s tube denudation was used to cause the chest thoracic aorta of the rabbit damaged.The animals were killed after tunica intima damaged lw.2w,4w ,then extracted the chest thoracic aorta constitution of the rabbit..The changes of blood vessel morphology were observed by HE staining. The expression of PDGF-B at different stage was assayed in situ hybridization.The transcription and protein expression of PDGF-B mRNA were detected by the methods of RT-PCR and western immunoblot.Result: The protein expression of PDGF-B was phancro-reinforcement after earlier period(lw-2w) of tunica intima damaged.(P<0.01)During 3w and 4w the expression of PDGF-B was reduced gradually.HE staining manifested that the mecrosis and abscissiou of endothelium and demi-endometium were caused by injurying.The inter-elasticity blade broke and thrombo formatted.Afer inuried one week,the endometrium thickened and consisted of cells.Most of them is SMCa-actin male.The foam cell,lymphocyte and a little reutrality can be seen occasionally.The cells near to the intraluminal still keep hyperplasia hypertrophy after several weeks.The spread of indometrium male cells were reduced clearly in one week of medication group.A little feeble male cells were seen in uveal tract and still keep nesistant level after several weeks. In situ hybridization.assay: PDGF-B can express at different stage. The expression of a litter male cells can be seen in the normal intima. The spread of the male cells in intima were increased clearly after one week of injury .A little feeble male cells were seen in uveal tract and still keep nesistant level after several weeks. The expression of the TCM group were more weakened than western group.RT-PCR manifest that the expression of the control group was more than normal group at first week after chest thoracic aorta injuried. Compared the 4l week with the first two weeks, the expression was recovered gradually. It indicated MRNA had the growing tendency during the earlier period of tunica intima damaged. While QMTcould prevent the transcription of MRAN in earlier period of chest tunica intima damaged.Western immunoblot assay: The expression of PDGF-B in the control group was more than normal group at first week after chest thoracic aorta injuried. Compared the 4th week with the first two weeks, the expression was reduced clearly.Conclusion: PDGF-B is the major regulation an control factor during the earlier period of proliferation of tunica intima. QMT can prevent the rote of PDGF-B. we can deduce that the blood platelet is activated during the earlier period of injury. PDGF-B is collected in the injuried locus byendothelial cell and histocyte to induce the PDGF-B to produce a pinnacle at earlier period. These grouth factors start the angio-blood muscle proliferation through autocrine and paracrine, These are related to the blood stagnant of TCM. Metabolite simulates gene abnomal expression and induces protooncogene expression, then excrete some cells and body fluide and cause constition abnormally hyperplasia and restenosis. Reinforcing QI actirating the blood and eliminating the stagnant QMTcan have intervene role both in cell and gene level. It can pierosis and regenerate the damaged endotheliurn, at the same time it can prevent the platelet around and inflammation bleeding damaged vessel collecting. QMT can be simultaneous treatment of QI and blood, and reinforcing. Both Qi and Yin. It can not only reinforcing deficiency and tre更多还原