冠心Ⅱ号方成分配伍对心肌梗塞大鼠主要基因表达的影响
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摘要
心肌缺血是临床上最常见的心肌损伤原因,多由于冠状动脉粥样硬化所引起。当心肌供血受限或心肌对氧和营养物质需求过盛,都能造成心肌中氧和营养物质的缺乏;临床上常见为冠状动脉病变引起心肌供血不足,造成心肌严重功能障碍,甚至坏死。西医在治疗上主要采取降低心肌耗氧量和增加缺血区的血液供应,尽管可供选择的药物较为广泛,然而治疗效果并不理想,而且存在一定的副作用,长期服用,病人多难以接受。随着活血化瘀类中药在防治此类疾病良好疗效的显现,并且较小的副作用,充分显示其良好的应用前景。冠心Ⅱ号方就是此类药物的代表,它是20世纪70年代研发的中药复方,由丹参、川芎、红花、赤芍、降香5味中药组成,药味数目适当,组方严谨,疗效确切,其针对的胸痹心痛证与现代医学相通之处较多,且其单味药药效、植物化学及其成分动力学研究积累较多,比较适合作为中药复方方剂配伍现代研究的选题。多年的临床及药理实验研究结果表明:冠心Ⅱ号具有调节血液循环功能,增加冠脉血流量及心肌营养血流量,改善血液流变性、血凝状态及血小板功能,预防血栓形成及动脉粥样硬化,对垂体后叶素和结扎冠状动脉诱发的心肌缺血有明显保护作用。本研究通过自主研制的心血管相关寡核苷酸芯片研究心肌梗塞模型基因表达的时序变化以及冠心Ⅱ号及其成分配伍对心血管相关基因表达的影响,从而为心肌梗塞的病理以及冠心Ⅱ号的药理的研究提供参考,建立起系统生物学技术与药理学相结合的方剂复杂组效关系与配伍规律的研究技术平台,为中药方剂配伍理论提供新思路、新方法。
通过心脏特异基因的筛选及心血管疾病相关基因的选择,我们确定了1285个受试基因,设计并合成特异性的寡核苷酸后点制到氨基修饰的玻璃基片上,制成心血管相关寡核苷酸芯片。经过质量检测,各点浓度、大小均一,形状规则,样品自杂交显示相关性较高,因此适合后续的研究。选用结扎左冠脉前降支致心肌梗塞的大鼠模型研究心肌梗死后1天、3天、7天、15天以及30天大鼠心血管相关基因的改变,发现相对于假手术组,结扎后第3天基因变化最为剧烈;1天、3天和7天基因变化趋于一致,而与后面两个时间点的基因变化存在较大差异,而前期药理学检测表明,结扎后期心室壁明显变薄,而且存在大面积的疤痕,说明在疤痕形成期的基因变化较疤痕稳定期剧烈,而且差异表达的基因共性较少。心肌梗塞模型中与脂肪酸代谢密切相关的PPAR代谢通路中的各基因在几个受试时间点均受到了不同程度的抑制,而参与脂肪酸代谢通路中的各基因在结扎后一天表现出强烈抑制,而3天后上调的基因比例有所升高,这有可能是大鼠自身的调节引起的。心脏特异表达的基因在模型组各个时间点中的变化表明,在第30天,特异表达基因大部分下调,说明在结扎后30天,大鼠的心脏功能受到了明显的抑制,这与前期的药理学检测结果一致。给予冠心Ⅱ号及其各成分以后,基因的变化呈现出与模型相反的表达趋势,说明我们的药物对心血管相关基因具有保护作用。梗死早期发生变化的基因主要是由药物刺激引起的,而梗死中期(7天)大部分基因的表达通过药物干预后恢复到正常水平,但是到了梗死后期(30天),单独由药物作用引起变化的基因比例又大大增加,因此可以认为梗死早期为药物治疗期,梗死中期为药物起效阶段,而在梗死后期,可能就是一个恢复期或者说巩固期。汤剂组的基因表达变化在前面的时间点(1d和7d)与血分药中药配伍组(红花、丹参、赤芍)的基因变化比较相近,而在第30天的时候与降香的基因变化相近,因此我们认为,在梗死的早期,在汤剂中发挥主要作用的是血分药中药配伍的成分,可能对疤痕的形成具有一定的抑制作用,而到了梗死后期,降香作为理气为主的药在汤剂中发挥了更大的作用,因此气分药(川芎、降香)可能对疤痕的稳定具有一定的效果。冠心Ⅱ号及其各组分均能使间隙连接蛋白(Cx32)的表达在一定程度上恢复到正常水平,提示冠心Ⅱ号可能通过调控Cx32的表达,进而调控细胞周期使得细胞增殖。前期的药理学检测结果表明,冠心Ⅱ号能够促进新生血管的形成,但是在本研究中,人们广泛关注的与血管新生相关的基因(如VEGF、FGF等)在我们的给药组中并为得到很好的效果,这说明冠心Ⅱ号促进血管新生可能存在另外的途径,这有待于进一步的研究。
Cardiac infarction is one of the main causes of death in both developing and developed countries over past decades.Currently available approaches for treating patients with this disease are not satisfied.Traditional Chinese medicines(TCMs) have been increasingly paid attention to.Guanxin No.2 decoction(GXⅡ)is a well researched decoction,and is widely used to treat with the cornary artery disease (CAD)patients in China and other Asian countries clinically.But the pathogenesis of CAD is still blurry,and the mechanism of the decoction is also not clear.The main purpose of this research is to detect the time related changes of the expression of cardiac genes,the mechanism of the protection of GXⅡon the disease,and the effect of the single component of GXⅡ.
A rat cardiovascular related oligonucleotide microarray was constructed.The genes waere selected from the literatures in which they were reported as candidate genes, and business cardiovascular microarrays,and some related databases.Furthermore, the most important part of this gene set was a number of heart specific genes which was detected by chi-square test from the UniGene database.The microarray has been tested by self-self RNA hybridization.Data shows the correlation was satisfied,so our microarray suited for the research.
The microarray was used to examine the gene expression pattern of the rat myocardial infarction(MI)model,which was carried out by ligating the proximal left anterior descending coronary artery.More differentially expressed(DE)genes were found after 3 days of the ligation,and the DE genes were clustered together in shorter time points,i.e.1 day,3 days,and 7 days after the experiment,and they were more different from day 15,and day 30.Former pharmacological experiments showed a plaque was formed after the ligation and expanded to a larger part with time goes by, so the genes which participated in the plaque forming and the genes which expressed after the plaque formed were quite different.The DE genes that expressed all through the time have been researched.Among these genes,a number of them which participated in the PPAR signaling pathway were inhibited,and genes which participated in fatty acid metabolism were significantly down regulated at the 1~(st)day after the ligation.So during the myocardial infarction,the fatty acid metabolism was significantly inhibited.The analysis of heart specific genes in the MI model revealed that at the 30~(th)day after infarction,most of the genes were down regulated,which implicated that the function of the heart was extremely destroyed.This was similar to the pharmacological results we carried out earlier.
After the administration of GXⅡand its components,most of the cardiac genes showed an opposite expression compared to the models,which implied the protection effects of the decoction and its components.
According to our classification of the genes,their early changes were response to the medicine,and in the process of time,more and more genes have been regulated to their normal level,but at the 30~(th)day after infarction,the genes were regulated solely by the medicine again.We then classified the phases of the administration of decoction to three parts:the early phase was the therapeutic time,and the next was the recovery phase,and the consolidated phase.The gene expression of the GXⅡgroup was similar to the Xue group at the 1~(st)day and the 7~(th)day,but it was closer to Jiangxiang,which belonged to Qi group according to the classification of Chinese traditional doctors.So we inferred that during the plaque formation phase,the Xue group of the decoction revealed a strong therapeutic effect.But after the formation, the Qi group showed a regulated effect.
The decoction and its components could regulate the expression of Cx32 to a normal expression,which implied that the mechanisms of the proliferation effect of the decoction may be regulating the expression of Cx32 and then inhibiting the elongation of the cell cycle,and increase the proliferation.
Formerly pharmacological experiments showed the GXⅡhad an effect of angiogenesis,but the known genes in which participated the process,such as VEGF and FGF,were not increased in our dataset.This implies that the angiogenesis initiated by GXⅡand its components might not be induced by the up regulation of VEGF and FGF,other mechanisms should be considered.
通过心脏特异基因的筛选及心血管疾病相关基因的选择,我们确定了1285个受试基因,设计并合成特异性的寡核苷酸后点制到氨基修饰的玻璃基片上,制成心血管相关寡核苷酸芯片。经过质量检测,各点浓度、大小均一,形状规则,样品自杂交显示相关性较高,因此适合后续的研究。选用结扎左冠脉前降支致心肌梗塞的大鼠模型研究心肌梗死后1天、3天、7天、15天以及30天大鼠心血管相关基因的改变,发现相对于假手术组,结扎后第3天基因变化最为剧烈;1天、3天和7天基因变化趋于一致,而与后面两个时间点的基因变化存在较大差异,而前期药理学检测表明,结扎后期心室壁明显变薄,而且存在大面积的疤痕,说明在疤痕形成期的基因变化较疤痕稳定期剧烈,而且差异表达的基因共性较少。心肌梗塞模型中与脂肪酸代谢密切相关的PPAR代谢通路中的各基因在几个受试时间点均受到了不同程度的抑制,而参与脂肪酸代谢通路中的各基因在结扎后一天表现出强烈抑制,而3天后上调的基因比例有所升高,这有可能是大鼠自身的调节引起的。心脏特异表达的基因在模型组各个时间点中的变化表明,在第30天,特异表达基因大部分下调,说明在结扎后30天,大鼠的心脏功能受到了明显的抑制,这与前期的药理学检测结果一致。给予冠心Ⅱ号及其各成分以后,基因的变化呈现出与模型相反的表达趋势,说明我们的药物对心血管相关基因具有保护作用。梗死早期发生变化的基因主要是由药物刺激引起的,而梗死中期(7天)大部分基因的表达通过药物干预后恢复到正常水平,但是到了梗死后期(30天),单独由药物作用引起变化的基因比例又大大增加,因此可以认为梗死早期为药物治疗期,梗死中期为药物起效阶段,而在梗死后期,可能就是一个恢复期或者说巩固期。汤剂组的基因表达变化在前面的时间点(1d和7d)与血分药中药配伍组(红花、丹参、赤芍)的基因变化比较相近,而在第30天的时候与降香的基因变化相近,因此我们认为,在梗死的早期,在汤剂中发挥主要作用的是血分药中药配伍的成分,可能对疤痕的形成具有一定的抑制作用,而到了梗死后期,降香作为理气为主的药在汤剂中发挥了更大的作用,因此气分药(川芎、降香)可能对疤痕的稳定具有一定的效果。冠心Ⅱ号及其各组分均能使间隙连接蛋白(Cx32)的表达在一定程度上恢复到正常水平,提示冠心Ⅱ号可能通过调控Cx32的表达,进而调控细胞周期使得细胞增殖。前期的药理学检测结果表明,冠心Ⅱ号能够促进新生血管的形成,但是在本研究中,人们广泛关注的与血管新生相关的基因(如VEGF、FGF等)在我们的给药组中并为得到很好的效果,这说明冠心Ⅱ号促进血管新生可能存在另外的途径,这有待于进一步的研究。
Cardiac infarction is one of the main causes of death in both developing and developed countries over past decades.Currently available approaches for treating patients with this disease are not satisfied.Traditional Chinese medicines(TCMs) have been increasingly paid attention to.Guanxin No.2 decoction(GXⅡ)is a well researched decoction,and is widely used to treat with the cornary artery disease (CAD)patients in China and other Asian countries clinically.But the pathogenesis of CAD is still blurry,and the mechanism of the decoction is also not clear.The main purpose of this research is to detect the time related changes of the expression of cardiac genes,the mechanism of the protection of GXⅡon the disease,and the effect of the single component of GXⅡ.
A rat cardiovascular related oligonucleotide microarray was constructed.The genes waere selected from the literatures in which they were reported as candidate genes, and business cardiovascular microarrays,and some related databases.Furthermore, the most important part of this gene set was a number of heart specific genes which was detected by chi-square test from the UniGene database.The microarray has been tested by self-self RNA hybridization.Data shows the correlation was satisfied,so our microarray suited for the research.
The microarray was used to examine the gene expression pattern of the rat myocardial infarction(MI)model,which was carried out by ligating the proximal left anterior descending coronary artery.More differentially expressed(DE)genes were found after 3 days of the ligation,and the DE genes were clustered together in shorter time points,i.e.1 day,3 days,and 7 days after the experiment,and they were more different from day 15,and day 30.Former pharmacological experiments showed a plaque was formed after the ligation and expanded to a larger part with time goes by, so the genes which participated in the plaque forming and the genes which expressed after the plaque formed were quite different.The DE genes that expressed all through the time have been researched.Among these genes,a number of them which participated in the PPAR signaling pathway were inhibited,and genes which participated in fatty acid metabolism were significantly down regulated at the 1~(st)day after the ligation.So during the myocardial infarction,the fatty acid metabolism was significantly inhibited.The analysis of heart specific genes in the MI model revealed that at the 30~(th)day after infarction,most of the genes were down regulated,which implicated that the function of the heart was extremely destroyed.This was similar to the pharmacological results we carried out earlier.
After the administration of GXⅡand its components,most of the cardiac genes showed an opposite expression compared to the models,which implied the protection effects of the decoction and its components.
According to our classification of the genes,their early changes were response to the medicine,and in the process of time,more and more genes have been regulated to their normal level,but at the 30~(th)day after infarction,the genes were regulated solely by the medicine again.We then classified the phases of the administration of decoction to three parts:the early phase was the therapeutic time,and the next was the recovery phase,and the consolidated phase.The gene expression of the GXⅡgroup was similar to the Xue group at the 1~(st)day and the 7~(th)day,but it was closer to Jiangxiang,which belonged to Qi group according to the classification of Chinese traditional doctors.So we inferred that during the plaque formation phase,the Xue group of the decoction revealed a strong therapeutic effect.But after the formation, the Qi group showed a regulated effect.
The decoction and its components could regulate the expression of Cx32 to a normal expression,which implied that the mechanisms of the proliferation effect of the decoction may be regulating the expression of Cx32 and then inhibiting the elongation of the cell cycle,and increase the proliferation.
Formerly pharmacological experiments showed the GXⅡhad an effect of angiogenesis,but the known genes in which participated the process,such as VEGF and FGF,were not increased in our dataset.This implies that the angiogenesis initiated by GXⅡand its components might not be induced by the up regulation of VEGF and FGF,other mechanisms should be considered.
引文
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60.吴晓霞,智光,吴加金.反武作用因子中血清反应因子变化与心力衰竭关系的研究.中华心血管病杂志,2001.29:51-3.
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65.Giles,EJ.The vascular endothelial growth factor(VEGF)signaling pathway:a therapeutic target in patients with hematologic malignancies.Oncologist,2001.6 Suppl 5:32-9.
66.Stein,I.,A.Itin,et al.Translation of vascular endothelial growth factor mRNA by internal ribosome entry:implications for translation under hypoxia.Mol Cell Biol,1998.18(6):3112-9.
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72.Peichev,M.,A.J.Naiyer,et al.Expression of VEGFR-2 and AC133 by circulating human CD34(+)cells identifies a population of functional endothelial precursors.Blood,2000.95(3):952-8.
73.Casscells,W.Growth factor therapies for vascular injury and ischemia.Circulation,1995.91(11):2699-702.
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75.Feucht,M.,B.Christ,and J.Wilting.VEGF induces cardiovascular malformation and embryonic lethality.Am J Pathol,1997.151(5):1407-16.
76.Zhou,M.,R.L.Sutliff,et al.Fibroblast growth factor 2 control of vascular tone.Nat Med,1998.4(2):201-7.
77.Hollon,T.Researchers and regulators reflect on first gene therapy death.Nat Med,2000.6(1):6.