【作者】 徐冰；

【导师】 陈立新；

【作者基本信息】 北京中医药大学 ， 中西医结合临床， 2012， 博士

【摘要】 目的以统计学和数据挖掘方法,从冠心病不稳定性心绞痛患者临床信息中归纳证候类型和证候要素,并研究其与血清中粘附分子P-选择素,VCAM-1浓度变化的关系,从无监督的机器分析数据方法中,获得较客观的更深层次隐含关系。通过对人心脏微血管内皮细胞(HCMEC)进行缺糖缺氧和复糖复氧造模,模拟心绞痛发作时缺血对血管内皮的损伤机制,研究黄芪多糖对内皮损伤时粘附分子(P-选择素,VCAM-1)表达的干预作用和对P38和NF-Kb通路的信号转导机制。方法1.运用Kohonen网络聚类方法,对265例不稳定性心绞痛(UAP)患者的临床症状聚类分析,获得不稳定性心绞痛(UAP)的临床常见证候分型；运用因子分析的方法对UAP的常见证候要素进行归纳总结；运用QUEST决策树算法,探索证候要素和粘附分子P-选择素,VCAM-1的内在联系。2.通过免疫组化、免疫蛋白印迹和荧光实时定量PCR的方法,模拟冠心病心绞痛缺血发作时的病理机制,对HCMEC进行缺糖缺氧和复糖复氧处理,观察黄芪多糖干预缺血再灌注损伤模型HCMEC下P-选择素、VCAM-1的蛋白表达和mRNA转录水平变化,以及P38和NF-Kb的磷酸化活性水平。结果1.UAP证候分型：运用Kohonen网络聚类方法,UAP证候聚为9个证型,按构成比排列依次为心血瘀阻(20.99%)、气虚血瘀夹痰(14.89%)、阳虚血瘀(11.83%)、心气亏虚(11.45%)、气阴两虚(10.69%)、痰浊壅滞(10.69%)、气郁(滞)化火(热)(9.16%)、痰热壅滞(6.49%)、心阳亏虚(3.82%)。2.UAP证候要素：采用因子分析方法分析UAP患者临床症状,得到具有中医理论意义的7个公因子(证候要素),按构成比排列依次为血瘀(47.33%),气虚(38.55%)、痰浊(27.10%)、热蕴(20.61%)、气滞(16.03%)、阴虚(15.27%)、阳虚(14.89%)；57种组合型证候要素中,≥10例有6型,>10～≥5例有9型,>5～≥3例有11型,2例有10型,1例有21型,其中血瘀(15.85%)、气虚(12.83%)、气虚血瘀(7.17%)型最多。3.UAP证候类型与粘附分子关系：各组证候类型的P-selectin、VCAM-1浓度之间有统计学差异(P<0.01), P-selectin浓度以气虚血瘀夹痰组<血瘀组<阳虚血瘀组<痰浊或兼化热组<气滞或兼化热组<痰浊组<气阴两虚组<气虚<阳虚；与气虚血瘀夹痰组比较,除血瘀组外的其他各组均有统计学差异(P<0.05,P<0.01),与气虚组比较,除气阴两虚、阳虚组外其他各组均有统计学差异(P<0.05, P<0.01)。VCAM-1浓度以气虚血瘀夹痰<痰浊或兼化热<血瘀组<气滞或兼化热组<阳虚血瘀<痰浊组<气阴两虚组<气虚<阳虚；与气虚血瘀夹痰组比较,除心血瘀阻、气郁(滞)化火(热)、痰热蕴结外的其他各组均有统计学差异(P<0.05,P<0.01),与气虚组比较,除心气亏虚、气阴两虚、痰浊阻滞外的其他各型均有统计学差异(P<0.05,P<0.01)。UAP证候要素与粘附分子关系(QUEST算法)：气虚与低浓度P-selectin值有关,与VCAM-1的浓度关系不明显；瘀血与高浓度P-selectin值有关,与VCAM-1浓度有一定关系,但关系较复杂；痰浊与P-selectin的浓度关系不明显,与高浓度VCAM-1值有关；热蕴与一定范围较高浓度的VCAM-1有关,与P-selectin的浓度关系不明显；阳虚与低浓度P-selectin、VCAM-1值均有关；阴虚和气滞与P-selectin、VCAM-1的浓度关系不明显。即P-selectin在瘀血存在时呈高表达,气虚、阳虚时呈低表达；VCAM-1在瘀血、热蕴、痰浊存在时呈高表达,阳虚时呈低表达。4.损伤模型粘附分子及P38和NF-κb基因转录水平和蛋白表达：模拟冠心病心绞痛缺血发作时的病理机制,对人心脏微血管内皮细胞(HCMEC)进行缺糖缺氧和复糖复氧处理后,P-selectin、VCAM-1蛋白表达和1nRNA转录水平比正常组明显增高(P<0.05,P<0.01),P-P38和其下游靶点ATF-2、NF-κb下游靶点iκb的mRNA基因转录水平比正常组明显增高(P<0.01)。5.黄芪多糖干预损伤后粘附分子及P38和NF-κb基因转录水平和蛋白表达：HCMEC的P-selectin、VCAM-1蛋白表达和mRNA转录水平较模型组比较明显下调(P<0.05, P<0.01), P-P38、ATF-2、iκb的mRNA基因转录水平较模型组有显著下降(P<0.01),与NF-κb抑制剂PDTC和P38抑制剂SB203580比较无统计学差异(P>0.05)。结论1.UAP证候类型以心血瘀阻和气虚血瘀夹痰型最为多见。2.UAP证候要素为血瘀、气虚、痰浊、热蕴、气滞、阴虚、阳虚；以组合型证候要素居多,组合规律复杂多样,其中血瘀、气虚、气虚血瘀型最多。3. P-selectin、VCAM-1在虚实夹杂和实证时表达较高,以气虚血瘀夹痰证最高,在虚证时表达偏低,以阳气亏虚的浓度最低。QUEST算法提示,P-selectin的浓度直接反映瘀血的程度,间接反映痰浊的程度；VCAM-1的浓度直接反映痰浊程度,间接反映瘀血、热蕴程度；热蕴对增加P-selectin、VCAM-1浓度起促进作用；气滞对P-selectin、VCAM-1浓度的影响小于痰、瘀对二者的影响。4.正常HCMEC中P38和NF-κb活性受到相对抑制,使P-selectin、VCAM-1处于低表达、低转录水平,损伤后P38和NF-κb活性明显增强,使P-selectin、VCAM-1表达和转录明显增高,加重细胞损伤。5.黄芪多糖能下调HCMEC的P-selectin、VCAM-1表达及转录,具有浓度依赖性；对P38和NF-κb活性起到一定的抑制作用,抑制效果与PDTC和SB203580比较无差异；对P38通路和NF-κb通路的信号转导有共同抑制作用,通过抑制两通路的活性,使依靠这两条通路转录基因和表达蛋白的粘附分子(P-selectin、VCAM-1)水平被有效降低,阻断粘附分子介导的内皮细胞和白细胞粘附后,白细胞释放大量细胞毒性介质对内皮的损伤。本研究UAP者以血瘀、气虚多见,血清中sP-selectin、sVCAM-1虚证时偏低表达,虚实夹杂和实证时较高表达；经缺氧再灌注损伤造模后,高度表达的P-selectin、VCAM-1经黄芪多糖干预后浓度明显下降,推测以益气为主的黄芪,兼有活血的功效,在UAP的治疗中起到了非常重要的作用。更多还原

【Abstract】 ObjectiveTo generalize syndrome patterns and syndrome factors in unstable angina pectoris coro-nary heart disease patients by statistical and data mining methods and study links between it, the adhesion molecule of P-selectin in serum and concentration variations of VCAM-1. Futherly capture more objective and implicit relations by unsupervised machinery data analy-sis.With the help of ischemia-reperfusion model of human cardiac microvascular endothelial cell to imitate the damage of ischemia of angina pectoris onset on vascular endothelial cell and study the intervention effect of Astragalus Polysaccharide on adhesion molecule such as P-selectin and VCAM-1and its signal transduction mechanism of P38and NF-kb.Method1. Kohonen network clustering was adopted to analyze the symptoms of265unstable angina pectoris (UAP) cases and thus received the common syndrome pattern of UAP. Factor analysis was used to summarize and generalize the syndrome factors. At last, Decision Tree Algorithm was applied for detection of inner connections between syndrome factors, P-selectin and VCAM-1.2. Human cardiac microvascular endothelial cell (HCMEC) was managed by ischemia-reperfusion to imitate the damage of ischemia of angina pectoris onset on vascular endothelial cell. Variations of protein expression of P-selectin and VCAM-1as well as transcription level of mRNA, phosphorylation of P38and NF-Kb in HCMEC in ischemia-reperfusion model intervened by Astragalus Polysaccharide with the help of immunohistochemistry, western botting and quantitative real time PCR assay.Result1. UAP syndrome pattern:According to Kohonen network clustering, the syndrome pattern of UAP were divided into9catigories as below:heart-blood stasis (20.99%), qi deficiency with bood stasis plus phlegm (14.89%), yang deficiency with blood stasis (11.83%), heart-qi deficiency (11.45%), qi and yin deficiency (10.69%), phlegm-turbid obstruction (10.69%), depressed qi transforming into heat (9.16%), phlegm-heat stagnation (6.49%), heart yang deficiency (3.82%)。2. UAP syndrome factor:According to factor analysis, the analysis results of symptoms of UAP patients was received seven common factors (symptom factors) that were blood stasis (47.33%), qi dificiency (38.55%), phlegm turbid (27.10%), heat retention (20.61%) qi stagnation (16.03%), yin deficiency (15.27%) and yang deficiency (14.89%).Forthe57kinds of cyndrome pattern matches; more than ten cases had six factors; five to ten cases had nine factors; three to five cases had eleven factors; two cases had ten factors; one case had twenty-one factors. Among these mathes, bood stasis (15.85%), qi deficiency (12.83%) and qi deficiency with blood stasis (7.17%) were the most frequent patterns.3. The relation between UAP syndrome pattern and adhesion molecules:There were significant differences between P-selectin and VCAM-1concentration of different syndrome patterns (P<0.01). The P-selectin level from lowest to highest in turn:qi deficiency with bood stasis plus phlegm, blood stasis, yang deficiency with blood stasis, phlegm-heat stagnation, depressed qi transforming into heat, phlegm-turbid obstruction, qi and yin deficiency and qi deficiency and yang deficiency. Except for blood stasis group, all other groups had statistical differentces in contrast to qi deficiency with bood stasis plus phlegm group (P<0.05or P<0.01). Besides qi and yin deficiency and yang deficiency group, all other groups showed significant differences in comparison to qi deficiency group. The VCAM-1level from lowest to highest in turn:qi deficiency with bood stasis plus phlegm, phlegm-heat stagnation, blood stasis, depressed qi transforming into heat, yang deficiency with blood stasis, phlegm-turbid obstruction, qi and yin deficiency, qi deficiency and yang deficiency (P<0.05, P<0.01) Except blood stasis, depressed qi transforming into heat and phlegm-heat stagnation group, all other groups had statistical differentces in contrast to qi deficiency with bood stasis plus phlegm group (P<0.05or P<0.01). Besides qi and yin deficiency, phlegm-turbid obstruction and ya deficiency group, all other groups showed significant differences in comparison to qi deficiency group (P＜0.05, P＜0.01)The relation between UAP syndrome factor and adhesion molecules (QUEST algo-rithm):Qi deficiency is related to low P-selectin level rather than VCAM-1level; blood stasis is related to high P-selectin and VCAM-1level; phlegm turbid is linked to high VCAM-1; heat retention is linked to high VCAM-1concentration; yang deficiency is related to low P-selectin level; yin deficiency and qi stagnation had nothing to do with P-selectin and VCAM-1level. Thus, P-selectin had a high expression in blood stasis syndrome while had a low expression in yang deficiency and qi deficiency syndrome; VCAM-1expressed highly in blood stasis, heat retention and phlegm turbid syndrome pattern while expressed low in yang deficiency syndrome.4. Protein expression and mRNA transcription level of adhesion molecules and P38、 NF-Kb by ischemia-reperfusion:It was observed that protein expression as well as mRNA transcription level of P-selectin and VCAM-1were higher than normal group (P＜0.05, P<0.01). While the mRNA transcription level of P-P38and its target ATF-2as well as NF-Kb and its target ixb were significantly elevated in contrast to normal group (P<0.01)5. Protein expression and mRNA transcription level of adhesion molecules and P38、 NF-Kb after intervention by Astragalus Polysaccharide:Protein expression as well as mRNA transcription level of P-selectin and VCAM-1of model group were obviously down-regulated than control group (P＜0.05, P<0.01). While the mRNA transcription level of P-P38, ATF-2and kb mRNA dropped drastically than model group (P<0.01). However, in comparison to PDTC, the inhibitor of NF-Kb and to SB203580which is the inhibitor of P38, there was no statistical difference (P>0.05)Conclusion1. In UAP patients, heart-blood obstruction and qi deficiency with bood stasis plus phlegm sydrome were the most common ones.2. The syndrome factors of UAP were blood stasis, qi dificiency, phlegm turbid, heat retention, qi stagnation, yin deficiency and yang deficiency and they were commonly observed in combinated forms. The rule of combination was complicated and blood stasis, qi deficiency and qi deficiency with blood stasis were the most popular ones.3. P-selectin and VCAM-1had a high expression in intermingled deficiency and exces-sive syndrome and excessive syndromes, especially in qi deficiency with bood stasis plus phlegm syndrome. In additon, they had a low expression in deficiency syndromes and their expression were lowest in yang and qi deficiency synreome pattern. Thus it was speculated that the concentration of P-selectin reflected the degree of blood stasis directly while reflected the degree of phlegm-turbid indirectly. As for VCAM-1concentration, it mirrored phlegm-turbid directly while mirrored blood stasis and heat retention level indirectly. Heat retention promoted the density of P-selectin and VCAM-1and its effect was greater than the force of qi stagnation.4. In HCMEC under normal cultivation circumstance, the activity of P38and NF-Kb were inhibited and P-selectin and VCAM-1were in low expression and transcription level. After treated by ischemia-reperfusion, activity of P38and NF-Kb in HCMEC enhanced sub-stantially. In the mean time, expression and transcription level of P-selectin and VCAM-1promoted significant, causing further damage cellular injury.5. After intervened by Astragalus Polysaccharide, protein expression as well as mRNA transcription level of P-selectin and VCAM-1in HCMEC down-regulated obviously which saved as inhibtor of P38and NF-Kb with similar effects as PDTC and SB203580. Thus, it was speculated that Astragalus Polysaccharide was of impressive effects on signaling pathway of P38together with signal transduction of NF-Kb. Additionally, the concentration-dependent effect was achieved by declined the activity of pathways mentioned above leading to dropped level of related adhesion molecules as P-selectin and VCAM-1. Finally, the adhesion mole-cule mediated endothelial cell and white cell adhesion was therefore blocked and thus white cells were arrested from releasing cytotoxic mediators to damage the endothelial cells.In the study of UAP patients, blood stasis and qi dificiency were the most common ones. P-selectin and VCAM-1had a high expression in intermingled deficiency and excessive syn-drome and excessive syndromes. In additon, they had a low expression in deficiency syndromes. After hypoxia reperfusion injury, the high expression of P-selectin, VCAM-1concentrations decreased significantly after intervention by astragalus polysaccharide, presumably astragalus have both supplementing qi and activating blood circulation, and plays a very important role in the treatment of UAP.更多还原