鹅冰舒冠滴丸对血管内皮细胞脂质过氧化损伤保护作用研究
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摘要
第一部分鹅冰舒冠滴丸临床疗效观察及对冠心病心绞痛病人脂质过氧化指标的影响
鹅冰舒冠滴丸是导师程丑夫教授根据传统中医药理论和自身长期临床实践经验研制的治疗冠心病心绞痛的中药新药。本品有芳香通脉、益气豁痰、化瘀止痛的功效,适应于冠心病心绞痛阳气亏虚、痰瘀阻滞患者。临床研究采用随机双盲对照试验对本品种疗效进行研究观察确证,并对冠心病心绞痛病人脂质过氧化指标进行了观察,以了解鹅冰舒冠滴丸对冠心病心绞痛病人脂质过氧化指标的影响。
目的:确证鹅冰舒冠滴丸治疗冠心病心绞痛阳气亏虚、痰瘀阻滞证疗效,并了解其对冠心病心绞痛病人脂质过氧化指标的影响。
方法:临床研究采用随机分组、双盲双模拟、阳性药物平行对照试验方法,选择符合冠心病心绞痛临床诊断和中医阳气亏虚、痰瘀阻滞证诊断,且每周心绞痛发作≥2次患者为受试对象,阳性对照药物选择麝香保心丸,用药方法:试验组,鹅冰舒冠滴丸5丸/次,同时服用麝香保心丸模拟剂2丸/次,每日3次。餐后半小时服用;对照组,麝香保心丸2丸/次,同时服用鹅冰舒冠滴丸模拟剂5丸/次,每日3次。餐后半小时服用。试验性治疗一月后,客观评价其临床疗效。并观察血浆内皮素(ET-1)、丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性等指标治疗前后变化,分析其与疗效关系。
结果:试验结果,共观察87例患者,试验组45例,对照组42例,两组用药前资料无显著差异,分组均衡性良好,有可比性。鹅冰舒冠滴丸组(试验组)心绞痛疗效为显效26例(57.78%)、有效15例(33.33%),总有效率91.11%;心电图疗效显效14例(32.56%)、有效20例(46.51%),总有效率79.07%;中医证候疗效显效24例(53.33%)、有效18例(40.00%),总有效率93.33%,均高于对照组,但差异无统计学意义(P＞0.05)。试验组硝酸甘油平均停减率高于对照组(69.25%,58.87%),但统计学分析组间差异无统计学意义(P=0.0708)。并发现治疗后随着病情好转,患者脂质过氧化反应的终产物丙二醛(MDA)浓度有显著降低,而氧自由基的主要清除剂超氧化物歧化酶(SOD)活性较治疗前显著升高(P＜0.01),内皮细胞分泌的重要血管活性物质、反映内皮功能不全指标的内皮素(ET-1)也有显著降低。
结论:鹅冰舒冠滴丸对冠心病心绞痛有良好治疗作用,其对抗过氧化脂质及改善血管内皮细胞功能作用可能是临床疗效的作用机理之一。
第二部分鹅冰舒冠滴丸对ox-LDL所致血管内皮细胞损伤的保护作用研究
为进一步明确本品种对抗过氧化脂质对血管内皮细胞损伤的作用,采用现代细胞分子生物学,以细胞凋亡、内皮细胞分泌血管活性物质NO、ET、t-PA、PAI等为指标,进行了本品种对氧化修饰低密度脂蛋白(ox-LDL)所致培养血管内皮细胞损伤模型保护作用的研究。
目的:研究鹅冰舒冠滴丸对ox-LDL所致血管内皮细胞损伤的保护作用。
方法:采用MTT法检测鹅冰舒冠滴丸对体外培养人脐静脉内皮细胞株VEC-304的毒性作用,以细胞生长不受影响的最大浓度和这一浓度的1/2量作为的实验浓度。
根据文献报道,以终浓度200ug/ml的ox-LDL造成血管内皮细胞损伤模型,在造模前或造模同时加入鹅冰舒冠滴丸溶液作为处理因素,模拟临床使用鹅冰舒冠滴丸治疗冠心病心绞痛时对冠心病危险因素引起的内皮损伤的预防或治疗作用,检测内皮细胞凋亡情况及细胞培养上清液中NO、ET、t-PA、PAl-1等内皮细胞损伤指标,以了解鹅冰舒冠滴九对ox-LDL所致血管内皮细胞损伤的保护作用。
分组处理:将传代生长于12孔板的内皮细胞分为:①阴性对照组:用DMEM完全培养液(含10%小牛血清)37℃孵育24h;②模型组:用含终浓度200ug/ml的ox-LDL的DMEM完全培养液37℃孵育24h,造成细胞损伤、诱导凋亡;③鹅冰舒冠滴丸保护组:分别用含鹅冰舒冠滴丸1000ug/ml、500ug/ml的DMEM完全培养液培养2h后,加入终浓度为200ug/ml的ox-LDL孵育24h;④鹅冰舒冠滴丸试验组:分别以含鹅冰舒冠滴丸1000ug/ml、500ug╱ml及ox-LDL终浓度为200ug/ml的DMEM完全培养液孵育24h。
细胞凋亡检测采用荧光细胞染色法及流式细胞仪技术;NO测定采用硝酸还原酶法;内皮素(ET)测定采用放免法;Ⅰ型纤溶酶原抑制剂(PAl-1)、组织型纤溶酶原激活物(t-PA)检测采用发色底物法。
结果:荧光显微镜观察阴性对照组细胞染色质在核内分布均匀。ox-LDL作用24h后,可观察到早、中、晚期各种典型的凋亡细胞,细胞核染色质形成大小不同的凝聚块,溢裂断开分散到细胞质中并形成凋亡小体,呈现细胞凋亡时典型的染色质凝聚。鹅冰舒冠滴丸各组细胞凋亡明显减少。
经流式细胞仪检测阴性对照组VEC细胞凋亡率为1.67%。而模型组经200μg╱mlox-LDL诱导VEC细胞24h后,其凋亡率则明显上升,达到32.28%。G1期前出现亚G1峰,即凋亡峰(AP)。与模型组相比,鹅冰舒冠滴丸各组的凋亡率均明显下降(13.82%～17.43%,P＜0.05),保护组凋亡率较试验组及更低(P＜0.05),不同剂量间无显著性差异(P＞0.05)。
细胞培养液中内皮细胞分泌血管活性物质检测结果,模型组NO、ET-1、PAI均有增高,而NO╱ET-1比、t-PA下降,鹅冰舒冠滴丸可在一定程度对抗ox-LDL所致NO、ET-1、PAI增高和NO/ET-1比、t-PA的下降,多数情况下保护组作用更为明显。
结论:鹅冰舒冠滴丸对氧化修饰低密度脂蛋白所致内皮细胞凋亡有明显保护作用,并能增加ox-LDL损伤条件下内皮细胞NO分泌量,降低内皮素,升高NO/ET-1比,改善内皮细胞损伤形成的促凝因素,初步证实了鹅冰舒冠滴丸对oxLDL所致内皮细胞损伤有较好保护作用,对冠心病治疗有积极意义。
Part 1.The observation of the Ebingxuguan Drop Pill's treatment effects and the influence of superoxide of the coronary heart disease.
The Ebingxuguan Drop Pill(SGDW) is a new medicine for the coronary heart disease patient of TCM(traditional Chinese medicine) which was developed by professor Chengchoufu,based on his distance clinic practices and the theory of TCM.SGDW has the efficacy of accelera the blood block,increase the Qi' function then resolve the phlegm obstruct,and lighten the pain.It should be recommended to the coronary angina that is QI weak and phlegm block.This research adopting the random compared,double blind trail to make sure the treatment effects and the affection to the coronary heart disease patient's superoxidize.
Purpose:Make sure the clinical affection of the SGDW use for the patient with certain coronary heart disease,then get further definite evidence of the influence of the coronary heart disease patient's oxidize.
Method:The research adopted random,double blind,double imitate,the masculine medicine parallelism trial,choused the patients which matched the diagnoses of coronary angina pectoris and TCM,and paroxysm of angina more then 2 times every week.The compare drug is Xexiangbaoxi Pill.
Dose:the patients of test group use 5 pills/times Ebingshuguan Pill and 2 pills/times Xexiangbaoxi Pill,3 times everyday and half an hour after meal each time;the control group,2 pills/times Xexiangbaoxi Pill and 5 pills/ times Ebingshuguan Pill,3 times everyday and half an hour after meal each time.A month later,examine the ET-1,MDA and SOD' activity to make sure the effect.
Result:The experiment observed 87 patients totally,the test group 45,the control group 42,the result showed that the two groups with good balanced before the experiment.The test group:The effect for the angina is 26 example (57.78%),valid 15,the total efficient is 91.11%;The electrocardiogram curative effect shows the effect is 14 example(32.56%),valid 20,the total efficient is 79.07%;for the Zheng of the TCM the effect 24 example (53.33%),valid 18,total efficient 93.33%,all were higher than the control group,but the difference did not show any statistic meaning(P＞0.05).And the result show a lot patients with Ebingshuguan Drop Pill stop or reduce the dose of nitric acid glycerin,the ratio is 69.25%and 58.87%,a little lighter then the compare patients,but with no statistics meaning(P=0.0708).With the better of the patient's condition,the concentration of the MDA which is the last outcome of the oxidize action of fat dropped lower,and the activity of the SOD which was the main elimination for oxygen-derived flee radicals increases a lot than before(P＜0.01);and ET-1,the impartment vein active substance which is excreted by endothelia cell,which can reflect the function of the endothelia cell's function,fall remarkable.
Conclusion:The Ebingshuguan Drop Pill has good affection on treating the coronary angina pectoris,it's resisting of oxidize fat and the meliorate of the endothelia cell's function may be one of the mechanisms of the clinical curative effect.
Part 2.The research of Ebingshuguan Drop Pill's affection on the damage of vessels endothelia cell which is caused by ox-LDL
For make sure the function of the SGDW's infection on the damage of vessels endothelia cell that which is caused by ox-LDL,adopted molecular biology's solution,take apoptosis,NO,ET,t- PA,PAI etc as the index sign,carry on the research of the protection of the damage of vessels endothelia cell that which is caused by ox-LDL.
Purpose:Study the Ebingshuguan Drop Pill's protection of the damage of vessels ndothelia cell,which is caused by ox-LDL.
Method:Adopted MTT method examine the SGDW' s toxicity to the vessels endothelia cell VEC—304 which is trained outside the human body,take the 1/2 concentration that the biggest density which uninfluenced the cell as the test dose.
According to the report,take the model of the damage of vessels endothelia cell which is caused by 200ug/ml eventually ox-LDL,joining the solution of the SGDW before or build the mold as the treatment factor,imitate the clinical usage of the SGDW to treatment the damage of vessels endothelia cell that caused by the dangerous factor of coronary angina pectoris.
Examine apoptosis,NO,ET,t- PA,PAI etc,working out the effect ion of the SGDW.
Divide group:divided the cell grew in the 12 knotholes board into 4 parts:
①:Bland group:Hatch 24 hours in DMEM developing liquid(contain 10% calf serum) at the temp at 37℃;②Model group:Hatch 24 hours in DMEM developing liquid which contains 200ug/ml ox-LDL at the temp at 37℃,resulting in the cell hurt;Ebingshuguan Drop Pill protection set:Each hatch 2 hours in DMEM developing liquid which contains 1000ug/ml and 500ug/ml Ebingshuguan Drop Pill solution,then hatch 24 hours in DMEM developing liquid which contains 200ug/ml ox-LDL;④Ebingshuguan Pill group:Each hatch 24 hours in DMEM developing liquid contained 200ug/ml ox-LDL and 1000ug/ml or 500ug/ml Ebingshuguan Drop Pill solution. The apoptosis examination adopted immunocytochemical method and flow cytometry techniques;The measurement of NO adopted the nitric acid revivification enzyme method;The ET measurement adopted radiation immunity method;the PAI-1 and the t-PA examination adoption chromomeric funds method.
Result:The fluorescence microscope observed the bland group cell dyes distribute uniformity.After the ox-LDL function 24 hours,can observe various cell of typical model of early,medium or later period apoptosis,and can detected the condensation of apoptotic nuclear,overflowing into the cell and formatted small bodys,present the typical model of dyeing quality coagulate which used can be fund in apoptosis.The decrease of apoptosis in the test group is improved obviously.
The result showed that the apoptosis of the bland group was 1.67%,the model group is 32.28%,the G1 apex(AP) appeared before the g1 period of time. Compared with the model group,the rate of cell demise treat group descend(13.82%～17.43%,P＜0.05) obviously,the protection group was more lower than the test group(P＜0.05),but no different in each subgroup. The examination of the active substance excreted by vessels endothelia cell in the cell development liquid showed the NO,ET-1,PAI of model group all increased,but the NO/ET-1,the t- PA descended,the Ebingshuguan Drop Pill can resist the increase of NO,ET-1,PAI and descend of NO/ET-1,t- PA, caused by ox- LDL,and under most circumstances the protect set's function more obviously.
Conclusion:The Ebingshuguan Drop Pill can protect the vessels endothelia cell apoptosis caused by ox-LDL,and increase the excretion of the endothelia cell under the ox- LDL condition,lowering endothelia element,confirmed the Ebingshuguan Drop Pill's protection to the injury of endothelia cell caused by ox- LDL condition.
鹅冰舒冠滴丸是导师程丑夫教授根据传统中医药理论和自身长期临床实践经验研制的治疗冠心病心绞痛的中药新药。本品有芳香通脉、益气豁痰、化瘀止痛的功效,适应于冠心病心绞痛阳气亏虚、痰瘀阻滞患者。临床研究采用随机双盲对照试验对本品种疗效进行研究观察确证,并对冠心病心绞痛病人脂质过氧化指标进行了观察,以了解鹅冰舒冠滴丸对冠心病心绞痛病人脂质过氧化指标的影响。
目的:确证鹅冰舒冠滴丸治疗冠心病心绞痛阳气亏虚、痰瘀阻滞证疗效,并了解其对冠心病心绞痛病人脂质过氧化指标的影响。
方法:临床研究采用随机分组、双盲双模拟、阳性药物平行对照试验方法,选择符合冠心病心绞痛临床诊断和中医阳气亏虚、痰瘀阻滞证诊断,且每周心绞痛发作≥2次患者为受试对象,阳性对照药物选择麝香保心丸,用药方法:试验组,鹅冰舒冠滴丸5丸/次,同时服用麝香保心丸模拟剂2丸/次,每日3次。餐后半小时服用;对照组,麝香保心丸2丸/次,同时服用鹅冰舒冠滴丸模拟剂5丸/次,每日3次。餐后半小时服用。试验性治疗一月后,客观评价其临床疗效。并观察血浆内皮素(ET-1)、丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性等指标治疗前后变化,分析其与疗效关系。
结果:试验结果,共观察87例患者,试验组45例,对照组42例,两组用药前资料无显著差异,分组均衡性良好,有可比性。鹅冰舒冠滴丸组(试验组)心绞痛疗效为显效26例(57.78%)、有效15例(33.33%),总有效率91.11%;心电图疗效显效14例(32.56%)、有效20例(46.51%),总有效率79.07%;中医证候疗效显效24例(53.33%)、有效18例(40.00%),总有效率93.33%,均高于对照组,但差异无统计学意义(P＞0.05)。试验组硝酸甘油平均停减率高于对照组(69.25%,58.87%),但统计学分析组间差异无统计学意义(P=0.0708)。并发现治疗后随着病情好转,患者脂质过氧化反应的终产物丙二醛(MDA)浓度有显著降低,而氧自由基的主要清除剂超氧化物歧化酶(SOD)活性较治疗前显著升高(P＜0.01),内皮细胞分泌的重要血管活性物质、反映内皮功能不全指标的内皮素(ET-1)也有显著降低。
结论:鹅冰舒冠滴丸对冠心病心绞痛有良好治疗作用,其对抗过氧化脂质及改善血管内皮细胞功能作用可能是临床疗效的作用机理之一。
第二部分鹅冰舒冠滴丸对ox-LDL所致血管内皮细胞损伤的保护作用研究
为进一步明确本品种对抗过氧化脂质对血管内皮细胞损伤的作用,采用现代细胞分子生物学,以细胞凋亡、内皮细胞分泌血管活性物质NO、ET、t-PA、PAI等为指标,进行了本品种对氧化修饰低密度脂蛋白(ox-LDL)所致培养血管内皮细胞损伤模型保护作用的研究。
目的:研究鹅冰舒冠滴丸对ox-LDL所致血管内皮细胞损伤的保护作用。
方法:采用MTT法检测鹅冰舒冠滴丸对体外培养人脐静脉内皮细胞株VEC-304的毒性作用,以细胞生长不受影响的最大浓度和这一浓度的1/2量作为的实验浓度。
根据文献报道,以终浓度200ug/ml的ox-LDL造成血管内皮细胞损伤模型,在造模前或造模同时加入鹅冰舒冠滴丸溶液作为处理因素,模拟临床使用鹅冰舒冠滴丸治疗冠心病心绞痛时对冠心病危险因素引起的内皮损伤的预防或治疗作用,检测内皮细胞凋亡情况及细胞培养上清液中NO、ET、t-PA、PAl-1等内皮细胞损伤指标,以了解鹅冰舒冠滴九对ox-LDL所致血管内皮细胞损伤的保护作用。
分组处理:将传代生长于12孔板的内皮细胞分为:①阴性对照组:用DMEM完全培养液(含10%小牛血清)37℃孵育24h;②模型组:用含终浓度200ug/ml的ox-LDL的DMEM完全培养液37℃孵育24h,造成细胞损伤、诱导凋亡;③鹅冰舒冠滴丸保护组:分别用含鹅冰舒冠滴丸1000ug/ml、500ug/ml的DMEM完全培养液培养2h后,加入终浓度为200ug/ml的ox-LDL孵育24h;④鹅冰舒冠滴丸试验组:分别以含鹅冰舒冠滴丸1000ug/ml、500ug╱ml及ox-LDL终浓度为200ug/ml的DMEM完全培养液孵育24h。
细胞凋亡检测采用荧光细胞染色法及流式细胞仪技术;NO测定采用硝酸还原酶法;内皮素(ET)测定采用放免法;Ⅰ型纤溶酶原抑制剂(PAl-1)、组织型纤溶酶原激活物(t-PA)检测采用发色底物法。
结果:荧光显微镜观察阴性对照组细胞染色质在核内分布均匀。ox-LDL作用24h后,可观察到早、中、晚期各种典型的凋亡细胞,细胞核染色质形成大小不同的凝聚块,溢裂断开分散到细胞质中并形成凋亡小体,呈现细胞凋亡时典型的染色质凝聚。鹅冰舒冠滴丸各组细胞凋亡明显减少。
经流式细胞仪检测阴性对照组VEC细胞凋亡率为1.67%。而模型组经200μg╱mlox-LDL诱导VEC细胞24h后,其凋亡率则明显上升,达到32.28%。G1期前出现亚G1峰,即凋亡峰(AP)。与模型组相比,鹅冰舒冠滴丸各组的凋亡率均明显下降(13.82%～17.43%,P＜0.05),保护组凋亡率较试验组及更低(P＜0.05),不同剂量间无显著性差异(P＞0.05)。
细胞培养液中内皮细胞分泌血管活性物质检测结果,模型组NO、ET-1、PAI均有增高,而NO╱ET-1比、t-PA下降,鹅冰舒冠滴丸可在一定程度对抗ox-LDL所致NO、ET-1、PAI增高和NO/ET-1比、t-PA的下降,多数情况下保护组作用更为明显。
结论:鹅冰舒冠滴丸对氧化修饰低密度脂蛋白所致内皮细胞凋亡有明显保护作用,并能增加ox-LDL损伤条件下内皮细胞NO分泌量,降低内皮素,升高NO/ET-1比,改善内皮细胞损伤形成的促凝因素,初步证实了鹅冰舒冠滴丸对oxLDL所致内皮细胞损伤有较好保护作用,对冠心病治疗有积极意义。
Part 1.The observation of the Ebingxuguan Drop Pill's treatment effects and the influence of superoxide of the coronary heart disease.
The Ebingxuguan Drop Pill(SGDW) is a new medicine for the coronary heart disease patient of TCM(traditional Chinese medicine) which was developed by professor Chengchoufu,based on his distance clinic practices and the theory of TCM.SGDW has the efficacy of accelera the blood block,increase the Qi' function then resolve the phlegm obstruct,and lighten the pain.It should be recommended to the coronary angina that is QI weak and phlegm block.This research adopting the random compared,double blind trail to make sure the treatment effects and the affection to the coronary heart disease patient's superoxidize.
Purpose:Make sure the clinical affection of the SGDW use for the patient with certain coronary heart disease,then get further definite evidence of the influence of the coronary heart disease patient's oxidize.
Method:The research adopted random,double blind,double imitate,the masculine medicine parallelism trial,choused the patients which matched the diagnoses of coronary angina pectoris and TCM,and paroxysm of angina more then 2 times every week.The compare drug is Xexiangbaoxi Pill.
Dose:the patients of test group use 5 pills/times Ebingshuguan Pill and 2 pills/times Xexiangbaoxi Pill,3 times everyday and half an hour after meal each time;the control group,2 pills/times Xexiangbaoxi Pill and 5 pills/ times Ebingshuguan Pill,3 times everyday and half an hour after meal each time.A month later,examine the ET-1,MDA and SOD' activity to make sure the effect.
Result:The experiment observed 87 patients totally,the test group 45,the control group 42,the result showed that the two groups with good balanced before the experiment.The test group:The effect for the angina is 26 example (57.78%),valid 15,the total efficient is 91.11%;The electrocardiogram curative effect shows the effect is 14 example(32.56%),valid 20,the total efficient is 79.07%;for the Zheng of the TCM the effect 24 example (53.33%),valid 18,total efficient 93.33%,all were higher than the control group,but the difference did not show any statistic meaning(P＞0.05).And the result show a lot patients with Ebingshuguan Drop Pill stop or reduce the dose of nitric acid glycerin,the ratio is 69.25%and 58.87%,a little lighter then the compare patients,but with no statistics meaning(P=0.0708).With the better of the patient's condition,the concentration of the MDA which is the last outcome of the oxidize action of fat dropped lower,and the activity of the SOD which was the main elimination for oxygen-derived flee radicals increases a lot than before(P＜0.01);and ET-1,the impartment vein active substance which is excreted by endothelia cell,which can reflect the function of the endothelia cell's function,fall remarkable.
Conclusion:The Ebingshuguan Drop Pill has good affection on treating the coronary angina pectoris,it's resisting of oxidize fat and the meliorate of the endothelia cell's function may be one of the mechanisms of the clinical curative effect.
Part 2.The research of Ebingshuguan Drop Pill's affection on the damage of vessels endothelia cell which is caused by ox-LDL
For make sure the function of the SGDW's infection on the damage of vessels endothelia cell that which is caused by ox-LDL,adopted molecular biology's solution,take apoptosis,NO,ET,t- PA,PAI etc as the index sign,carry on the research of the protection of the damage of vessels endothelia cell that which is caused by ox-LDL.
Purpose:Study the Ebingshuguan Drop Pill's protection of the damage of vessels ndothelia cell,which is caused by ox-LDL.
Method:Adopted MTT method examine the SGDW' s toxicity to the vessels endothelia cell VEC—304 which is trained outside the human body,take the 1/2 concentration that the biggest density which uninfluenced the cell as the test dose.
According to the report,take the model of the damage of vessels endothelia cell which is caused by 200ug/ml eventually ox-LDL,joining the solution of the SGDW before or build the mold as the treatment factor,imitate the clinical usage of the SGDW to treatment the damage of vessels endothelia cell that caused by the dangerous factor of coronary angina pectoris.
Examine apoptosis,NO,ET,t- PA,PAI etc,working out the effect ion of the SGDW.
Divide group:divided the cell grew in the 12 knotholes board into 4 parts:
①:Bland group:Hatch 24 hours in DMEM developing liquid(contain 10% calf serum) at the temp at 37℃;②Model group:Hatch 24 hours in DMEM developing liquid which contains 200ug/ml ox-LDL at the temp at 37℃,resulting in the cell hurt;Ebingshuguan Drop Pill protection set:Each hatch 2 hours in DMEM developing liquid which contains 1000ug/ml and 500ug/ml Ebingshuguan Drop Pill solution,then hatch 24 hours in DMEM developing liquid which contains 200ug/ml ox-LDL;④Ebingshuguan Pill group:Each hatch 24 hours in DMEM developing liquid contained 200ug/ml ox-LDL and 1000ug/ml or 500ug/ml Ebingshuguan Drop Pill solution. The apoptosis examination adopted immunocytochemical method and flow cytometry techniques;The measurement of NO adopted the nitric acid revivification enzyme method;The ET measurement adopted radiation immunity method;the PAI-1 and the t-PA examination adoption chromomeric funds method.
Result:The fluorescence microscope observed the bland group cell dyes distribute uniformity.After the ox-LDL function 24 hours,can observe various cell of typical model of early,medium or later period apoptosis,and can detected the condensation of apoptotic nuclear,overflowing into the cell and formatted small bodys,present the typical model of dyeing quality coagulate which used can be fund in apoptosis.The decrease of apoptosis in the test group is improved obviously.
The result showed that the apoptosis of the bland group was 1.67%,the model group is 32.28%,the G1 apex(AP) appeared before the g1 period of time. Compared with the model group,the rate of cell demise treat group descend(13.82%～17.43%,P＜0.05) obviously,the protection group was more lower than the test group(P＜0.05),but no different in each subgroup. The examination of the active substance excreted by vessels endothelia cell in the cell development liquid showed the NO,ET-1,PAI of model group all increased,but the NO/ET-1,the t- PA descended,the Ebingshuguan Drop Pill can resist the increase of NO,ET-1,PAI and descend of NO/ET-1,t- PA, caused by ox- LDL,and under most circumstances the protect set's function more obviously.
Conclusion:The Ebingshuguan Drop Pill can protect the vessels endothelia cell apoptosis caused by ox-LDL,and increase the excretion of the endothelia cell under the ox- LDL condition,lowering endothelia element,confirmed the Ebingshuguan Drop Pill's protection to the injury of endothelia cell caused by ox- LDL condition.
引文
[1]程丑夫,谭元生,胡宏,等。舒冠滴丸对麻醉犬心脏血流动力学及心肌耗氧量的影响。中药药理与临床,2000;16(3):24-26
[2]谭圣娥,谭元生,胡宏,等。舒冠滴九对麻醉犬急性心肌缺血、心肌梗塞及血浆ET、CGRP的影响。中药药理与临床,2000;16(4):26-28
[3]金朝辉。鹅冰舒冠滴九治疗冠心病心绞痛的实验研究。硕士论文。湖南中医学院,2002。
[4]韩启德,丈允镒,主编。血管生物学。北京医科大学、中国协和医科大学联合出版社。1997年9月,第一版。P24-39;P205-213。
[5]俞梦越,吴维力,等。内皮功能不全与冠心病。心血管病学进展,2002;23(4):193-199
[6]Lusis AJ.Atherosclerosis.Nature,2000,407:233 241.
[7]Ross R.Atherosclerosis an inflammatory disease.N Eng J Med,1999,340:115-126.
[8]李江津,内皮细胞凋亡与冠心病。中国组织化学与细胞化学杂志。2002,11(2):212
[9]Dimmeler S,Zeiher AM.Endothelial cell apoptosis in angiogenesis and vessel regression.Circulation Research 2000,87:434-439
[10]Harada shiba M,Kinoshita M,Kamido H,et al.oxidized low density lipoprotein induces apoptosis in cultured human umbilica vein endothelial cells by common and unique mechanisms.J Biol Chem 1998.273:9681-9687
[11]卫生部《中药新药·临床研究指导原则》第一辑
[12]郑筱萸主编。《中药新药临床研究指导原则》(试行)。中国医药科技出版社,2002年5月,第1版。北京。
[13]贝政平.3200个内科疾病诊断标准.第1版.北京:北京科学技术出版社,1996
[14]不稳定性心绞痛诊断和治疗建议。中华医学会心血管病学分会,中华心血管病杂志编辑委员会。中华心血管病杂志,2002.28(6):409
[15]成都中医学院。《金匮要略选读》。上海科技出版社,1980年6月,第1版。
[16]明·张介宾编著。《类经》。人民卫生出版社,1980年4月,第1版第2次印刷。
[17]宋·太平惠民和剂局编。《太平惠民和剂局方》。人民卫生出版社,1985年10月,第1版。
[18]清·吴谦等编。《医宗金鉴》。人民卫生出版社,1985年7月,第2版。
[19]清·陈士铎著,程丑夫评述。《<石室秘录>评述》。湖南科技出版社,1991年3月,第1版。
[20]国家中医药管理局《中华本草》编委会。《中华本草》。上海科技出版社,1999年5月,第1版。
[21]沈绍功,诊治冠心病的新思路。中国中医急症,1999;8(2):51。
[22]Burke AP,Farb A,Malcom GT,et al.[J].N Engl J Med,1997,336:1276-1282.
[23]Bertrand ME,Simoons ML,Fox KA,et al.Management of acute coronary syndromes in patients presenting without persistent ST segment elevation[J].Eur Heart J,2002,23(23):1801 1840
[24]Feng B,Yao PM,Li Y,etal.The endoplasmic reticulum is the site of cholesterol induced cytotoxicity in macrophages.Nat Cell Biol,2003,5:781 792.
[25]Steinberg D.A the rogenesis in perspective:hypercholesterolem ia and inflammation as partners in crime.NatMed,2002,8:1211 1217
[26]Napoli C,D'A miento FP,Mancini FP,etal.Fatty streak formation occurs in human fetal aortas and is greatly enhanced by maternal hypercholesterolem ia:intimal accumulation of low density lipoprotein and its oxidation precede monocyte recruitment into early atherosclerotic lesions.J Clin Invest,1997,100:2680 2690.
[27]张瞥,等。老年冠心病痰瘀辨证血清脂蛋白谱检测意义。实用中西医结合杂志,1997;10(17):1645。
[28]骆雷鸣。稳定冠状动脉粥样硬化斑块新策略。国外医学内科学分册,2000;27(9):369。
[29]Bombeli T,Karsan A,Tait JF,et al.Apoptotic vascular endothelial cells become procoagulant.Blood 1997,89:2429-2442
[2]谭圣娥,谭元生,胡宏,等。舒冠滴九对麻醉犬急性心肌缺血、心肌梗塞及血浆ET、CGRP的影响。中药药理与临床,2000;16(4):26-28
[3]金朝辉。鹅冰舒冠滴九治疗冠心病心绞痛的实验研究。硕士论文。湖南中医学院,2002。
[4]韩启德,丈允镒,主编。血管生物学。北京医科大学、中国协和医科大学联合出版社。1997年9月,第一版。P24-39;P205-213。
[5]俞梦越,吴维力,等。内皮功能不全与冠心病。心血管病学进展,2002;23(4):193-199
[6]Lusis AJ.Atherosclerosis.Nature,2000,407:233 241.
[7]Ross R.Atherosclerosis an inflammatory disease.N Eng J Med,1999,340:115-126.
[8]李江津,内皮细胞凋亡与冠心病。中国组织化学与细胞化学杂志。2002,11(2):212
[9]Dimmeler S,Zeiher AM.Endothelial cell apoptosis in angiogenesis and vessel regression.Circulation Research 2000,87:434-439
[10]Harada shiba M,Kinoshita M,Kamido H,et al.oxidized low density lipoprotein induces apoptosis in cultured human umbilica vein endothelial cells by common and unique mechanisms.J Biol Chem 1998.273:9681-9687
[11]卫生部《中药新药·临床研究指导原则》第一辑
[12]郑筱萸主编。《中药新药临床研究指导原则》(试行)。中国医药科技出版社,2002年5月,第1版。北京。
[13]贝政平.3200个内科疾病诊断标准.第1版.北京:北京科学技术出版社,1996
[14]不稳定性心绞痛诊断和治疗建议。中华医学会心血管病学分会,中华心血管病杂志编辑委员会。中华心血管病杂志,2002.28(6):409
[15]成都中医学院。《金匮要略选读》。上海科技出版社,1980年6月,第1版。
[16]明·张介宾编著。《类经》。人民卫生出版社,1980年4月,第1版第2次印刷。
[17]宋·太平惠民和剂局编。《太平惠民和剂局方》。人民卫生出版社,1985年10月,第1版。
[18]清·吴谦等编。《医宗金鉴》。人民卫生出版社,1985年7月,第2版。
[19]清·陈士铎著,程丑夫评述。《<石室秘录>评述》。湖南科技出版社,1991年3月,第1版。
[20]国家中医药管理局《中华本草》编委会。《中华本草》。上海科技出版社,1999年5月,第1版。
[21]沈绍功,诊治冠心病的新思路。中国中医急症,1999;8(2):51。
[22]Burke AP,Farb A,Malcom GT,et al.[J].N Engl J Med,1997,336:1276-1282.
[23]Bertrand ME,Simoons ML,Fox KA,et al.Management of acute coronary syndromes in patients presenting without persistent ST segment elevation[J].Eur Heart J,2002,23(23):1801 1840
[24]Feng B,Yao PM,Li Y,etal.The endoplasmic reticulum is the site of cholesterol induced cytotoxicity in macrophages.Nat Cell Biol,2003,5:781 792.
[25]Steinberg D.A the rogenesis in perspective:hypercholesterolem ia and inflammation as partners in crime.NatMed,2002,8:1211 1217
[26]Napoli C,D'A miento FP,Mancini FP,etal.Fatty streak formation occurs in human fetal aortas and is greatly enhanced by maternal hypercholesterolem ia:intimal accumulation of low density lipoprotein and its oxidation precede monocyte recruitment into early atherosclerotic lesions.J Clin Invest,1997,100:2680 2690.
[27]张瞥,等。老年冠心病痰瘀辨证血清脂蛋白谱检测意义。实用中西医结合杂志,1997;10(17):1645。
[28]骆雷鸣。稳定冠状动脉粥样硬化斑块新策略。国外医学内科学分册,2000;27(9):369。
[29]Bombeli T,Karsan A,Tait JF,et al.Apoptotic vascular endothelial cells become procoagulant.Blood 1997,89:2429-2442