益气活血中药复方治疗CVB₃病毒性心肌炎的实验研究

【作者】 何伟；

【导师】 张明雪；

【作者基本信息】 辽宁中医药大学 ， 中医内科学， 2009， 博士

【摘要】 研究目的:病毒性心肌炎是指由多种病毒感染引起,以心肌细胞变性、坏死、间质炎性细胞浸润和纤维渗出为主要改变的心肌疾病。其致病病毒以柯萨奇B组病毒（CVB）最为主要,而在CVB的六个血清分型中,CVB₃具有强嗜心性,因此,当前主要针对CVB₃感染建立病毒性心肌炎模型来开展各项研究,本课题选择病毒性心肌炎作为研究对象的主要原因是,本病自身有很高的危害性,如小儿及成人的各个年龄阶段均可累及患病,急性期过后,又有演变为扩张型心肌病的可能性,同时,也可引起小范围爆发流行,而近年病毒性心肌炎的发病率更是呈现上升趋势,因此,针对CVB₃病毒性心肌炎的治疗机制研究显得迫在眉睫。本课题以新生2—3d大鼠体外心肌细胞培养建立CVB₃感染的VMC模型;通过Hoechst33258荧光检测心肌细胞凋亡;免疫组化法检测心肌细胞间缝隙连接蛋白Cx43、细胞骨架—肌动蛋白（actin）、心肌肌钙蛋白I（cTnI）表达;透射电镜观察CVB₃心肌炎细胞感染模型心肌细胞功能、形态学改变;激光扫描共聚焦显微镜实时动态检测心肌细胞游离Ca²⁺变化及细胞间缝隙连接通讯功能。试图验证益气活血中药复方具有减轻CVB₃病毒性心肌炎心肌细胞免疫损伤;抑制心肌细胞凋亡发生;调节细胞内游离Ca²⁺浓度;改善细胞间缝隙连接通讯;修复心肌细胞骨架及超微结构的作用,进一步证实益气活血中药复方是治疗CVB₃病毒性心肌炎的有效方剂,益气活血法是治疗本病的有效方法。材料与方法:①益气活血中药复方细胞毒性测定:取复苏的Hela细胞,经培养瓶传代后,以每孔0.2ml接种到96孔细胞培养板中,置37℃,5%二氧化碳培养箱中培养,当Hela细胞呈单层贴壁生长时,测益气活血中药复方最大无毒剂量。用DMEM培养液将益气活血中药复方注射液从原液开始配制成11个浓度。每孔0.2ml,每个浓度设4个复孔,另设4个加正常生长液的对照孔。分别于24h、48h观察细胞生长状态,参考正常对照孔细胞形态,以不引起细胞病变的最大药物稀释浓度为最大无毒浓度（TD₀）。②CVB₃病毒毒力滴定:于96孔培养板中制备单层生长的Hela细胞,弃去培养板中的生长液,以HBSS液洗涤各培养孔2次。用含2%胎牛血清的维持液,将CVB₃病毒以10倍浓度从10^-1稀释到10^-8,每孔0.1ml,接种于96孔培养板中,每个浓度设4个复孔,另设4个正常对照孔,各加维持液0.1ml,置于37℃,5%二氧化碳培养箱中培养,分别于24h、48h观察细胞病变情况。③益气活血中药复方对CVB₃致心肌细胞死亡的抑制实验（MTT法）:取对数生长期心肌细胞,每孔加入0.2ml半数组织感染量浓度CVB₃病毒吸附心肌细胞2h。吸弃含病毒培养液,以含2%胎牛血清的维持液轻轻洗涤一遍后,加入从药物最大无毒剂量起始稀释8个浓度的含益气活血中药复方的生长液,每孔0.2ml,每个浓度设4个复孔,另设1个调零孔。于48h后,每孔加入0.18ml DMEM培养液,再加入0.02ml MTT溶液,放入培养箱继续培养4h,小心吸弃孔内培养液,每孔加入0.15ml DMSO,置振荡器上振荡8min,于酶联免疫检测仪490 nm处测量各孔的吸光值（OD）。④益气活血中药复方对CVB₃病毒性心肌炎心肌细胞凋亡的影响:正常分离培养乳鼠心肌细胞,接种于24孔培养板做细胞爬片,48h后取单层贴壁生长达70—80%的正常组、病毒组、中药干预组心肌细胞玻片,以PBS冲洗2次,每孔加入甲醇冰醋酸固定10min,PBS冲洗2次,每孔加入Hoechst33258染色液1ml（5μg/ml）,避光反应15min,PBS冲洗3次,每次3min,滴加0.05ml抗荧光淬灭封片剂做封片处理,于荧光显微镜下观察。⑤益气活血中药复方对CVB₃病毒性心肌炎心肌细胞cTnI、Gx43、actin表达的影响:采用SABC免疫组化法检测cTnI、Cx43、actin表达:用3%H₂O₂孵育长有细胞的玻片10min,PBS冲洗。滴加正常山羊血清封闭液,室温孵育15分钟,倾去,勿洗。滴加1:100比例稀释的一抗,37℃温育2h,PBS冲洗,滴加生物素化二抗,37℃孵育30min。PBS冲洗3次,每次5min。滴加SABC复合物,37℃湿盒孵育30min。PBS冲洗3次,每次5min,DAB显色,自来水冲洗,苏木素复染,脱水、透明处理,树脂封片,镜下观察。⑥透射电镜检测益气活血中药复方对CVB₃病毒性心肌炎心肌细胞超微结构的影响:正常培养心肌细胞72h后,用HBSS液冲洗正常组、病毒组、中药组心肌细胞,0.25%的胰酶消化3—5min,收集细胞至1.5mlEP离心管中,1500rpm离心10min,弃上清,以2.5%戊二醛固定,送电镜检查。⑦荧光漂白恢复（FRAP）检测CVB₃病毒性心肌炎心肌细胞间缝隙连接通讯功能:于单层贴壁生长达70—80%的心肌细胞中,加入以Ca²⁺、Mg²⁺PBS配成10μg/ml 6—CFDA的应用液,于37℃,5%CO₂培养箱中避光孵育30min,取出后PBS漂洗后,加入适量含Ca²⁺、Mg²⁺的PBS后,上共聚焦显微镜检测。⑧益气活血中药复方对CVB₃病毒性心肌炎心肌细胞游离钙浓度的影响:于共聚焦专用培养皿中正常培养乳鼠心肌细胞,于37℃,5%CO₂培养箱中避光负载Fluo-3 AM钙离子荧光探针30min,hanks漂洗后,上共聚焦显微镜检测。结果:①益气活血中药复方细胞毒性测定:益气活血中药复方最大无毒剂量为7.813mg/ml。②CVB₃病毒毒力滴定:CVB₃病毒50%培养细胞感染量为1×10^-4.5。③益气活血中药复方对CVB₃致心肌细胞死亡的抑制实验（MTT法）:益气活血中药复方最大无毒剂量干预CVB₃攻击后的心肌细胞时,其吸光度值亦最大,与其它稀释度组比较,差异显著,P＜0.001,证实益气活血中药复方最大无毒剂量具有明显的抑制CVB₃致心肌细胞死亡作用。④益气活血中药复方对CVB₃病毒性心肌炎心肌细胞凋亡的影响:正常组心肌细胞核呈圆形、卵圆形,未见凋亡细胞核,而100×镜下观察病毒组心肌细胞核,呈现出成片的亮蓝色,400×镜下观察可见细胞核形态不完整,呈碎片状分布,也可见分叶状细胞核。中药组心肌细胞核多呈圆形,未见明显的凋亡细胞核形态,提示益气活血中药复方可抑制CVB₃感染后细胞凋亡发生,具有保护心肌作用。⑤益气活血中药复方对CVB₃病毒性心肌炎心肌细胞cTnI表达的影响:免疫组化检测心肌肌钙工（cTnI）表达结果显示,正常组cTnI未见有明显阳性棕褐染色。病毒感染组cTnI呈强阳性（+++）表达,棕褐色阳性信号多且色深,与正常组阳性目标平均光密度比较,差异显著,P＜0.05。中药干预组cTnI呈弱阳性（+）表达,水平明显下降,与病毒组比较,差异显著,P＜0.01。⑥益气活血中药复方对CVB₃病毒性心肌炎心肌细胞Cx43表达的影响:免疫组化检测Connexin 43蛋白发现,正常组心肌细胞呈强阳性（+++）表达,而病毒组Cx43呈极弱阳性（±）表达,与正常组阳性目标平均光密度比较,差异极其显著,P＜0.001。中药治疗组Cx43呈阳性（++）表达,与病毒组比较,差异显著,P＜0.05。⑦益气活血中药复方对CVB₃病毒性心肌炎心肌细胞actin表达的影响:免疫组化检测肌动蛋白（actin）结果显示,正常组心肌细胞actin呈强阳性（+++）表达。病毒组actin呈弱阳性（+）表达,与正常组比较,差异显著,P＜0.05。中药治疗组actin呈阳性（++）表达,与病毒组比较,P＜0.01。⑧透射电镜检测益气活血中药复方对CVB₃病毒性心肌炎心肌细胞超微结构的影响:CVB₃感染组与正常组心肌细胞相比,细胞器损伤严重,表明CVB₃病毒性心肌炎模型建立成功,而中药干预组心肌细胞形态及细胞器结构接近正常组,与模型组比较差异明显,表明益气活血中药复方能防御并减轻CVB₃造成的心肌损伤,具有修复损伤心肌细胞超微结构的作用。⑨荧光漂白恢复（FRAP）检测CVB₃病毒性心肌炎心肌细胞间缝隙连接通讯功能:正常心肌细胞间缝隙连接通讯功能良好,病毒组心肌细胞漂白后荧光恢复率显著低于正常组心肌细胞,差异明显,P＜0.05,中药组漂白后荧光恢复率明显升高,与病毒组比较差异显著,P＜0.01。⑩益气活血中药复方对CVB₃病毒性心肌炎心肌细胞游离钙浓度的影响:益气活血中药复方对正常心肌细胞静息状态内钙无明显影响,P＞0.05,而能提高CVB₃攻击的心肌细胞内钙离子浓度,差异明显,P＜0.05。结论:1.以新生大鼠原代心肌细胞体外培养方法,成功建立了CVB₃攻击的心肌细胞感染模型。2.本实验从免疫、分子、蛋白及细胞超微结构等层次水平上,揭示益气活血中药复方防治病毒性心肌炎心肌损伤机制,体现了中医药学着眼于“整体”的辨治理念。3.通过对细胞凋亡、细胞内游离Ca²⁺、细胞间缝隙连接通讯、Cx43、actin、cTnI及心肌细胞超微结构等进行功能学检测和形态学观察,证实了以益气活血药为主组成的中药复方,具有多作用、多层次、多靶点效应。4.本实验进一步证实了益气活血法是治疗VMC的有效法则,而益气活血中药复方是治疗VMC的有效方剂,提示本方在VMC的治疗中具有广阔的应用前景。更多还原

【Abstract】 Purpose:Viral myocarditis refers to infection caused by a variety of in order to myocardial cell degeneration and necrosis,interstitial inflammatory cell infiltration and fibrous exudation for cardiac disease,major changes occur. Its pathogenicity to Coxsackie virus group B virus（CVB） is the main,and in the six CVB serotypes in,CVB₃ Anopheles strong mind,therefore,the current principal for the establishment of viral infection in CVB₃ myocarditis model to carry out the research,the subject of viral myocarditis to choose research subjects as a main reason is that this disease has a high self-harm,such as children and adults of all ages can be involved stage disease,acute period, and into a dilated the possibility of cardiomyopathy at the same time,can also be caused by small-scale outbreaks,and viral myocarditis in recent years the incidence of the rise is,therefore,against CVB₃ viral myocarditis treatment mechanism appears imminent.Subject to the 2-3d newborn rats in vitro neonatal rat myocardial CVB₃ infection in cell culture to establish the VMC model;Hoechst33258 fluorescence detection through cardiomyocyte apoptosis;immunohistochemical detection of myocardial cell connexin Cx43,cytoskeleton - actin（actin）,cardiac troponin I（cTnl） expression;TEM cells infected with CVB₃ myocarditis model of cardiac cell function and morphological changes.Test the transform of Ca²⁺ and the communication function between cell gap junction in myocardialcell dynamic by SLCM in dynamic state.Yiqihuoxue attempt to verify the compound traditional Chinese medicine to relieve myocardial CVB₃ viral myocarditis cell immune injury: inhibition of cardiomyocyte apoptosis occurred;to adjust Ca²⁺ and to improve the inter-cell gap junctional intercellular communication;repair myocardial ultrastructure of the cytoskeleton and the role of,and further confirmed traditional Chinese medicine compound Yiqihuoxue is the treatment of viral myocarditis in CVB₃ valid prescription,Yiqihuoxue treatment of this disease is an effective way. Material and method:①Yiqihuoxue cytotoxicity determination of compound Chinese medicine:Recovery from Hela cells by passage culture bottles after inoculation with 0.2ml of each hole to 96 hole cell culture plate,the home 37℃,5%carbon dioxide incubator to cultivate,when Hela cells were adherent monolayer growth,the measurement Yiqihuoxue largest non-toxic dose of Chinese medicine compound.DMEM culture medium with the traditional Chinese medicine compound Yiqihuoxue injection started from the stock solution concentration of the preparation 11.Each hole 0.2ml,each concentration for 4-hole complex,plus an additional four control normal growth and pore fluid.In 24h,48h observation of cell growth state,a reference hole normal cells to the cytopathic effect is not caused by the largest concentration of drug dilution largest concentration of non-toxic（TD₀）.②CVB₃ virus virulence titration:In 96-well plate in the preparation of single-layer growth of Hela cells,jumped to the growth plate in the liquid to wash the culture fluid HBSS hole 2.Containing 2%fetal bovine serum with the maintenance of fluid,the CVB₃ virus to 10-fold dilution concentrations from 10^-1 to 10^-8,each hole 0.1ml,inoculated in 96-well plate,each concentration for 4-hole complex,another hole four normal controls,the increase in the maintenance of liquid 0.1ml,at 37℃,5%carbon dioxide incubator to cultivate, respectively 24h,48h observation cell lesions.③Yiqihuoxue Chinese herbal compound on CVB₃-induced myocardial cell death inhibition（MTT method）:Logarithmic phase from myocardial cells,0.2ml of each hole by adding half the volume of the concentration of tissue infections myocytes CVB₃ virus adsorption 2h.Suction disposable culture medium containing the virus, containing 2%fetal bovine serum to the maintenance of gently washing liquid on it,adding the largest non-toxic drug dosage from the initial dilution of the concentration of 8 Yiqihuoxue Chinese herbal compound containing the growth solution,0.2 per hole ml,each concentration for 4-hole complex,a separate zero-hole.In 48h,each hole by adding 0.18ml DMEM culture medium,then add 0.02ml MTT solution into incubator to continue to foster 4h,carefully suction hole disposable culture medium,each hole by adding 0.15ml DMSO,the oscillation oscillator home 8min,in enzyme-linked immunosorbent assay detector 490 nm of the hole measured absorbance（OD）.④Yiqihuoxue Chinese herbal compound on CVB₃ viral myocarditis the apoptosis of myocardial cells:Isolation and culture of normal neonatal rat myocardial cells,inoculated in 24-hole cell culture plate so climbing film,48h after the adherent monolayer of Health check up to 70-80% of the normal group,the virus group,the intervention group of Chinese medicine myocytes glass to PBS wash 2,each hole by adding glacial acetic acid methanol fixed 10min,PBS washed 2 times,each hole Stain Hoechst33258 adding 1ml （5μg/ml）,light response 15min,PBS washed 3 times,each time 3min,dropping anti-0.05ml fluorescence quenching letters do mount tablet treatment,observed under the fluorescence microscope.⑤Yiqihuoxue Chinese herbal compound on CVB₃ viral myocarditis myocardial cells cTnI,Cx43,actin expression:Using SABC immunohistochemical method was used to detect cTnI,Cx43,actin expression: incubation with 3%H2O2 cells long slide 10min,PBS wash.Dropwise closed normal goat serum solution,incubated at room temperature for 15 minutes to dump,do not wash.Dropwise a 1:100 dilution of first antibody,37℃and incubated 2h, PBS wash,dropwise biotinylated second antibody,37℃incubated 30min.PBS washed 3 times,each time 5min.SABC complex dropwise,37℃wet box incubated 30min.PBS washed 3 times,each time 5min,DAB color,tap water rinse,hematoxylin staining,dehydration,transparent processing,resin mount,were observed.⑥Yiqihuoxue TEM testing of Chinese medicine compound of CVB₃ viral myocarditis ultrastructure of myocardial cells:Cultured myocardial cells to normal 72h after washing with normal HBSS solution,the virus group,the Chinese Medicines Board of myocardial cells,0.25%trypsin digestion of the 3-5min,the collection of cells to centrifuge tube 1.5mlEP in,1500rpm centrifugal 10min,the supernatant discarded in order to 2.5%glutaraldehyde fixed power microscope.⑦FRAP testing CVB₃-induced myocardial cell communication function cell gap junction:viral myocarditis cardiac myocyte check up to 70-80%add to 10μg/ml 6—CFDA: application liquor washed by Ca2+,Mg2+ PBS,37℃incubated 5%carbon dioxide 30min.After that washed by Ca2+,Mg2+ PBS again,then test it by FRAP.⑧Yiqihuoxue Chinese herbal compound on CVB₃ myocardial cells Ca2+:put culture of normal neonatal rat myocardial cells,at 37℃,incubator to cultivate box under Fluo-3 AM Ca2+ by fluorescent probe,after hank srinsing put it on SLCM to test.Results:①Yiqihuoxue cytotoxicity determination of compound Chinese medicine: Yiqihuoxue Chinese medicine to determine the maximum non-toxic dose of compound 7.813mg/ml。②CVB₃ virus virulence titration:CVB₃ virus,50%of cultured cells infected with 1×10^-4.5。③Yiqihuoxue Chinese herbal compound on CVB₃-induced myocardial cell death inhibition（MTT method）:Yiqihuoxue largest non-toxic dose of Chinese medicine compound CVB₃ intervention of myocardial cells after the attack,the absorbance values the most,with other dilution group,a significant difference,P＜0.001,confirmed that the largest Chinese herbal compound Yiqihuoxue with non-toxic dose inhibited CVB₃-induced myocardial cell death role.④Yiqihuoxue Chinese herbal compound on CVB₃ viral myocarditis the apoptosis of myocardial cells:Normal myocardial nuclei were round,oval,no apoptotic nuclei,and 100×were observed myocardial cell virus group,showing a blue light into tablets,400×microscope observation shows that incomplete nuclear morphology,was debris-like distribution,but also lobulated nucleus can be seen.Cardiac nuclear medicine mostly round,no obvious nuclear morphology of apoptosis,suggesting that traditional Chinese medicine compound Yiqihuoxue CVB₃ infected cells can inhibit apoptosis,myocardial protective role.⑤Yiqihuoxue Chinese herbal compound on CVB₃ viral myocarditis cTnI expression of cell:Immunohistochemical detection of cardiac muscle Ca I（cTnI） expression showed that the normal group there was no apparent cTnI positive brown staining. CTnI-infected group showed strong positive（+++） expression of more positive signals tan color and depth,with the normal group the average optical density of positive goal difference significant,P＜0.05.CTnI Chinese intervention group were weakly positive（+） expression levels were significantly decreased compared with the virus,a significant difference,P＜0.01.⑥Yiqihuoxue Chinese herbal compound on CVB₃ viral myocarditis cardiac myocyte expression of Cx43: Immunohistochemical detection of Connexin 43 protein found in normal myocardial cells were strongly positive（+++） expression of Cx43 and the virus was very weakly positive（±） expression,and the normal group the average optical density of positive goals,the difference is extremely significant,P＜0.001.Chinese medicine treatment group Cx43-positive（++） expression,and virus group,a significant difference,P＜0.05.⑦Yiqihuoxue Chinese herbal compound on CVB₃ viral myocarditis cardiac myocyte expression of actin:Immunohistochemical detection of actin（actin） showed that normal myocardial cells were strongly positive（+++） actin expression.Actin virus was weakly positive（+） expression, compared with the normal group,significant difference,P＜0.05.Chinese medicine treatment group actin-positive（++） expression,and virus group,P＜0.01.⑧Transmission electron microscopy detection of Chinese herbal compound Yiqihuoxue CVB₃ viral myocarditis ultrastructure of myocardial cells:CVB₃ infection of myocardial cells with normal compared to cell injury,indicating that CVB₃ viral myocarditis model the success of the intervention group and the Chinese form of myocardial cells and cell structure close to the normal group, compared with the model group differences,indicating that Chinese Yiqihuoxue and defense compound can reduce the myocardial damage caused by CVB₃,repair damage with the role of myocardial ultrastructure.⑨FRAP testing CVB₃-induced myocardial cell communication function cell gap junction:normal group’s cell gap junction communication was better than model group.P＜0.05.Chinese Yiqihuoxue was also better than model groupP＜0.01.⑩Yiqihuoxue Chinese herbal compound on CVB₃ myocardial cells Ca²⁺:The effect of Yiqihuoxue Chinese herbal compound on Ca²⁺ in normal neonatal rat myocardial cells was not obviously,P＞0.05,but it can improve the model group,P＜0.05.Conclusion:1.To primary neonatal rat cardiac myocytes in vitro methods,the establishment of the myocardial cells attack CVB₃ infection model. 2.Immune from this experiment,molecular,protein and cell levels,such as the level of ultrastructure revealed Yiqihuoxue control of Chinese medicine composed of viral myocarditis myocardial injury mechanisms,reflects the traditional Chinese medicine focus on the "overall" idea of the Diagnosis and Treatment.3.Through apoptosis,CVB₃-induced myocardial cell communication function and cell gap junction Cx43,actin,cTnI and myocardial cell ultrastructure such as functional testing and morphological observation,confirmed the drug mainly in the composition of Yiqihuoxue Chinese medicine compound has a multi-role, multi-level,multi-target effect.4.The experiments further confirmed Yiqihuoxue VMC is an effective treatment of the law,and medicine Yiqihuoxue VMC compound is an effective prescription treatment,suggesting that this treatment in the VMC has broad application prospects.更多还原