益气活血中药复方影响病毒性心肌炎心肌细胞基因表达的实验研究

【作者】 刘峻；

【导师】 张明雪；

【作者基本信息】 辽宁中医药大学 ， 中医内科学， 2011， 博士

【摘要】 研究目的:病毒性心肌炎（Viral myocarditis,VMC）是临床上最常见的感染性心肌病,它是由嗜心性病毒感染所引起的心肌细胞变性、坏死和间质炎性细胞浸润以及纤维渗出等为主要改变的心肌疾病,可进一步并发严重的心律失常、心力衰竭、心源性休克甚至猝死,亦可演变为扩张型心肌病。导致病毒性心肌炎的病毒中,最为主要是柯萨奇B组病毒（CVB）,在CVB的六个血清分型中,CVB3具有强嗜心性,所以,近来开展的各项研究主要是针对以CVB3感染所建立的病毒性心肌炎模型。因为VMC自身具有极高的危害性,所以本课题选择病毒性心肌炎作为研究的主要对象,由于小儿及成人的各个年龄段均可感染患病,急性期后,又有可能进一步演变为扩张型心肌病,并且VMC还可引起小范围的爆发流行,特别是近年来病毒性心肌炎的发病率更是呈现上升态势,所以,针对CVB₃病毒性心肌炎的治疗机制的研究更加显得尤为重要。本研究是在中医脏腑理论学说及伤寒、温病学说等研究的基础上,结合多年的临床诊治经验,发现“气虚血瘀”是病毒性心肌炎发生的主要内在因素,并据此精心筛选出由黄芪、白参、郁金、丹参、麦冬等药组成的益气活血中药复方来治疗病毒性心肌炎,具有显著的抗心律失常和减轻心肌损伤等作用,但该中药复方的具体作用机制尚不明确。因此,本研究拟建立CVB3心肌细胞感染模型,利用改良的抑制性消减杂交（suppression subtractive hybridization ,SSH）等分子生物学技术,来探讨受CVB₃攻击的宿主细胞中与益气活血中药复方观察组中趋化因子CXCL12、Sash1、small inducible cytokine A2、ribosomal protein S4基因的表达变化,以期进一步从基因水平揭示益气活血中药复方调控VMC的作用靶标和途径信息,为深入研究该方在病毒性心肌炎治疗过程中所具有的生物学效应和意义提供新的理论基础和实验依据,从而进一步证实益气活血法是治疗病毒性心肌炎的有效方法,益气活血中药复方是治疗CVB₃病毒性心肌炎的有效方剂。材料与方法:1实验材料1.1实验动物新生2-3天wistar乳鼠（辽宁中医药大学实验动物中心负责提供）。1.2实验药品及相关试剂特级胎牛血清（灏洋生物,天津）、高糖DMEM（Gibco,美国）、硫酸链霉素（美罗大药厂,大连）、青霉素钠（华北制药股份有限公司,石家庄）、碳酸氢钠（天津市氨基酸公司,天津）、MTT（Sigma,美国）、胰蛋白酶（Invitrogen,美国）、DMSO（Sigma,美国）、PBS（博士德,武汉）等。ANT（santa,美国）、PFP（santa,美国）、α-MHC（abcam,美国）、DAB显色试剂盒（博士德,武汉）、SABC免疫组化（过氧化物酶）试剂盒（博士德,武汉）、Triton X-100（Solarbio,北京）、抗原修复液（博士德,武汉）。2实验方法2.1益气活血中药复方的细胞毒性测定:把复苏的Hela细胞提取出来,然后经培养瓶传代,接种到96孔的细胞培养板中,每孔0.2ml,放置于37℃,5%二氧化碳培养箱中进行培养,当出现呈单层贴壁生长的Hela细胞时,测量益气活血中药复方的最大无毒剂量。将益气活血中药复方注射液用DMEM培养液从原液开始配制成11个浓度。每个浓度设置4个复孔,每孔0.2ml,另外设4个对照孔加入正常的生长液。观察细胞生长状态（分别于24h和48h）,参考正常对照孔的细胞形态,最大无毒剂量（TD0）是以不引起细胞病变的最大药物稀释浓度。2.2 CVB₃病毒毒力滴定:在96孔的培养板中制备单层生长的Hela细胞,弃掉生长液,并且各培养孔用HBSS液洗涤2次。将CVB₃病毒以10倍浓度从10-1用含2%胎牛血清的维持液稀释到10-8,接种于96孔的培养板中,每孔0.1ml,每个浓度设4个复孔,另外设4个正常孔对照,每孔加入0.1ml维持液,放置于37℃,5%二氧化碳培养箱中进行培养,观察细胞的病变情况（分别于24h和48h）。2.3益气活血中药复方对CVB₃所致心肌细胞死亡的抑制实验（MTT法）:将50%组织感染量浓度的CVB₃病毒与提取后的对数生长期心肌细胞混合0.2ml/孔,吸附心肌细胞2h。在加入含有益气活血中药复方的生长液之前,需保证该生长液已被稀释8个浓度。核算方法以药物最大无毒剂量为标尺。4个复孔对应一个浓度,在剂量上,设置值为0.2ml/孔,每个浓度还设有1个调零孔。DMEM培养液需在四十八个小时之后才能添加,剂量为0.18ml/孔,一切就绪后,方可放入MTT溶液0.02ml。另外MTT溶液需要再在培养箱中一定时限,即4小时。吸弃孔内培养液,加入DMSO,每孔0.15ml,置于振荡器上,振荡8min,在酶联免疫检测仪490 nm处测量各孔的吸光值（OD）。2.4益气活血中药复方对CVB₃病毒性心肌炎心肌细胞趋化因子CXCL12、SASH1、MCP-1、Rps4基因表达的影响:利用改良的抑制性消减杂交技术（suppression subtractive hybridization,SSH）,分离益气活血中药复方观察组和大鼠心肌细胞CVB₃感染组差异表达的基因,并通过荧光RT-PCR对上述结果进行验证。3实验结果:3.1CVB₃病毒毒力滴定:50%培养细胞CVB₃病毒感染量为1×10^-4.5。3.2益气活血中药复方细胞毒性测定:益气活血中药复方的最大无毒浓度是7.813mg/ml。3.3益气活血中药复方对CVB₃所导致的心肌细胞死亡的抑制实验（MTT法）:CVB₃攻击后的心肌细胞被益气活血中药复方最大无毒剂量干预时,其吸亮度值也最大,和其他稀释度组相比,差异显著,（P<0.0010）,从而可以证实益气活血中药复方的最大无毒剂量具有显著的抑制CVB₃所致心肌细胞死亡的作用。3.4益气活血中药复方对CVB₃病毒性心肌炎心肌细胞趋化因子CXCL12、SASH1、MCP-1、Rps4基因表达的影响:改良的SSH结果显示益气活血中药复方观察组中趋化因子CXCL12、SASH1、MCP-1、Rps4基因的表达比CVB₃病毒组中高,这一结果通过荧光RT-PCR得到验证。实验结论:1.利用体外培养的方法,成功建立了被CVB₃攻击的新生乳鼠原代心肌细胞的感染模型。2.通过观察益气活血中药复方对CVB₃病毒性心肌炎心肌细胞趋化因子CXCL12、SASH1、MCP-1、Rps4基因表达的影响,进一步证实了以益气活血药为主组成的中药复方,具有多靶点、多层面和多作用的效果。3.本课题从更深层次论证了益气活血法对病毒性心肌炎疗效的确切性,而益气活血中药复方是诊治病毒性心肌炎的有效方剂,揭示本方在VMC的治疗中具有广阔的应用前景。更多还原

【Abstract】 Objective: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.With that in mind, this research will establish a CVB3 mycocarditis cell infection model, through certain molecular biological technologies like the improved suppression subtractive hybridization （SSH）, in order to explore the genetic expressive change of the chemotatic factors such as CXCL12、Sash1、small inducible cytokine A2、ribosomal protein S4, which are located in the attacked host cell and in the treatment group that use the method of Chinese Herbal boosting qi and enlivening blood formula （YiQi HuoXue Chinese Herbal Compound）. The objective is to release the regulating function and the mode of Chinese Herbal boosting qi and enlivening blood formula to VMC, so as to further explore its biological effect and significance in the treatment of VMC, and to provide new theoretical basis and experimental basis. The research will also go further in showing that boosting qi and enlivening blood is a proven formula for treatment of VMC. Material and method:1.Research Materials1.1 Experimental Animals Newborn 2-3d wistar rats （provided by LiaoNing Chinese Medical College Department of Experimental Animals）1.2 Experimental Medicine and Reagents High Glucose DMEM（Gibco , USA） , Specialize fetal bovine serum（HaoYang Biological, TianJin）, Penicillin （North China Pharmaceutical Co., Ltd., Shijiazhuang）, Streptomycin sulfate （Merro Pharma, Dalian）,Sodium bicarbonate （Tianjin Amino Acid Company, Tianjin）,MTT（Sigma, USA）, DMSO（Sigma, USA）, Trypsin（Invitrogen, USA）, PBS（Boster, WuHan）. PFP （Santa, USA）, ANT （Santa, USA）, a-MHC（Abcam, USA）, SABC immunohistochemistry （peroxidase）Kit （Boster, Wuhan）, Antigen retrieval solution （Boster, Wuhan）, Chromogenic DAB kit （Boster, Wuhan）, Triton X-100（Solarbio, Beijing）.2.Research Methods2.1 Yiqihuoxue 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）.2.2 CVB3 Virulence Titration: In the 96-well plate dispose of the mono-layer Hella cell growth and wash each well twice with the HBSS solution. Using 2%fetal bovine serum as suspension, add ten concentrations of CVB3 virus diluted from 10-1 to 10-8. Each well with 0.1ml. On the 96 well plate, each concentration will be placed into four wells and four additional wells as a control. Each solution will be kept at 0.1ml and placed into a 5% Carbon Dioxide incubator and kept at 37oC. Cellular pathology will be observed at 24h and 48h. observation cell lesions.2.3 Yiqihuoxue 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）.2.4 the influence of YiQi HuoXue Chinese Herbal Compound to the genetic expression of Viral myocarditis,（VMC） chemotatic factors such as CXCL12, Sash1, MCP-1, ribosomal protein S4: through the improved suppression subtractive hybridization （SSH）, the rat cardiac muscle cell CVB3 infection group and the gene that has a differential expression in YiQi HuoXue Chinese Herbal Compound group will be demeshed. The result wil be proved through FL RT-PCR.3 Experimental Results:3.1 Determination of Boosting Qi and Enlivening Blood Formula cytotoxicity: the maximum non-toxic dose for the formula is 7.813mg/ml.3.2 CVB3 virulence titration: the infection rate of 50% cultured CVB3 virus cells is 1×10^-4.5. 3.3 Boosting qi and enlivening blood formula inhibition of myocardial cell death from CVB3 virus（MTT method）:use of maximum non-toxic dose boosting qi and enlivening blood formula on post-attack myocardial cells, had the largest absorption value, and in comparison with other dilutions there was a clear difference, p<0.001. This shows that the maximum non-toxic dose of boosting qi and enlivening blood formula has a clear inhibitory effect on CVB3 induced myocardial cell death.3.4the influence of YiQi HuoXue Chinese Herbal Compound to the genetic expression of Viral myocarditis,（VMC） chemotatic factors such as CXCL12, Sash1, MCP-1, ribosomal protein S4: The results of SSH showed that genetic expression of CXCL12, Sash1, MCP-1, ribosomal protein S4 in treatment group were higher than that in CVB3 infection group. The results of fluorescent RT-PCR agreed with that of SSH.Conclusion:1.To primary neonatal rat cardiac myocytes in vitro methods, the establishment of the myocardial cells attack CVB3 infection model.2.Through observing the influence of YiQi HuoXue Chinese Herbal Compound to the genetic expression of Viral myocarditis,（VMC） chemotatic factors such as CXCL12, Sash1, MCP-1, ribosomal protein S4, the multi-functional, multistrata, multi-targeting effect of YiQi HuoXue Chinese Herbal Compound have been confirmed.3.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