基因芯片技术与缺血性脑损伤的基础研究

缺血性脑损伤通常导致神经元死亡或功能障碍,从而引发脑功能障碍.细胞死亡或功能障碍往往伴随或源自特定基因表达的变化.因此,确定参与缺血性脑损伤过程及损伤后细胞修复及塑型过程的基因,对损伤机制及基于该损伤机制而采取适当的治疗措施是至关重要的.基因芯片技术是20世纪90年代发展起来的一项前沿生物技术,它从根本上改变了生理学和生物技术的观念和效率[1].其应用高密度的显微阵列进行大批量的基因筛选,只有巨大的功能信息处理能力,近年来在缺血性脑损伤的研究中得以应用,为该领域研究提供了丰富信息.     

Comparison of the Network Structural Characteristics of Calcium Signaling Pathway in Cerebral Ischemia after Intervention by Different Components of Chinese Medicine

Objective:To explore the network control mechanism of the calcium signaling pathway in cerebral ischemic injury after intervention by the main components of Qingkailing(清开灵),i.e.Baicalin,Jasminoidin and their combination.Methods:Thirty mice were randomly divided into 5 groups,a baicalin group,a Jasminoidin group,a baicalin plus Jasminoidin group,a nimodipine group,and a model group(n=6).The global cerebral ischemia-reperfusion mouse model was established.The mice were administrated respectively by injection of baicalin,Jasminoidin,mixture of baicalin and Jasminoidin,and nimodipine into the caudal vein,with the model group given no any drug.Three hours after operation,the brain was removed and sectioned.After calculation of cerebral ischemic area by 2,3,5-triphenyltetrazolium staining,the percentage of infarct volume was calculated.The total RNA of the mouse brain tissue was extracted to obtain the whole genome expression profile,and the differentially expressed genes related to the calcium signaling pathway was analyzed with Bayesian network structures.Results:Compared with the model group,the ischemic area was significantly reduced in the baicalin group,the Jasminoidin group,the Baicalin plus Jasminoidin group(all P<0.05).The ischemic area in the baicalin plus Jasminoidin group was smaller than the other three groups(all P<0.01).In the gene regulatory network structures of calcium signaling pathway,the average length and equitability were the highest in the baicalin plus Jasminoidin group,followed by the nimodipine group.Conclusion:Compared with a single component,combination of Baicalin and Jasminoidin can more obviously intervene in the overall expression of calcium signaling pathway,and the mechanism is related with the aggregation characteristic of the gene expression network.     

缺血性中风血瘀证患者MTHFR基因多态性研究

目的探讨N^5，N^10亚甲基四氢叶酸还原酶（MTHFR）基因C677T多态性与缺血性中风病血瘀证易感性的关系。方法收集缺血性中风病血瘀证患者84例（病例组），缺血性中风病非血瘀证患者143例（对照组），抽外周静脉血提取DNA，采用聚合酶链反应-限制性内切酶片段长度多态性（PCR-RFLP）检测MTHFR基因C677T多态性类型。结果2组MTHFR基因C677T基因型频率分布差异有统计学意义（χ^2=12．618；d．f．=2，P=0．002），突变型等位基因频率病例组高于对照组（分别为39．29％和24．48％），TT基因型与缺血性中风血瘀证发生显著相关（OR=3．730；95％CI为1．229～11．318；P=0．014）。结论MTHFR基因TT型可能是缺血性中风病血瘀证发病的易感危险因素之一。     

三皮汤对小鼠溃疡性结肠炎模型IL-2和IL-10表达的影响

目的:观察三皮汤对溃疡性结肠炎(UC)小鼠的疗效及探讨其治疗机制。方法:40只BALB/C小鼠随机分成正常组、模型组、柳氮磺胺吡啶(SASP)组和三皮汤组,每组10只。予以恶唑酮建立模型后,三皮汤组和SASP组分别给予三皮汤和SASPig,每天观察小鼠体重、大便、饮食等情况,10天后处死小鼠,称脾重,取其病变结肠观察组织学改变,并采用酶联免疫吸附法(ELISA)测定各组小鼠血清白细胞介素(IL)-2及IL-10的含量。结果:三皮汤组和SASP组小鼠症状较模型组有明显改善;三皮汤组和SASP组小鼠脾组织重量较模型组有所增加;三皮汤组IL-2含量高于模型组(P<0.01),IL^-10含量低于模型组(P<0.01)。结论:三皮汤对UC有确切疗效,且能升高血清IL-2及降低IL-10的含量,调整细胞因子间的网络平衡,改善机体免疫功能,控制肠道炎症。     

中医药治疗缺血性脑卒中研究进展

缺血性脑卒中是中老年人常见病、多发病之一。我国每年新增脑卒中患者150万～180万,其中缺血性脑卒中约占43%～65%。本病中医多责之于气血失调、痰瘀作祟,属于＂中风＂范畴。中医药治疗本病具有广阔的前景,发挥了一定作用,现就中医药治疗急慢性脑梗塞综述如下。     

清开灵有效组分药理通路的多样性分析

目的比较清开灵有效组分BA(黄芩苷)、JA(栀子苷)、CA(胆酸)与CM(珍珠母)在治疗脑缺血过程中药理通路变化机制。方法将脑缺血模型小鼠随机分为BA、JA、CA、CM和M(模型组),每组15只,脑缺血后2h予以相应药物治疗,24h后断头取脑,抽提小鼠脑海马组织的总RNA,利用与脑缺血相关的374个基因的cDNA芯片检测基因表达谱变化。将所有数据标准化处理后,以Arraytrack软件为平台,选取BA与M,JA与M,CA与M和CM与M两组间t检验得出的差异基因(P<0.05,Foldchange>1.5),将差异表达基因按照Genebank ID上传到GeneGo数据库,选取P<0.05的信号通路,选取各组分P值最小的前2位药理通路分析不同组分的药效机制。结果BA、JA、CA、CM与M组比较后差异基因数量分别为46、50、54和30条,根据这4个组分差异基因相似度最大的前2位信号通路可看出JA、CA、CM都参与凋亡与存活-TNFR1信号通路的调控,另外BA表现为对G蛋白信号和A2A受体信号的调控作用,CA表现为调节NMDA依赖性的LTP的调节。结论清开灵组分发挥药效作用具有多样性特点,BA主要表现为抗凋亡,JA主要表现为抑制细胞凋亡和促进缺血后脑保护等方面,CA侧重于抑制钙离子内流,抗神经元变异方面,但CM效果不甚理想。     

黄芩苷、栀子苷及其配伍治疗局灶性脑缺血的海马基因表达：一项基因网络功能分析

The compound traditional Chinese medicine Qingkailing,which is an ingredient used to treat cerebral ischemia,has been limited to studies concerning single genes or single pathways.Interactions and pharmacological mechanisms of the compound ingredients(baicalin and jasminoidin) remain poorly understood.In the present study,baicalin and jasminoidin,as well as the combination,were used to separately treat mouse models of cerebral ischemia.cDNA microarray analyses of 374 cerebral ischemia-related genes were utilized to determine changes in gene-expression profiles.Arraytrack 3.40 and Ingenuity Pathway Analysis(IPA) databases were utilized to analyze changes in gene molecular functions and network path functions.Baicalin or jasminoidin alone effectively reduced infarct area,and the combination resulted in significantly better outcomes.IPA showed inhibited cell apoptosis in the baicalin group and Ca2+ channel regulation in the jasminoidin group.The combination of baicalin and jasminoidin activated HTR3A and F5 expression,regulated Ca2+ channels,activated kappa light polypeptide gene enhancer inhibitor IKBKG in B cells to control IkB kinase/nuclear factor-kB cascade,suppressed activation of inflammatory cytokine interleukin-6 receptors and activated transduction of guanine-nucleotide-binding protein(G protein) signal.Results suggested that the combination of baicalin and jasminoidin resulted in similar molecular mechanisms to baicalin and jasminoidin alone.However,novel pharmacological actions of compatibility were detected,demonstrating significant protection against cerebral ischemia.     

用主成分分析探索不同中药组分配伍干预脑缺血的基因表达模式

目的比较清开灵主要组分黄芩苷、栀子苷及其配伍干预脑缺血损伤的基因表达模式。方法12只4周龄小鼠随机分为黄芩苷组(BA组)、栀子苷组(JA组)、栀子苷+黄芩苷组(BJ组)、尼莫地平组(NM组),建立全脑缺血再灌注小鼠模型后,各组分别给予尾静脉注射黄芩苷、栀子苷、栀子苷+黄芩苷以及尼莫地平。抽提小鼠脑组织的总RNA,制备小鼠全基因组表达谱,对其进行基因本体论功能分类,构建相关系数矩阵,对差异表达的细胞黏附相关基因进行主成分分析。结果获取16463个小鼠脑测全基因组表达谱,筛选出32个差异表达的细胞黏附相关基因。主成分分析结果显示,4组的前2个主成分的累积贡献度为100%,BJ组总得分大于单一组分组,NM组对细胞黏附相关基因干预的总得分高于BA和JA组。各次检测的主成分分布显示,NM组和3个中药组均距离较远,BJ组和BA组的距离均近于JA组。结论黄芩苷、栀子苷及其配伍均能有效干预细胞黏附相关基因,配伍后的干预对黏附相关功能基因的影响大于单一组分组,其基因表达模式和黄芩苷相近。     

清开灵有效组分调节小鼠海马缺血相关基因的共性与多样性分析

目的:比较清开灵组分黄芩苷(BA),栀子苷(JA),胆酸(CA)与珍珠母(CM)在治疗海马缺血过程中基因表达变化机制。方法:75只6周龄小鼠随机分为BA,JA,CA,CM,M(模型组),每组15只,建立海马缺血再灌注小鼠模型,2 h后实验组分别经尾静脉按3 mL.kg-1体重注射相应药物,24 h后断头取脑制作冠状切片,TTC(氯化四唑)染色,计算不同组别梗死体积百分比;同时抽提小鼠海马组织的总RNA,利用与脑缺血相关的374个基因的cDNA芯片检测基因表达谱变化,采用Ar-raytrack软件选取P0.05,Fold change1.5的差异基因,根据差异基因的GO功能探讨药效作用特点。结果:除CM组外,BA,JA,CA与模型组比较均能有效减少海马缺血梗塞面积(P0.05)。BA,JA,CA与CM比较差异基因数量分别为41条(上调24,下调17),22条(上调13,下调9),11条(上调8,下调3),它们共同抑制Myb基因的表达。结论:BA,JA,CA产生药效结果不仅有共同的基因作用靶点和作用机制,同时还存在多样性的特点。