Establishment of diagnostic system on Kampo Medicine with gene analysis.

用荧光差异显示和DNA芯片方法鉴定经丹参或汉庚木托处理后表达发生变化的基因。首先，通过荧光差异显示进行分析。使用从受试者制备的RNA（作为遗传信息信使的核酸）作为模板，通过用荧光染料标记的oligo（dT）引物的逆转录反应合成cDNA。将锚引物和任意序列引物用于聚合酶链反应以获得扩增的基因产物（在荧光差异显示中，聚合酶链反应发生的次数与引物的数量一样多）。对扩增的基因产物进行电泳以筛选汉方药物治疗前后表达发生变化的基因，然后进行基因分离、克隆和测序以鉴定目的基因。接下来，进行DNA芯片方法。对于每种汉方药物，验证了这些基因表达变化的测量，并鉴定了汉方药物应答者的特定基因表达模式。将表达随汉方药物特异性变化的基因分组，以确定与汉方药物药理作用相关的基因组。发现一些基因在这两种汉方药物的应答者中具有特征性表达。一组具有两种汉方药物应答者特征性表达谱的基因在用药后未显示其表达变化，表明基因的表达谱和序列的影响。特别是在对阿托伐他汀的反应者中，一些基因显示出强烈暗示与疗效相关的表达谱。这些结果表明，药物治疗前的基因表达谱可能会识别出可能从汉方药物治疗中受益的患者。

Fluorescent differential display and DNA chip methods were used to identify genes whose expression changed after treatment with Kososan or Hangekobokuto. First, analysis was performed by fluorescent differential display. RNA (nucleic acids functioning as a messenger of genetic information) prepared from subjects were used as a template to synthesize cDNA by reverse transcription reaction with a fluorescent dye-labeled oligo (dT) primer. The anchor primer and arbitrary sequence primers were used for a polymerase chain reaction to obtain amplified gene products (in fluorescent differential display, polymerase chain reactions occurred as many times as the number of primers). The amplified gene products were subjected to electrophoresis to screen genes whose expression changed before and after Kampo medication, followed by gene isolation, cloning, and sequencing for identification of the genes of interest. Next, a DNA chip method was performed. For each Kampo medicine, measurement of the change in expression of these genes was validated, and specific gene expression patterns in responders to Kampo medication were identified. Genes whose expression specifically changed with each Kampo medication were classified into groups to identify groups of genes related to the pharmacological effects of Kampo medicines.Some genes were found to be characteristically expressed in responders to each of these two Kampo medicines. A group of genes with expression profiles characteristic of responders to each of the two Kampo medicines showed no change in their expression after medication, suggesting influences of expression profiles and sequences of the genes. Particularly in responders to Kososan, some genes showed expression profiles strongly suggestive of an association with efficacy. These results suggest that gene expression profiling before medication may identify patients who are likely to benefit from Kampo medication.