肾阴虚证和肾阳虚证基因表达谱的比较研究
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摘要
【研究背景与目的】
中医“证”是疾病发生过程中不同阶段病因病机的高度概括,既然同一证有共同的临床表现和病理机制,那么其肯定有共同的物质基础,而这种物质基础很有可能反应在基因或基因组水平上。肾所藏之精禀受于父母,是构成胚胎发育的原始物质,其与现代医学描述的遗传物质(DNA)具有一定的同一性。因此,从基因组方面深入研究肾虚证的分子机制对于阐明肾虚证本质有着重要意义。肾阴虚证和肾阳虚证是中医学的基本证候,历代医家对肾阴虚证和肾阳虚证的理论与防治研究都颇为重视。肾阴虚证和肾阳虚证作为疾病的某一阶段的主要矛盾,必然受到“病”这一基本矛盾的影响。正是由于不同疾病的特异性,决定了不同疾病相同中医证候之间的差异;而同一疾病不同中医证侯之间也存在差异。只有通过对这些差异的研究,进而归纳出证的一般规律,才有可能对肾阴虚证和肾阳虚证有更全面的解析。因此,“病证结合”是研究肾阴虚证和肾阳虚证差异基因表达的重要思路。
本课题组在先前的肾阴虚证相关基因研究过程中,通过西医辨病与中医辨证相结合,从糖尿病、慢性肾炎、狼疮性肾病以及亚健康状态等肾阴虚证入手,应用RNA微量扩增、抑制性消减杂交、基因克隆等技术和方法,成功地构建了肾阴虚证的cDNA文库;结果发现,上述不同病种肾阴虚证的消减文库中存在明显差异,而亚健康肾阴虚证的消减文库与共同文库很相似。这提示构建的肾阴虚证cDNA消减文库必定受到了疾病的干扰,即筛选出的部分差异基因可能与该疾病有关却与肾阴虚证无关,亚健康肾阴虚证的消减文库受到的干扰最小。这可能是因为亚健康是介于健康和疾病之间的中间状态,虽然未达到疾病,但已表现为中医证候,属于功能性改变。这提示亚健康对于中医证候本质研究有着非常重要的意义。
基因芯片技术的出现是生物信息分析领域的一个革命性的里程碑,它具有高通量、微型化和自动化的特点,已成为高效、快速、大规模获取相关生物信息的重要手段。作为一种新兴的生物技术,基因芯片一出现即引起世界的广泛关注。目前,基因芯片技术已广泛应用于中医药研究的许多领域。怎么把西医的病与中医的证统一起来呢?基因表达谱也许是重要的连结点。因此,基因表达谱可为中医证候本质研究提供前所未有的契机。
综上所述,本研究拟在前期研究的基础上,采用“病证结合”的方法,应用基因芯片技术,开展肾阴虚证和肾阳虚证基因表达谱的比较研究,寻找肾阴虚证和肾阳虚证的差异表达基因,探讨肾阴虚证和肾阳虚证在基因水平可能的发生机制,为肾阴虚证和肾阳虚证的本质研究提供科学依据。
【方法】
1、采用“病证结合”方法,选取IgA肾病以及亚健康状态肾阴虚证和肾阳虚证患者作为研究对象,确诊为肾阴虚证和肾阳虚证患者,作为实验组;选择健康志愿者作为正常对照组。
2、收集样本,制备样品,运用基因芯片技术进行芯片杂交和扫描分析,研究肾阴虚证和肾阳虚证的基因表达谱。根据基因芯片分析结果,随机选择肾阴虚证和肾阳虚证的差异表达基因,进行RT-PCR验证。
3、结合MAS软件分析,并使用检索互联网生物学公共数据库(http://www.genesifter.net/web/index.html)提供的检索程序对差异表达基因进行分析。对差异表达基因进行GO注释(http://www.geneontology.org),并利用KEGG(kyotoencyclopedia of genes and genom,网址http://www.genome.ad.jp)、BioCarta(http://www.bioCarta.com)和GenMAPP(http://www.genmapp.org)作pathway分析。
【结果】
1、肾阴虚证组和正常对照组间存在差异基因表达特征图谱。获得差异表达基因共79条。其中,表达水平上调(Ratio值大于2)的有6条,表达水平下调(Ratio值小于0.5)的有73条。这些差异表达的基因按功能分析进行大体分类,主要涉及到免疫功能、新陈代谢、蛋白质生物合成、氧化应激、遗传信息过程、离子通道、蛋白质氨基酸去磷酸化、细胞凋亡、细胞信号转导有关等方面;这些差异表达的基因涉及到多个信号转导通路,如轴突导向信号转导通路、蛋白磷酸酶信号转导通路、细胞质囊泡信号转导通路、G蛋白信号转导通路、钙信号转导通路、丝裂酶原活化蛋白激酶信号转导通路等。
2、肾阳虚证组和正常对照组间存在差异基因表达特征图谱。获得差异表达基因共75条。其中,表达水平上调(Ratio值大于2)的有22条,表达水平下调(Ratio值小于0.5)的有53条。这些差异表达的基因按功能分析进行大体分类,主要涉及到免疫应答、代谢通路、信号通路、细胞因子、细胞凋亡、细胞受体、细胞骨架和运功、氧化应激、离子通道(钙、镁、锌、铜)、DNA结合、转录因子、蛋白质翻译合成等方面;这些差异表达的基因涉及到多个信号转导通路,如粘着连接通路、细胞因子间受体相互作用通路、MAPK(丝裂酶原活化蛋白激酶)信号转导通路、Wnt信号转导通路、钙信号转导通路、激活蛋白受体信号通路、胰岛素信号通路、镁离子结合信号通路、TGF-β信号转导通路等。
3、通过比较分析,肾阴虚证组和肾阳虚证组基因表达谱存在明显的差异,全部的差异表达基因有145条,详细分析见上述第1、2条。肾阴虚证组、肾阳虚证组与正常对照组比较,二者共同的差异表达基因有9条如CA8(碳酸酐酶Ⅷ)、SYPL1(突触泡蛋白样1)、PHF14(PHD指蛋白14)、ARHGAP18(Rho GTP激活蛋白18)、PYROXD1(吡啶核苷酸氧化还原酶1)、RECQL(RecQ蛋白样、DNA解旋酶Q1样)、OSTF1(破骨细胞刺激因子1)、SEC23B(Sec23同源物B)、231418_at等。主要涉及到免疫功能、新陈代谢、细胞周期、骨骼发育、DNA修复、蛋白质的合成等方面的功能。另外一条基因231418_at功能尚不明确。
【结论】
1、基因芯片是研究中医证候相关基因的比较理想的一种技术方法。
2、采用“病证结合”,开展肾阴虚证和肾阳虚证基因表达谱的比较研究,初步获得肾阴虚证的相关基因,共79条;初步获得肾阳虚证的相关基因,共75条。
3、肾阴虚证与肾阳虚证差异表达基因存在明显不同,提示可以为肾阴虚证和肾阳虚证的中医辨证分型提供科学依据;与正常对照组比较,肾阴虚证与肾阳虚证存在共同的差异表达基因,表明肾阴虚证与肾阳虚证都存在肾虚,这不但为研究中医证侯本质提供科学的思路与方法,也为探讨肾阴虚证和肾阳虚证在基因水平的发生机制及进一步研制肾阴虚证和肾阳虚证的基因芯片奠定基础。
[ Background]
The syndrome of Traditional Chinese Medicine is the summarization of etiopathogenisis in different stage of diseases. Since same symptome have common clinical manifestation and pathomechanism, then they have common material foundation, but this material probably concerned with gene or genome. The essence in kidney which come from essential substance for reproduction of the parents, is the hyle of embryonic development, it identity with DNA in modern medicine. Therefor, it is significant to study the molecular mechanism of nephrasthenia syndrome from the aspect of genome, in elucidation substance of nephrasthenia syndrome. As both of the basic syndrome, Kidney-yin deficiency and Kidney-yang deficiency is, family history of Kidney-yin deficiency and Kidney-yang deficiency medical certification and control of the theory are quite seriously. Syndrome of Kidney-yin deficiency and syndrome of Kidney-yang deficiency reflects functions of the overall body immediately, this basic contradiction is the "disease" of the interference. The difference between syndrome of Kidney-yin deficiency and syndrome of Kidney-yang deficiency in different diseases is because of the specificity of diseases. Only to study the difference and to induce general rule of syndrome, then we have all round knowledge of syndrome of Kidney-yin deficiency and syndrome of Kidney-yang deficiency. Accordingly, it is necessary to combine disease with syndrome to research syndrome of Kidney-yin deficiency and syndrome of Kidney-yang deficiency of differential gene expression.
cDNA Lib of Kidney-yin deficiency (including diabetes, chronic nephritis, lupus nephropathia and sub-health)was successfully set up in our prior study works by some techniques and methods, such as RNA microamount-amplification, differential hybridization and gene cloning. The results showed that Kidney-yin deficiency of the different diseases in the subtractive library card there is an obvious difference, and Kidney-yin deficiency sub-health subtractive library card is very similar to the common library. This prompted the construction of subtractive cDNA library of Kidney-yin deficiency and evidence of the disease must be interference. That is, differences in selected parts of the gene may be associated with the disease and Kidney-yin deficiency of the card has nothing to do with the interference of the disease, the most minimum disruption is Kidney-yin deficiency of sub-health in subtractive library card. This may be due to sub-health between health and disease is intermediate, although the disease does not meet, but has shown Chinese medicine syndromes are functional changes, it is most pure syndromes in the nature. This indicates that it is very important significance of the sub-health for the essence of TCM syndrome research.
The technology of gene chip is a revolutionary milestone of bioinformatics analysis field, It has a high-throughput, miniaturization and automation features, has become a highly efficient, rapid and large-scale access to information related to an important means of biological. As an emerging bio-technology, gene chip is widespread concern around the world. At present, The technology of gene chip has been widely used in many areas of TCM research. How the disease to Western medicine and the syndrome of traditional Chinese medicine are unified together? Gene expression may be an important link point. Therefore, gene expression offered unprecedented opportunity for research the essence of TCM Syndrome.
With the method of disease combined with syndrome and the technology of gene chip, to complete the comparative study of gene expression of Kidney-yin deficiency and Kidney-yang deficiency. Accordingly, the objective of the study is to find differential expression gene of Kidney-yin deficiency and Kidney-yang deficiency and to approach the mechanism of Kidney-yin deficiency and Kidney-yang deficiency in the level of gene.
[Methods]
1、Using "disease combined with syndrome", the patients with Kidney-yin deficiency and Kidney-yang deficiency of sub-health or IgA nephropathy were divided into experimental group. Those healthy people were divided into normal control group.
2、Collected cases and preparation samples, used of gene chip technology for chip hybridization and scanning analysis. Study on gene expression profiles of Kidney-yin deficiency and Kidney-yang deficiency. According to the results of gene chip analysis, randomly selected differential expressed genes of Kidney-yin deficiency and Kidney-yang deficiency for RT-PCR validation.
3、Combination of MAS software, analysis differential gene expression tosearch the internet using public biological databases by http://www.genesifter. net/web/index.html and http://www.geneontology.org. Pathway is analysised by KEGG(kyotoencyclopedia of genes and genome, http://www.genome.ad.jp ) and BioCarta(http://www.bioCarta.com) and GenMAPP (http://www.genmapp.org) .
[Results]
1、There is characteristic profiles of differential expressed genes between Kidney-yin deficiency group and normal control group. Among a total of 79 differential expressed genes. Increase the expression level (Ratio values greater than 2) has six, Reduced expression level (Ratio value of less than 0.5) there is the 73 .These differentially expressed genes in their analysis according to the functions of the general classification. Mainly genes related to immune function, metabolism, protein biosynthesis, oxidative stress, the process of genetic information, ion channels, protein amino acid phosphorylation go, apoptosis, cell signal transduction. These differentially expressed genes involves a number of signal transduction pathway, such as, Axon guidance pathway、Phosphoprotein phosphatase activity pathway、Cytoplasmic vesicle pathway、G Protein Signaling pathway、Calcium signaling pathway、MAPK signaling pathway, etc.
2、There is characteristic profiles of differential expressed genes between Kidney-yang deficiency group and normal control group. Among a total of 75 differential expressed genes. Increase the expression level (Ratio values greater than 2) has 22, Reduced expression level (Ratio value of less than 0.5) there is the53 . Mainly genes related to immune response, metabolic pathways, signal transduction pathways, Cytokines, apoptosis, cell receptors, and cytoskeleton, oxidative stress, Ion channels (calcium, magnesium, zinc, copper), DNA binding, transcription factor, protein synthesis Translate . These differentially expressed genes involves a number of signal transduction pathway, such as, Adherens junction Pathway、Cytokine-cytokine receptor interaction pathway、MAPK signaling pathway (mitogen - activated protein kinase)、Wnt signaling pathway、Calcium signaling pathway、Receptor signaling protein activity、Trypsin activity pathway、Magnesium ion binding pathway、TGF-beta signaling pathway etc.
3、There was a clear difference of gene expression profiles of Kidney-yin deficiency and Kidney-yang deficiency through a comparative analysis. All of the differential expressed genes has 145, detailed analysis see the above paragraph of article 1,2. Among of Kidney-yin deficiency group、Kidney-yang deficiency group and normal control group, the common differential expressed genes has 9. Including CA8、SYPL1、PHF14、ARHGAP18、PYROXD1、RECQL、OSTF1、SEC23B、231418_at etc. These genes related to immune function, metabolism, Cell cycle, bone development, DNA repair,protein biosynthesis, etc. 231418_at gene function is not clear.
[Conclusions]
1、Gene-chip is a more ideal method of studying TCM Syndrome associated genes.
2、Using "disease combined with syndrome", carryed out the comparative research on differential gene expression of Kidney- yin deficiency and Kidney- yang deficiency. There is a initial evidence of Kidney-yin deficiency-related genes, a total of 79. There is a initial evidence of Kidney-yang deficiency-related genes, a total of 75.
3、There is differential gene expression between Kidney-yin deficiency and Kidney-yang deficiency, tips for TCM Syndrome types of Kidney-yin deficiency and Kidney-yang deficiency with evidence and scientific basis. Compared with normal control group, there is differential gene expression in common of Kidney-yin deficiency and Kidney-yang deficiency, show that there is performance of Kidney deficiency between Kidney-yin deficiency and kidney-yang deficiency, To provide a scientific ideas and methods for reveal the essence of TCM. It was the foundation on to explore the Kidney-yin deficiency and Kidney-yang deficiency mechanism in the gene level and further development of Kidney-yin deficiency and Kidney-yang deficiency of the gene chip.
中医“证”是疾病发生过程中不同阶段病因病机的高度概括,既然同一证有共同的临床表现和病理机制,那么其肯定有共同的物质基础,而这种物质基础很有可能反应在基因或基因组水平上。肾所藏之精禀受于父母,是构成胚胎发育的原始物质,其与现代医学描述的遗传物质(DNA)具有一定的同一性。因此,从基因组方面深入研究肾虚证的分子机制对于阐明肾虚证本质有着重要意义。肾阴虚证和肾阳虚证是中医学的基本证候,历代医家对肾阴虚证和肾阳虚证的理论与防治研究都颇为重视。肾阴虚证和肾阳虚证作为疾病的某一阶段的主要矛盾,必然受到“病”这一基本矛盾的影响。正是由于不同疾病的特异性,决定了不同疾病相同中医证候之间的差异;而同一疾病不同中医证侯之间也存在差异。只有通过对这些差异的研究,进而归纳出证的一般规律,才有可能对肾阴虚证和肾阳虚证有更全面的解析。因此,“病证结合”是研究肾阴虚证和肾阳虚证差异基因表达的重要思路。
本课题组在先前的肾阴虚证相关基因研究过程中,通过西医辨病与中医辨证相结合,从糖尿病、慢性肾炎、狼疮性肾病以及亚健康状态等肾阴虚证入手,应用RNA微量扩增、抑制性消减杂交、基因克隆等技术和方法,成功地构建了肾阴虚证的cDNA文库;结果发现,上述不同病种肾阴虚证的消减文库中存在明显差异,而亚健康肾阴虚证的消减文库与共同文库很相似。这提示构建的肾阴虚证cDNA消减文库必定受到了疾病的干扰,即筛选出的部分差异基因可能与该疾病有关却与肾阴虚证无关,亚健康肾阴虚证的消减文库受到的干扰最小。这可能是因为亚健康是介于健康和疾病之间的中间状态,虽然未达到疾病,但已表现为中医证候,属于功能性改变。这提示亚健康对于中医证候本质研究有着非常重要的意义。
基因芯片技术的出现是生物信息分析领域的一个革命性的里程碑,它具有高通量、微型化和自动化的特点,已成为高效、快速、大规模获取相关生物信息的重要手段。作为一种新兴的生物技术,基因芯片一出现即引起世界的广泛关注。目前,基因芯片技术已广泛应用于中医药研究的许多领域。怎么把西医的病与中医的证统一起来呢?基因表达谱也许是重要的连结点。因此,基因表达谱可为中医证候本质研究提供前所未有的契机。
综上所述,本研究拟在前期研究的基础上,采用“病证结合”的方法,应用基因芯片技术,开展肾阴虚证和肾阳虚证基因表达谱的比较研究,寻找肾阴虚证和肾阳虚证的差异表达基因,探讨肾阴虚证和肾阳虚证在基因水平可能的发生机制,为肾阴虚证和肾阳虚证的本质研究提供科学依据。
【方法】
1、采用“病证结合”方法,选取IgA肾病以及亚健康状态肾阴虚证和肾阳虚证患者作为研究对象,确诊为肾阴虚证和肾阳虚证患者,作为实验组;选择健康志愿者作为正常对照组。
2、收集样本,制备样品,运用基因芯片技术进行芯片杂交和扫描分析,研究肾阴虚证和肾阳虚证的基因表达谱。根据基因芯片分析结果,随机选择肾阴虚证和肾阳虚证的差异表达基因,进行RT-PCR验证。
3、结合MAS软件分析,并使用检索互联网生物学公共数据库(http://www.genesifter.net/web/index.html)提供的检索程序对差异表达基因进行分析。对差异表达基因进行GO注释(http://www.geneontology.org),并利用KEGG(kyotoencyclopedia of genes and genom,网址http://www.genome.ad.jp)、BioCarta(http://www.bioCarta.com)和GenMAPP(http://www.genmapp.org)作pathway分析。
【结果】
1、肾阴虚证组和正常对照组间存在差异基因表达特征图谱。获得差异表达基因共79条。其中,表达水平上调(Ratio值大于2)的有6条,表达水平下调(Ratio值小于0.5)的有73条。这些差异表达的基因按功能分析进行大体分类,主要涉及到免疫功能、新陈代谢、蛋白质生物合成、氧化应激、遗传信息过程、离子通道、蛋白质氨基酸去磷酸化、细胞凋亡、细胞信号转导有关等方面;这些差异表达的基因涉及到多个信号转导通路,如轴突导向信号转导通路、蛋白磷酸酶信号转导通路、细胞质囊泡信号转导通路、G蛋白信号转导通路、钙信号转导通路、丝裂酶原活化蛋白激酶信号转导通路等。
2、肾阳虚证组和正常对照组间存在差异基因表达特征图谱。获得差异表达基因共75条。其中,表达水平上调(Ratio值大于2)的有22条,表达水平下调(Ratio值小于0.5)的有53条。这些差异表达的基因按功能分析进行大体分类,主要涉及到免疫应答、代谢通路、信号通路、细胞因子、细胞凋亡、细胞受体、细胞骨架和运功、氧化应激、离子通道(钙、镁、锌、铜)、DNA结合、转录因子、蛋白质翻译合成等方面;这些差异表达的基因涉及到多个信号转导通路,如粘着连接通路、细胞因子间受体相互作用通路、MAPK(丝裂酶原活化蛋白激酶)信号转导通路、Wnt信号转导通路、钙信号转导通路、激活蛋白受体信号通路、胰岛素信号通路、镁离子结合信号通路、TGF-β信号转导通路等。
3、通过比较分析,肾阴虚证组和肾阳虚证组基因表达谱存在明显的差异,全部的差异表达基因有145条,详细分析见上述第1、2条。肾阴虚证组、肾阳虚证组与正常对照组比较,二者共同的差异表达基因有9条如CA8(碳酸酐酶Ⅷ)、SYPL1(突触泡蛋白样1)、PHF14(PHD指蛋白14)、ARHGAP18(Rho GTP激活蛋白18)、PYROXD1(吡啶核苷酸氧化还原酶1)、RECQL(RecQ蛋白样、DNA解旋酶Q1样)、OSTF1(破骨细胞刺激因子1)、SEC23B(Sec23同源物B)、231418_at等。主要涉及到免疫功能、新陈代谢、细胞周期、骨骼发育、DNA修复、蛋白质的合成等方面的功能。另外一条基因231418_at功能尚不明确。
【结论】
1、基因芯片是研究中医证候相关基因的比较理想的一种技术方法。
2、采用“病证结合”,开展肾阴虚证和肾阳虚证基因表达谱的比较研究,初步获得肾阴虚证的相关基因,共79条;初步获得肾阳虚证的相关基因,共75条。
3、肾阴虚证与肾阳虚证差异表达基因存在明显不同,提示可以为肾阴虚证和肾阳虚证的中医辨证分型提供科学依据;与正常对照组比较,肾阴虚证与肾阳虚证存在共同的差异表达基因,表明肾阴虚证与肾阳虚证都存在肾虚,这不但为研究中医证侯本质提供科学的思路与方法,也为探讨肾阴虚证和肾阳虚证在基因水平的发生机制及进一步研制肾阴虚证和肾阳虚证的基因芯片奠定基础。
[ Background]
The syndrome of Traditional Chinese Medicine is the summarization of etiopathogenisis in different stage of diseases. Since same symptome have common clinical manifestation and pathomechanism, then they have common material foundation, but this material probably concerned with gene or genome. The essence in kidney which come from essential substance for reproduction of the parents, is the hyle of embryonic development, it identity with DNA in modern medicine. Therefor, it is significant to study the molecular mechanism of nephrasthenia syndrome from the aspect of genome, in elucidation substance of nephrasthenia syndrome. As both of the basic syndrome, Kidney-yin deficiency and Kidney-yang deficiency is, family history of Kidney-yin deficiency and Kidney-yang deficiency medical certification and control of the theory are quite seriously. Syndrome of Kidney-yin deficiency and syndrome of Kidney-yang deficiency reflects functions of the overall body immediately, this basic contradiction is the "disease" of the interference. The difference between syndrome of Kidney-yin deficiency and syndrome of Kidney-yang deficiency in different diseases is because of the specificity of diseases. Only to study the difference and to induce general rule of syndrome, then we have all round knowledge of syndrome of Kidney-yin deficiency and syndrome of Kidney-yang deficiency. Accordingly, it is necessary to combine disease with syndrome to research syndrome of Kidney-yin deficiency and syndrome of Kidney-yang deficiency of differential gene expression.
cDNA Lib of Kidney-yin deficiency (including diabetes, chronic nephritis, lupus nephropathia and sub-health)was successfully set up in our prior study works by some techniques and methods, such as RNA microamount-amplification, differential hybridization and gene cloning. The results showed that Kidney-yin deficiency of the different diseases in the subtractive library card there is an obvious difference, and Kidney-yin deficiency sub-health subtractive library card is very similar to the common library. This prompted the construction of subtractive cDNA library of Kidney-yin deficiency and evidence of the disease must be interference. That is, differences in selected parts of the gene may be associated with the disease and Kidney-yin deficiency of the card has nothing to do with the interference of the disease, the most minimum disruption is Kidney-yin deficiency of sub-health in subtractive library card. This may be due to sub-health between health and disease is intermediate, although the disease does not meet, but has shown Chinese medicine syndromes are functional changes, it is most pure syndromes in the nature. This indicates that it is very important significance of the sub-health for the essence of TCM syndrome research.
The technology of gene chip is a revolutionary milestone of bioinformatics analysis field, It has a high-throughput, miniaturization and automation features, has become a highly efficient, rapid and large-scale access to information related to an important means of biological. As an emerging bio-technology, gene chip is widespread concern around the world. At present, The technology of gene chip has been widely used in many areas of TCM research. How the disease to Western medicine and the syndrome of traditional Chinese medicine are unified together? Gene expression may be an important link point. Therefore, gene expression offered unprecedented opportunity for research the essence of TCM Syndrome.
With the method of disease combined with syndrome and the technology of gene chip, to complete the comparative study of gene expression of Kidney-yin deficiency and Kidney-yang deficiency. Accordingly, the objective of the study is to find differential expression gene of Kidney-yin deficiency and Kidney-yang deficiency and to approach the mechanism of Kidney-yin deficiency and Kidney-yang deficiency in the level of gene.
[Methods]
1、Using "disease combined with syndrome", the patients with Kidney-yin deficiency and Kidney-yang deficiency of sub-health or IgA nephropathy were divided into experimental group. Those healthy people were divided into normal control group.
2、Collected cases and preparation samples, used of gene chip technology for chip hybridization and scanning analysis. Study on gene expression profiles of Kidney-yin deficiency and Kidney-yang deficiency. According to the results of gene chip analysis, randomly selected differential expressed genes of Kidney-yin deficiency and Kidney-yang deficiency for RT-PCR validation.
3、Combination of MAS software, analysis differential gene expression tosearch the internet using public biological databases by http://www.genesifter. net/web/index.html and http://www.geneontology.org. Pathway is analysised by KEGG(kyotoencyclopedia of genes and genome, http://www.genome.ad.jp ) and BioCarta(http://www.bioCarta.com) and GenMAPP (http://www.genmapp.org) .
[Results]
1、There is characteristic profiles of differential expressed genes between Kidney-yin deficiency group and normal control group. Among a total of 79 differential expressed genes. Increase the expression level (Ratio values greater than 2) has six, Reduced expression level (Ratio value of less than 0.5) there is the 73 .These differentially expressed genes in their analysis according to the functions of the general classification. Mainly genes related to immune function, metabolism, protein biosynthesis, oxidative stress, the process of genetic information, ion channels, protein amino acid phosphorylation go, apoptosis, cell signal transduction. These differentially expressed genes involves a number of signal transduction pathway, such as, Axon guidance pathway、Phosphoprotein phosphatase activity pathway、Cytoplasmic vesicle pathway、G Protein Signaling pathway、Calcium signaling pathway、MAPK signaling pathway, etc.
2、There is characteristic profiles of differential expressed genes between Kidney-yang deficiency group and normal control group. Among a total of 75 differential expressed genes. Increase the expression level (Ratio values greater than 2) has 22, Reduced expression level (Ratio value of less than 0.5) there is the53 . Mainly genes related to immune response, metabolic pathways, signal transduction pathways, Cytokines, apoptosis, cell receptors, and cytoskeleton, oxidative stress, Ion channels (calcium, magnesium, zinc, copper), DNA binding, transcription factor, protein synthesis Translate . These differentially expressed genes involves a number of signal transduction pathway, such as, Adherens junction Pathway、Cytokine-cytokine receptor interaction pathway、MAPK signaling pathway (mitogen - activated protein kinase)、Wnt signaling pathway、Calcium signaling pathway、Receptor signaling protein activity、Trypsin activity pathway、Magnesium ion binding pathway、TGF-beta signaling pathway etc.
3、There was a clear difference of gene expression profiles of Kidney-yin deficiency and Kidney-yang deficiency through a comparative analysis. All of the differential expressed genes has 145, detailed analysis see the above paragraph of article 1,2. Among of Kidney-yin deficiency group、Kidney-yang deficiency group and normal control group, the common differential expressed genes has 9. Including CA8、SYPL1、PHF14、ARHGAP18、PYROXD1、RECQL、OSTF1、SEC23B、231418_at etc. These genes related to immune function, metabolism, Cell cycle, bone development, DNA repair,protein biosynthesis, etc. 231418_at gene function is not clear.
[Conclusions]
1、Gene-chip is a more ideal method of studying TCM Syndrome associated genes.
2、Using "disease combined with syndrome", carryed out the comparative research on differential gene expression of Kidney- yin deficiency and Kidney- yang deficiency. There is a initial evidence of Kidney-yin deficiency-related genes, a total of 79. There is a initial evidence of Kidney-yang deficiency-related genes, a total of 75.
3、There is differential gene expression between Kidney-yin deficiency and Kidney-yang deficiency, tips for TCM Syndrome types of Kidney-yin deficiency and Kidney-yang deficiency with evidence and scientific basis. Compared with normal control group, there is differential gene expression in common of Kidney-yin deficiency and Kidney-yang deficiency, show that there is performance of Kidney deficiency between Kidney-yin deficiency and kidney-yang deficiency, To provide a scientific ideas and methods for reveal the essence of TCM. It was the foundation on to explore the Kidney-yin deficiency and Kidney-yang deficiency mechanism in the gene level and further development of Kidney-yin deficiency and Kidney-yang deficiency of the gene chip.
引文
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