电针深刺“腰突五穴”有效治疗腰椎间盘突出症的数字基因表达谱及分子网络研究
发布时间：2018-08-22 14:44
【摘要】：研究目的：1.从转录组学层面探讨腰椎间盘突出症(Lumbar disc herniation, LDH)的分子发病机制。2.从转录组学层面揭示电针(Electro-acupuncture, EA)深刺“腰突五穴”治疗LDH的分子疗效机制。研究方法：1.核糖核酸测序(Ribonucleic Acid Sequencing, RNA-seq)样本临床筛选：①分组与治疗：依据诊断标准、纳入标准和排除标准,临床纳入LDH组患者30例。对其进行6次EA深刺“腰突五穴”治疗：即采用直径0.35mm,长75mmm的无菌毫针直刺患侧大肠俞、关元俞及L4、L5、S1夹脊穴,得气后辅以电针治疗,留针25分钟。根据治疗前后所采集的日本骨科协会评估治疗分数(Japanese Orthopaedic Association Score, JOA)腰椎功能评分计算治疗改善率,筛选出治疗改善率排名前8位,且评级均为“良”及以上的患者进行测序。依据纳入标准和排除标准,纳入与上述LDH组测序患者年龄、性别匹配的8例健康对照组受试者,不做治疗处理。②样本采集：LDH组分别于治疗前及6次治疗后采集外周静脉全血,健康对照组纳入后即采集外周静脉全血。选择LDH组测序患者治疗前后的血样与健康对照组受试者的血样,对每例样本单独提取白细胞总核糖核酸(Total Ribonucleic Acid, Total RNA),记为Group 1(LDH组治疗前)、Group 2(LDH组治疗后)和Group 3(健康对照组)。2. RNA-seq研究：①测序流程：首先对上述样本白细胞total RNA进行制备与鉴定,其次进行mRNA的分离与定量,最后构建mRNA文库并上机运算。根据上机结果,分析整个组的表达情况,得出三组数字基因表达谱(Digital Gene Expression Profiling,DGE)。②测序数据分析：比较三组基因表达情况,筛选出在发病和治疗过程中调节方向相反的显著差异基因；对上述基因进行基因本体(Gene Ontology,GO)、京都基因和基因组百科全书(Kyoto Encyclopedia of Genes and Genomes, KEGG)以及Ingenuity Pathway Analysis (IPA)分析,从转录组学层面探讨LDH的分子发病机制与EA深刺“腰突五穴”治疗LDH的分子疗效机制。3.定量反转录-聚合酶链式反应(Quantitative Reverse Transcription-Polymerase Chain Reaction, qRT-PCR)验证：采用qRT-PCR技术对RNA-seq结果进行验证,保证研究的准确性和可靠性。方法如下：首先对total RNA进行反转录,设计qPCR引物并进行扩增条件测试,其次进行qPCR上机,最后生成数据并进行分析。研究结果：1.测序样本临床筛选结果：筛选出了LDH组基因测序的8名患者,纳入年龄、性别匹配的8名健康受试者；采集了测序受试者外周静脉全血样本,分别提取白细胞totalRNA,标为Group1(LDH组治疗前),Group2(LDH组治疗后)及Group3(健康对照组)。2. RNA-seq研究结果：①差异基因筛选结果：Group 1与Group 3对比,得出120个LDH发病相关的差异基因,Group 1与Group 2比,得出76个差异基因。这两部分差异基因中有17个基因重合,且在发病和治疗过程中的表达方向相反。分析该17个差异基因与LDH分子发病机制和EA深刺“腰突五穴”治疗LDH的分子疗效机制相关。②差异基因分析结果：对发病和疗效机制相关的17个差异基因进行GO、KEGG、IPA分析,结果(1)GO富集结果提示与水分平衡相关。(2)KEGG未富集到相关通路。(3)IPA结果：A.通路富集结果提示与白细胞介素17(Interleukin, IL-17)信号转导通路相关,且通路中位于细胞核位置的核心基因为CCL2。分析该基因表达上调可引发并加重LDH的疼痛症状,并通过该通路引发并加重LDH的免疫炎症反应。B.分子网络结果提示富集到了一个与炎症反应、免疫疾病相关的分子网络,且差异基因CCL2、IFI27与非差异基因TNF、IFNG位于网络的中心,具有互作关系。分析CCL2、IFI27基因表达上调通过该网络可引发和加重LDH的疼痛、腰椎间盘退行性变和免疫炎症反应。EA深刺“腰突五穴”治疗LDH的分子疗效机制与LDH分子发病机制中各基因表达调节的方向正好相反。3. qRT-PCR验证结果：对RNA-seq结果中四个重要基因CCL2、IFI27、IFNG、TNF进行qRT-PCR验证,结果表明IFI27、CCL2表达差异较为明显,IFNG.TNF的表达差异不明显,与RNA-seq结果相同。这证实了研究的准确性与可靠性。研究结论：1.LDH部分分子发病机制：①腰椎间盘细胞水分失衡会导致退行性变,从而引发LDH。②CCL2基因表达上调,可引发并加重LDH的疼痛症状。③CCL2基因表达上调,可影响IL-17信号转导通路,引发和加重LDH的免疫炎症反应。④CCL2、IFI27基因表达上调后,通过与TNF、BFNG基因的互作网络,引发和加重LDH的疼痛、腰椎间盘退行性变与免疫炎症反应。2.EA深刺“腰突五穴”治疗LDH的分子疗效机制：①EA深刺“腰突五穴”可在腰椎间盘细胞水转运的过程中起到积极作用,延缓退行性变。②EA深刺“腰突五穴”通过下调CCL2基因表达,缓解LDH的疼痛症状。③EA深刺“腰突五穴”通过下调CCL2基因表达,来调节IL-17信号转导通路,以减轻LDH的免疫炎症反应。④EA深刺“腰突五穴”可下调CCL2、IFI27基因表达,影响与TNF、IFNG等基因的互作网络,减轻LDH的免疫炎症反应,缓解疼痛,延缓腰椎间盘退行性变。
[Abstract]:Objective: 1. To explore the molecular pathogenesis of lumbar disc herniation (LDH) from the transcriptome level. 2. To reveal the molecular therapeutic mechanism of electro-acupuncture (EA) deep needling "five points of lumbar disc herniation" for LDH from the transcriptome level. Methods: 1. Ribonucleic acid Sequencing (RNA Sequencing) - seq) sample clinical screening: 1. grouping and treatment: according to diagnostic criteria, inclusion criteria and exclusion criteria, 30 cases of LDH patients were included in the clinical group. According to the Japanese Orthopaedic Association Score (JOA) score of lumbar vertebral function before and after treatment, the treatment improvement rate was calculated, and the top 8 patients with "good" and above were selected for sequencing. Eight healthy control subjects matched with the age and sex of the LDH group were enrolled without treatment. 2. Sample collection: The whole blood of peripheral vein was collected in LDH group before and after 6 times of treatment, and the whole blood of peripheral vein was collected in healthy control group immediately after the LDH group was enrolled. Total Ribonucleic Acid (Total RNA) was separately extracted from each sample and recorded as Group 1 (before treatment in LDH group), Group 2 (after treatment in LDH group) and Group 3 (healthy control group). 2. RNA-seq study: 1. Sequencing process: First, the total RNA of white blood cells in the above samples was prepared and identified, and then carried out. The isolation and quantification of mRNA, and finally the construction of the mRNA library and computer operations. According to the results of the computer, analysis of the expression of the whole group, three groups of digital Gene Expression Profiling (DGE). 2 Sequencing data analysis: Comparing the three groups of gene expression, screening out the disease and treatment process in the opposite direction of regulation of the obvious. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Ingenuity Pathway Analysis (IPA) were used to study the molecular pathogenesis of LDH and the molecular therapy of EA deep needling "five points of lumbar process" for LDH. Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) was used to verify the results of RNA-seq to ensure the accuracy and reliability of the study. Methods: First, total RNA was retranscribed, qPCR primers were designed and amplified. Results: 1. Sequencing sample clinical screening results: 8 patients with LDH genome sequencing were selected, including 8 healthy subjects with age and sex matching; whole blood samples of peripheral vein were collected, and total RNA of white blood cells was extracted and labeled Gro. RNA-seq results: 1. Differential gene screening results: Group 1 compared with Group 3, 120 differential genes associated with LDH, Group 1 compared with Group 2, and 76 differential genes. 17 of the two differential genes overlapped and were at the onset of the disease. The 17 differentially expressed genes were correlated with the molecular pathogenesis of LDH and the molecular therapeutic mechanism of EA deep needling "five points of lumbar process" in the treatment of LDH. (2) KEGG was not enriched in the related pathway. (3) IPA results: A. pathway enrichment results suggested that interleukin 17 (IL-17) signal transduction pathway was related, and the core gene in the pathway was CCL2. Upregulation of KEGG expression could induce and aggravate pain symptoms of LDH, and through this pathway triggered and induced by the LDH. B. Molecular network results suggest that there is a molecular network involved in inflammation and immune diseases, and the differential genes CCL2, IFI27 and non-differential genes TNF, IFNG are located at the center of the network, and there is an interaction between them. The molecular therapeutic mechanism of EA deep needling "five points of lumbar process" in the treatment of LDH is opposite to the regulation of gene expression in the molecular pathogenesis of LDH. 3. QRT-PCR results: Four important genes CCL2, IFI27, IFNG, TNF in RNA-seq results were verified by qRT-PCR. The results showed that IFI27, CCL2 table The expression of IFNG. TNF was not significantly different from that of RNA-seq. This confirms the accuracy and reliability of the study. Research conclusions: 1. Partial molecular pathogenesis of LDH: 1. Water imbalance in lumbar intervertebral disc cells leads to degeneration, which leads to the up-regulation of LDH. 2 CCL2 gene expression, which can lead to and aggravate the pain of LDH. Up-regulation of CCL2 gene expression can affect IL-17 signal transduction pathway and induce and aggravate the immunoinflammatory reaction of LDH. After up-regulation of CCL2 and IFI27 gene expression, the pain of LDH, the degeneration of lumbar intervertebral disc and the immunoinflammatory reaction of LDH can be induced and aggravated through the interaction network with TNF and BFNG genes. 2. EA pricks deeply the "five points of lumbar process" to treat LDH. Therapeutic mechanism: 1) EA deep needling "five points of lumbar process" can play an active role in the process of water transport of lumbar intervertebral disc cells, delay degeneration. 2 EA deep needling "five points of lumbar process" can alleviate LDH pain symptoms by down-regulating the expression of CCL2 gene. 3 EA deep needling "five points of lumbar process" can regulate IL-17 signal transduction by down-regulating the expression of CCL2 gene. (4) EA deep needling "five points of lumbar process" can down-regulate the expression of CCL2 and IFI27 genes, affect the interaction network with TNF, IFNG and other genes, alleviate the immunoinflammatory reaction of LDH, relieve pain and delay the degeneration of lumbar intervertebral disc.
【学位授予单位】：北京中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R246.9
[Abstract]:Objective: 1. To explore the molecular pathogenesis of lumbar disc herniation (LDH) from the transcriptome level. 2. To reveal the molecular therapeutic mechanism of electro-acupuncture (EA) deep needling "five points of lumbar disc herniation" for LDH from the transcriptome level. Methods: 1. Ribonucleic acid Sequencing (RNA Sequencing) - seq) sample clinical screening: 1. grouping and treatment: according to diagnostic criteria, inclusion criteria and exclusion criteria, 30 cases of LDH patients were included in the clinical group. According to the Japanese Orthopaedic Association Score (JOA) score of lumbar vertebral function before and after treatment, the treatment improvement rate was calculated, and the top 8 patients with "good" and above were selected for sequencing. Eight healthy control subjects matched with the age and sex of the LDH group were enrolled without treatment. 2. Sample collection: The whole blood of peripheral vein was collected in LDH group before and after 6 times of treatment, and the whole blood of peripheral vein was collected in healthy control group immediately after the LDH group was enrolled. Total Ribonucleic Acid (Total RNA) was separately extracted from each sample and recorded as Group 1 (before treatment in LDH group), Group 2 (after treatment in LDH group) and Group 3 (healthy control group). 2. RNA-seq study: 1. Sequencing process: First, the total RNA of white blood cells in the above samples was prepared and identified, and then carried out. The isolation and quantification of mRNA, and finally the construction of the mRNA library and computer operations. According to the results of the computer, analysis of the expression of the whole group, three groups of digital Gene Expression Profiling (DGE). 2 Sequencing data analysis: Comparing the three groups of gene expression, screening out the disease and treatment process in the opposite direction of regulation of the obvious. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Ingenuity Pathway Analysis (IPA) were used to study the molecular pathogenesis of LDH and the molecular therapy of EA deep needling "five points of lumbar process" for LDH. Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) was used to verify the results of RNA-seq to ensure the accuracy and reliability of the study. Methods: First, total RNA was retranscribed, qPCR primers were designed and amplified. Results: 1. Sequencing sample clinical screening results: 8 patients with LDH genome sequencing were selected, including 8 healthy subjects with age and sex matching; whole blood samples of peripheral vein were collected, and total RNA of white blood cells was extracted and labeled Gro. RNA-seq results: 1. Differential gene screening results: Group 1 compared with Group 3, 120 differential genes associated with LDH, Group 1 compared with Group 2, and 76 differential genes. 17 of the two differential genes overlapped and were at the onset of the disease. The 17 differentially expressed genes were correlated with the molecular pathogenesis of LDH and the molecular therapeutic mechanism of EA deep needling "five points of lumbar process" in the treatment of LDH. (2) KEGG was not enriched in the related pathway. (3) IPA results: A. pathway enrichment results suggested that interleukin 17 (IL-17) signal transduction pathway was related, and the core gene in the pathway was CCL2. Upregulation of KEGG expression could induce and aggravate pain symptoms of LDH, and through this pathway triggered and induced by the LDH. B. Molecular network results suggest that there is a molecular network involved in inflammation and immune diseases, and the differential genes CCL2, IFI27 and non-differential genes TNF, IFNG are located at the center of the network, and there is an interaction between them. The molecular therapeutic mechanism of EA deep needling "five points of lumbar process" in the treatment of LDH is opposite to the regulation of gene expression in the molecular pathogenesis of LDH. 3. QRT-PCR results: Four important genes CCL2, IFI27, IFNG, TNF in RNA-seq results were verified by qRT-PCR. The results showed that IFI27, CCL2 table The expression of IFNG. TNF was not significantly different from that of RNA-seq. This confirms the accuracy and reliability of the study. Research conclusions: 1. Partial molecular pathogenesis of LDH: 1. Water imbalance in lumbar intervertebral disc cells leads to degeneration, which leads to the up-regulation of LDH. 2 CCL2 gene expression, which can lead to and aggravate the pain of LDH. Up-regulation of CCL2 gene expression can affect IL-17 signal transduction pathway and induce and aggravate the immunoinflammatory reaction of LDH. After up-regulation of CCL2 and IFI27 gene expression, the pain of LDH, the degeneration of lumbar intervertebral disc and the immunoinflammatory reaction of LDH can be induced and aggravated through the interaction network with TNF and BFNG genes. 2. EA pricks deeply the "five points of lumbar process" to treat LDH. Therapeutic mechanism: 1) EA deep needling "five points of lumbar process" can play an active role in the process of water transport of lumbar intervertebral disc cells, delay degeneration. 2 EA deep needling "five points of lumbar process" can alleviate LDH pain symptoms by down-regulating the expression of CCL2 gene. 3 EA deep needling "five points of lumbar process" can regulate IL-17 signal transduction by down-regulating the expression of CCL2 gene. (4) EA deep needling "five points of lumbar process" can down-regulate the expression of CCL2 and IFI27 genes, affect the interaction network with TNF, IFNG and other genes, alleviate the immunoinflammatory reaction of LDH, relieve pain and delay the degeneration of lumbar intervertebral disc.
【学位授予单位】：北京中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R246.9
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