電針深刺“腰突五穴”有效治療腰椎間盤突出癥的數(shù)字基因表達譜及分子網(wǎng)絡(luò)研究
發(fā)布時間：2018-08-22 14:44
【摘要】：研究目的：1.從轉(zhuǎn)錄組學層面探討腰椎間盤突出癥(Lumbar disc herniation, LDH)的分子發(fā)病機制。2.從轉(zhuǎn)錄組學層面揭示電針(Electro-acupuncture, EA)深刺“腰突五穴”治療LDH的分子療效機制。研究方法：1.核糖核酸測序(Ribonucleic Acid Sequencing, RNA-seq)樣本臨床篩選：①分組與治療：依據(jù)診斷標準、納入標準和排除標準,臨床納入LDH組患者30例。對其進行6次EA深刺“腰突五穴”治療：即采用直徑0.35mm,長75mmm的無菌毫針直刺患側(cè)大腸俞、關(guān)元俞及L4、L5、S1夾脊穴,得氣后輔以電針治療,留針25分鐘。根據(jù)治療前后所采集的日本骨科協(xié)會評估治療分數(shù)(Japanese Orthopaedic Association Score, JOA)腰椎功能評分計算治療改善率,篩選出治療改善率排名前8位,且評級均為“良”及以上的患者進行測序。依據(jù)納入標準和排除標準,納入與上述LDH組測序患者年齡、性別匹配的8例健康對照組受試者,不做治療處理。②樣本采集：LDH組分別于治療前及6次治療后采集外周靜脈全血,健康對照組納入后即采集外周靜脈全血。選擇LDH組測序患者治療前后的血樣與健康對照組受試者的血樣,對每例樣本單獨提取白細胞總核糖核酸(Total Ribonucleic Acid, Total RNA),記為Group 1(LDH組治療前)、Group 2(LDH組治療后)和Group 3(健康對照組)。2. RNA-seq研究：①測序流程：首先對上述樣本白細胞total RNA進行制備與鑒定,其次進行mRNA的分離與定量,最后構(gòu)建mRNA文庫并上機運算。根據(jù)上機結(jié)果,分析整個組的表達情況,得出三組數(shù)字基因表達譜(Digital Gene Expression Profiling,DGE)。②測序數(shù)據(jù)分析：比較三組基因表達情況,篩選出在發(fā)病和治療過程中調(diào)節(jié)方向相反的顯著差異基因；對上述基因進行基因本體(Gene Ontology,GO)、京都基因和基因組百科全書(Kyoto Encyclopedia of Genes and Genomes, KEGG)以及Ingenuity Pathway Analysis (IPA)分析,從轉(zhuǎn)錄組學層面探討LDH的分子發(fā)病機制與EA深刺“腰突五穴”治療LDH的分子療效機制。3.定量反轉(zhuǎn)錄-聚合酶鏈式反應(yīng)(Quantitative Reverse Transcription-Polymerase Chain Reaction, qRT-PCR)驗證：采用qRT-PCR技術(shù)對RNA-seq結(jié)果進行驗證,保證研究的準確性和可靠性。方法如下：首先對total RNA進行反轉(zhuǎn)錄,設(shè)計qPCR引物并進行擴增條件測試,其次進行qPCR上機,最后生成數(shù)據(jù)并進行分析。研究結(jié)果：1.測序樣本臨床篩選結(jié)果：篩選出了LDH組基因測序的8名患者,納入年齡、性別匹配的8名健康受試者；采集了測序受試者外周靜脈全血樣本,分別提取白細胞totalRNA,標為Group1(LDH組治療前),Group2(LDH組治療后)及Group3(健康對照組)。2. RNA-seq研究結(jié)果：①差異基因篩選結(jié)果：Group 1與Group 3對比,得出120個LDH發(fā)病相關(guān)的差異基因,Group 1與Group 2比,得出76個差異基因。這兩部分差異基因中有17個基因重合,且在發(fā)病和治療過程中的表達方向相反。分析該17個差異基因與LDH分子發(fā)病機制和EA深刺“腰突五穴”治療LDH的分子療效機制相關(guān)。②差異基因分析結(jié)果：對發(fā)病和療效機制相關(guān)的17個差異基因進行GO、KEGG、IPA分析,結(jié)果(1)GO富集結(jié)果提示與水分平衡相關(guān)。(2)KEGG未富集到相關(guān)通路。(3)IPA結(jié)果：A.通路富集結(jié)果提示與白細胞介素17(Interleukin, IL-17)信號轉(zhuǎn)導(dǎo)通路相關(guān),且通路中位于細胞核位置的核心基因為CCL2。分析該基因表達上調(diào)可引發(fā)并加重LDH的疼痛癥狀,并通過該通路引發(fā)并加重LDH的免疫炎癥反應(yīng)。B.分子網(wǎng)絡(luò)結(jié)果提示富集到了一個與炎癥反應(yīng)、免疫疾病相關(guān)的分子網(wǎng)絡(luò),且差異基因CCL2、IFI27與非差異基因TNF、IFNG位于網(wǎng)絡(luò)的中心,具有互作關(guān)系。分析CCL2、IFI27基因表達上調(diào)通過該網(wǎng)絡(luò)可引發(fā)和加重LDH的疼痛、腰椎間盤退行性變和免疫炎癥反應(yīng)。EA深刺“腰突五穴”治療LDH的分子療效機制與LDH分子發(fā)病機制中各基因表達調(diào)節(jié)的方向正好相反。3. qRT-PCR驗證結(jié)果：對RNA-seq結(jié)果中四個重要基因CCL2、IFI27、IFNG、TNF進行qRT-PCR驗證,結(jié)果表明IFI27、CCL2表達差異較為明顯,IFNG.TNF的表達差異不明顯,與RNA-seq結(jié)果相同。這證實了研究的準確性與可靠性。研究結(jié)論：1.LDH部分分子發(fā)病機制：①腰椎間盤細胞水分失衡會導(dǎo)致退行性變,從而引發(fā)LDH。②CCL2基因表達上調(diào),可引發(fā)并加重LDH的疼痛癥狀。③CCL2基因表達上調(diào),可影響IL-17信號轉(zhuǎn)導(dǎo)通路,引發(fā)和加重LDH的免疫炎癥反應(yīng)。④CCL2、IFI27基因表達上調(diào)后,通過與TNF、BFNG基因的互作網(wǎng)絡(luò),引發(fā)和加重LDH的疼痛、腰椎間盤退行性變與免疫炎癥反應(yīng)。2.EA深刺“腰突五穴”治療LDH的分子療效機制：①EA深刺“腰突五穴”可在腰椎間盤細胞水轉(zhuǎn)運的過程中起到積極作用,延緩?fù)诵行宰儭＂贓A深刺“腰突五穴”通過下調(diào)CCL2基因表達,緩解LDH的疼痛癥狀。③EA深刺“腰突五穴”通過下調(diào)CCL2基因表達,來調(diào)節(jié)IL-17信號轉(zhuǎn)導(dǎo)通路,以減輕LDH的免疫炎癥反應(yīng)。④EA深刺“腰突五穴”可下調(diào)CCL2、IFI27基因表達,影響與TNF、IFNG等基因的互作網(wǎng)絡(luò),減輕LDH的免疫炎癥反應(yīng),緩解疼痛,延緩腰椎間盤退行性變。
[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.
【學位授予單位】：北京中醫(yī)藥大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：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.
【學位授予單位】：北京中醫(yī)藥大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R246.9
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