miR-27b在脊柱结核中的表达及其对花生四烯酸细胞色素P450信号通路的影响

发布时间：2018-05-18 19:49

本文选题：miR-27b + 花生四烯酸CYP450途径　；参考：《昆明医科大学》2017年硕士论文

【摘要】：[研究背景及目的]脊柱结核(STB)是发病率最高的肺外结核,致残率较高。对于结核分枝杆菌在结核中的病理过程尚未完全清楚。近期研究表明:结核分枝杆菌感染巨噬细胞的过程中,可通过某种途径控制了某些miRNA的表达,对其细胞活性和功能进行调控。最终实现在巨噬细胞内寄生和长期存活,在一定条件下复制增多而引发结核病。花生四烯酸代谢途径CYP450代谢途径是近期发现的一条新途径,在人体中的生物化学和病理学意义还未认识和阐明。人体血液中单核细胞和巨噬细胞内表达的CYP450同工酶主要为CYP450 1b1[15]。相关研究表明:花生四烯酸细胞色素P450代谢途径产生的环氧化二十碳三稀甘油酸(EETs)可通过多种途径抑制细胞凋亡。结合杆菌抑制巨噬细胞凋亡可能与该途径有关。在我们的前期基因芯片研究发现miR-27b的表达在脊柱结核患者中有显著变化,进一步的实验结果显示:miR-27b在结核杆菌诱导巨噬细胞凋亡中同样有显著变化,表明miR-27b在结核杆菌感染过程中发挥重要作用,其可通过调节巨噬细胞凋亡增多从而影响结核病的发病过程,但其具体发病机制尚不清楚。经过生物信息学分析发现CYP450 1b1可能是miR-27b的一个靶基因。故我们推测结核杆菌可能通过调控miR-27b及其靶基因CYP450的表达,调节花生四烯酸途径使EETs减少而抑制单个核细胞的凋亡,从而达到影响脊柱结核的病理过程。本研究对这一假设进一步验证。[材料和方法]1、miR-27b及预测靶基因CYP450 lbl在脊柱结核临床样本中的表达:根据患者临床表现,影像学资料和相关的实验室检查,结合穿刺活检或手术取材病理组织学检查结果设立脊柱结核病例;病例组(n=30)和对照组(n=12),在抗痨治疗实施前抽取外周血标本5mL,加入Ficoll试剂后进行梯度离心,获取单个核细胞。对照组人群来源于我院健康体检中心健康自愿者,同法获得外周血单个核细胞 peripheral blood mononuclear cell(PBMC)。再提取总 RNA 及 microRNA 并进行反转录后对样本中mRNACYP450 lbl及miR-27b进行RT-PCR检测,对其△△CT值进行统计学分析(p0.05有统计学意义),并通过分析miR-27b和CYP4501bl对脊柱结核检测的特异性和敏感性,建立受试者操作特征曲线(ROC曲线),利用曲线下面积指标(AUC)评价其对脊柱结核的诊断效率。2、靶基因关系的验证:采用荧光素酶报告基因验证miR-27b与细胞色素P450lb1(CYP 4501bl)的基因-靶基因关系。3、miR-27b对花生四烯酸CYP450代谢途径对巨噬细胞凋亡的影响验证:a、miR-27bmimics转染:Thp-1诱导分化为巨噬细胞。使用Lipofectamine2000法将目的基因miR-27b mimics进行转染,根据转染量分设为空白对照组,2.5ul,5ul,1 Oul共4组。并将FAM control mimics进行转染,设立阴性对照组,同样根据转染浓量分为空白对照组。b、流式细胞仪检测miR-27bmimics转染巨噬细胞后的凋亡水平。c、miR-27b mimics转染后CYPlbl基因表达水平:取转染了 miR-27b mimics不同浓度的巨噬细取总RNA,变性琼脂糖凝胶电泳检测RNA质量后进行反转录。转录后进行RT-PCR反,计算不同转染浓度巨噬细胞中CYP450lb1基因的AACT后进行统计学分析(p0.05有统计学意义)。d、Western blot检测CYP450 lbl表达水平。e、ELSIA检测EET表达量。[结果]1、临床样本检测:实验组CYP450 lbl基因RT-PCR检测△ △ CT值与健康对照组相比表达升高(1.76±0.69/1.18±0.27),miR-27b基因RT-PCR检测△△(CT值与健康对照组相比表达升高表达降低(0.92±0.22/1.18±0.32),两者都呈现差异性表达(p0.05)。miR-27b与CYP4501bl检测脊柱结核患者的敏感性分别为0.67%及0.585,特异性分别为83%及91%,AUC分别为0.786及0.814。2、荧光素酶报告基因显示:当表达miR-27b的质粒同表达CYP4501bl基因3' UTR的野生型质粒共同转染巨噬细胞时,荧光素酶的表达值降低,同对照组相比降低(6.469 ±0.886/8.901±0.935),具有显著性差异(p0.05),而当将11个碱基序列,即假定的结合位点突变之后,由于过表达的miR-27b不能再特异结合到荧光素酶报告基因载体上,即无法调控荧光素酶报苦基因的表达,所以荧光素酶的表达含量同对照组相比(12.305±1.518/10.807±2.287)无显著差异(p0.05)。以上结果表明,miR-27b可以下调巨噬细胞中CYP4501b1基因的表达,CYP4501b1基因是miR-27b的靶基因。3、miR-27b功能验证:转染miR-27bmimics基因空对照组,2.5ul组,5ul组,10ul组的巨噬细胞凋亡率分别为0%,9.4%,6.8%,8.4%,结果显示miR-27b表达量增加可促进巨噬细胞凋亡。转染miR-27b mimics基因空对照,2.5ul,5ul,10ul浓度的巨噬细胞的CYP450 1b1基因PCR检测△△CT值值分别为1,0.36,0.15,0.02,结果显示随着miR-27b转录浓度升高,CYP450 1b1基因表达量减少。Western blot检测转染后巨噬细胞CYP4501b1达水平降低。ElISA检测结果显示空对照组,2.5ul组,5ul组,10ul组的EEt-14、15 表达量分别为 20.748pg/ml,11.106pg/ml,13.336pg/ml,9.727pg/ml。随着miR-27b转录浓度的升高EEt-14、15表达量下降。[结论]1、脊柱结核病人外周血单个核细胞中miR-27b表达量降低,CYP4501b1.基因表达量增高与健康人群对照组间存在明显的差异。miR-27b及CYP4501b1可能成为脊柱结核早期诊断分子标记物之一。2、miR-27b与CYP4501b1基因间存在基因-靶基因关系。3、miR-27b表达降低,巨噬细胞中CYP4501b1基因表达量升高,其下游产物EETs表达量升高,凋亡率降低。综上所述,miR-27b的靶基因为CYP4501b1,在结核分枝杆菌(MTB)病程中miR-27b的表达降低,其作用是增加靶基因CYP4501b1的表达,通过花生四烯酸途径降低EEts的表达使PBMC的凋亡率减少,从而影响结核分枝杆菌的病理过程。
[Abstract]:[background and purpose] spinal tuberculosis (STB) is the highest incidence of extrapulmonary tuberculosis. The rate of disability is high. The pathological process of Mycobacterium tuberculosis in tuberculosis is not completely clear. Recent studies have shown that in the process of Mycobacterium tuberculosis infection of macrophages, the expression of certain miRNA can be controlled by some way and its cell activity The CYP450 metabolic pathway of the peanut four enoic acid metabolism pathway is a new pathway discovered recently. The biochemical and pathological significance in human body has not yet been recognized and clarified. The CYP450 isozymes expressed in macrophages are mainly CYP450 1b1[15]. related studies. It is suggested that the epoxidation of twenty carbon three dilute glyceric acid (EETs) produced by the metabolic pathway of peanut four enoic acid cytochrome P450 can inhibit apoptosis through a variety of pathways. The microchip study found that the expression of miR-27b has a significant change in the patients with spinal tuberculosis. Further experimental results show that miR-27b also has significant changes in the apoptosis of macrophages induced by Mycobacterium tuberculosis, indicating that miR-27b plays an important role in the process of Mycobacterium tuberculosis infection, which can regulate the increase of macrophage apoptosis and influence tuberculosis. The pathogenesis of the disease, but its specific pathogenesis is not clear. Through bioinformatics analysis, CYP450 1B1 may be a target gene of miR-27b. Therefore, we speculate that the Mycobacterium tuberculosis may regulate the expression of miR-27b and its target gene CYP450, regulate the decrease of EETs and inhibit the apoptosis of mononuclear cells by the peanut four enoic acid pathway. This study further verified this hypothesis. [material and methods]1, miR-27b, and predictive target gene CYP450 LBL are expressed in clinical specimens of spinal tuberculosis: according to clinical manifestations, imaging data, and related laboratory tests, junction biopsy or surgical histopathological examinations Cases of spinal tuberculosis were set up; case group (n=30) and control group (n=12) were used to extract 5mL from peripheral blood before the treatment of anti tuberculosis treatment. After adding Ficoll reagent, gradient centrifugation was used to obtain mononuclear cells. The control group was derived from the healthy volunteers in the health check-up center of our hospital, and the peripheral blood mononucl of peripheral blood mononuclear cells was obtained by the same method. Ear cell (PBMC). Then the total RNA and microRNA were extracted and the RT-PCR detection of mRNACYP450 LBL and miR-27b in the samples was carried out. The value of delta delta CT was statistically analyzed (P0.05 was statistically significant). ROC curve), using the area index under the curve (AUC) to evaluate the diagnostic efficiency of the spinal tuberculosis,.2, the target gene relationship verification: using luciferase reporter gene to verify the relationship between miR-27b and cytochrome P450lb1 (CYP 4501bl) gene target gene.3, miR-27b on the effect of miR-27b on the apoptosis of macrophage by the peanut four enacylic acid CYP450 metabolism pathway: A, MI R-27bmimics transfection: Thp-1 induced differentiation into macrophages. The target gene miR-27b mimics was transfected by Lipofectamine2000 method. The transfection amount was divided into blank control group, 2.5ul, 5ul, 1 Oul, and FAM control mimics was transfected and negative control group was set up. The same transfection concentration was divided into blank control group.B, flow finer The apoptosis level of miR-27bmimics after transfected macrophages was detected by cytosgraph, and the expression level of CYPlbl gene after transfection of miR-27b mimics: the total RNA was obtained by transfection of different concentrations of miR-27b mimics, and the denatured agarose gel electrophoresis was used to reverse transcription of RNA, and then RT-PCR inverse was carried out to calculate C macrophages in different transfection concentrations. The AACT of the YP450lb1 gene was statistically analyzed (P0.05 was statistically significant).D, Western blot was used to detect CYP450 LBL expression level.E, and ELSIA to detect EET expression. [results]1, clinical samples detection: the expression of delta delta detection in experimental group was higher than that in healthy control group (1.76 + 0.27). The expression of CT was decreased (0.92 + 0.22/1.18 + 0.32) compared with the healthy control group (0.92 + 0.22/1.18 + 0.32). The sensitivity of both.MiR-27b and CYP4501bl for the detection of spinal tuberculosis was 0.67% and 0.585 respectively, the specificity was 83% and 91%, the AUC was 0.786 and 0.814.2 respectively, and the luciferase reporter gene showed that the table was on the table. When miR-27b plasmid co transfected macrophages with CYP4501bl gene 3'UTR, the expression of luciferase was reduced (6.469 + 0.886/8.901 + 0.935) compared with the control group (P0.05), and when the 11 base sequences, that is, the assumed binding site mutation, due to the over expressed miR-27b The expression of luciferase reporter gene could not be specifically combined with the luciferase reporter gene carrier, so the expression of luciferase was not significantly different from that of the control group (12.305 + 1.518/10.807 + 2.287) (P0.05). The above results showed that miR-27b could express the CYP4501b1 gene in the following macrophages, CYP4501b1 The gene is the target gene of miR-27b,.3, miR-27b function verification: the apoptosis rate of macrophages in the miR-27bmimics gene empty control group, 2.5ul group, 5ul group and 10ul group is 0%, 9.4%, 6.8%, 8.4% respectively. The results show that the increase of miR-27b expression can promote the apoptosis of macrophages. The value of the CYP450 1B1 gene PCR detection was 1,0.36,0.15,0.02. The results showed that the expression of CYP450 1B1 gene was reduced by.Western blot and the CYP4501b1 level of macrophage after transfection was decreased with the increase of miR-27b transcriptional concentration. The result of.ElISA detection was 20.. 748pg/ml, 11.106pg/ml, 13.336pg/ml, 9.727pg/ml. decreased with the increase of miR-27b transcriptional concentration. [conclusion]1, the expression of miR-27b in peripheral blood mononuclear cells in patients with spinal tuberculosis decreased, and there was a significant difference between the CYP4501b1. gene expression and the control group of the healthy population.MiR-27b and CYP4501b1 may become the spinal column. One of the early diagnosis molecular markers of tuberculosis.2, the gene target gene relationship between miR-27b and CYP4501b1 gene is.3, the expression of miR-27b is reduced, the expression of CYP4501b1 gene in the macrophage is increased, the downstream product EETs expression is increased, and the apoptosis rate is reduced. In summary, the target group of miR-27b is m in the course of Mycobacterium tuberculosis (MTB). The expression of iR-27b is reduced, and its effect is to increase the expression of the target gene CYP4501b1. The decrease of the expression of EEts through the arachidic acid pathway reduces the apoptosis rate of PBMC, thus affecting the pathological process of Mycobacterium tuberculosis.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R529.2

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