本文選題：miR-27b + 花生四烯酸CYP450途徑�。� 參考：《昆明醫(yī)科大學》2017年碩士論文

【摘要】：[研究背景及目的]脊柱結核(STB)是發(fā)病率最高的肺外結核,致殘率較高。對于結核分枝桿菌在結核中的病理過程尚未完全清楚。近期研究表明:結核分枝桿菌感染巨噬細胞的過程中,可通過某種途徑控制了某些miRNA的表達,對其細胞活性和功能進行調控。最終實現(xiàn)在巨噬細胞內寄生和長期存活,在一定條件下復制增多而引發(fā)結核病。花生四烯酸代謝途徑CYP450代謝途徑是近期發(fā)現(xiàn)的一條新途徑,在人體中的生物化學和病理學意義還未認識和闡明。人體血液中單核細胞和巨噬細胞內表達的CYP450同工酶主要為CYP450 1b1[15]。相關研究表明:花生四烯酸細胞色素P450代謝途徑產生的環(huán)氧化二十碳三稀甘油酸(EETs)可通過多種途徑抑制細胞凋亡。結合桿菌抑制巨噬細胞凋亡可能與該途徑有關。在我們的前期基因芯片研究發(fā)現(xiàn)miR-27b的表達在脊柱結核患者中有顯著變化,進一步的實驗結果顯示:miR-27b在結核桿菌誘導巨噬細胞凋亡中同樣有顯著變化,表明miR-27b在結核桿菌感染過程中發(fā)揮重要作用,其可通過調節(jié)巨噬細胞凋亡增多從而影響結核病的發(fā)病過程,但其具體發(fā)病機制尚不清楚。經過生物信息學分析發(fā)現(xiàn)CYP450 1b1可能是miR-27b的一個靶基因。故我們推測結核桿菌可能通過調控miR-27b及其靶基因CYP450的表達,調節(jié)花生四烯酸途徑使EETs減少而抑制單個核細胞的凋亡,從而達到影響脊柱結核的病理過程。本研究對這一假設進一步驗證。[材料和方法]1、miR-27b及預測靶基因CYP450 lbl在脊柱結核臨床樣本中的表達:根據患者臨床表現(xiàn),影像學資料和相關的實驗室檢查,結合穿刺活檢或手術取材病理組織學檢查結果設立脊柱結核病例;病例組(n=30)和對照組(n=12),在抗癆治療實施前抽取外周血標本5mL,加入Ficoll試劑后進行梯度離心,獲取單個核細胞。對照組人群來源于我院健康體檢中心健康自愿者,同法獲得外周血單個核細胞 peripheral blood mononuclear cell(PBMC)。再提取總 RNA 及 microRNA 并進行反轉錄后對樣本中mRNACYP450 lbl及miR-27b進行RT-PCR檢測,對其△△CT值進行統(tǒng)計學分析(p0.05有統(tǒng)計學意義),并通過分析miR-27b和CYP4501bl對脊柱結核檢測的特異性和敏感性,建立受試者操作特征曲線(ROC曲線),利用曲線下面積指標(AUC)評價其對脊柱結核的診斷效率。2、靶基因關系的驗證:采用熒光素酶報告基因驗證miR-27b與細胞色素P450lb1(CYP 4501bl)的基因-靶基因關系。3、miR-27b對花生四烯酸CYP450代謝途徑對巨噬細胞凋亡的影響驗證:a、miR-27bmimics轉染:Thp-1誘導分化為巨噬細胞。使用Lipofectamine2000法將目的基因miR-27b mimics進行轉染,根據轉染量分設為空白對照組,2.5ul,5ul,1 Oul共4組。并將FAM control mimics進行轉染,設立陰性對照組,同樣根據轉染濃量分為空白對照組。b、流式細胞儀檢測miR-27bmimics轉染巨噬細胞后的凋亡水平。c、miR-27b mimics轉染后CYPlbl基因表達水平:取轉染了 miR-27b mimics不同濃度的巨噬細取總RNA,變性瓊脂糖凝膠電泳檢測RNA質量后進行反轉錄。轉錄后進行RT-PCR反,計算不同轉染濃度巨噬細胞中CYP450lb1基因的AACT后進行統(tǒng)計學分析(p0.05有統(tǒng)計學意義)。d、Western blot檢測CYP450 lbl表達水平。e、ELSIA檢測EET表達量。[結果]1、臨床樣本檢測:實驗組CYP450 lbl基因RT-PCR檢測△ △ CT值與健康對照組相比表達升高(1.76±0.69/1.18±0.27),miR-27b基因RT-PCR檢測△△(CT值與健康對照組相比表達升高表達降低(0.92±0.22/1.18±0.32),兩者都呈現(xiàn)差異性表達(p0.05)。miR-27b與CYP4501bl檢測脊柱結核患者的敏感性分別為0.67%及0.585,特異性分別為83%及91%,AUC分別為0.786及0.814。2、熒光素酶報告基因顯示:當表達miR-27b的質粒同表達CYP4501bl基因3' UTR的野生型質粒共同轉染巨噬細胞時,熒光素酶的表達值降低,同對照組相比降低(6.469 ±0.886/8.901±0.935),具有顯著性差異(p0.05),而當將11個堿基序列,即假定的結合位點突變之后,由于過表達的miR-27b不能再特異結合到熒光素酶報告基因載體上,即無法調控熒光素酶報苦基因的表達,所以熒光素酶的表達含量同對照組相比(12.305±1.518/10.807±2.287)無顯著差異(p0.05)。以上結果表明,miR-27b可以下調巨噬細胞中CYP4501b1基因的表達,CYP4501b1基因是miR-27b的靶基因。3、miR-27b功能驗證:轉染miR-27bmimics基因空對照組,2.5ul組,5ul組,10ul組的巨噬細胞凋亡率分別為0%,9.4%,6.8%,8.4%,結果顯示miR-27b表達量增加可促進巨噬細胞凋亡。轉染miR-27b mimics基因空對照,2.5ul,5ul,10ul濃度的巨噬細胞的CYP450 1b1基因PCR檢測△△CT值值分別為1,0.36,0.15,0.02,結果顯示隨著miR-27b轉錄濃度升高,CYP450 1b1基因表達量減少。Western blot檢測轉染后巨噬細胞CYP4501b1達水平降低。ElISA檢測結果顯示空對照組,2.5ul組,5ul組,10ul組的EEt-14、15 表達量分別為 20.748pg/ml,11.106pg/ml,13.336pg/ml,9.727pg/ml。隨著miR-27b轉錄濃度的升高EEt-14、15表達量下降。[結論]1、脊柱結核病人外周血單個核細胞中miR-27b表達量降低,CYP4501b1.基因表達量增高與健康人群對照組間存在明顯的差異。miR-27b及CYP4501b1可能成為脊柱結核早期診斷分子標記物之一。2、miR-27b與CYP4501b1基因間存在基因-靶基因關系。3、miR-27b表達降低,巨噬細胞中CYP4501b1基因表達量升高,其下游產物EETs表達量升高,凋亡率降低。綜上所述,miR-27b的靶基因為CYP4501b1,在結核分枝桿菌(MTB)病程中miR-27b的表達降低,其作用是增加靶基因CYP4501b1的表達,通過花生四烯酸途徑降低EEts的表達使PBMC的凋亡率減少,從而影響結核分枝桿菌的病理過程。
[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.
【學位授予單位】：昆明醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R529.2

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