筛选类风湿关节炎中糖代谢关键基因的研究

发布时间：2018-01-04 21:29

本文关键词：筛选类风湿关节炎中糖代谢关键基因的研究　出处：《山东大学》2016年硕士论文　论文类型：学位论文

【摘要】：类风湿关节炎(Rheumatoid arthritis, RA)是一种自生免疫性疾病,主要出现滑膜炎症、血管翳形成和随后的关节损伤等病理改变。糖代谢是机体内最主要的代谢途径之一,主要是指葡萄糖在降解的过程,包括糖酵解、有氧氧化和磷酸戊糖代谢途径。糖酵解参与是氨基酸、脂类、核苷酸类三大代谢,并能产生三磷酸腺苷(Adenosine Triphosphate, ATP)。糖代谢不仅为机体提供能量,还通过信号通路,信号网络来调节多种生理过程。糖代谢在糖尿病、中风、精神分裂症和药物滥用等疾病中发挥重要作用。有研究表明,糖代谢包括糖酵解这一过程,在RA的发病机制和发生发展中起着重要作用。有研究认为,糖酵解活动增强,在RA的发病机制中,可能发挥着重要作用。既往主要是从免疫学和遗传学的角度进行研究RA的相关致病机制,对于糖代谢如何参与影响RA发生发展尚无深入研究。而能否从糖代谢角度思考RA这一过程,我们的很多研究成果和最近的研究都提供了新的视角。PCR Array是一项敏感和可靠的基因表达分析技术,不仅具有实时荧光定量PCR技术较好的准确性和灵敏度,同时还具有微列阵的高通量优势,可以进行信号转导途径、生理过程或与疾病相关的一组基因网络分析。本研究利用PCRArray法检测胶原诱导型关节炎(Collagen induced arthritis, CIA)大鼠的糖代谢相关基因,旨在检测糖代谢的关键酶在RA中的表达水平改变,并阐明作用,进一步探索糖代谢在类风湿性关节炎的作用.进一步探讨糖代谢和类风湿关节炎的相关性。方法：取牛II型胶原诱导大鼠(CIA组)和正常对照大鼠(NCR组)膝关节滑膜组织,提取总RNA,采用Rat Glucose Metabolism RT2 ProfilerTM PCR Array筛选表达不一致基因；采用RT-PCR法对CIA组和NCR组进行mRNA水平的检测,采用RT-PCR法和Western blot法检测类风湿关节炎(RA组)和骨关节炎(OA组)滑膜组织中基因mRNA水平和蛋白水平的表达。收集类风湿关节炎血清(RAB组)和正常献血者血清(NCB组),采用ELISA法检测血清中的表达水平。取膝关节滑膜组织原代培养FLS；采用人工合成的Si-RNA-PGK1干扰剂特异性抑制PGK1在RAFLS中的表达,阴性Si-RNA为阴性对照组,瞬时转染24 h后,采用RT-PCR法和Western blot法检测抑制效率,采用CCK-8、 Transwell、流式细胞术、ELISA等方法检测干扰PGK1表达后对细胞增殖、侵袭、迁移、细胞周期和相关炎性因子分泌的影响。结果PCR Array检测显示,CIA组烯醇化酶(ENO1)、己糖激酶2(HK2)、磷酸甘油酸激酶-1(PGK1)较NCR组表达上调(P0.05),磷酸烯醇式丙酮酸羧激酶-1(PCK1)和丙酮酸脱氢酶激酶-4(PDK4)表达下调(P0.05); RT-PCR法和Western blot法检测显示,RA组和OA组相比, ENO1和PGK1表达上调(P0.05), ELISA法检测结果显示,RAB组和NCB组相比,PGK1表达升高(P0.05)。转染特异性Si-RNA-PGK1后, 各干扰组与阴性对照组相比,Si-RNA-PGK1干扰剂转染24 h时,PGK1 mRNA水平抑制效果显著(P0.05),RAFLS的增殖受到抑制(P0.05), 细胞的侵袭能力下降(P0.05),IFN-γ和IL-1β分泌降低(P0.05)。结论：牛Ⅱ型胶原诱导大鼠和类风湿关节炎滑膜组织中糖代谢关键酶ENO1和PGK1表达增高,类风湿关节炎血液中PGK1表达增加,均提示类风湿关节炎中糖代谢活动增强,导致相关基因的表达异常。这说明糖代谢中的关键酶PGK1参与了细胞异常增殖、迁移以及IFN-γ和IL-1 p等炎症因子的分泌表达,说明PKK1对RA炎症因子表达及RAFLS的生物学功能有影响。
[Abstract]:Rheumatoid arthritis (Rheumatoid arthritis RA) is a kind of spontaneous autoimmune disease, mainly inflammation of synovium, pannus formation change and subsequent joint injury pathology. Glucose metabolism is one of the most important metabolic pathways in the body, mainly refers to the process of glucose degradation, including glycolysis and aerobic oxidation and the pentose phosphate pathway is involved in glycolysis. Amino acids, lipids, nucleotides three metabolism, and can produce ATP (Adenosine Triphosphate, ATP). Glucose metabolism not only provides energy for the body, but also through the signal pathway, signal network to regulate a variety of physiological processes. Glucose metabolism in diabetes, stroke, schizophrenia play an important effect of disease and drug abuse and other diseases. Studies have shown that glucose metabolism including glycolysis in this process plays an important role in the pathogenesis of RA and development. Some studies suggested that the glycolytic activity The dynamic enhancement in the pathogenesis of RA, may play an important role. It is mainly related to the pathogenesis of RA from the perspective of immunology and genetics, the sugar metabolism influence how to participate in the occurrence and development of RA. There is no in-depth study and whether this process on RA from the angle of glucose metabolism, many of our research results and recent studies have provided a new perspective.PCR Array expression analysis technique is a sensitive and reliable gene, not only has the accuracy and sensitivity of real-time fluorescence quantitative PCR technology is good, but also has the advantages of high-throughput microarray, can signal transduction pathways, analysis of physiological processes associated with disease or a group of gene network. The detection of collagen induced arthritis by PCRArray (Collagen induced arthritis, CIA) glucose metabolism related genes in rats, to detect the key enzyme in sugar metabolism The expression level of RA in the change, and to elucidate the role, to further explore the effect of glucose metabolism in rheumatoid arthritis. To further explore the correlation between glucose metabolism and rheumatoid arthritis. Methods: bovine type II collagen induced rats (CIA group) and normal control rats (group NCR) of knee joint synovial tissue, extraction of total RNA, Rat Glucose Metabolism RT2 ProfilerTM PCR by Array gene expression screening is not consistent; detection of mRNA level of CIA group and NCR group by RT-PCR method, rheumatoid arthritis was detected by RT-PCR method and Western blot method (group RA) and osteoarthritis (OA) gene expression level of mRNA and protein in synovial tissue the serum was collected. Rheumatoid arthritis (RAB group) and normal blood donors serum (NCB group), the expression levels in serum were detected by ELISA. The knee joint synovial tissue cultured FLS; using Si-RNA-PGK1 as artificial interference The expression of agent specific inhibition of PGK1 in RAFLS, negative Si-RNA as negative control group, 24 h after transient transfection, using RT-PCR method and Western blot method to detect the inhibition efficiency, using CCK-8, Transwell, flow cytometry, the expression of ELISA interference detection method PGK1 on cell proliferation, invasion, migration, secretion of cells cycle and related inflammatory factors. PCR Array results showed that group CIA enolase (ENO1), hexokinase 2 (HK2), phosphoglycerate kinase -1 (PGK1) than group NCR (P0.05), expression of phosphoenolpyruvate carboxykinase kinase -1 (PCK1) and pyruvate dehydrogenase kinase -4 (PDK4) expression of RT-PCR (P0.05); display detection method and Western blot method, RA group compared with OA group, expression of ENO1 and PGK1 (P0.05), ELISA assay showed that RAB group compared with NCB group, the expression of PGK1 increased (P0.05). After transfection of specific Si-RNA-PGK1, the interference group and the negative Compared with the control group, Si-RNA-PGK1 interference was transfected 24 h, significant inhibitory effect of PGK1 mRNA (P0.05), RAFLS (P0.05) inhibited proliferation, decreased cell invasion ability (P0.05), IFN- IL-1 and gamma beta secretion (P0.05). Conclusion: the increased expression of rat synovial tissue in rheumatoid arthritis and the key enzyme ENO1 and PGK1 induced by bovine type II collagen, rheumatoid arthritis and blood PGK1 expression increased, suggesting metabolic activity in rheumatoid arthritis related gene expression leads to enhanced anomaly. This shows that PGK1 is the key enzyme in sugar metabolism is involved in cell proliferation, migration and secretion expression of IFN- gamma and IL-1 P inflammation the influence factor of the biological function of PKK1 RA on the expression of inflammatory factors and RAFLS.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R593.22

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