TXNDC5通过胰岛素信号途径参与类风湿关节炎病变过程的研究

发布时间：2018-06-03 12:01

本文选题：类风湿关节炎 + TXNDC5　；参考：《山东大学》2017年硕士论文

【摘要】：研究背景:类风湿关节炎(rheumatoid arthritis,RA)患病率为0.35%,是一种慢性系统性自身免疫性疾病,主要临床表现为关节损伤和滑膜炎症,组织病理学特征为关节滑膜细胞增殖、细胞浸润能力增强、细胞凋亡降低、血管翳形成和大量炎性细胞浸润。RA导致患者关节活动明显受限甚至致躯体残疾,严重威胁着人类的健康。但是该病的致病机制尚不完全明确,治疗药物也仅以抗炎药物为主。因此,RA开展相关的细胞和分子机制的研究对RA的诊治具有重要意义。硫氧还蛋白 5(thioredoxin domain containing protein 5,TXNDC5)基因编码的蛋白质属于蛋白质二硫键异构酶家族,可催化二硫键的重排,具有抗氧化、促进血管形成,参与细胞炎症等多种生物功能。我们的前期研究发现,TXNDC5在RA患者的滑膜组织和血液中高表达,其编码基因是RA的遗传易感基因。我们的实验动物学研究证明,TXNDC5转基因小鼠更易于被胶原Ⅱ诱导出关节炎。因此我们建议,TXNDC5在RA发病过程中发挥重要作用,虽然其具体致病机制尚不清楚。本课题研究的目的就是探索TXNDC5如何参与、影响RA的病变过程。近年的基因组学及其他分子生物学研究显示,TXNDC5是糖尿病易感基因,在糖尿病发病过程中发挥重要作用。糖尿病是一种由于胰岛素分泌缺陷或胰岛素作用障碍所致的以高血糖为特征的代谢性疾病,常伴有胰岛素抵抗或胰岛素相关信号通路的异常。其他人的研究证明,TXNDC5不仅能催化胰岛素二硫键的还原,减少胰岛素的合成,而且可以降低胰岛素与其受体的结合活性,加剧糖尿病病情。流行病学调查也显示,RA患者中2型糖尿病的患病风险增高,患者机体胰岛素抵抗增强。另外,大量研究提示,RA的炎症活动及炎症介质影响糖代谢、胰岛素信号通路及胰岛素抵抗相关通路,进而提高了 RA患者罹患糖尿病的风险。鉴于RA与糖尿病患病风险的关系以及TXNDC5在两种疾病中的重要作用,提示我们TXNDC5有可能通过胰岛素相关信号通路参与RA的发生发展。因此,本课题计划探索TXNDC5是否通过胰岛素信号途径参与RA病变过程,分析TXNDC5与胰岛素抵抗、胰岛素信号通路相关基因之间的关系。本课题也计划在分子水平上了解RA与糖尿病之间的内在联系。目的:类风湿关节炎滑膜细胞(rheumatoid arthritis synovial cells,RASFs)是 RA关节滑膜中的重要组成成分,有显著增强的增殖能力,可分泌多种炎症因子和生长因子,促进RA滑膜炎和关节组织破坏过程。为了研究TXNDC5对RA的致病机制和作用,本课题首先用小分子RNA抑制RASFs中TXNDC5的表达,然后观察细胞的增殖、迁移和凋亡变化情况。同时,用PCRarray分析anti-TXNDC5 siRNA处理的RASFs,筛选TXNDC5在RASFs中参与胰岛素抵抗或者胰岛素信号通路的相关基因,然后用Real-time PCR、ELISA、Western blot等方法验证PCR array结果。PCRarray是近几年兴起的快速筛选信号途径和疾病关联基因的技术。该技术把一信号途径或一疾病所用相关基因装载在一芯片上,通过Real-time PCR技术一次性筛选到目的基因。方法:采用瞬时转染的方法将针对TXNDC5小干扰RNA(siRNA)导入在RASFs中,抑制TXNDC5的表达,然后采用Real-time PCR和Western blot技术检测TXNDC5的mRNA和蛋白表达水平;采用CCK8增殖实验、细胞迁移实验以及流式细胞术分别检测抗TXNDC5 siRNA处理组(anti-TXNDC5 siRNA组)RASFs的增殖、迁移和凋亡,对照组包括空白对照组、阴性对照组(AllstarsiRNA处理组);采用 insulin resistance RT2 Profiler PCR Array 和 insulin signaling pathway RT2 Profiler PCR Array 分别分析 anti-TXNDC5 siRNA 转染的 RASFs 和 Allstar siRNA转染的RASFs,通过比较相关基因的差异表达发现TXNDC5的下游调控基因;采用 Real-time PCR 和 ELISA 法验证 PCR array 结果;采用 Real-time PCR和Western blot检测RA和骨关节炎(Osteoarthritis,OA)滑膜组织中IGFBP1(insulin like growth factor binding protein 1)的表达水平。结果:实验结果显示,抑制TXNDC5的表达后,RASFs细胞的增殖(P=0.003)和迁移能力(P=0.002)明显降低,凋亡明显增加(P=0.006);PCRArray在anti-TXNDC5 siRNA组和Allstar siRNA组中共检测到5个差异表达的基因,其中 insulin resistance RT2 Profiler PCR Array 筛选出 IGF-1(insulin like growth factor-1)、PCK1(phosphoenolpyruvate carboxykinase 1)、SLC2A4(solute carrier family 2 member 4)和 IL1Rl(interleukin 1 receptor type 1)基因,insulin signaling pathway RT2 Profiler PCR Array 筛选出 IGFBP1 基因。Real-time PCR 验证上述结果,证实 IGFBP1 在 anti-TXNDC5 siRNA 组中显著增高(P=0.005),而 IGF-1、PCK1、SLC2A4和IL1R1的表达在两组间差异没有统计学意义;ELISA证实IGFBP1的蛋白水平在anti-TXNDC5 siRNA转染RASFs的细胞培养液中显著增高(P0.001);由于有文献报道 IGFBP3(insulin like growth factor binding protein 3)在RASFs中表达,并在IGF信号途径中发挥重要作用,而且IGFBP3与IGFBP1的功能十分相似,都是IGF-1重要的结合蛋白,我们也用Real-time PCR检测IGFBP3的表达水平。结果显示,IGFBP3的表达在两组间没有差异;Real-time PCR分析了人RA和OA组织中IGFBP1的表达,结果显示RA滑膜组织中IGFBP1的mRNA表达明显低于OA滑膜组织(P=0.011)。同样,Western blot结果显示RA组织中IGFBP1的蛋白表达较OA组织中明显降低(P=0.014)。实验结果显示,抑制RASFs中TXNDC5的表达后,IGFBP1的表达量增高,而IGF-1和IGFBP3的表达并未发生变化。Western blot结果显示相对于OA滑膜组织,RA滑膜组织中IGFBP1表达降低。结论:以上结果显示,RASFs中TXNDC5被抑制后,IGFBP1的表达增加。我们前期工作已证明RA滑膜组织中高表达TXNDC5。因此,本研究结果建议,RASFs高表达TXNDC5可能会抑制IGFBP1的表达。由于IGFBP与IGF-1结合可以抑制IGF活性。因此,我们的结果还建议RA滑膜中高表达的TXNDC5通过抑制1GFBP1表达增加IGF-1/IGFBP1比值而提高IGF-1的活性。已有报道,IGF-1具有促炎和抑制细胞凋亡的作用,并且还能促进多种细胞的增殖、分化及迁移。本研究发现抑制TXNDC5表达后,RASFs细胞增殖、迁移能力降低,凋亡率增加。这可能是由于增高的IGFBP1抑制了 IGF-1的活性致使细胞的行为发生改变。总之,结合以前的工作和本研究的结果,我们建议TXNDC5参与RA病变过程如下:TXNDC5在RA滑膜细胞中高表达,通过抑制IGFBP1表达而不是IGFBP3刺激IGF-1活性,从而增强RASFs增殖、浸润能力并抑制细胞凋亡,结果促进RA的病程进展。本研究在RA滑膜组织中发现IGFBP1低表达也证明了该建议。另外,有人也发现糖尿病患者血液中IGFBP1表达水平明显异常、IGF-1活性明显变化。本研究的结果也建议TXNDC5以及对IGFBP1表达的调控可能是RA与糖尿病之间内在联系的重要分子机制。TXNDC5负向调控IGFBP1表达以及影响RASFs细胞功能的发现,为研究RA的发病机制提供了新的思路。
[Abstract]:Background: the prevalence of rheumatoid arthritis (RA) is 0.35%. It is a chronic systemic autoimmune disease. The main clinical manifestations are joint injury and synovial inflammation. Histopathological features are joint synovial cell proliferation, cell infiltration capacity, cell apoptosis, pannus formation and large number of inflammatory cells. Infiltration of.RA leads to significantly limited joint activity and even physical disability, which is a serious threat to human health. However, the pathogenesis of the disease is not completely clear, and the treatment drugs are mainly anti inflammatory drugs. Therefore, the study of RA related cellular and molecular mechanisms is of great significance to the diagnosis of RA. Thioredoxin 5 (thioredoxi N domain containing protein 5, TXNDC5) gene encoded proteins belong to the protein two sulfur bond isomerase family, which can catalyze the rearrangement of the two sulfur bond, have antioxidant activity, promote angiogenesis, and participate in many biological functions such as cell inflammation. Our previous study found that TXNDC5 was highly expressed in the synovial tissue and blood of RA patients and its coding base Our experimental zoological studies have shown that TXNDC5 transgenic mice are more prone to be induced by collagen II to induce arthritis. Therefore, we suggest that TXNDC5 play an important role in the pathogenesis of RA, although its specific pathogenesis is not clear. The purpose of this study is to explore how TXNDC5 participates in the effect of TXNDC5 and affects RA. The process of disease. In recent years, genomics and other molecular biology studies have shown that TXNDC5 is a susceptible gene for diabetes and plays an important role in the pathogenesis of diabetes. Diabetes is a metabolic disease characterized by hyperglycemia due to insulin secretion defect or insulin action disorder, often accompanied by insulin resistance or islets. Other people's studies have shown that TXNDC5 can not only catalyze the reduction of insulin two sulfur bonds, reduce insulin synthesis, but also reduce the binding activity of insulin to its receptors and aggravate the condition of diabetes. Epidemiological investigation also shows that the risk of type 2 diabetes is higher in patients with RA and the islets of the patients' body islets. In addition, a large number of studies have suggested that inflammatory activities and inflammatory mediators of RA affect glucose metabolism, insulin signaling pathways and insulin resistance related pathways, and thus enhance the risk of diabetes in RA patients. In view of the relationship between RA and the risk of diabetes and the important role of TXNDC5 in the two diseases, we suggest that we have TXNDC5 It is possible to participate in the development of RA through insulin signaling pathway. Therefore, we plan to explore whether TXNDC5 participates in the RA process through insulin signaling pathway, and analyzes the relationship between TXNDC5 and insulin resistance, and the relationship between insulin signaling pathway related genes. The subject also plans to understand the internal relationship between RA and diabetes at the molecular level. Objective: rheumatoid arthritis synovial cells (RASFs) is an important component of the synovial synovium of the RA joint. It has a significant enhancement of proliferation ability, can secrete a variety of inflammatory factors and growth factors, promote the process of RA synovitis and joint tissue destruction. In order to study the pathogenesis and effect of TXNDC5 to RA, the pathogenesis and effect of TXNDC5 are studied. Firstly, we use small molecule RNA to suppress the expression of TXNDC5 in RASFs, and then observe the proliferation, migration and apoptosis of cells. Meanwhile, we use PCRarray to analyze RASFs treated by anti-TXNDC5 siRNA and select the related genes involved in insulin resistance or insulin signaling pathway in RASFs, and then Real-time PCR, ELISA, and ELISA. OT and other methods verify the PCR array results.PCRarray is a rapid screening signal pathway and the technology of disease associated genes in recent years. The technology loaded a signal pathway or a disease related genes on a chip and screened the target gene by Real-time PCR technology. Method: the transient transfection method will be directed against TXND. C5 small interference RNA (siRNA) was introduced into RASFs to inhibit the expression of TXNDC5. Then Real-time PCR and Western blot techniques were used to detect mRNA and protein expression levels of TXNDC5. Proliferation, migration and apoptosis were detected by CCK8 proliferation, cell migration and flow cytometry, respectively. The control group, including the blank control group and the negative control group (AllstarsiRNA treatment group), used insulin resistance RT2 Profiler PCR Array and insulin signaling pathway RT2 Profiler. The downstream regulatory genes of DC5; Real-time PCR and ELISA methods were used to verify the PCR array results. Real-time PCR and Western blot were used to detect the expression level of RA and osteoarthritis (Osteoarthritis and osteoarthritis) synovial tissues. Cell proliferation (P=0.003) and migration ability (P=0.002) were significantly decreased, and apoptosis was significantly increased (P=0.006); PCRArray in anti-TXNDC5 siRNA group and Allstar siRNA group detected 5 differentially expressed genes, and insulin resistance RT2 Profiler. Carboxykinase 1), SLC2A4 (solute carrier family 2 member 4) and IL1Rl (interleukin 1 receptor type 1). The expression of IL1R1 was not statistically significant between the two groups; ELISA demonstrated that the protein level of IGFBP1 was significantly increased in the cell culture solution of anti-TXNDC5 siRNA transfected RASFs (P0.001), and that IGFBP3 (insulin like growth factor) was expressed in the anti-TXNDC5 siRNA and played an important role in the signaling pathway, The function of IGFBP3 and IGFBP1 is very similar, all of which are important binding proteins of IGF-1. We also use Real-time PCR to detect the expression level of IGFBP3. The results show that the expression of IGFBP3 is not different between the two groups; Real-time PCR analyses the expression of IGFBP1 in RA and OA tissues. Synovial tissue (P=0.011). Similarly, Western blot results showed that the expression of IGFBP1 protein in RA tissue was significantly lower than that in OA tissue (P=0.014). The results showed that the expression of IGFBP1 increased after inhibition of the expression of TXNDC5 in RASFs, while IGF-1 and IGFBP3 expression did not change, and the synovial membrane was shown to be relative to the synovial membrane. The expression of IGFBP1 in the tissue was reduced. Conclusion: the above results showed that the expression of IGFBP1 increased after the inhibition of TXNDC5 in RASFs. Our previous work has proved that the high expression of TXNDC5. in the RA synovial tissues has been proved. The results suggested that the high expression of TXNDC5 in RASFs may inhibit the expression of IGFBP1. The binding of IGFBP to IGF-1 can inhibit the activity of IGF. Therefore, the activity of IGFBP and IGF-1 can be suppressed. Our results also suggest that the high expression of TXNDC5 in the RA synovial membrane improves the activity of IGF-1 by inhibiting 1GFBP1 expression and increasing the IGF-1/IGFBP1 ratio. It has been reported that IGF-1 has the role of proinflammatory and inhibiting apoptosis, and also promotes the proliferation, differentiation and migration of multiple cells. This study found that the proliferation of RASFs cells was inhibited after the inhibition of TXNDC5 expression. Decrease in mobility and increase the rate of apoptosis. This may be due to the increased activity of IGFBP1 that inhibits the activity of IGF-1 causing changes in cell behavior. In conclusion, combined with previous work and the results of this study, we suggest that TXNDC5 participate in the process of RA lesions as follows: TXNDC5 is highly expressed in RA synovial cells by inhibiting the expression of IGFBP1, not IGFBP3 spines. Stimulated IGF-1 activity, thus enhancing RASFs proliferation, infiltration capacity and inhibiting apoptosis, results in the progress in the course of RA. The discovery of low expression of IGFBP1 in the RA synovial tissue also proved the suggestion. In addition, some people also found that the level of IGFBP1 expression in the blood of diabetic patients was obviously abnormal and the IGF-1 activity was significantly changed. The results of this study were also found in this study. It is suggested that TXNDC5 and the regulation of IGFBP1 expression may be an important molecular mechanism of the intrinsic link between RA and diabetes,.TXNDC5 negative regulation of IGFBP1 expression and the discovery of the function of RASFs cells, which provide a new idea for the study of the pathogenesis of RA.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R593.22

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