【作者】 李健；

【导师】 常晓天；

【作者基本信息】 山东大学 ， 临床检验诊断学， 2017， 硕士

【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(P<0.001);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。更多还原

【Abstract】 Background:Rheumatoid arthritis(rheumatoid arthritis,RA)prevalence was 0.35%,is a chronic systemic autoimmune disease,and the clinical manifestations are joint damage and synovial inflammation.The main histopathological characteristics of RA are articular synovial cell proliferation,apoptosis,cell infiltration ability enhancement,pannus formation,and a large number of inflammatory cells infiltrations.Patients with RA significantly restricted in joint activities and even cause a physical disability,which threat to human health.But the pathogenic mechanism of RA is not fully clear,and anti-inflammatory drugs are main treatment.Therefore,to carry out the related research about cellular and molecular mechanism of RA is very important for the diagnosis and treatment of RA.Thioredoxin domain containing protein 5(TXNDC5)which encode by TXNDC5 genes belongs to the family of key protein disulfide isomerase,catalytic disulfide bond rearrangement,antioxidant,promoting angiogenesis,participate in a variety of biological functions such as cell inflammation.Our previous studies have found that TXNDC5 were increased in the synovial tissue and blood of patients with RA,and its coding gene is genetic susceptibility genes of RA.Our experimental research proves that are easier to be collagen Ⅱ TXNDC5 transgenic mice induced arthritis.So we suggest that TXNDC5 play an important role in the pathogenesis process of RA,although the specific pathogenic mechanism is not clear.The purpose of this research is to explore TXNDC5 how to participate in the process of RA.In recent years,genomics and other molecular biology found that,TXNDC5 is diabetes susceptibility genes,and play an important role in the process of diabetes.Diabetes is a kind of the insulin secretion defect or insulin disorder characterized by hyperglycemia due to the action of metabolic disease,often accompanied by insulin resistance or abnormal insulin signaling pathway.Others TXNDC5 can not only show that the catalytic reduction of insulin and reduce insulin synthesis,but also can reduce insulin and its receptor binding activity,aggravate diabetes.Epidemiological investigation also showed that RA patients increased risk of type 2 diabetes,the body’s resistance to insulin in patients with increased.In addition,a large number of studies suggest that inflammatory activity of RA can affect the metabolism of sugar,insulin signaling pathways and inflammatory mediators of insulin resistance related pathways,thus increasing the risk of RA patients suffering from diabetes.Given the relationship between RA and diabetes,and the important function of TXNDC5 in the two diseases,suggests TXNDC5 may be involved in development of RA through the insulin signaling pathway.Therefore,this topic plans to explore whether TXNDC5 through insulin signaling pathways involved in RA disease process,analysis the relationship between TXNDC5 and insulin resistance,insulin signaling pathway associated genes.This topic also plans to understand the inner relationship between RA and diabetes at the molecular level.Objective:Rheumatoid arthritis synovial cells(RASFs)is an important component in RA synovial joints,have significantly enhanced proliferation ability,can secrete a variety of inflammatory cytokines and growth factors.promote RA synovial inflammation and joint tissue destruction process.In order to study the relationship between TXNDC5 and pathogenic mechanism of RA,we inhibited TXNDC5 in RASFs with small molecular RNA,and then observe changes in cell proliferation,migration and apoptosis.At the same time,use the PCR array screening TXNDC5 related genes involved in insulin resistance or insulin signaling pathway in RASFs,and then use the Real-time PCR,ELISA and Western blot to confirm PCR array results.PCR array is a technology to rapidly screen genes in signaling pathways associated with disease.The technique used by a signaling pathways or a disease related gene loaded on a chip.It can quickly screen the purpose gene through the Real-time PCR technology.Methods:adopt the method of transient transfection to treat RASFs with anti-TXNDC5 small interfering RNA(siRNA),inhibit TXNDC5 expression.and then the Real-time PCR and Western blot technique to detect TXNDC5 mRNA and protein expression level;Using CCK8 proliferation experiment,cell migration and flow cytometry respectively to detect resistance TXNDC5 siRNA treatment group(anti-TXNDC5 siRNA group)RASFs proliferation,migration and apoptosis.Conrtrol group including the blank control group,negative control group(Allstar siRNA treatment group);Using insulin resistance RT2 Profiler PCR Array and insulin signaling pathway RT2 Profiler PCR Array respectively analysis anti-TXNDC5 siRNA transfection RASFs and Allstar siRNA transfection RASFs,found TXNDC5 downstream regulation genes by comparing the difference between the related gene expression;The Real-time PCR and ELISA method validation PCR array rsesults;The Real-time PCR and Western blot detection s IGFBP1(IGF binding protein 1)and IGFBPI(IGF binding protein 3)expression level in RA and Osteoarthritis(Osteoarthritis,OA)synovial tissue.Results:the experimental results showed that inhibiting TXNDC5 expression.RASFs cell proliferation(P=0.003)and migration ability significantly decrease(P=0.002),apoptosis increased significantly(P=0.006);Between the anti-TXNDC5 siRNA group and Allstar siRNA group,insulin resistance RT2 Profiler PCR Array detected IGF-1(insulin like growth factor 1),PCKI(phosphoenolpyruvate carboxykinase 1),SLC2A4(solute carrier family member 2 and 4)and IL1R1(interleukin 1 receptor type I)gene,insulin signaling pathway RT2 Profiler PCR Array detected IGFBPI(insulin like gro,wth factor binding protein 1)gene.Furthermore,Real-time PCR confirmed IGFBPI increased significantly(P=0.005)in anti-TXNDC5 siRNA group,and IGF-1,PCK1,SLC2A4 and IL1R1 expression has no statistical significance difference between the two groups;ELISA confirm IGFBP1 protein levels increased significantly(P<0.001)in cell culture of anti-TXNDC5 siRNA group.There were studies reported that IGFBP3 expressed(insulin like growth factor binding protein 3)in RASFs,and play an important role in IGF signal pathway.IGFBP3 and IGFBP1 are all important IGF-1 binding protein,and their function is very similar.So we detected IGFBP3 expression with Real-time PCR.According to the results,IGFBP3 expression has no difference between the two groups.Real-time PCR analysis IGFBP1 expression in the organization of RA and OA,the results show that RA synovial tissues IGFBPI mRNA expression is significantly lower than OA synovial tissue(P=0.011).Similarly,Western blot results show that the RA organization IGFBP1 protein expression is significantly decreased in OA group(P=0.014).The experimental results show that the inhibition TXNDC5 in RASFs,IGFBP1 expression quantity increased,while the expression of IGF-1 and IGFBP3 did not change.Western blot results show that compared with OA synovial tissue,IGFBP1 expression is reduced in RA synovial tissues.Conclusion:these results indicate that.inhibiting TXNDC5 in RASFs,IGFBP1 expression increased.Our preliminary work has proved TX:NDC5 were highly expressed in RA synovial tissue.Therefore.this research results suggested that high RASFs express TXNDC5 may inhibit IGFBP1 expression.Because IGFBP combined with IGF-1 can inhibit the activity of IGF.Thus.our results also suggest that RA synovial increased TXNDC5 by inhibiting IGFBP 1 expression of higher the ratio of IGF-1/IGFBP1,and inhibit the activity of IGF-1.Has been reported,IGF-1 have the function of the proinflammatory and inhibiting cell apoptosis,and it also can promote a variety of cell proliferation,differentiation and migration.After this study found that inhibit TXNDC5 expression,RASFs cell proliferation,migration ability is reduced,apoptosis rate increased.This may be due to higher IGFBP1 inhibits the activity of IGF-1,so cells behavior have changed.In a word,combined with the previous work and the results of this study,we recommend TXNDC5 involved in RA disease process is as follows:increased TXNDC5 in RA synovial cells stimulate the IGF-1 to stimulate RASFs proliferation,infiltrating ability enhancement,and inhibiting apoptosis,the results promote the progression of RA by inhibiting IGFBP1 instead of IGFBP3 expression activity.This study found IGFBP1 lower expression in RA synovial tissue also proved the proposal.In addition,some people also found that people with diabetes blood IGFBP1 expression level apparently unusual,IGF-1 activity changes obviously.The results of this study also suggests TXNDC5 and IGFBP1 expression of regulation may be inner link between RA and diabetes one of the important molecular mechanism.TXNDC5 negative control IGFBP1 expression and found that affect RASFs cell function which offers a new way for studying the pathogenesis of RA.更多还原