【作者】 赵霞；

【导师】 纪春岩；

【作者基本信息】 山东大学 ， 内科学（血液病）（专业学位）， 2017， 博士

【摘要】 研究背景:淋巴瘤是源于淋巴造血系统的恶性肿瘤。随着诊疗手段的不断发展,患者的生存期和生活质量得到了很大改善,仍有很大一部分淋巴瘤患者会出现复发或耐药,甚至导致死亡。淋巴瘤属于免疫系统肿瘤,其发生是多因素、多环节、多基因相互作用的结果,它与遗传、免疫微环境的异常、抑癌基因的失活、细胞凋亡和细胞异常增殖等有关。最近研究显示,肿瘤细胞在发生发展或被化疗药物杀伤时,均可产生炎症小体。炎症小体是参与机体免疫的重要组成部分,NLRP3炎症小体是目前研究得最清楚的炎症小体,在肿瘤的发生发展和化疗耐药中占据重要地位。当外来病原体入侵或者体内细胞损伤和死亡时,NLRP3可招募ASC和caspase-1组装成炎症小体,炎症小体通过NLRP3/caspase轴释放大量的IL-1β和IL-18等炎性细胞因子以促进肿瘤发生。IL-18是炎症小体的主要效应分子,可以通过诱导PD-1的表达来抑制NK细胞的生长,促进肿瘤生长和转移。单核苷酸多态性(SNPs)可影响肿瘤炎症微环境,与肿瘤的发生、发展密切相关。NLRP3炎症小体相关基因的SNPs与多种癌症的发生发展有关。NLRP3炎症小体及其产物可以直接促进肿瘤细胞的生长以及耐药的产生,炎症反应的抑制或其产物的中和对癌变和肿瘤进展具有较强的抑制作用,靶向NLRP3炎症小体有望成为肿瘤治疗的重要手段。研究目的:研究淋巴瘤患者NLRP3炎症小体相关基因的多态性和表达,并评估其临床意义,利用淋巴瘤细胞系进行功能试验进一步探索其功能并阐明其在淋巴瘤中的作用机制。研究方法:1、淋巴瘤患者NLRP3相关基因SNPs检测:选取390例淋巴瘤患者和385例健康对照者为研究对象,刮取淋巴瘤患者骨髓涂片,抽取健康对照者EDTA抗凝全血,然后应用DNA样本提取试剂盒提取DNA,PCR(探针法)检测IL-18(rs1946518)、IL-1β(rs16944)、CARD8(rs2043211)、NFκB 94 ins/del(rs28362491)各基因分型。2、淋巴瘤患者及正常对照的NLRP3相关基因的表达:选取68例淋巴瘤患者(46例新诊断的患者和22例缓解患者)和40例健康对照提取外周血mRNA,反转录成cDNA,设计引物,应用RT-PCR方法检测淋巴瘤患者和正常对照者的外周血单个核细胞中 IL-18、IL-1β、caspase-1、NLRP3、ASC、NFκB 基因 mRNA水平的表达。应用ELISA方法检测淋巴瘤患者和健康对照外周血清中IL-18、IL-1β的水平。免疫组织化学方法检测淋巴瘤患者淋巴瘤组织病理切片及正常淋巴结组织切片中NLRP3相关基因IL-18、NLRP3、ASC的表达。3、在淋巴瘤细胞系Pfeiffe中加入LPS+ATP处理后,应用Western blot法检测ASC及NFκB蛋白的表达水平,以GAPDH作内参。4、设置地塞米松浓度梯度,观察NLRP3炎症小体活化对Pfeiffer细胞药物敏感性影响。实验分组设计为对照组、LPS+ATP组、DEX组、DEX+LPS+ATP组,收集各组细胞和上清液进行相关指标检测。应用ELISA方法测定各组细胞培养上清液中IL-18和IL-1β的浓度,应用RT-PCR方法检测各组细胞IL-18、IL-1β、caspase-1、NLRP3、c-myc、TP53、bcl-2、bax 基因 mRNA 水平的表达。5、在Pfeiffer培养体系中加入IL-18和抗IL-18培养48h。实验分组设计为对照组、DEX组、IL-18+DEX组、抗IL-18+DEX组,收集各组细胞进行相关指标检测。应用RT-PCR方法检测各组细胞c-myc、TP53、bcl-2、bax基因mRNA水平的表达。6、CCK8方法检测各组细胞增殖情况,并进行药敏分析;根据细胞周期试剂盒说明应用流式细胞术进行细胞周期分析;使用Annexin V/PI凋亡检测试剂盒流式细胞术分析各组细胞凋亡情况。7、统计学分析:将连续变量和分类变量用方差分析或非参数Kruskal-Wallis检验进行比较。标准卡方检验用于测试Hardy-Weinberg平衡的基因型频率。通过计算优势比(OR)和相应的95%置信区间(CI)来评估基因型与疾病风险之间的关联。使用Mann-Whitney U检验进行RT-PCR和ELISA检测的各组结果之间的比较。Kaplan-Meier曲线用于描述存活。进行细胞实验三次,定量数据表示为平均值±标准差,使用非配对t检验进行统计学分析。P<0.05被认为具有统计学意义。研究结果:一、NLRP3炎症小体相关基因SNPs与淋巴瘤的易感性及预后的相关性研究1、炎症小体NLRP3相关基因多态性与淋巴瘤的易感性:淋巴瘤患者IL-18(rsl946518)和NFκB 94 ins/del(rs28362491)基因型分布与正常对照具有显著性差异。2、L-18(rs1946518)和 NFκB94ins/del(rs28362491)的等位基因与淋巴瘤的易感性:携带 IL-18(rs1946518)等位基因“G”和 NFκB94ins/del(rs28362491)等位基因“ins”者淋巴瘤患病风险均显著增加。3、NLRP3炎症小体基因多态性与淋巴瘤患者临床指标相关性:IL-18(rsl946518)基因型与LDH水平相关,CARD8(rs2043211)基因型与性别、B症状和结外浸润部位相关,NFκB 94 ins/del(rs28362491)与LDH水平、分型和骨髓浸润相关。4、IL-18(rs1946518)和 NFκB 94 ins/del(rs28362491)与 B 细胞非霍奇金淋巴瘤(B-NHL)易感性:IL-18(rs1946518)与T-NHL和B-NHL易感性均具有显著相关性。而NFκB 94 ins/del(rs28362491)仅与B-NHL的易感性显著相关。携带IL-18等位基因“G”者,T-NHL和B-NHL患病风险均显著增加。携带NF-kB等位基因“ins”者,B-NHL患病风险显著增加。5、CARD8(rs2043211)的AA基因型及LDH水平较高的淋巴瘤患者具有较短的生存期。二、NLRP3炎症小体相关基因在淋巴瘤患者中的表达及相关功能研究1、NLRP3炎症小体相关基因表达:NLRP3相关基因IL-18、IL-1β、caspase-1、NLRP3和NFκB的mRNA表达在淋巴瘤患者中均明显高于正常人,但治疗后缓解淋巴瘤患者中只有IL-18 mRNA表达明显低于新诊断淋巴瘤患者,其它基因表达未见显著性差异。IL-18(rs1946518)、NFκB 94 ins/del(rs28362491)、IL-1β(rs16944)基因不同基因型间mRNA表达均无显著性差异。2、淋巴瘤患者外周血血清IL-18以及IL-1β水平:新诊断的淋巴瘤患者的血清IL-18水平显著高于对照组,化疗缓解后较化疗前显著降低。而新诊断的淋巴瘤患者血清IL-1β水平显著高于对照组,但化疗缓解后与化疗前相比没有显著变化,IL-18(rs1946518)GT基因型患者的血清IL-18水平显著高于GG基因型。3、淋巴瘤病理组织中NLRP3相关基因表达:淋巴瘤患者ASC和NLRP3表达明显高于正常对照,IL-18表达略高于正常对照。4、NLRP3炎症小体活化验证:用LPS刺激6h,ATP作用1h后,Pfeiffer细胞ASC和NFκB的蛋白表达水平升高,NLRP3相关基因IL-18、caspase-1、IL-1β、NLRP3的mRNA表达均升高,培养上清液中IL-18和IL-1β水平显著升高,均提示NLRP3炎症小体被活化。5、NLRP3炎症小体的活化增强了 Pfeiffer细胞对地塞米松的耐药性:LPS+ATP处理细胞后,地塞米松对Pfeiffer细胞的抑制率下降,显著提高了地塞米松的IC50 值。6、NLRP3炎症小体减弱地塞米松对细胞增殖的抑制作用:地塞米松可抑制pfeiffer细胞增殖,NLRP3炎症小体的活化促进细胞周期从G1进入S期,通过上调c-myc mRNA表达,减弱了地塞米松对细胞周期的影响,从而促进细胞增殖。7、NLRP3炎症小体抑制地塞米松诱导的细胞凋亡作用:地塞米松可诱导pfeiffer细胞凋亡,NLRP3炎症小体的活化通过上调bcl-2和下调Bax从而抑制细胞凋亡,减弱地塞米松的促凋亡作用。8、重组人IL-18(rhIL-18)调节NLRP3炎症小体对Pfeiffer细胞的生物学作用:加入rhIL-18后c-myc mRNA和bcl-2 mRNA表达显著增加,而TP53 mRNA和bax mRNA表达显著降低,从而促进细胞增殖,抑制细胞凋亡,抑制地塞米松的抗肿瘤作用,而加入抗IL-18后作用相反。结论:1、IL-18(rs1946518)和 NFκB94ins/del(rs28362491)基因多态性是影响淋巴瘤易感性的相关因素,CARD8(rs2043211)基因多态性对淋巴瘤的生存期有较大影响。2、淋巴瘤患者NLRP3相关基因表达高于正常人,NLRP3炎症小体通过其下游基因尤其是IL-18的作用,促进淋巴瘤细胞增殖,抑制淋巴瘤细胞凋亡,减弱地塞米松的抗肿瘤作用。3、IL-18可活化NLRP3,促进淋巴瘤细胞增殖,抑制淋巴瘤细胞凋亡,减弱地塞米松的抗肿瘤作用。抗IL-18作用与之相反。4、NLRP3炎症小体可能通过其效应分子IL-18促进淋巴瘤的发生发展。更多还原

【Abstract】 Background:Lymphoma is a malignant tumor derived from lymphoid hematopoietic system.With the continuous development of medical treatment,the survival and quality of life of patients have been greatly improved.However,there are still many lymphoma patients that will relapse or be resistant to chemotherapy,and even lead to death.Lymphoma is an immune system tumor,and the occurrence of tumors is a multi-factor,multi-link,multi-gene interaction results which are related to genetic,immune microenvironment abnormalities,apoptosis,tumor suppressor gene inactivation and abnormal cell proliferation.Recent studies have shown that tumor cells in the development or chemotherapy treatment could produce inflammasome which was regarded as an important part of immunity.NLRP3 inflammasome is the most studied inflammasome which occupies an important position in the occurrence,development and chemoresistance of tumor.Inflammasome can be recruited,assembled and activated by exogenous pathogen invasion or in vivo cell damage and death.Inflammasomes release a large number of IL-1 and IL-18 and other inflammatory cytokines through the NLRP3/caspase axis to promote tumorigenesis.IL-18 is the main effector molecule of NLRP3 inflammasome which could inhibit the growth of NK cells to promote tumor growth and metastasis by inducing the expression of PD-1.Single nucleotide polymorphisms(SNPs)affect the inflammatory microenvironment nesting malignant tumors,and SNPs may also provide a direct effect on the malignant cells.Genetic SNPs associated with NLRP3 inflammasome were found to be related to the development of various cancers.NLRP3 inflammasome and its products could directly promote the growth of tumor cells and drug resistance.The inhibition of inflammatory response or the neutralization of its products has a far-reaching inhibitory effect on canceration and tumor progression,therefore targeting NLRP3 inflammasome is expected to become an important means of tumor therapy.Objective:The aim of this study was to investigate the polymorphism and expression of NLRP3 inflammasome in patients with lymphoma and evaluate its clinical significance,and further to explore its mechanism in lymphoma by using functional experiment of lymphoma cell line.Methods:1.SNPs of NLRP3-related gene in lymphoma patients:390 patients with lymphoma and 385 healthy controls were selected.Bone marrow smear was taken from patients with lymphoma.EDTA anticoagulated whole blood was extracted from healthy controls,and then extracted by DNA sample extraction kit DNA.PCR(FISH)were used to detect the genotypes.2.The expression of NLRP3-related genes in lymphoma patients and controls:Sixty eight patients with lymphoma(46 newly diagnosed patients and 22 patients with remission)and 40 healthy controls were used to extract peripheral blood mRNA and reverse transcribed into cDNA.The expressions of IL-18,IL-1β,caspase-1,NLRP3,ASC and NFκB mRNA in peripheral blood mononuclear cells of patients with lymphoma and controls were detected by RT-PCR.The levels of IL-18 and IL-1β in peripheral blood of patients with lymphoma and controls were detected by ELISA.The protein expressions of IL-18,NLRP3 and ASC in lymphoma tissues and normal lymph nodes were detected by immunohistochemistry.3.The expression levels of ASC and NFκB protein were detected by western blot after adding LPS and ATP in Pfeiffer culture system,while GAPDH was used as internal reference.4.Pfeiffer cells were added with different concentrations of dexamethasone or after adding LPS and ATP,and then were detected for drug sensitivity.The experimental groups were divided into control,LPS+ATP,DEX and DEX+LPS+ATP.The cells and supernatants were collected for determination.The mRNA expressions of IL-18,IL-1β,caspase-1,NLRP3,c-myc,TP53,bcl-2 and bax in cells were detected by RT-PCR.The levels of IL-18 and IL-β in supernatant were measured by ELISA.5.IL-18 and anti-IL-18 were added to Pfeiffer culture system for 48h.The experimental group was divided into control,DEX,IL-18+DEX and anti-IL-18+DEX.The expressions of c-myc,TP53,bcl-2 and bax mRNA were detected by RT-PCR.6.CCK8 method was used to detect the cell proliferation and the drug sensitivity analysis.According to the cell cycle kit,cell cycle analysis was performed by flow cytometry.Apoptosis of each group was detected using flow cytometry by Annexin V/PI apoptosis detection kit.7.Statistical analysis:The continuous and categorical variables were compared by ANOVA or nonparametric Kruskal-Wallis test.The standard chi-square test was used to test the genotype frequencies of Hardy-Weinberg equilibrium.The association between genotype and disease risk was assessed by calculating the odds ratio(OR)and the corresponding 95%confidence interval(CI).The Mann-Whitney U test was used to perform mRNA and ELISA analysis to compare the expression of each group.The Kaplan-Meier curve was used to describe survival.The cell experiments were tested three times,and the quantitative data were expressed as the mean ±SD deviation and analyzed using the unpaired t test.P<0.05 was considered statistically significant.Results:1.Relationship between SNPs of NLRP3 inflammasome and susceptibility and prognosis of lymphoma(1)The genotype distribution of IL-18(rs1946518)and NF∈B 94 ins/del(rs28362491)in lymphoma patients was significantly different from that in normal controls.(2)The risk of lymphoma carrying IL-18(rsl 946518)allele "G" or NFκB 94 ins/del(rs28362491)allele "ins" was significantly increased.(3)IL-18(rsl946591)genotype was associated with LDH levels.CARD8(rs2043211)genotype was associated with gender,B symptoms and extranodal infiltration.And NFκB 94 ins/del(rs28362491)genotype was associated with LDH levels,typing and bone marrow infiltration.8(4)IL-18(rsl946518)gene and NFκB 94 ins/del(rs28362491)gene were significantly associated with susceptibility to B-NHL.IL-18(rs 1946518)gene was also significantly associated with susceptibility to T-NHL.The risk of T-NHL or B-NHL was significantly increased in the carriers with IL-18 allele "G",and the NFκB allele "ins" was associated with a significant increase in the risk of B-NHL.(5)Lymphoma patients with CARD8(rs2043211)AA genotype or higher LDH level have a poor survival.2.Expressions of NLRP3 inflammasome-related genes in patients with lymphoma and their related functions(1)The mRNA expressions of IL-18,IL-1β,caspase-1,NLRP3 and NFκB of patients with lymphoma were significantly higher than those of controls.However,only the expression of IL-18 mRNA in lymphoma patients after chemotherapy remission was lower than that in newly diagnosed lymphoma patients.(2)IL-18 serum levels in newly diagnosed lymphoma patients were significantly higher than those in controls,and decreased after chemotherapy.The serum levels of IL-1β in patients with newly diagnosed lymphoma were significantly higher than those in controls,but showed no significant change with those after chemotherapy.The serum levels of IL-18 in IL-18(rsl946518)GT genotype were significantly higher than those in GG genotype.(3)The protein expressions of ASC and NLRP3 in lymphoma tissues were significantly higher than those in controls,and the protein expressions of IL-18 in lymphoma patients were slightly higher than those in controls.(4)After adding LPS for 6 hours and ATP for 1 hour,the protein expression levels of ASC and NFκB were increased in Pfeiffer.Moreover,the expressions of IL-18,caspase-1,IL-1β and NLRP3 mRNA were increased,and the levels of IL-18 and IL-1β in culture supernatant were significantly increased,which suggested that NLRP3 inflammasome was activated.(5)After being treated with LPS and ATP,the inhibitory rate of dexamethasone on Pfeiffer cells decreased,and the IC50 value of dexamethasone increased significantly.So the activation of NLRP3 inflammasome enhanced the resistance of Pfeiffer cells to dexamethasone.(6)Dexamethasone inhibited the proliferation of Pfeiffer cells.The activation of NLRP3 inflammasome promoted the cell cycle from G1 to S phase,reduced the effect of dexamethasone on cell cycle,andpromoted the cell proliferation by increasing the expression of c-myc mRNA.(7)Dexamethasone induced the Pfeiffer cell apoptosis.The activation of NLRP3 inflammasome inhibited cell apoptosis and weakened the anti-tumor effect of dexamethasone by up-regulation of bcl-2 and down-regulation of bax.(8)The expressions of c-myc and bcl-2 mRNA were significantly increased,while the expressions of TP53 and bax mRNA were significantly decreased after rhIL-18 was added,which promoted the cell proliferation,inhibited the cell apoptosis,and inhibited the anti-tumor effect of dexamethasone.Moreover,anti-IL-18 antibody has the according opposite effects.Conclusion:1.The gene polymorphisms of IL-18(rs1946518)and NFκB 94 ins/del(rs28362491)were associated with the increasing susceptibility of lymphoma.The CARD8(rs2043211)polymorphism had a great effect on the survival of lymphoma.2.The expressions of NLRP3-related genes in patients with lymphoma were higher than those in normal subjects.NLRP3 inflammasome could promote the proliferation of lymphoma cells,inhibit the apoptosis of lymphoma cells,and reduce the anti-tumor effect of dexamethasone by its downstream genes such as IL-1β and IL-18.3.IL-18 could activate the NLRP3 inflammasome,promote the proliferation of lymphoma cells,inhibit the apoptosis of lymphoma cells,and reduce the anti-tumor effect of dexamethasone.Anti-IL-18 could play the opposite effect.4.NLRP3 inflammasome contributes to the susceptibility of lymphoma and plays a carcinogenic role through its effector cytokine IL-18.更多还原