原发免疫缺陷病DOCK8缺陷综合征及IKBKB缺陷疾病的发病机制探讨
发布时间：2018-07-29 08:07
【摘要】：第一部分DOCK8缺陷状态下滤泡辅助T细胞产生IL-21调节Ig E的机制研究背景:高Ig E综合征(Hyper-Ig E syndrome,HIES)是一组常染色体遗传的原发性免疫缺陷病,其中常染色体隐性遗传(AR)的DOCK8免疫缺陷综合症(Dedicator of cytokinesis 8immunodeficiency syndrome,DIDS;OMIM 243700)是一种以特应性皮炎、反复、慢性皮肤病毒感染和呼吸道感染症状、血清lg E升高和嗜酸性粒细胞增多为特征的原发性免疫缺陷疾病。DOCK8基因定位于人类9号染色体p24.3,其编码的DOCK8蛋白可激活Rho-GTP酶,从而调节诸多细胞功能,尤其是肌动蛋白细胞骨架调控、影响细胞迁移活动。DOCK8缺陷患者罹患联合免疫缺陷,但B细胞介导的抗体缺陷最为突出,其中尤以Ig E水平急剧升高较为明显,目前机制未知。目的:分析中国的DOCK8缺陷综合征患儿临床表现及免疫功能,采用临床样本与DOCK8-KO小鼠模型探究DOCK8蛋白缺陷对Ig E水平的影响及其机制。方法:采用高通量测序及流式细胞术对疑似高Ig E综合征的患儿进行诊断,确立DOCK8缺陷诊断后进行淋巴细胞免疫功能评估,分析患儿及健康同龄对照外周血滤泡辅助T细胞数量和功能改变,以及其相关功能分子IL-21水平,实时荧光定量检测Bcl-6及Blimp-1 m RNA表达。运用TALEN技术建立DOCK8-KO小鼠模型并对其免疫功能进行评估,分析滤泡辅助T细胞数量功能改变,以及其相关功能分子ICOS、PD-1、Bcl-6及IL-21水平,对DOCK8-KO小鼠进行IL-21替代治疗并观察其对免疫功能的修复效应。进一步构建OVA诱导哮喘DOCK8-KO小鼠模型并对其进行IL-21补充治疗,解析其治疗机制。结果:1.7例DOCK8免疫缺陷患儿,2男5女,高Ig E综合征NIH评分均高于40分,表现为反复顽固的病毒感染,肺炎,血清Ig E异常增高和嗜酸性粒细胞增多等现象。针对DOCK8单基因的高通量测序分析显示7例患儿发生不同程度的大片段缺失突变、移码突变或错义突变,均为尚未报道过的新发突变。其中P1患儿为19-48号外显子杂合性大片段缺失并伴有移码突变(c.5842 del G,c 5843CA p.A1948fs X1953),P2患儿11号外显子纯合性缺失,12-33号外显子杂合性缺失,P3-4患儿为移码突变(c.3152del G p.S1051fs X1093)和错义突变(c.5175GC p.E1725D),P5患儿为2号外显子纯合性缺失,1号,3-39号外显子杂合性缺失,P6患儿为7号外显子纯合性缺失,8-10号外显子杂合性缺失,P7患儿为移码突变(c.1278-1279 del TG p.V427fs X435)。7例患儿完成流式细胞术及Western检测,外周血单个核细胞均无DOCK8蛋白表达。患儿外周血CD4+T细胞、记忆B细胞和记忆T细胞均有不同程度降低。T细胞删除环(TRECs)、淋巴细胞增殖水平和NK细胞的细胞毒杀伤功能均明显低于正常同龄儿童。外周血调节性B细胞水平低下,而调节性T细胞,CD4+T细胞产生细胞因子IL-17、IL-4及IFN-γ水平均正常。DOCK8患儿滤泡辅助T细胞占CD4+T细胞比率与正常人相比无统计学差异,但绝对值却明显低于正常对照。尤其是Tfh细胞分泌的IL-21明显下降。2.(1)运用TALEN技术建立DOCK8-KO小鼠,DOCK8-KO小鼠较WT小鼠的血清Ig E、血液及脾脏中的Ig E+B细胞有升高趋势,并有统计学差异。DOCK8-KO小鼠的滤泡辅助T细胞相关功能分子出现不同程度的下降,其中细胞因子IL-21水平下降极为明显,Tfh细胞分化的主要调节转录因子Bcl-6,以及Tfh细胞的功能分子PD-1、ICOS水平均不同程度降低。(2)采用传统剂量四分之一的低剂量OVA成功诱导DOCK8-KO小鼠哮喘模型,模型小鼠出现明显的肺功能异常,血清Ig E水平升高,嗜酸性粒细胞增多等过敏特征。OVA诱导的DOCK8-KO及WT小鼠Ig E+B细胞水平均有升高现象,但前者升高的水平是后者的数倍,其中血清总Ig E及OVA特异性Ig E水平升高尤为明显。经OVA诱导的DOCK8-KO小鼠IL-21水平随之进一步下降,而经OVA诱导的WT小鼠则没有明显改变。另外,OVA诱导的DOCK8-KO小鼠脾脏记忆性Tfh细胞,Tfh细胞分化的主要调节转录因子Bcl-6,以及Tfh细胞的功能分子PD-1、ICOS均出现明显降低。3.(1)使用重组小鼠IL-21细胞因子(20ng/d×3-6天)对DOCK8-KO小鼠进行替代治疗,发现KO小鼠血清Ig E水平几乎能够回复到正常水平,脾脏Tfh细胞及其分化的主要调节转录因子Bcl-6,以及Tfh细胞的功能分子PD-1和ICOS均能不同程度的回升至接近正常水平,治疗效应十分明显。(2)采用IL-21治疗OVA诱导的小鼠模型,WT小鼠的Ig E水平虽有回复,但总体治疗效果不如DOCK8-KO小鼠明显。经OVA诱导的DOCK8-KO小鼠经过IL-21治疗其Ig E水平能够回复到正常水平,并且Tfh细胞及其主要调节转录因子Bcl-6,以及Tfh细胞的功能分子PD-1,ICOS均能不同程度的回升至正常水平。结论:DOCK8缺陷引起Tfh细胞发育分化障碍,IL-21分泌降低,可能是导致B细胞对过敏原反应失控,产生大量功能性Ig E的根本原因。第二部分IKKβ缺陷对NF-κB入核的影响背景:IKBKB基因突变是一种特殊的联合免疫缺陷病,以反复细菌、病毒、真菌感染为主要表现,大部分病人会出现不同程度鹅口疮、支气管炎及肺炎,并伴有慢性腹泻、生长发育迟缓、脑出血、癫痫、脐炎、脐带脱落延迟等现象。IKBKB基因定位于人类8号染色体p11.2,其编码的IKKβ蛋白是IκB kinase的亚单位之一,主要参与NF-κB通路激活过程,活化的IKK磷酸化IκBα(NF-κB的抑制剂),磷酸化的IκBα不断降解,使其对NF-κB的抑制功能减弱,后者则可以自由出入细胞核并激活参与炎症及免疫反应的各种基因。目的:分析一例IKBKB突变患儿临床表现、蛋白表达、基因突变及免疫功能。探讨395位点氨基酸磷酸化对NF-κB入核影响及通路功能的影响及机制。方法:患儿男,17岁,生后2月龄开始出现反复呼吸道感染,腹泻及低体重。采用高通量测序发现基因突变,并用一代测序验证,采用流式细胞术及免疫印迹法检测IKKβ蛋白。采用流式细胞术分析淋巴细胞功能,EMSA方法检测核内NF-κB水平。制备395位点定点变异细胞系,以去除该位点磷酸化,探讨其对IKKβ蛋白稳定性的影响机制。结果:高通量测序显示IKBKB基因发生错义突变(c.1183TC,p.Y395H),为尚未报道的新发突变。一代测序验证确认为该位点突变,流式细胞术及免疫印迹法均未能检测到IKKβ蛋白表达。免疫功能评估中发现患儿记忆B细胞极度低下,调节性T细胞缺如,淋巴细胞增殖水平明显受损。调节性B细胞升高,而CD4+T细胞表达细胞因子IL-17、IL-4、IL-21及IFN-γ均处于正常下限,NK细胞的细胞毒杀伤功能及TCRVβ多样性正常。患儿细胞核内NF-κB蛋白较正常人明显降低,提示基因突变导致进入细胞核的NF-κB水平减少。进一步发现395位点突变导致磷酸化位点消失后IKKβ降解速率增快,蛋白稳定性下降。结论:确诊1例极为罕见的PID病例——IKBKB缺陷,为全球范围内报道的第10例患儿。IKKβ的395位点发生的错义突变为尚未报道过的新发突变。首次阐明该位点是IKKβ蛋白重要的磷酸化位点,由酪氨酸突变为组氨酸造成的氨基酸改变会导致磷酸化位点丢失,降解增快,从而最终影响NF-κB入核。
[Abstract]:In the first part of the DOCK8 deficiency, follicular assisting T cells to produce the mechanism of IL-21 regulation of Ig E: high Ig E syndrome (Hyper-Ig E syndrome, HIES) is a group of autosomal primary immunodeficiency diseases, including the autosomal recessive inheritance (AR) Yndrome, DIDS; OMIM 243700) is a primary immunodeficiency disease characterized by atopic dermatitis, recurrent, chronic skin virus infection and respiratory infection. The.DOCK8 gene of serum LG E and eosinophils is a primary immunodeficiency disease, which is located on the human chromosome 9 of human chromosome p24.3, and its encoded DOCK8 protein activates the Rho-GTP enzyme, thus regulating many of them. The cell function, especially the actin cytoskeleton regulation, affects the joint immunodeficiency in the cell migration.DOCK8 deficiency patients, but the B cell mediated antibody defects are most prominent, especially the rapid rise of Ig E level, and the present mechanism is unknown. Objective: to analyze the clinical manifestation and immunity of children with DOCK8 deficiency syndrome in China. Function, using clinical samples and DOCK8-KO mouse model to explore the effect of DOCK8 protein deficiency on the level of Ig E and its mechanism. Methods: high throughput sequencing and flow cytometry were used to diagnose children with suspected high Ig E syndrome, and the lymphocyte immune function evaluation was established after the diagnosis of DOCK8 defect, and the children and the healthy same age control were analyzed. The number and function of T cells were assisted by peripheral blood follicles, as well as the IL-21 level of its related functional molecules, and the expression of Bcl-6 and Blimp-1 m RNA were detected by real time fluorescence. The TALEN technique was used to establish the DOCK8-KO mouse model and evaluate its immune function. The function of the follicle assisted T cells was changed, and the related functional molecules ICOS, PD-1, Bcl were analyzed. At the level of -6 and IL-21, the DOCK8-KO mice were treated with IL-21 replacement therapy and their effects on the immune function were observed. Further construction of OVA induced asthma DOCK8-KO mice model and IL-21 supplementation were carried out to analyze the therapeutic mechanism. Results: 1.7 cases of DOCK8 immunodeficiency children, 2 men and 5 women, and high Ig E syndrome NIH score were all higher than 40 points. For repeated stubborn virus infection, pneumonia, abnormal increase of serum Ig E and eosinophils. High throughput sequencing analysis of DOCK8 single gene showed that 7 children had different degrees of large fragment deletion, code mutation or missense mutation, all of which were unreported new mutations. Among them, children with P1 were exon 19-48. C.5842 del G, C 5843CA p.A1948fs X1953, loss of homozygosity in exon 11 of P2 children, loss of heterozygosity in exon 12-33 of children with P2 (c.3152del G, c.3152del G p.S1051fs) and missense mutations were found in children with exon 2, 1 and 3-39. The heterozygosity of exon was absent, the children of P6 were homozygous deletion of exon 7 and heterozygosity in exon 8-10, and P7 children were transferred code mutation (c.1278-1279 del TG p.V427fs X435) with.7 cases complete flow cytometry and Western detection, and peripheral blood mononuclear cells had no DOCK8 egg white expression. The.T cell deletion ring (TRECs) was reduced to some extent, the proliferation level of lymphocyte and the cytotoxic function of NK cells were significantly lower than those of normal age children. The regulatory B cells in peripheral blood were low, and regulatory T cells, CD4+T cells produced cytokine IL-17, IL-4 and IFN- gamma water were normal.DOCK8 children with follicular auxiliary T. There was no statistical difference in the ratio of cell CD4+T cells to normal people, but the absolute value was significantly lower than that of normal controls. Especially, the IL-21 secreted by Tfh cells significantly decreased.2. (1) using TALEN technology to establish DOCK8-KO mice, the Ig E of the DOCK8-KO mice and the Ig E+B cells in the blood and spleen, and the statistical difference between the DOCK8-KO mice and the WT mice. The follicle assisted T cell related functional molecules in different.DOCK8-KO mice decreased to varying degrees, in which the level of cytokine IL-21 decreased significantly, the main regulating transcription factor Bcl-6 of Tfh cell differentiation, and the PD-1 of Tfh cells, the level of ICOS were reduced in varying degrees. (2) the low dose OVA of traditional dose 1/4 was used. The DOCK8-KO mouse asthma model was successfully induced. The model mice had obvious abnormal pulmonary function, the level of serum Ig E increased, the eosinophil increased, and the level of Ig E+B cells in DOCK8-KO and WT mice induced by.OVA was increased, but the level of the former was several times that of the latter, and the serum total Ig E and OVA specificity Ig. The level of horizontal elevation was particularly obvious. The IL-21 level of DOCK8-KO mice induced by OVA was further decreased, while the WT mice induced by OVA did not change obviously. In addition, the OVA induced DOCK8-KO mouse spleen memory Tfh cells, the main regulating transcription factor Bcl-6 of the Tfh cell differentiation, and the functional molecular PD-1 of the Tfh cells were significantly reduced. 3. (1) the recombinant mouse IL-21 cytokine (20ng/d x 3-6 days) was used to replace the DOCK8-KO mice. It was found that the serum Ig E level of the KO mice could almost revert to the normal level. The spleen Tfh cells and the main regulating transcription factor Bcl-6 of the splenic Tfh cells, as well as the Tfh cell functional molecules PD-1 and ICOS were all able to recover to the normal level to the normal level. Level, treatment effect is very obvious. (2) the use of IL-21 to treat OVA induced mouse model, WT mice Ig E level although there is a response, but the overall treatment effect is not as obvious as DOCK8-KO mice. OVA induced DOCK8-KO mice after IL-21 treatment of Ig E level can be back to normal levels, and Tfh cells and the main regulation of transcription factors, And the functional molecules of Tfh cells, PD-1, and ICOS can rise to the normal level in varying degrees. Conclusion: DOCK8 defects cause the development and differentiation of Tfh cells and the decrease of IL-21 secretion, which may be the root cause of the uncontrolled reaction of the B cells to the allergen reaction and the production of a large number of functional Ig E. The background of the influence of the second part IKK beta defect on NF- nuclear B is: Gene mutation is a special kind of joint immunodeficiency disease, which is characterized by repeated bacteria, virus and fungal infection. Most patients have different degrees of thrush, bronchitis and pneumonia, accompanied by chronic diarrhea, growth retardation, cerebral hemorrhage, epilepsy, cord inflammation, and delay of umbilical cord abscission. The.IKBKB gene is located in human 8. P11.2, its encoded IKK beta protein is one of the subunits of I kappa B kinase, which is mainly involved in the activation process of NF- kappa B pathway, and activated IKK phosphorylation I kappa B alpha (NF- kappa B). Objective: to analyze the clinical manifestation, protein expression, gene mutation and immune function of a patient with IKBKB mutation. The effect and mechanism of the 395 site amino acid phosphorylation on NF- kappa B nucleation and pathway function. Methods: children, 17 years old, 2 month old after birth, recurrent respiratory infection, diarrhea and low weight. High throughput sequencing IKK beta protein was detected by flow cytometry and immunoblotting. Flow cytometry and immunoblotting were used to detect IKK beta protein. Flow cytometry was used to analyze lymphocyte function and EMSA method was used to detect the level of NF- kappa B. The site directed mutated cell line of 395 loci was prepared to remove the phosphorylation of the site and the mechanism of its effect on the stability of IKK beta protein was discussed. Result: high throughput sequencing showed that the IKBKB gene occurred missense mutation (c.1183TC, p.Y395H), which was a new mutation that had not been reported. One generation sequencing verification confirmed the mutation of the site. The expression of IKK beta protein was not detected by flow cytometry and immunoblotting. The immune function evaluation found that the children's memory B cells were extremely low, and the regulatory T cells were absent. The level of lymphocyte proliferation was significantly impaired. Regulatory B cells were elevated, while CD4+T cells expressed cytokine IL-17, IL-4, IL-21 and IFN- gamma in the normal lower limit. The cytotoxic function of NK cells and the diversity of TCRV beta were normal. The protein of NF- kappa B in the nucleus of the children was lower than that of the normal human, suggesting that the gene mutation leads to NF- kappa B. IKK beta degradation rate increased rapidly and protein stability decreased after the 395 site mutation resulted in the disappearance of phosphorylation sites. Conclusion: 1 cases of extremely rare cases of IKBKB were diagnosed, and the missense mutation of the 395 site of.IKK beta in tenth cases of.IKK was reported worldwide for the first time. This site is an important phosphorylation site for IKK beta protein. The amino acid change caused by mutation of tyrosine to histidine leads to the loss of phosphorylation sites and the rapid degradation of the amino acid, which ultimately affects the nucleation of NF- kappa B.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R725.9
本文编号：2152047
[Abstract]:In the first part of the DOCK8 deficiency, follicular assisting T cells to produce the mechanism of IL-21 regulation of Ig E: high Ig E syndrome (Hyper-Ig E syndrome, HIES) is a group of autosomal primary immunodeficiency diseases, including the autosomal recessive inheritance (AR) Yndrome, DIDS; OMIM 243700) is a primary immunodeficiency disease characterized by atopic dermatitis, recurrent, chronic skin virus infection and respiratory infection. The.DOCK8 gene of serum LG E and eosinophils is a primary immunodeficiency disease, which is located on the human chromosome 9 of human chromosome p24.3, and its encoded DOCK8 protein activates the Rho-GTP enzyme, thus regulating many of them. The cell function, especially the actin cytoskeleton regulation, affects the joint immunodeficiency in the cell migration.DOCK8 deficiency patients, but the B cell mediated antibody defects are most prominent, especially the rapid rise of Ig E level, and the present mechanism is unknown. Objective: to analyze the clinical manifestation and immunity of children with DOCK8 deficiency syndrome in China. Function, using clinical samples and DOCK8-KO mouse model to explore the effect of DOCK8 protein deficiency on the level of Ig E and its mechanism. Methods: high throughput sequencing and flow cytometry were used to diagnose children with suspected high Ig E syndrome, and the lymphocyte immune function evaluation was established after the diagnosis of DOCK8 defect, and the children and the healthy same age control were analyzed. The number and function of T cells were assisted by peripheral blood follicles, as well as the IL-21 level of its related functional molecules, and the expression of Bcl-6 and Blimp-1 m RNA were detected by real time fluorescence. The TALEN technique was used to establish the DOCK8-KO mouse model and evaluate its immune function. The function of the follicle assisted T cells was changed, and the related functional molecules ICOS, PD-1, Bcl were analyzed. At the level of -6 and IL-21, the DOCK8-KO mice were treated with IL-21 replacement therapy and their effects on the immune function were observed. Further construction of OVA induced asthma DOCK8-KO mice model and IL-21 supplementation were carried out to analyze the therapeutic mechanism. Results: 1.7 cases of DOCK8 immunodeficiency children, 2 men and 5 women, and high Ig E syndrome NIH score were all higher than 40 points. For repeated stubborn virus infection, pneumonia, abnormal increase of serum Ig E and eosinophils. High throughput sequencing analysis of DOCK8 single gene showed that 7 children had different degrees of large fragment deletion, code mutation or missense mutation, all of which were unreported new mutations. Among them, children with P1 were exon 19-48. C.5842 del G, C 5843CA p.A1948fs X1953, loss of homozygosity in exon 11 of P2 children, loss of heterozygosity in exon 12-33 of children with P2 (c.3152del G, c.3152del G p.S1051fs) and missense mutations were found in children with exon 2, 1 and 3-39. The heterozygosity of exon was absent, the children of P6 were homozygous deletion of exon 7 and heterozygosity in exon 8-10, and P7 children were transferred code mutation (c.1278-1279 del TG p.V427fs X435) with.7 cases complete flow cytometry and Western detection, and peripheral blood mononuclear cells had no DOCK8 egg white expression. The.T cell deletion ring (TRECs) was reduced to some extent, the proliferation level of lymphocyte and the cytotoxic function of NK cells were significantly lower than those of normal age children. The regulatory B cells in peripheral blood were low, and regulatory T cells, CD4+T cells produced cytokine IL-17, IL-4 and IFN- gamma water were normal.DOCK8 children with follicular auxiliary T. There was no statistical difference in the ratio of cell CD4+T cells to normal people, but the absolute value was significantly lower than that of normal controls. Especially, the IL-21 secreted by Tfh cells significantly decreased.2. (1) using TALEN technology to establish DOCK8-KO mice, the Ig E of the DOCK8-KO mice and the Ig E+B cells in the blood and spleen, and the statistical difference between the DOCK8-KO mice and the WT mice. The follicle assisted T cell related functional molecules in different.DOCK8-KO mice decreased to varying degrees, in which the level of cytokine IL-21 decreased significantly, the main regulating transcription factor Bcl-6 of Tfh cell differentiation, and the PD-1 of Tfh cells, the level of ICOS were reduced in varying degrees. (2) the low dose OVA of traditional dose 1/4 was used. The DOCK8-KO mouse asthma model was successfully induced. The model mice had obvious abnormal pulmonary function, the level of serum Ig E increased, the eosinophil increased, and the level of Ig E+B cells in DOCK8-KO and WT mice induced by.OVA was increased, but the level of the former was several times that of the latter, and the serum total Ig E and OVA specificity Ig. The level of horizontal elevation was particularly obvious. The IL-21 level of DOCK8-KO mice induced by OVA was further decreased, while the WT mice induced by OVA did not change obviously. In addition, the OVA induced DOCK8-KO mouse spleen memory Tfh cells, the main regulating transcription factor Bcl-6 of the Tfh cell differentiation, and the functional molecular PD-1 of the Tfh cells were significantly reduced. 3. (1) the recombinant mouse IL-21 cytokine (20ng/d x 3-6 days) was used to replace the DOCK8-KO mice. It was found that the serum Ig E level of the KO mice could almost revert to the normal level. The spleen Tfh cells and the main regulating transcription factor Bcl-6 of the splenic Tfh cells, as well as the Tfh cell functional molecules PD-1 and ICOS were all able to recover to the normal level to the normal level. Level, treatment effect is very obvious. (2) the use of IL-21 to treat OVA induced mouse model, WT mice Ig E level although there is a response, but the overall treatment effect is not as obvious as DOCK8-KO mice. OVA induced DOCK8-KO mice after IL-21 treatment of Ig E level can be back to normal levels, and Tfh cells and the main regulation of transcription factors, And the functional molecules of Tfh cells, PD-1, and ICOS can rise to the normal level in varying degrees. Conclusion: DOCK8 defects cause the development and differentiation of Tfh cells and the decrease of IL-21 secretion, which may be the root cause of the uncontrolled reaction of the B cells to the allergen reaction and the production of a large number of functional Ig E. The background of the influence of the second part IKK beta defect on NF- nuclear B is: Gene mutation is a special kind of joint immunodeficiency disease, which is characterized by repeated bacteria, virus and fungal infection. Most patients have different degrees of thrush, bronchitis and pneumonia, accompanied by chronic diarrhea, growth retardation, cerebral hemorrhage, epilepsy, cord inflammation, and delay of umbilical cord abscission. The.IKBKB gene is located in human 8. P11.2, its encoded IKK beta protein is one of the subunits of I kappa B kinase, which is mainly involved in the activation process of NF- kappa B pathway, and activated IKK phosphorylation I kappa B alpha (NF- kappa B). Objective: to analyze the clinical manifestation, protein expression, gene mutation and immune function of a patient with IKBKB mutation. The effect and mechanism of the 395 site amino acid phosphorylation on NF- kappa B nucleation and pathway function. Methods: children, 17 years old, 2 month old after birth, recurrent respiratory infection, diarrhea and low weight. High throughput sequencing IKK beta protein was detected by flow cytometry and immunoblotting. Flow cytometry and immunoblotting were used to detect IKK beta protein. Flow cytometry was used to analyze lymphocyte function and EMSA method was used to detect the level of NF- kappa B. The site directed mutated cell line of 395 loci was prepared to remove the phosphorylation of the site and the mechanism of its effect on the stability of IKK beta protein was discussed. Result: high throughput sequencing showed that the IKBKB gene occurred missense mutation (c.1183TC, p.Y395H), which was a new mutation that had not been reported. One generation sequencing verification confirmed the mutation of the site. The expression of IKK beta protein was not detected by flow cytometry and immunoblotting. The immune function evaluation found that the children's memory B cells were extremely low, and the regulatory T cells were absent. The level of lymphocyte proliferation was significantly impaired. Regulatory B cells were elevated, while CD4+T cells expressed cytokine IL-17, IL-4, IL-21 and IFN- gamma in the normal lower limit. The cytotoxic function of NK cells and the diversity of TCRV beta were normal. The protein of NF- kappa B in the nucleus of the children was lower than that of the normal human, suggesting that the gene mutation leads to NF- kappa B. IKK beta degradation rate increased rapidly and protein stability decreased after the 395 site mutation resulted in the disappearance of phosphorylation sites. Conclusion: 1 cases of extremely rare cases of IKBKB were diagnosed, and the missense mutation of the 395 site of.IKK beta in tenth cases of.IKK was reported worldwide for the first time. This site is an important phosphorylation site for IKK beta protein. The amino acid change caused by mutation of tyrosine to histidine leads to the loss of phosphorylation sites and the rapid degradation of the amino acid, which ultimately affects the nucleation of NF- kappa B.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R725.9
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