阿尔茨海默病患者ApoE基因多态性与IL-6及TNF-α的相关性研究.pdf

临床和实验医学杂志2023年8 月第2 2 卷第16 期4结论Natl Acad Sci U S A,2018,115(27):E6356-E6365.10 Esteban O,Markiewicz CJ,Blair RW,et al.fMRIPrep:a robust pre-本研究分析了特发性耳鸣患者经过2 4周窄带声治processing pipeline for functional MRIJ.Nat Methods,2019,16疗前后脑沟深度的变化发现，窄带声音疗法改变了大脑(1):111-116.一些重要脑区及皮层的沟深。具有沟深特征的表面形11 Klein A,Ghosh SS,Bao FS,et al.Mindboggling morphometry of hu-态学分析法可以提供更微妙的局部形态学变化解释耳man brains J.PLoS Comput Biol,2017,13(2):e1005350.12 Glasser MF,Coalson TS,Robinson EC,et al.A multi-modal parcellation鸣治疗的机制。尤其值得注意的是，长期的窄带声音治of human cerebral cortexJ.Nature,2016,536(7615):171-178.疗对耳鸣患者的恢复有正性的临床意义。13 Lv H,Liu C,Wang Z,et al.Altered functional connectivity of the thal-amus in tinnitus patients is correlated with symptom alleviation after参考文献sound therapyJ.Brain Imaging Behav,2020,14(6):2668-2678.1 Conlon B,Langguth B,Hamilton C,et al.Bimodal neuromodulation14 He Q,Turel O,Wei L,et al.Structural brain differences associatedcombining sound and tongue stimulation reduces tinnitus symptoms in awith extensive 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receives infor-mation about motor outputJ.Sci Adv,2019,5(7):eaaw5388.19 Natu VS,Arcaro MJ,Barnett MA,et al.Sulcal Depth in the MedialVentral Temporal Cortex Predicts the Location of a Place-Selective Re-gion in Macaques,Children,and AdultsJJ.Cereb Cortex,2021,31(1):48-61.20 Cheng S,Xu G,Zhou J,et al.A Multimodal Meta-Analysis of Struc-tural and Functional Changes in the Brain of Tinnitus J.Front HumNeurosci,2020,14:28.21 Krick CM,Grapp M,Daneshvar-Talebi J,et al.Cortical reorgani-zation in recent-onset tinnitus patients by the Heidelberg Model of Mu-sic TherapyJ.Front Neurosci,2015,9:49.(收稿日期：2 0 2 3-0 4-2 9)D0I:10.3969/j.issn.1671-4695.2023.16.003阿尔茨海默病患者ApoE基因多态性与 IL-6 及 TNF-的相关性研究薛萌李芳高莉娜侯保华李鹏472000；2 河南省理工大学河南焦作4540 0 0；3郑州金域临床检验中心河南郑州450 0 0 0）【摘要】目的探究ApoE基因多态性与阿尔茨海默病（AD患者的炎症因子水平以及临床表现的相关性。方法回顾性收集2 0 2 1年9月至2 0 2 3年1月就诊于黄河三门峡医院神经内科的AD患者6 2 例，另选取健康体检中心的50名健康体检者作为对照组。根据认知量表(ADAS-Cog)评分结果将 AD组分为轻度组(AD-1 组，n=29):ADAS-Cog评分 2 0 分,中重度组（AD-2组，n=33)：A D A S-Co g 评分2 0 分。观察AD-1组,AD-2组和对照组患者的ApoE基因型及血清IL-6和 TNF-水平,并分析ApoE基因多态性与 IL-6 和TNF-水平的相关性。结果 AD-2 组基金项目：河南省科技厅2 0 2 2 年重点科技项目（编号：2 2 2 10 2 310 340）；河南省教育厅2 0 2 2 年高校重点科研项目（编号：2 2 A310015）文章编号:16 7 1-46 9 5(2 0 2 3)16-16 8 9-0 4焦晓园韦晨曦”（1黄河三门峡医院神经内科河南三门峡:1690:的3/4基因型比例以及4等位基因比例分别为39.39%、2 8.7 9%，高于AD-1组（13.7 9%、12.0 7%）和对照组（10.0 0%、7.0 0%）,差异均有统计学意义（P0.05）。8 4基因携带组的ADAS-Cog评分、IL-6、T NF-水平分别为（2 2.154.97)分、（18 0.16 46.49）pg/mL、（6 8.8 17.7 0）p g/m L,均高于s4基因非携带组（18.97 5.2 2）分、（147.2 944.73）p g/m L、（6 3.8 46.15）p g/m LI，差异均有统计学意义（P0.05）。A D 患者的ADAS-Cog评分与IL-6水平以及TNF-水平呈正相关（r=0.491、0.30 0,P 0.0 0 1）。结论ApoE基因多态性参与AD患者认知功能损害,其中84等位基因作用更为显著,这一过程可能通过调控炎症反应实现。【关键词】阿尔茨海默病ApoE基因炎症因子基因多态性ApoE gene polymorphism and expression of IL-6 and TNF-in Alzheimers disease.XUE Meng,LI Fang,GAO Li-na,et al.Depart-ment of Neurology,Huanghe Sanmenxia Hospital,Sanmenxia Henan 472000,China.Abstract】O b j e c t i v e T o e x p l o r e t h e c o r e l a t i o n b e t w e e n A p o E g e n e p o l y mo r p h i s m a n d t h e l e v e l o f i n f l a mma t o r y c y t o k i n e s a n d c l i n i c a lmanifestations in patients with Alzheimers disease(AD).Methods Sixty-two AD patients who visited the Neurology of Huanghe SanmenxiaHospital from September 2021 to January 2023 were retrospectively collected,and 50 health examinees from the health examination center were se-lected as the control group.According to the results of the Alzheimers Disease Assessment Scale(ADAS-Cog),the AD group was divided intomild group(AD-1 group,n=29):ADAS Log score 20 points,moderate to severe group(AD-2 group,n=33):ADAS Log score 20points.The ApoE genotypes,the levels of serum IL-6 and TNF-were compared in AD-1 group,AD-2 group and control group,and thecorrelation between ApoE gene polymorphism and levels of IL-6 and TNF-was analyzed.Results The proportion of s3/4 genotype and 4 al-leles in AD-2 group were 39.39%and 28.79%,respectively,which were higher than that in AD-1 group(13.79%and 12.07%)and thecontrol group(10.00%and 7.00%),the differences were statistically significant(P 0.05).ADAS-Cog scores,IL-6,TNF-in the 84gene carrier group were(22.15 4.97)points,(180.16 46.49)pg/mL,(68.81 7.70)pg/mL,respectively,which were higher than thosein the 84 gene non-carrier group(18.97 5.22)points,(147.29 44.73)pg/mL,(63.84 6.15)pg/mL,the differences were statisti-cally significant(P 0.05).ADAS-Cog scores in AD patients were positively correlated with IL-6 and TNF-levels(r=0.491,0.300,P 6 5岁；(3签署知情同意书。排除标准：（1)患有脑血管疾病、帕金森综合征、脑肿瘤、脑积水、头外伤等其他器质性神经系统疾病；（2)患有抑郁症等其他精神疾病；（3）患有其他严重的全身系统疾病；（4)正在服用抗胆碱能作用的药物。1.3方法1.3.1ApoE基因检测采用血液提取DNA样本,使用ApoE基因分型荧光PCR法检测,使用人类ApoE基ADAS-Cog)评分结果将 AD 组分为轻度组（AD-1组，n=29）:A D A S-C o g 评分0.05）,具有可比性。见表1。本研究经黄河三门峡医院伦理委员会批准（批号：ky2022082)。表1各组受试者基线资料年龄男性受教育年限艮吸烟史饮酒史组别例数(岁,x)例(%)(年,xs)例(%)对照组5069.70 3.53AD-1组2970.14 3.99AD-2组3370.61 4.24X?/值0.719P值0.6981.2丝例(%)20(40.00)7.10 2.7817(58.62)7.59 2.2115(45.45)6.39 2.502.5773.5700.2760.16824(48.00)17(58.62)21(63.64)2.1350.34427(54.00)15(51.72)18(54.55)0.0560.972临床和实验医学杂志2 0 2 3年8 月第2 2 卷第16 期因2/3/4基因分型试剂盒（厦门人瑞生物医药科技有限公司）进行检测。1.3.2认知水平评估所有AD患者均由1名有经验的神经内科医师采用阿尔茨海默病评定量表一ADAS-Cog评估其认知水平1.3.3血液学检测所有受试者均于清晨空腹状态下采集静脉血液,检测血常规、肝功能、肾功能、电解质、血脂、血糖、血清炎症因子。采用酶联免疫吸附试验检测血清白细胞介素（interleukin,IL）-6 及肿瘤坏死因子(tumor necrosis factor alpha,TNF-）水平,试剂盒购自杭州联科生物技术股份有限公司。1.4统计学处理采用SPSS26.0软件对本研究的数据进行统计学分析,计量资料以均数标准差（xs）表示，组间比较采用t检验；计数资料以例或百分率（%）表示，组间比较采用检验；采用Spearman相关性分析认知功能与IL-6及 TNF-之间的关系；P0.05为差异有统计学意义。2结果2.1基因型分布对照组、AD-1组及AD-2组基因分布均符合HardyW e i n b e r g 平衡定律（=3.817，P=0.576;x=8.987,P=0.110;x=9.141,P=0.104），表明该群体具有代表性。AD-2组的3/4基因型比例高于对照组和AD-1组,8 3/3基因型比例低于对照组和AD-1组,差异有统计学意义（P0.05）。见表2。表2 各组ApoE基因型分布组别例数82/3对照组507(14.00)2(4.00)AD-1组293(10.34)3(10.34)AD-2组333(9.09)X?值P值注：与对照组比较，b与AD-1组比较，P0.05。2.2等位基因频率分布AD-2组的4等位基因比例高于对照组和AD-1组,差异有统计学意义（P0.05)。见表3。表3各组等位基因分布【例（%)】组别例数对照组50AD-1组29AD-2 组33X?值P值注：与对照组比较，b与AD-1组比较，P0.05。2.3ADAS-cog评分与 IL-6 及 TNF-水平4基因携带组的ADAS-Cog评分、IL-6、T NF-水平均高于 4 基因非携带组,差异均有统计学意义（P0.05)。见表4。:1691 表 4AD患者 8 4携带组与非携带组的ADAS-cog评分及血清学指标比较（xs）ADAS-Cog组别例数评分(分）84基因携带组722.15 4.9784基因非携带组718.97 5.22t值-2.416P值0.0192.4AD患者认知水平与IL-6及TNF-水平相关性AD 患者的ADAS-Cog评分与IL-6与TNF-水平呈正相关（r=0.491、0.30 0,P 0.0 5)。3讨论ApoE基因编码ApoE蛋白,参与脂质运输以及大脑中A聚集和清除相关的过程,并在星形胶质细胞中大量表达。ApoE等位基因已被证明以不同的方式影响脑脂质运输、神经炎症、葡萄糖代谢、神经元信号和线粒体功能。大量证据支持ApoEs4与大脑A聚集之间存在联系，甚至有报道称ApoE4可直接促进A原纤维的形成7 ,Castellano等8 将ApoE/A复合物注射到野生型小鼠大脑中发现，相较于ApoE2和ApoE3,ApoE84的存在显著减慢了A在大脑中的清除速率;以上机制均导致A在脑中蓄积,加重了 AD患者的病理及临床表现。本研究探讨了ApoE基因型分布、认知功能障碍与血清炎症水平的关系,结果发现,AD-2组的 8 4等位基因频率高于对照组和AD-1组,提示ApoE基因多态性节例(%)参与AD患者认知功能损害,其中 4 等位基因作用更82/483/336(72.00)19(65.52)6(18.18)11(33.33)ab18.3740.00382839(9.00)84(84.00)6(10.34)45(77.59)9(13.64)38(57.58)17.8160.001IL-6(pg/mL)180.16 46.49 147.29 44.73-2.8090.00783/4为显著。8 4等位基因携带组患者的炎症因子水平明显5(10.00)高于对照组，且与ADAS-Cog评分呈正相关，这提示4(13.79)AD患者的认知功能障碍可能与炎症反应相关。Jin13(39.39)b等9 在一项针对8 0 岁以上的老年人的研究中发现，ApoE s4基因型与认知障碍之间存在显著的关联；在另一项散发 AD 的研究中发现,ApoE 4 等位基因的存在加重了区域神经退行性变，ApoE 4携带者疾病进展速度也更快10 ,这支持了本研究的结果。然而另一项研究却发现ApoE基因型与认知水平未表现出显著的关联性,这可能是由于研究对象的年龄存在差异，本研究中84纳人的均为6 5岁以上AD患者，一项Meta分析表明7(7.00)ApoE与认知功能障碍之间的关联程度随平均年龄增加7(12.07)19(28.79)bTNF-(pg/mL)68.81 7.7063.84 6.152.8230.006而增加,在年长的老年人中更为明显,这可以解释不同研究结果之间存在差异的原因。本研究结果表明,AD 患者 IL-6和 TNF-水平与ADAS-Cog评分呈正相关,且ApoE 4 携带组的 IL-6、T NF-水平高于非携带组,因此笔者推测 ApoE84等位基因可能影响AD的认知功能,且可能通过炎症因子途径发挥作用。一项10 年的纵向研究表明,外周血 IL-6在晚发型AD患者的脑脊液和血清中均升高,并且可以有效预测认知功能下降12 ,这支持了本研究1692 结果。ApoEs4通过调控IL-6水平,使AD患者血脑屏障通透性增高，并可直接导致tau蛋白过度磷酸化,加重了 AD 患者大脑的病理改变13。TNF-是 AD 的另一种促炎细胞因子,在炎症反应过程中的细胞因子级联启动和调节中发挥核心作用14;TNF-通过上调分泌酶的产量和分泌酶活性从而加重AD患者大脑中的A病理，同时,A可通过激活转录因子直接刺激小胶质细胞产生 TNF,从而进一步加重炎症反应15。在动物实验中发现,神经元与ApoE4阳性胶质细胞共培养可明显提高TNF分泌水平,并显著降低神经元活力，而神经元与ApoE基因敲除的胶质细胞共同培养时的活力更强,分泌的TNF-水平也更低16 。以上研究均可支持本研究结果,说明了炎症因子水平可能加重了AD患者的病理改变,这一改变可能由 ApoE基因调控,且ApoEs4等位基因的影响更为显著。4结论综上所述,ApoEs4 基因的存在加重了AD患者的认知障碍,这一过程可能是通过炎症因子发挥作用。本研究仍存在一定局限性，本研究的样本量较小，且尚未进行长期随访，未来仍需要进一步扩大样本量，并进行纵向研究,评估炎症因子以及 ApoE基因对于 AD 患者认知障碍及炎症水平的长期影响。参考文献1任汝静，殷鹏，王志会，等中国阿尔茨海默病报告2 0 2 1J诊断学理论与实践，2 0 2 1，2 0（4）：317-337.2 Scheltens P,De Strooper B,Kivipelto M,et al.Alzheimers diseaseJ.Lancet(London,England),2021,397(10284):1577-1590.3Serrano-Pozo A,Das S,Hyman BT.ApoE and Alzheimers disease:advances in genetics,pathophysiology,and therapeutic approachesJ.Lancet Neurol,2021,20(1):68-80.4 Kinney JW,Bemiller SM,Murtishaw AS,et al.Inflammation as a cen-tral mechanism in Alzheimers disease J.Alzheimers Dementia,2018,4:575-590.Journal of Clinical and Experimental Medicine Vol.22,No.16 Aug.2023Med,2011,3(89):89ra57.9JJin X,He W,Zhang Y,et al.Association of ApoE 4 genotype and lif-estyle with cognitive function among Chinese adults aged 80 years andolder:A cross-sectional study J.PLoS Med,2021,18(6):e1003597.10 Oveisgharan S,Buchman AS,Yu L,et al.ApoE 8284 genotype,inci-dent AD and MCI,cognitive decline,and AD pathology in older adultsJ.Neurology,2018,90(24):e2127-e2134.1l Davies G,Armstrong N,Bis JC,et al.Genetic contributions to varia-tion in general cognitive function:a meta-analysis of 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h。采用MTT法测定U266细胞、RPMI8226细胞的增殖情况，采用AnnexinV-FITC/PI染色试剂盒检测U266细胞、RP-MI8226细胞凋亡情况,并采用蛋白质印迹法测定Bax、Bc l-2 蛋白表达。结果处理48 h后，随着冬凌草甲素浓度（1050 mol/L)的增加,U266细胞、RPMI8226细胞的细胞增殖抑制率、细胞凋亡率均显著升高，差异均有统计学意义（P*通讯作者：祝龙,E-mail:文章编号：16 7 1-46 9 5(2 0 2 3)16-16 9 2-0 4