摘要
研究目的：收集2011年和2012年重庆医科大学附属儿童医院的乙脑病例，采用双抗夹心ELISA法测定空白组、乙脑毒损脑络、毒陷心包组血中IL-10、TNF-α和IFN-γ的表达水平，观察其有无统计学差异,探讨（1）乙脑不同中医证型与IL-10、TNF-α和IFN-γ水平的关系；（2）IL-10、TNF-α和IFN-γ在乙脑的脑组织损伤过程中可能的作用机制；（3）探究乙脑不同阶段症状与细胞因子的关联性，揭示其本质与物质基础。为今后流行性乙型脑炎分型与治疗提供客观依据，为中医标准化进程提供新思路。
采集2011和2012年重庆医科大学附属儿童医院流行性乙型脑炎患者（乙脑）的标本，收集乙脑毒损脑络、毒陷心包证临床资料，采集与检测理化指标、筛选中医证候易感基因相关的试验标本。采用Taqman法测定基因分型，统计不同组别间的多态性，旨在找到IL-10启动子区域中的位置-592和IFN-γ启动子区域中的位置-1616与乙脑患者疾病易感性、分型和预后的关联性。
研究方法：
全部84例患者均来源于2011年6月至2012年9月重庆医科大学附属儿童医院传染科住院部和门诊患儿。研究组包括毒损脑络组患者30例（男17，女13），平均年龄6.62±4.01岁；毒陷心包组患者30例（男18，女12），平均年龄5.16±2.79岁。对照组为同期于门诊体检的患者24例（男14，女10），平均年龄5.58±3.20岁。全部受检者于入院后或次日清晨空腹抽取肘静脉血4ml（用EDTA抗凝），在3000r/min的条件下离心后，分离出上层血清分装后置于-800C冰箱冷冻保存。用双抗体夹心ELISA法测定人外周血IL-10、TNF-α和IFN-γ的含量，人白介素10ELISA试剂盒、人肿瘤坏死因子αELISA试剂盒、人γ干扰素ELISA试剂盒由博士德提供。检测在武汉市第一医院生物实验室内按试剂说明完成。全部数据以±S表示，计数资料的比较采用卡方检验，计量资料的比较采t检验及方差分析。
研究中的所有受试者（n：297）为2011年6月到2012年9月乙脑流行季节的知情同意的患者。其中，有66例为毒损脑络型患者（根据临床诊断及IgM抗体阳性诊断），39例为毒陷心包型患者（根据临床诊断及IgM抗体阳性诊断），乙脑患者均来自重庆医科大学附属儿童医院传染科住院部患儿，另外192例为空白组患者，均来自重庆医科大学附属儿童医院门诊体检儿童。全部受检者于入院后或次日清晨空腹抽取肘静脉血4ml（用EDTA抗凝）置于-80℃冰箱冷冻保存。使用康为世纪通用型柱式基因组提取试剂盒，在武汉市一医院生物实验室提取基因组DNA，用TaqMan探针荧光PCR方法测定基因分型，引物和荧光探针由金唯智公司设计合成。对测序结果进行统计分析，该数据用卡方检验（χ2）使用标准的统计软件,p<0.05有统计学意义。基因型频率采用Hardy-Weinberg平衡试验法统计。使用PLINK软件来计算基因型和等位基因频率的χ2值，比值比和95％置信区间的值。
研究结果
①毒损脑络、毒陷心包组各与空白组血清中IL-10水平对比，毒损脑络、毒陷心包组血清中IL-10含量均明显高于空白组，二者比较具有显著差异(P<0.05)。毒陷心包组血清中IL-10含量明显高于毒损脑络组，二者有显著差异。
②毒损脑络、毒陷心包组与空白组血清中IFN-γ水平对比，毒损脑络、毒陷心包组血清中IFN-γ含量均明显高于空白组，二者比较具有显著差异(P<0.05)。毒陷心包组血清中IFN-γ含量明显高于毒损脑络组，二者有显著差异。
③毒陷心包组的意识障碍严重程度明显高于毒损脑络组，二者有显著差异。
④毒陷心包组的意识障碍持续时间明显高于毒损脑络组，二者有显著差异。
⑤将60例患者意识障碍程度与其周围血中IL-10水平进行Pearson等级相关检验，两者的相关系数为0.276，P=0.032<0.05具有显著相关。
⑥将60例患者的意识障碍程度与其周围血中IFN-γ水平进行Pearson等级相关检验，两者的相关系数为0.424，P=0.001<0.01具有显著相关。
⑦IFN-γ启动子区域-1616位点TT纯合子频率与TC加CC的频率的比较在乙脑组（即所有乙脑患者，包括毒损脑络组与毒陷心包组）和空白对照组间频率分布有统计学意义（TT vs TC+CC:Χ2=4.79，P=0.0287**＜0.05）。
⑧IL-10启动子区域-592位点CC纯合子频率与CA加AA的频率在乙脑组（即所有乙脑患者，包括毒损脑络组与毒陷心包组）和空白对照组比较时频率分布有统计学意义（CC vs CA+AA:Χ2=3.93，P=0.0475**）,另外，等位基因频率C比A的频率在乙脑组（即所有乙脑患者，包括毒损脑络组与毒陷心包组）和空白对照组间比较，频率分布有统计学意义（C vs A:Χ2=5.344，P=0.021**＜0.05，OR=0.638，95％CI=0.44-0.94）。
⑨等位基因频率C比A的频率在毒损脑络组和空白对照组间频率分布有统计学意义（C vs A:Χ2=4.045，P=0.044**＜0.05，OR=0.634，95％CI=0.41-0.99）。
研究结论
1. IL-10、IFN-γ水平在毒损脑络型和毒陷心包证型比较时，含量有统计学意义，提示IL-10、IFN-γ可作为临床分型的依据，为中医卫气营血传变提供依据，并可作为感染程度、脑损伤指标之一运用于临床。
2.IL-10具有抑制炎症因子，防治细胞过度损伤的功能，因此IL-10水平可间接反映炎症的严重程度，本实验反映了IL-10水平与意识障碍程度有显著相关性，提示IL-10能反映脑组织损伤程度和炎症的严重程度，故可作为临床辨证分型的依据。
3．本实验反映了IFN-γ水平与意识障碍程度有显著相关性，提示IFN-γ能反映出脑组织损伤程度，亦能提示邪入营血,内扰心神，因此可以作为临床辨证分型、判断疾病轻重的依据。
4. IFN-γ的-1616位点纯合子TT基因型更不容易患流行性乙型脑炎；
5.IL-10的-592A等位基因的受试者更容易患流行性乙型脑炎，纯合子CC基因型更不容易患乙脑。在个性化医疗的时代，了解基因突变和传染病的易感性之间的联系是很有必要的，这些信息有助于医生了解病人疾病可能的预后，可提前采取适当的治疗措施。
Objective:
JE cases which collected in2011and2012, using double-antibodysandwich ELISA to measure control group JE dusunnaoluo group、duxianxinbao group expression in blood IL-10, TNF-α and IFN-γ in level,whether the statistical difference was observed.To explore (1) JE possiblemechanism of IL-10、TNF-α and IFN-γ in the process of brain tissue damage;(2) correlation between different JE syndromes and IL-10、TNF-α and IFN-γ;(3) explore the relevance of the different stages of the symptoms ofencephalitis and cytokines, revealing its essence and material basis. Toprovide an objective basis for the future of Japanese encephalitis andtreatment，and to provide new ideas for TCM standardization process.
The collection in2011in Children’s Hospital of Chongqing MedicalUniversity of Japanese encephalitis patients (JE) specimens collected braincollateral damage poison, poison trap pericardial syndrome clinical datacollection and testing physical and chemical indicators, screening syndromespredisposing genes associated test specimen. Using Taqman assay genotyping,to find statistical polymorphism among different groups, aims to revealrelevance between the IL-10promoter region position-592and IFN-γpromoter region prone position-1616and sensibility、type and outcome ofpatients with encephalitis.
Methods:
All84patients are Children have been hospitalized in Children's Hospitalof Chongqing Medical University, Department of Infectious Diseases fromJune2011to September2012. Study group that is toxic brain collateraldamage group of30patients (17male,13female), mean age6.62±4.01years old; poison trap pericardium group of30patients (18men,12women),mean age5.16±2.79years old. Study group is composed of the twogroups.Patients in the control group of24patients (14male,10female) overthe same period in outpatient examination, the average age of5.58±3.20years old. All subjects in the admission or the next morning after fastingvenous blood4ml (with EDTA anticoagulant), after centrifugationconditions under3000r/min separated serum aliquots into the upper800℃freezer saved. Double antibody sandwich ELISA assay in human peripheralblood IL-10、TNF-α levels and IFN-γ, human interleukin10ELISA kit、human tumor necrosis factor α ELISA kit and man interferon γ ELISA kitprovided by Boster. Detected in the First Hospital of Wuhan BiologicalLaboratory reagents according to the instructions to complete. All data are inform of±S said count data were compared using chi-square test tocompare measurement data mining t-test and analysis of variance.
All the study subjects (n:297) of JE epidemic season in June2011toSeptember2012JE patient informed consent. Of these,66cases of poisoningpatients with brain collateral damage (based on clinical diagnosis and IgMantibody positive diagnosis),39cases of patients with pericardial poison trap(based on clinical diagnosis and IgM antibody positive diagnosis), Japaneseencephalitis patients from Chongqing Medical University Department ofInfectious Diseases, Children's Hospital inpatient children, in addition to192cases for the control group of patients, all from the children's Hospital ofChongqing Medical University medical clinic for children. All subjects in the admission or the next morning after fasting venous blood4ml (with EDTAanticoagulant) at-80℃cryopreservation. Use Kangwei century universalpillar genome extraction kit to extract genomic DNA in a hospital in Wuhan,biological laboratories, measuring genotyping PCR using TaqMan probefluorescence methods, primers and fluorescent probes designed by Goldenintellectualism synthesis. Sequencing results were analyzed, the data isdetermined by chi-square test (χ2) using standard statistical software, p <0.05considered statistically significant. Genotype frequency is determined byHardy-Weinberg equilibrium statistical test method. Use PLINK software tocalculate the value of χ2value of genotype and allele frequencies, odds ratiosand95%confidence intervals.
Results:
①Brain collateral damage poison, toxic levels of depression-10ILpericardial contrast with the control group serum brain collateral damagepoison, poison trap pericardium in serum IL-10levels were significantlyhigher in comparison with a significant difference between the two (P <0.05).Poison trap pericardial serum levels of IL-10was significantly higher than thegroup of brain collateral damage poison, there is a significant differencebetween the two.
②Brain collateral damage poison, poison trap pericardium and controlgroups in serum IFN-γ levels of contrast, brain collateral damage poison,poison trap pericardium group serum IFN-γ levels were significantly higher incomparison with a significant difference between the two (P <0.05). Poisontrap pericardium group serum IFN-γ levels were significantly higher thanpoison brain collateral damage group, a significant difference between thetwo groups.
③Poison trap obstacle awareness pericardium group was significantly higherthan the severity of brain collateral damage toxin group, a significant difference between the two.
④Poison trap pericardium group was significantly higher than the duration ofconsciousness toxic brain collateral damage group, a significant differencebetween the two groups.
⑤Pearson rank correlation test will be the extent of60cases ofunconsciousness and IL10-level correlation coefficient was0.276, P=0.032<0.05significantly correlated.
⑥The extent of60cases of unconsciousness and IFN-γ levels Pearson rankcorrelation test, correlation coefficient was0.424, P=0.001<0.01significantly correlated.
⑦Between IFN-γ promoter region-1616loci TT homozygote frequency andthe frequency of TC and CC plus the JE group (ie, all Japanese encephalitispatients, including brain collateral damage poison and poison traps setpericardium group) and control group.frequency distribution was statisticallysignificant (TT vs TC+CC: Χ2=4.79, P=0.0287**).
⑧IL-10promoter region-592CC homozygotes in the frequency and thefrequency and JE CA plus AA group (ie all encephalitis patients, includingbrain collateral damage poison and poison traps set pericardium group)between the control group and the frequency distribution was statisticallysignificant (CC vs CA+AA: Χ2=3.93, P=0.0475**), in addition, thefrequency of a allele C ratio in the JE group (ie, all Japanese encephalitispatients, including toxic brain damage contact between the group and thepoison trap pericardium group) and control group was statistically significantfrequency distribution (C vs A: Χ2=5.344, P=0.021**, OR=0.638,95%CI=0.44-0.94).
⑨C allele frequency than the frequency of A in the brain collateral damageamong drug group and control group was statistically significant frequencydistribution (C vs A: Χ2=4.045, P=0.044**, OR=0.634,95%CI= 0.41-0.99).
Conclusion:
1. IL-10, IFN-γ levels in different syndromes content is statisticallysignificant, suggesting that IL-10, IFN-γ can be used as a basis for clinicalclassification for blood gas business in medical and public health provide thebasis for mass change, and as the extent of the infection, one used in clinicalbrain injury indicators.
2. IL-10inhibition of inflammatory cytokines play, prevent excessive damagecell function, and therefore IL10-levels can indirectly reflect the severity ofinflammation, the present study reflects the degree of IL10-levelconsciousness significant correlation can be seen IL-10can reflect the degreeof brain tissue damage and inflammation severity, it can be used as a clinicalsyndrome type basis.
3. The experiment reflects the IFN-γ levels and the degree of consciousnessthere is a significant correlation, we can see that IFN-γ can reflect the extentof brain damage, can also prompt evil blood into the camp, the disturbance ofmind, it can be used as a clinical syndrome type basis.
4. IFN-γ in-1616TT genotype homozygous loci not more susceptible toJapanese encephalitis;
5. IL-10-592A allele subjects more susceptible to encephalitis, nothomozygous CC genotype is more susceptible to encephalitis. In the era ofpersonalized medicine, gene mutations and susceptibility to understand thelink between infectious diseases is necessary, this information may helpdoctors understand the prognosis of patients with the disease, the treatmentmay take appropriate measures in advance.
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