李新成 王宏偉 申保生 宋新文

手足口病合并腦炎患兒腦脊液和血清細(xì)胞因子的測定及意義

李新成 王宏偉 申保生 宋新文

目的探討手足口病(HFMD)合并腦炎(VE)患兒腦脊液和血清腫瘤壞死因子α(TNF-α)、神經(jīng)元特異性烯醇化酶(NSE)及S100B的含量及其臨床意義。方法采用酶聯(lián)免疫吸附試驗(yàn)雙抗體夾心法對38例HFMD合并VE、40例單純HFMD和20例非神經(jīng)系統(tǒng)疾病患兒的腦脊液和血清TNF-α、NSE、S100B進(jìn)行檢測。結(jié)果HFMD合并VE組腦脊液和血清TNF-α、NSE、S100B水平高于單純HFMD組和對照組(P＜0.01), 且患兒腦脊液TNF-α、NSE、S100B與血清TNF-α、NSE、S100B含量呈正相關(guān)(r=0.75, 0.83, 0.81, P均<0.01)。單純HFMD組TNF-α水平高于對照組(P＜0.05), 且腦脊液TNF-α與血清TNF-α含量呈正相關(guān)(r=0.71, P＜0.01), 而NSE、S100B與對照組相比差異無統(tǒng)計(jì)學(xué)意義(P＞0.05)。結(jié)論腦脊液及血清TNF-α、NSE、S-100B水平檢測對手足口病合并腦炎的診斷具有一定的臨床意義, NSE、S100B可作為手足口病患者腦損傷的標(biāo)記物。

手足口??；腦炎；腫瘤壞死因子α；神經(jīng)元特異性烯醇化酶；S100B

手足口病(HFMD)是由腸道病毒引起的常見傳染病, 主要表現(xiàn)為發(fā)熱和手、足、口腔等部位皮疹, 輕型患者預(yù)后良好, 但合并病毒性腦炎(VE)后可引起神經(jīng)源性肺水腫和心肌損害, 嚴(yán)重的可導(dǎo)致死亡。腫瘤壞死因子α(TNF-α)由活化的單核-巨噬細(xì)胞分泌, 在機(jī)體免疫調(diào)節(jié)中發(fā)揮關(guān)鍵作用。研究表明神經(jīng)元特異性烯醇化酶(NSE)及S100B在反映腦損傷程度和預(yù)后方面具有重要價值。本研究擬通過檢測HFMD合并VE患兒血清和腦脊液中TNF-α、NSE及S100B水平,探討其在HFMD合并VE發(fā)病機(jī)制中的意義。

1 資料與方法

1.1一般資料 78例HFMD患兒為新鄉(xiāng)醫(yī)學(xué)院第一附屬醫(yī)院感染科住院患兒, 診斷均符合衛(wèi)生部《手足口病診療指南》(2010年版), 分為HFMD合并VE組(38例)和單純HFMD組(40例)。同時選擇無感染、腫瘤、神經(jīng)系統(tǒng)損害等而需外科手術(shù)的患兒作為對照組(20例)。各組患兒在性別、年齡等方面差異無統(tǒng)計(jì)學(xué)意義(P>0.05)。

1.2方法 各組患兒留取腦脊液2 ml及血清1 ml, -70℃保存?zhèn)錂z。TNF-α、NSE和及S100B應(yīng)用ELISA法(深圳晶美生物工程有限公司產(chǎn)品)檢測, 操作嚴(yán)格按照說明書進(jìn)行。

1.3統(tǒng)計(jì)學(xué)方法 數(shù)據(jù)通過SPSS17.0軟件分析, 計(jì)量資料以均數(shù)±標(biāo)準(zhǔn)差( x-±s)表示, 組間比較應(yīng)用單因素方差分析(one-way ANOVA)；相關(guān)性應(yīng)用Pearson相關(guān)分析。P＜0.05表示差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1各組腦脊液和血清TNF-α水平比較 單因素方差分析顯示3組腦脊液及血清TNF-α水平差異明顯(P＜0.01),其中單純HFMD組及HFMD合并VE組患兒腦脊液和血清TNF-α水平明顯高于對照組(P＜0.01), 且HFMD合并VE組患兒腦脊液及血清TNF-α水平高于單純HFMD組(P＜0.01)。見表1。

2.2各組腦脊液和血清NSE水平比較 單因素方差分析顯示3組腦脊液及血清NSE水平差異有統(tǒng)計(jì)學(xué)意義(P＜0.01),其中HFMD合并VE組患兒腦脊液和血清NSE水平高于單純HFMD組及對照組(P＜0.01), 但單純HFMD組患兒腦脊液及血清NSE水平與對照組相比差異無統(tǒng)計(jì)學(xué)意義(P＞0.05)。見表2。

2.3各組腦脊液和血清S100B水平比較 單因素方差分析顯示3組腦脊液及血清S100B水平差異有統(tǒng)計(jì)學(xué)意義(P＜0.01), 其中HFMD合并VE組患兒腦脊液和血清S100B水平高于單純HFMD組及對照組(P＜0.01), 但單純HFMD組患兒腦脊液及血清S100B水平與對照組相比差異無統(tǒng)計(jì)學(xué)意義(P＞0.05)。見表3。

表1 腦脊液和血清TNF-α水平比較( x-±s, μg/L)

表2 腦脊液和血清NSE水平比較( x-±s, μg/L)

表3 腦脊液和血清S100B水平比較( x-±s, μg/L)

2.4腦脊液與血清TNF-α、NSE、S100B水平間的相關(guān)分析 相關(guān)分析顯示HFMD患兒腦脊液TNF-α水平與血清TNF-α水平呈正相關(guān)(r=0.71, P＜0.01)；HFMD合并VE患兒腦脊液與血清TNF-α、NSE、S100B水平呈正相關(guān)(r=0.75, 0.83, 0.81, P均<0.01)。

3 討論

HFMD是由多種腸道病毒引起的急性傳染?。?］, 主要致病原是腸道病毒71型和柯薩奇病毒A組16型, 多發(fā)生于學(xué)齡前兒童, 大多數(shù)癥狀輕微, 預(yù)后良好, 但重癥病例多合并腦炎, 導(dǎo)致致命性損傷, 少數(shù)遺留后遺癥［2］。TNF-α是一種由多核巨細(xì)胞產(chǎn)生的具有廣泛生物學(xué)活性的細(xì)胞因子, 一方面它具有調(diào)節(jié)機(jī)體的免疫功能, 能使某些腫瘤細(xì)胞壞死；另一方面則介導(dǎo)炎癥過程、組織損傷、休克等病理生理反應(yīng)。本研究結(jié)果表明, HFMD患者腦脊液和血清TNF-α水平明顯高于對照組, 且腦脊液與血清TNF-α水平呈正相關(guān), 說明感染手足口病毒后, 病毒通過多種途徑啟動了炎癥反應(yīng)的瀑布反應(yīng), 而TNF-α在其中起了重要作用。本研究結(jié)果同時表明, 合并VE后的HFMD患兒腦脊液和血清中TNF-α水平明顯升高, 提示TNF-α通過激活細(xì)胞因子網(wǎng)絡(luò)系統(tǒng)而誘發(fā)全身炎性反應(yīng), 進(jìn)而破壞血腦屏障的完整性并增加其通透性, 上調(diào)誘導(dǎo)型一氧化氮合酶在神經(jīng)細(xì)胞的表達(dá), 誘導(dǎo)其凋亡, 從而引起中樞神經(jīng)系統(tǒng)廣泛性損害［3,4］。

NSE是γ型烯醇化酶, 特異性地存在于神經(jīng)元及神經(jīng)內(nèi)分泌細(xì)胞中, 而腦膠質(zhì)細(xì)胞和其他神經(jīng)組織中不含這種酶, 故稱NSE。NSE是一種大分子物質(zhì), 正常情況下血液和腦脊液中含量甚少。在顱腦損傷、腦梗死、腦出血、腦炎等［5,6］多種中樞神經(jīng)系統(tǒng)疾病中, 部分神經(jīng)元變性、壞死和崩解, NSE可釋放入腦脊液中, 同時血腦屏障的破壞及通透性升高, 導(dǎo)致NSE易于透過血腦屏障進(jìn)入腦脊液及血液,且神經(jīng)元損傷程度越重, 血腦屏障破壞程度越高, 腦脊液和血液中NSE水平就越高。因此, NSE是評判顱腦損傷程度和預(yù)后的一項(xiàng)特異性敏感生化指標(biāo)［7］。本研究結(jié)果表明, 單純HFMD患者腦脊液和血清NSE水平無升高, 而合并VE后腦脊液及血清NSE水平明顯升高, 且腦脊液與血清NSE水平呈正相關(guān), 提示因NSE特異性地存在于中樞神經(jīng)系統(tǒng), 故無合并腦炎的HFMD患兒含量無升高。而合并VE后由于手足口病毒的直接侵犯以及免疫性損害, 導(dǎo)致神經(jīng)系統(tǒng)炎癥反應(yīng),神經(jīng)元水腫、壞死和脫髓鞘［8］, 腦脊液NSE水平顯著升高。血腦屏障破壞及通透性增高, NSE透過血腦屏障滲漏到血液,血液中NSE水平也顯著增加。

S100B是一種酸性鈣結(jié)合蛋白, 其特異性地存在于中樞神經(jīng)膠質(zhì)細(xì)胞內(nèi), 具有調(diào)節(jié)細(xì)胞生長、能量代謝和參與細(xì)胞內(nèi)信號傳導(dǎo)的作用, 是膠質(zhì)細(xì)胞與神經(jīng)元相互作用的橋梁［9］。當(dāng)膠質(zhì)細(xì)胞和神經(jīng)元被破壞時, S100B蛋白就進(jìn)入腦脊液,被認(rèn)為是腦損傷程度和預(yù)后判斷的標(biāo)記物［10］。本研究結(jié)果表明, 單純HFMD患者腦脊液和血清S100B水平無升高, 而合并VE后腦脊液及血清S100B水平明顯升高, 且腦脊液與血清S100B水平呈正相關(guān), 提示因S100B特異性地存在于中樞神經(jīng)系統(tǒng), 故無合并腦炎的HFMD患兒含量無升高。而合并VE后S100B可能通過糖基化終產(chǎn)物受體(RAGE)依賴激活JNK通道, 同時激活NF-κB和AP-1, 誘發(fā)細(xì)胞因子瀑布鏈反應(yīng), 上調(diào)COX-2、IL-1β和TNF-α的表達(dá)在腦膠質(zhì)細(xì)胞的表達(dá), 引起神經(jīng)細(xì)胞的脫髓鞘改變、星形細(xì)胞的凋亡,參與大腦炎癥性疾病的病理生理過程, 腦脊液S100B水平顯著升高［11］。同時由于血腦屏障破壞及通透性增高, S100B透過血腦屏障滲漏到血液, 血液中NSE水平也顯著增加［12］。

本研究結(jié)果提示TNF-α與HFMD炎癥反應(yīng)密切相關(guān),而NSE及S100B在HFMD合并VE的顱腦損傷及血腦屏障破壞的發(fā)病機(jī)制中可能起重要作用。NSE及S100B可特異性地作為判斷HFMD是否合并VE的敏感指標(biāo), 從而指導(dǎo)臨床早期診斷及治療, 改善患兒的預(yù)后。

［1］ Lee JJ, Seah JB, Chow VT, et al.Comparative proteome analyses of host protein expression in response to Enterovirus 71 and Coxsackievirus A16 infections.J Proteomics, 2011, 74(10):2018-2024.

［2］ Griffiths MJ, Ooi MH, Wong SC, et al.In enterovirus 71 encephalitis with cardio-respiratory compromise, elevated interleukin 1β, interleukin 1 receptor antagonist, and granulocyte colonystimulating factor levels are markers of poor prognosis.J Infect Dis, 2012, 206(6):881-892.

［3］ Kushibiki S.Tumor necrosis factor-α-induced inflammatory responses in cattle.Anim Sci J, 2011, 82(4):504-511.

［4］ Hernández-Romero MC, Delgado-Cortés MJ, Sarmiento M, et al.Peripheral inflammation increases the deleterious effect of CNS inflammation on the nigrostriatal dopaminergic system.Neurotoxicology, 2012, 33(3):347-360.

［5］ Ahmad O, Wardlaw J, Whiteley WN.Correlation of levels of neuronal and glial markers with radiological measures of infarct volume in ischaemic stroke: a systematic review.Cerebrovasc Dis, 2012, 33(1):47-54.

［6］ Roka A, Kelen D, Halasz J, et al.Serum S100B and neuron-specific enolase levels in normothermic and hypothermic infants after perinatal asphyxia.Acta Paediatr, 2012, 101(3):319-323.

［7］ Hiihara T, Miyake T, Izumi S, et al.Serum and cerebrospinal fluid S100B, neuron-specific enolase, and total tau protein in acute encephalopathy with biphasic seizures and late reduced diffusion: a diagnostic validity.Pediatr Int, 2012, 54(1):52-55.

［8］ Zhang YC, Jiang SW, Gu WZ, et al.Clinicopathologic features and molecular analysis of enterovirus 71 infection: report of an autopsy case from the epidemic of hand, foot and mouth disease in China.Pathol Int, 2012, 62(8):565-570.

［9］ Villarreal A, Aviles Reyes RX, Angelo MF, et al.S100B alters neuronal survival and dendrite extension via RAGE-mediated NF-κB signaling.J Neurochem, 2011, 117(2):321-332.

［10］ Yardan T, Erenler AK, Baydin A, et al.Usefulness of S100B protein in neurological disorders.J Pak Med Assoc, 2011, 61(3):276-281.

［11］ Bianchi R, Kastrisianaki E, Giambanco I, et al.S100B protein stimulates microglia migration via RAGE-dependent upregulation of chemokine expression and release.J Biol Chem, 2011, 286(9):7214-7226.

［12］ Leis AA, Stokic DS, Petzold A.Glial S100B is elevated in serum across the spectrum of West Nile virus infection.Muscle Nerve, 2012, 45(6):826-830.

Clinical significance of determination of cerebrospinal fluid and serum cytokines in children with hand

foot and mouth disease and viral encephalitis

LI Xin-cheng, WANG Hong-wei, SHEN Bao-sheng, et al.Department of Infection Diseases, Huixian People's Hospital, Huixian, 453600, China

ObjectiveTo investigate content of cerebrospinal fluid and serum levels of tumor necrosis factorα(TNF-α), nenron specific enolase(NSE) and S100B in children with hand foot and mouth disease(HFMD)and viral encephalitis(VE).MethodsSeventy-eight cases of children with HFMD were divided into two groups: HFMD with VE case and HFMD only case.And 20 age-matched healthy children were enrolled in this study.The levels of TNF-α, NSE and S100B of cerebrospinal fluid and serum were measured bv enzyme-linked immunosorbent assay.ResultsCerebrospinal fluid and serum TNF-α, NSE and S100B levels of HFMD with VE group were significantly higher than HFMD only group and control group(P<0.01), and cerebrospinal fluid levels of TNF-α, NSE and S100B were positively correlated with the serum levels of TNF-α, NSE and S100B(r=0.75, 0.83, 0.81, P<0.01, respectively).Cerebrospinal fluid and serum TNF-αlevels of HFMD only group were significantly higher than control group(P<0.01), and cerebrospinal fluid levels of TNF-αwere positively correlated with the serum levels of TNF-α(r=0.71, P<0.01).Cerebrospinal fluid and serum NSE, S100B levels of HFMD only group were not higher than control group(P>0.05).ConclusionLevels of TNF-α, NSE and S100B in serum and cerebrospinal fluid was valuable for diagnosis in children with HFMD and VE.NSE and S-100B can be used as a marker for patients with brain injury in children with HFMD.

Hand foot and mouth disease; Encephalitis; Tumor necrosis factor-α; Nenron specific enolase; S100B

453600 河南省輝縣市人民醫(yī)院感染疾病科(李新成)；新鄉(xiāng)醫(yī)學(xué)院第一附屬醫(yī)院感染疾病科(王宏偉申保生 宋新文)