腫瘤壞死因子α、超敏C反應(yīng)蛋白在新生兒缺氧缺血性腦病中的變化及臨床意義

尚云，石計(jì)朋，楊衛(wèi)紅，馬慧敏，郭喜霞，唐成和

(新鄉(xiāng)醫(yī)學(xué)院第一附屬醫(yī)院，a.新生兒科,b.耳鼻喉科,c.兒內(nèi)科，河南 新鄉(xiāng)　453100)

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◇臨床醫(yī)學(xué)◇

腫瘤壞死因子α、超敏C反應(yīng)蛋白在新生兒缺氧缺血性腦病中的變化及臨床意義

尚云a，石計(jì)朋a，楊衛(wèi)紅a，馬慧敏b，郭喜霞c，唐成和a

(新鄉(xiāng)醫(yī)學(xué)院第一附屬醫(yī)院，a.新生兒科,b.耳鼻喉科,c.兒內(nèi)科，河南 新鄉(xiāng)453100)

目的檢測(cè)血清中腫瘤壞死因子α (TNF-α)，超敏C反應(yīng)蛋白(HsCRP)在新生兒缺氧缺血性腦病(HIE)中變化及意義，探討其相關(guān)性。方法選取臨床確診的74例HIE患兒(其中輕度31例，中度26例，重度17例；預(yù)后良好32例，預(yù)后不良42例)為觀察組，74例正常健康新生兒為對(duì)照組，于生后48 h應(yīng)用酶聯(lián)免疫吸附(ELISA)及放射免疫分析法檢測(cè)兩組新生兒血清TNF-α及HsCRP表達(dá)水平。結(jié)果與對(duì)照組比較，觀察組中TNF-α及HsCRP表達(dá)水平均顯著增高，TNF-α結(jié)果[(17.20±1.26 )vs( 97.00±5.97) ng·L-1](P<0.05)；HsCRP結(jié)果[(0.51±0.18)vs(11.93±1.91) mg·L-1](P<0.05)；輕度、中度、重度HIE中TNF-α及HsCRP表達(dá)隨著病情程度加重逐漸增高，組間兩兩比較，差異有統(tǒng)計(jì)學(xué)意義，TNF-α結(jié)果[輕度HIE組(31.37±3.28)vs中度HIE組(52.59±5.19)vs重度HIE組(102.65±7.81) ng·L-1](P<0.05)；HsCRP結(jié)果[輕度HIE組(4.63±0.69)vs中度HIE組(7.56±1.19)vs重度HIE組(12.92±3.25) mg·L-1](P<0.05)；預(yù)后良好、預(yù)后不良HIE中TNF-α及HsCRP表達(dá)預(yù)后不良患兒HIE組較預(yù)后良好增高，TNF-α結(jié)果[預(yù)后良好HIE組(44.32±4.84)vs預(yù)后不良HIE組(90.23±7.37) ng·L-1](P<0.05)；HsCRP結(jié)果[預(yù)后良好HIE組(5.99±0.99)vs預(yù)后不良HIE組(9.71±2.14) mg·L-1](P<0.05)。結(jié)論隨著病情程度加重及預(yù)后不良程度加重，TNF-α及HsCRP表達(dá)水平逐漸增高，提示TNF-α及HsCRP可能參與了HIE的病理生理過程，動(dòng)態(tài)監(jiān)測(cè)二者變化趨勢(shì)將有助于HIE的病情程度判斷及預(yù)后評(píng)估。

缺氧缺血,腦；腫瘤壞死因子α;C反應(yīng)蛋白質(zhì);嬰兒,新生

新生兒缺氧缺血性腦病(HIE)在圍產(chǎn)期神經(jīng)系統(tǒng)疾病中占有重要位置，每年約有30萬(wàn)殘疾兒童出現(xiàn)，成為危害我國(guó)兒童生活質(zhì)量的重大問題，且在臨床上部分新生兒因?yàn)榧皶r(shí)的復(fù)蘇而恢復(fù)正常，并未診斷為HIE，然而后期隨訪發(fā)現(xiàn)這些出生時(shí)需復(fù)蘇而未發(fā)展成HIE的所謂正常嬰兒均存在智商低下，部分有認(rèn)識(shí)障礙等后遺癥，因此，HIE仍是全球關(guān)注的課題[1]。目前HIE的發(fā)病機(jī)制尚不十分明確，但多項(xiàng)研究已證明炎性反應(yīng)、細(xì)胞因子釋放等在其發(fā)病機(jī)制中起重要作用[2-3]，同時(shí)發(fā)現(xiàn)腫瘤壞子因子α(TNF-α)及超敏C反應(yīng)蛋白(HsCRP)是HIE發(fā)生發(fā)展中關(guān)鍵的細(xì)胞因子及炎性蛋白之一，對(duì)于TNF-α及HsCRP與HIE疾病的輕重研究較多，較少見兩指標(biāo)與HIE疾病預(yù)后的關(guān)系，故本研究通過檢測(cè)TNF-α及HsCRP在不同程度、不同預(yù)后的HIE中的變化，探討兩者與HIE的程度及預(yù)后的相關(guān)性，為臨床早期診斷、判斷病情及評(píng)價(jià)預(yù)后提供理論依據(jù)，進(jìn)而減少腦損傷后遺癥、改善預(yù)后。

1　資料與方法

1.1臨床資料收集2011年2月到2013年2月新鄉(xiāng)醫(yī)學(xué)院第一附屬醫(yī)院兒科臨床確診的HIE患兒74例為觀察組，根據(jù)病情嚴(yán)重程度，將其分為輕度組(31例)，中度組(26例)，重組度(17例)，HIE病例均依據(jù)中華醫(yī)學(xué)會(huì)兒科學(xué)分會(huì)新生兒學(xué)組HIE臨床診斷標(biāo)準(zhǔn)和分度標(biāo)準(zhǔn)[4]，根據(jù)預(yù)后情況[5]，將其分為預(yù)后良好組(32例)，預(yù)后不良組(42例)，觀察組中男性42例，女性32例，均為足月兒，胎齡(39.6±2.7)周，體質(zhì)量(3 591±619)g，同時(shí)選擇新鄉(xiāng)醫(yī)學(xué)院第一附屬醫(yī)院同期分娩的74例正常健康足月新生兒為對(duì)照組，其中男性44例，女性30例，胎齡(39.5±2.9)周，體質(zhì)量(3 418±524)g，對(duì)照組均無(wú)窒息、感染、羊水、胎盤異常及宮內(nèi)窘迫病史，觀察組無(wú)合并其它疾病，兩組新生兒母親均無(wú)感染，無(wú)孕期合并癥，新生兒性別、胎齡、出生體質(zhì)量、分娩方式等方面差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)，本研究經(jīng)新鄉(xiāng)醫(yī)學(xué)院第一附屬醫(yī)院醫(yī)學(xué)倫理委員會(huì)批準(zhǔn)，患者均簽署知情同意書。

1.2方法及結(jié)果判定樣本采集：對(duì)照組及觀察組均分別于生后48 h采集其外周靜脈血3 mL，室溫下以3 000 r·min-1速度離心10 min，收集血清，放置于-20 ℃冰箱保存待用。試劑及標(biāo)本的檢測(cè)：TNF-α及HsCRP免疫試劑盒購(gòu)置LIFEKEY BioMeditech公司。采用酶聯(lián)免疫吸附(ELISA)及放射免疫分析方法(RIA)檢測(cè)，嚴(yán)格按照說(shuō)明書步驟進(jìn)行。

2　結(jié)果

2.1兩組血清中TNF-α及HsCRP檢測(cè)結(jié)果比較對(duì)照組和觀察組TNF-α分別為(17.20±1.26)、(97.00±5.97) ng·L-1，t=3.626；對(duì)照組和觀察組HsCRP分別為(0.51±0.18)、(11.93±1.91) mg·L-1；兩組比較差異有統(tǒng)計(jì)學(xué)意義(t=5.126,P<0.001),提示HIE觀察組血清中TNF-α、HsCRP表達(dá)均較正常健康對(duì)照組增強(qiáng),見圖1。

圖1　兩組血清中TNF-α、HsCRP檢測(cè)結(jié)果比較

2.2不同疾病程度HIE中TNF-α及HsCRP檢測(cè)結(jié)果比較輕、中、重度HIE組TNF-α分別為(31.37±3.28)、(52.59±5.19)、(102.65±7.81) ng·L-1； 輕、中、重度HIE組HsCRP分別為(4.63±0.69)、(7.56±1.19)、(12.92±3.25) mg·L-1；輕、中、重度HIE中TNF-α及HsCRP表達(dá)水平隨著病情程度加重增高，組間兩兩比較：輕度HIE組與中度HIE組TNF-α比較q=4.34，中度HIE組與重度HIE組TNF-α比較q=4.86，輕度HIE組與重度HIE組TNF-α比較q=6.68；輕度HIE組與中度HIE組HsCRP比較q=4.61，中度HIE組與重度HIE組HsCRP比較q=4.97，輕度HIE組與重度HIE組HsCRP比較q=6.37，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)，見圖2。

圖2　輕、中、重度組血清中TNF-α、HsCRP檢測(cè)結(jié)果比較

2.3預(yù)后良好組及預(yù)后不良組血清中TNF-α、HsCRP檢測(cè)結(jié)果比較預(yù)后良好HIE組和預(yù)后不良HIE組的TNF-α分別為：(44.32±4.84)、(90.23±7.37) ng·L-1，t=3.433；預(yù)后良好HIE組和預(yù)后不良HIE組的HsCRP分別為：(5.99±0.99)、(9.71±2.14) mg·L-1,t=2.396，預(yù)后良好HIE組及預(yù)后不良HIE組中TNF-α及HsCRP表達(dá)水平隨著預(yù)后不良程度加重增高，其中預(yù)后不良HIE組表達(dá)較高，差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)，見圖3。

3　討論

HIE是指圍產(chǎn)期缺氧窒息，導(dǎo)致腦的缺氧缺血性損害[6]。包括特征性的神經(jīng)病理及病理生理改變，并在臨床上出現(xiàn)一系列腦病的表現(xiàn)，如新生兒驚厥[7]，是新生兒腦病的最常見的原因之一[8]，占新生兒腦病的50%～80%[9]，部分病例可留有不同程度神經(jīng)系統(tǒng)后遺癥[10]，是導(dǎo)致新生兒死亡和嬰幼兒神經(jīng)系統(tǒng)功能障礙的主要原因之一[11-12]。隨著新生兒窒息復(fù)蘇及重癥監(jiān)護(hù)技術(shù)的提高，HIE的死亡率逐漸下降，然而其遠(yuǎn)期不同程度的神經(jīng)發(fā)育問題仍居高不降，故對(duì)于早期診斷及治療，預(yù)防神經(jīng)系統(tǒng)后遺癥尤為重要[13-14]。大量研究證明免疫系統(tǒng)尤其是炎性細(xì)胞因子與HIE的發(fā)生密切相關(guān)，腦缺血再灌注后，腦組織局部過度炎性反應(yīng)是造成腦損傷的重要原因，而細(xì)胞因子在這個(gè)病理過程中起著非常重要的作用，TNF-α是炎性反應(yīng)中主要因子之一[15]，腦脊液檢測(cè)顯示TNF-α在腦損傷早期即可產(chǎn)生，且其含量與神經(jīng)損傷的嚴(yán)重程度程正相關(guān)。研究證實(shí)TNF-α可誘導(dǎo)其它細(xì)胞因子如IL-6，IL-8和炎性介質(zhì)如血栓素A2產(chǎn)生，增強(qiáng)中性粒細(xì)胞及單核細(xì)胞的黏附作用，促進(jìn)內(nèi)皮細(xì)胞粘附分子產(chǎn)生及過度炎性反應(yīng)，增加血管通透性，促使腦水腫，誘導(dǎo)腦細(xì)胞調(diào)亡，加重腦缺血-再灌注損傷。本研究顯示急性期 HIE 組患兒血清TNF-α水平高于正常新生兒，且隨著 HIE 病情的加重，TNF-α水平有顯著性上升，與王團(tuán)美等[16]報(bào)道一致，但本研究還顯示，HIE中TNF-α水平隨預(yù)后不良程度增加而升高，說(shuō)明血清細(xì)胞因子TNF-α參與了新生兒HIE 腦損傷的過程，隨著HIE病情及預(yù)后不良加重，細(xì)胞因子水平顯著上升。

圖3　預(yù)后良好及不良HIE組血清中TNF-α、HsCRP檢測(cè)結(jié)果比較

CRP是20世紀(jì)30年代Tillett 和 Francis 首次發(fā)現(xiàn),因其能與肺炎雙球菌細(xì)胞壁C多糖發(fā)生沉淀反應(yīng)而得命。血清CRP是一種敏感的急性時(shí)相反應(yīng)蛋白[17],在胎兒時(shí)期,主要由肝細(xì)胞合成,且不能通過胎盤。新生兒期，CRP以微量形式存在于血清中,正常成人血清CRP含量亦較低,約99%人數(shù)小于8 mg·L-1，在炎性反應(yīng)或應(yīng)激狀態(tài)下,血清CRP在數(shù)小時(shí)內(nèi)急劇升高數(shù)十倍甚至上百倍[18]。個(gè)體病情緩解后,血清CRP又迅速降低。血清CRP常作為判斷細(xì)菌感染的早期指標(biāo)，它是機(jī)體重要防御物質(zhì)。臨床實(shí)驗(yàn)室采用高敏感的檢測(cè)技術(shù)，測(cè)定出的低濃度CRP，稱為高敏感或HsCRP[19]，因其有高靈敏度及準(zhǔn)確度的優(yōu)點(diǎn)，窒息引發(fā)腦缺氧缺血損傷后，HsCRP的改變對(duì)于HIE有重要臨床意義。本研究檢測(cè)了HsCRP在不同程度及預(yù)后不同HIE組中，發(fā)生后48 h HIE組HsCRP血清中濃度高于正常新生兒組，HIE損傷程度越重，HsCRP濃度越高，預(yù)后越差HIE患兒HsCRP濃度越高，提示HsCRP參與了與HIE有關(guān)的炎性免疫反應(yīng)，可以通過HsCRP數(shù)值變化了解患兒神經(jīng)系統(tǒng)是否損傷及損傷程度和炎性狀態(tài)，預(yù)測(cè)患兒預(yù)后情況。

TNF-α、HsCRP與HIE病程、預(yù)后密切相關(guān)，可以作為HIE的早期診斷、病情評(píng)估以及判斷預(yù)后的重要指標(biāo)之一，對(duì)于存在窒息缺氧患兒在臨床癥狀未出現(xiàn)之前可通過其二者的變化對(duì)患兒提前做出診斷，并采取相應(yīng)治療措施，預(yù)防神經(jīng)系統(tǒng)并發(fā)癥，提高人口素質(zhì)。

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Changes and clinical significance of tumor necrosis factor-α and high-sensitivity C-reactive protein in neonates with hypoxic-ischemic encephalopathy

SHANG Yun,SI Jipeng,YANG Weihong,et al

(TheFirstAffiliatedHospitalofXinxiangMedicalUniversity,Xinxiang,Henan453100,China)

ObjectiveTo investigate the potential roles of tumor necrosis factorα (TNF-α) and highsensitivity Creactive protein (HsCRP) in the progression and prognosis of neonatal hypoxicischemic encephalopathy(HIE).MethodsThis study was a clinical experimental study.Totally 74 cases of neonates with HIE diagnosed in the First Affiliated Hospital of Xinxiang Medical University From February 2011 to February 2013 were enrolled as observation group (mild 31 cases,moderate 26 cases,severe 17 cases;32 cases with good prognosis,42 poor prognosis),and another 74 cases of normal healthy neonates were selected as control group.The levels of TNF-α and HsCRP in all samples were measured 48 hours after being born by enzymelinked immunosorbent assay (ELISA) and radio immunoassay (RIA) method.ResultsThe data revealed significant upregulation of the serum levels of TNF-α and HsCRP in patients with HIE.The increase in the levels of these inflammatory mediators correlated with the severity of the disease and also had a positive correlation with the prognosis of the disease.Serum levels of TNF-α and HsCRP in HIE group were significantly higher than those in normal control group.TNF-α levels were 17.20±1.26vs97.00±5.97 ng·L-1(P<0.05)；HsCRP levels were 0.51±0.18vs11.93±1.91 mg·L-1(P<0.05).Serum levels of TNF-α and HsCRP in the moderate and severe patient groups were significantly higher compared with those in the mild group(P<0.05).Furthermore,there was a significant upregulation of the cytokines in the severe group compared with those in the moderate group(P<0.05).The differences among 3 groups were also significant ,and they were the highest with severe HIE,TNF-α levels(mild group 31.37±3.28vsmoderate group 52.59±5.19vssevere group 102.65±7.81 ng·L-1,P<0.05);HsCRP levels(mild group 4.63±0.69vsmoderate group 7.56±1.19vssevere group 12.92±3.25 mg·L-1,P<0.05).Comparison of the serum levels of TNF-α and HsCRP in patients with different prognoses.The levels of TNF-α,in patients with either a poor or good prognosis were further analyzed.Significant upregulation of the levels of TNF-α (90.23±7.37 ng·L-1) and HsCRP levels(9.71±2.14 mg·L-1,P<0.05) were observed in patients who had a poor prognosis compared with those in the patients who had a good prognosis,TNF-α (44.32±4.84 ng·L-1) and HsCRP (5.99±0.99 mg·L-1).ConclusionsWith the aggravation of the disease and poor prognosis aggravation,TNF-α and HsCRP expression increased,suggesting that TNF-α and HsCRP may be involved in the pathophysiological process of HIE,therefore,dynamic monitoring of both trends will help determine the severity of HIE and prognostic evaluation.

Hypoxia-ischemia,brain；Tumor necrosis factor-alpha;C-reactive protein;Infant,newborn

河南省衛(wèi)生廳資助項(xiàng)目(200804053)

唐成和，男，教授，碩士生導(dǎo)師，研究方向：新生兒腦損傷，E-mail：xxshangyun@163.com

10.3969/j.issn.1009-6469.2016.08.021

2016-04-18，

2016-06-30)