秦麗云，李　潔，陳海英

(1.山東省昌樂縣人民醫(yī)院，山東 昌樂 262400；2.中國醫(yī)科大學(xué)第四醫(yī)院，遼寧 沈陽 110032)

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S100A12和RAGE在子癇前期孕婦外周血及胎盤組織中的表達(dá)

秦麗云1，李潔1，陳海英2

(1.山東省昌樂縣人民醫(yī)院，山東 昌樂 262400；2.中國醫(yī)科大學(xué)第四醫(yī)院，遼寧 沈陽 110032)

目的探討S100鈣結(jié)合蛋白A12(S100A12)及晚期糖基化終末產(chǎn)物受體(RAGE)基因在子癇前期孕婦外周血及胎盤組織中的表達(dá)及意義。方法選取2014年2月至2015年3月在中國醫(yī)科大學(xué)第四醫(yī)院和昌樂縣人民醫(yī)院產(chǎn)科分娩的87例子癇前期孕婦分為非重度組(n=46)和重度組(n=41)，同期選取正常孕婦40例作為對(duì)照組。利用實(shí)時(shí)熒光定量PCR技術(shù)檢測外周血及胎盤組織中S100A12 mRNA及RAGE mRNA的表達(dá)。結(jié)果子癇前期孕婦外周血中S100A12 mRNA和RAGE mRNA相對(duì)表達(dá)量均高于對(duì)照組(t值分別為12.341和10.517，均P<0.05)，重度組子癇前期孕婦外周血中S100A12 mRNA和RAGE mRNA相對(duì)表達(dá)量均高于非重度組和對(duì)照組，且非重度組均高于對(duì)照組，差異均有統(tǒng)計(jì)學(xué)意義(t=1.980～8.191，均P<0.05)。子癇前期孕婦胎盤組織中S100A12 mRNA和RAGE mRNA相對(duì)表達(dá)量均高于對(duì)照組孕婦(t值分別為9.747和8.625，均P<0.05)，重度組孕婦胎盤組織中S100A12 mRNA和RAGE mRNA相對(duì)表達(dá)量均高于非重度組和對(duì)照組，且非重度組高于對(duì)照組，差異均有統(tǒng)計(jì)學(xué)意義(t=2.235～7.286，均P<0.05)。經(jīng)Pearson相關(guān)分析顯示，子癇前期孕婦外周血和胎盤組織中S100A12 mRNA相對(duì)表達(dá)量均與RAGE mRNA相對(duì)表達(dá)量呈正相關(guān)(r值分別為0.603和0.552，均P<0.05)。結(jié)論S100A12及RAGE基因在子癇前期孕婦外周血及胎盤組織中呈高表達(dá)，可能共同參與了子癇前期孕婦機(jī)體炎性反應(yīng)的發(fā)生。

子癇前期；外周血；胎盤組織；S100鈣結(jié)合蛋白A12；晚期糖基化終末產(chǎn)物受體

[Abstract]Objective To explore the expressions and significance of S100 calcium binding protein A12 (S100A12)and receptor for advanced glycation end products (RAGE) gene in peripheral blood and placenta tissue of pregnant women with preeclampsia. Methods Eighty-seven cases of pregnant women with preeclampsia delivering in Fourth Affiliated Hospital of China Medical University and People’s Hospital of Changle County were divided into non-severe group (n=46) and severe group (n=41). At the same time, 40 cases of healthy pregnant women were chosen as control group. Expressions of S100A12 mRNA and RAGE mRNA in peripheral blood and placenta tissues were detected by using real-time fluorescence quantitative PCR technology. Results The relative expression levels of S100A12 mRNA and RAGE mRNA in peripheral blood of pregnant women with preeclampsia were higher than those in the control group (tvalue was 12.341 and 10.517, respectively, bothP<0.05). The relative expression levels of S100A12 mRNA and RAGE mRNA in peripheral blood of the severe group were higher than the non-severe group and the control group, and those of non-severe group were higher than the control group. The differences were statistically significant (tvalue ranged 1.980-8.191, bothP<0.05). The relative expression levels of S100A12 mRNA and RAGE mRNA in placenta tissues of pregnant women with preeclampsia were higher than the control group (tvalue was 9.747 and 8.625, respectively, bothP<0.05). The relative expression levels of S100A12 mRNA and RAGE mRNA in placenta tissue of severe group were higher than non-severe group and the control group, and the relative expression levels of non-severe group were higher than the control group. The differences were statistically significant (tvalue ranged 2.235-7.286, bothP<0.05). Pearson correlation analysis showed that the relative expression levels of S100A12 mRNA in peripheral blood and placenta tissues of pregnant women with preeclampsia were positively correlated with the relative expression levels of RAGE mRNA (rvalue was 0.603 and 0.552, respectively, bothP<0.05).Conclusion High levels of expression of S100A12 and RAGE gene are detected in peripheral blood and placenta tissues of pregnant women with preeclampsia. S100A12 and RAGE may have jointly participated in the inflammatory response of pregnant women with preeclampsia.

子癇前期作為一種妊娠期高血壓疾病是造成孕產(chǎn)婦及圍產(chǎn)兒死亡的主要因素，發(fā)病率高達(dá)7%～13%[1]，其發(fā)病機(jī)制目前尚不明確。近年來研究發(fā)現(xiàn)，子癇前期可能是母體發(fā)生的過度炎性反應(yīng)，與患者免疫排除、胎盤組織局部缺血和缺氧等病理過程關(guān)系密切[2-3]。S100鈣結(jié)合蛋白A12(S100A12)作為鈣結(jié)合蛋白家族重要成員發(fā)揮促炎因子的作用，在調(diào)控炎性反應(yīng)中發(fā)揮著關(guān)鍵性作用[4]。晚期糖基化終末產(chǎn)物受體(receptor for advanced glycation end products，RAGE)作為S100A12結(jié)合受體，可通過激活細(xì)胞內(nèi)信號(hào)傳導(dǎo)通路參與全身及局部炎性疾病的發(fā)生[5]。目前，國內(nèi)外有關(guān)S100A12和RAGE在子癇前期孕婦發(fā)病中的意義少有報(bào)道。本研究嘗試對(duì)S100A12和RAGE基因在子癇前期孕婦外周血及胎盤組織中的表達(dá)進(jìn)行檢測，探討二者與子癇前期發(fā)病的關(guān)系，以期為子癇前期機(jī)制研究提供依據(jù)。

1資料與方法

1.1一般資料

選取2014年2月至2015年3月在中國醫(yī)科大學(xué)第四醫(yī)院和昌樂縣人民醫(yī)院產(chǎn)科分娩的共87例子癇前期孕婦，均符合子癇前期診斷標(biāo)準(zhǔn)[6]，年齡23～35歲，平均年齡(27.6±4.9)歲，根據(jù)患者病情嚴(yán)重程度分為非重度組和重度組；其中，非重度組46例，平均年齡(26.9±4.6)歲，重度組41例，平均年齡(28.1±5.4)歲，同期選取在產(chǎn)科進(jìn)行分娩的正常孕婦40例作為對(duì)照組，平均年齡(27.3±4.8)歲。3組孕婦均未合并其他并發(fā)癥，其年齡、孕次、產(chǎn)次一般情況比較均無顯著性差異(均P>0.05)。本研究通過醫(yī)院倫理委員會(huì)批準(zhǔn)，所有患者行知情同意。

1.2方法

1.2.1主要試劑和儀器

總RNA提取試劑盒購自美國Invitrogen公司，逆轉(zhuǎn)錄試劑盒購自Promega公司，SYBR Green PCR試劑盒購自美國ABI公司，S100A12和RAGE基因、內(nèi)參均由生工生物工程(上海)股份有限公司合成，實(shí)時(shí)熒光定量PCR儀購自美國Roche公司，紫外分光光度計(jì)購自美國Beckman公司，凝膠成像系統(tǒng)購自德國Vilber公司。

1.2.2標(biāo)本采集及處理

所有孕婦均于晨起抽取空腹肘靜脈血5mL，于3 000r/min離心10min，留取上清液保存于-20℃冰箱中。胎盤從產(chǎn)婦體內(nèi)娩出后，于胎盤母體面切取1cm×1cm×1cm胎盤組織，切除時(shí)要注意避開梗死、出血、鈣化區(qū)域，用冷生理鹽水將切取組織沖洗干凈，保存于-70℃冰箱。

1.2.3總RNA提取及逆轉(zhuǎn)錄

取胎盤組織進(jìn)行研磨后，利用總RNA提取試劑盒分別對(duì)外周血和胎盤組織中總RNA進(jìn)行提取，利用紫外分光光度計(jì)檢測獲得的RNA純度，以A260/A280≥1.8為合格標(biāo)本，利用逆轉(zhuǎn)錄試劑盒進(jìn)行逆轉(zhuǎn)錄為cDNA。

1.2.4利用實(shí)時(shí)熒光定量PCR技術(shù)檢測外周血及胎盤組織中S100A12 mRNA及RAGE mRNA表達(dá)

以cDNA作為模板，利用SYBR Green PCR試劑盒及PCR儀進(jìn)行PCR實(shí)驗(yàn)，以GAPDH為內(nèi)參，引物序列分別為S100A12的上游：5′-CACATTCCTGTGCATTCAGG-3′，下游：5′-TGCAAGCTCCTTTGTAAGCA-3′；RAGE的上游：5′-TTGGCGA￣GCCACTGGTGCTG-3′，下游：5′-TAGGACTGGTGGATGGCGGG￣TG-3′；GAPDH的上游：5′-GAGTCAACGGATTTGGTCGT-3′，下游：5′-GACAAGCTTCCCGTTCTCAG-3′。反應(yīng)條件：94℃預(yù)變性10min，90℃15s，64℃15s，70℃60s，連續(xù)進(jìn)行循環(huán)40次，每個(gè)標(biāo)本均設(shè)置3個(gè)平衡孔。對(duì)獲得的擴(kuò)增產(chǎn)物進(jìn)行凝膠電泳，利用Quantity One圖像分析軟件對(duì)電泳條帶分析，并獲得S100A12 mRNA和RAGE mRNA相對(duì)表達(dá)量。

1.3統(tǒng)計(jì)學(xué)方法

2結(jié)果

2.1各組孕婦外周血中S100鈣結(jié)合蛋白A12 mRNA和晚期糖基化終末產(chǎn)物受體 mRNA的相對(duì)表達(dá)量

子癇前期孕婦外周血中S100A12 mRNA和RAGE mRNA相對(duì)表達(dá)量均高于對(duì)照組，差異均有統(tǒng)計(jì)學(xué)意義(t值分別為12.341和10.517，均P<0.05)；重度組子癇前期孕婦外周血中S100A12 mRNA和RAGE mRNA相對(duì)表達(dá)量均高于非重度組和對(duì)照組，且非重度組均高于對(duì)照組，差異均有統(tǒng)計(jì)學(xué)意義(均P<0.05)，見表1和圖1。

表1　不同組孕婦外周血中S100A12 mRNA和RAGE mRNA相對(duì)表達(dá)量比較

圖1 S100A12 mRNA和RAGE mRNA在不同組孕婦外周血中表達(dá)

Fig.1The expressions of S100A12 mRNA and RAGE mRNA in maternal peripheral blood of different groups

2.2各組孕婦胎盤組織中S100鈣結(jié)合蛋白A12 mRNA和晚期糖基化終末產(chǎn)物受體 mRNA的相對(duì)表達(dá)量

子癇前期孕婦胎盤組織中S100A12 mRNA和RAGE mRNA相對(duì)表達(dá)量均高于對(duì)照組孕婦，差異均有統(tǒng)計(jì)學(xué)意義(t值分別為9.747和8.625，均P<0.05)；重度組孕婦胎盤組織中S100A12 mRNA和RAGE mRNA相對(duì)表達(dá)量均高于非重度組和對(duì)照組，且非重度組高于對(duì)照組，差異均有統(tǒng)計(jì)學(xué)意義(均P<0.05)，見表2和圖2。

表2　不同組孕婦胎盤組織中S100A12 mRNA和RAGE mRNA相對(duì)表達(dá)量

圖2 S100A12 mRNA和RAGE mRNA在不同組孕婦胎盤組織中表達(dá)

Fig.2The expressions of S100A12 mRNA and RAGE mRNA in placental tissues of different groups of pregnant women

2.3子癇前期孕婦外周血和胎盤組織中S100鈣結(jié)合蛋白A12 mRNA和晚期糖基化終末產(chǎn)物受體 mRNA表達(dá)的相關(guān)性

經(jīng)Pearson相關(guān)分析顯示，子癇前期孕婦外周血中S100A12 mRNA相對(duì)表達(dá)量與RAGE mRNA相對(duì)表達(dá)量呈正相關(guān)(r=0.603，P=0.000)，且胎盤組織中S100A12 mRNA相對(duì)表達(dá)量與RAGE mRNA相對(duì)表達(dá)量呈正相關(guān)(r=0.552，P=0.000)，見圖3。

圖3子癇前期孕婦外周血和胎盤組織中S100A12 mRNA和RAGE mRNA表達(dá)相關(guān)性(A：外周血，B：胎盤組織)

Fig.3The relationships between the expressions of S100A12 mRNA and RAGE mRNA in preeclampsia maternal peripheral blood and placental tissues(A:peripheral blood, B:placental tissue)

3討論

3.1炎性反應(yīng)在子癇前期發(fā)病中的作用

子癇前期是妊娠期特發(fā)性疾病，可對(duì)孕婦各器官系統(tǒng)及新生兒產(chǎn)生影響，該病發(fā)病機(jī)制較為復(fù)雜，目前尚未研究清楚。有研究認(rèn)為，胎盤是子癇前期發(fā)病的根源，胎盤滋養(yǎng)細(xì)胞功能異常致使血管重鑄而導(dǎo)致胎盤淺著床，從而引發(fā)胎盤發(fā)生缺血再灌注損傷，導(dǎo)致炎性反應(yīng)[7]，其提示炎性反應(yīng)在子癇前期發(fā)病中扮演重要角色。有研究利用內(nèi)毒素刺激的方式構(gòu)建了子癇前期動(dòng)物模型，且對(duì)抗炎治療有效[8]，進(jìn)一步說明過度的炎性反應(yīng)導(dǎo)致機(jī)體一系列損傷反應(yīng)是引發(fā)子癇前期的因素。

3.2 S100鈣結(jié)合蛋白A12在子癇前期孕婦外周血和胎盤組織中呈高表達(dá)

S100A12是一種重要的促炎因子，在機(jī)體炎性反應(yīng)及免疫防御中發(fā)揮重要作用，亦在細(xì)胞生長、分化、凋亡等生理過程中發(fā)揮調(diào)節(jié)作用[9]。有研究表明，S100A12可與鈣結(jié)合，刺激血管內(nèi)皮細(xì)胞黏附分子表達(dá)，加速炎性細(xì)胞激活及抗微生物作用的發(fā)揮[10]。本研究顯示，子癇前期孕婦外周血和胎盤組織中S100A12 mRNA相對(duì)表達(dá)量均高于對(duì)照組孕婦(均P<0.05)，重度組子癇前期孕婦外周血和胎盤組織中S100A12 mRNA相對(duì)表達(dá)量>非重度組>對(duì)照組，說明S100A12基因在子癇前期孕婦外周血及胎盤組織中出現(xiàn)高表達(dá)，且與患者病情有關(guān)，病情愈嚴(yán)重，相對(duì)表達(dá)量愈高，提示炎性反應(yīng)可能是引發(fā)子癇前期的重要病理基礎(chǔ)，與Blaauw等[11]研究結(jié)論一致。

3.3晚期糖基化終末產(chǎn)物受體與S100鈣結(jié)合蛋白A12可能共同參與了子癇前期孕婦炎性反應(yīng)過程

RAGE是具有重要生物功能的多配體受體，S100A12是其重要受體之一，S100A12和RAGE結(jié)合可激活磷脂肌醇(PKC)、絲裂原細(xì)胞外信號(hào)調(diào)節(jié)激酶(MEK)、鈣調(diào)蛋白激酶Ⅱ等信號(hào)通路，經(jīng)過一系列級(jí)聯(lián)反應(yīng)而導(dǎo)致炎性因子的表達(dá)，促進(jìn)炎性反應(yīng)的發(fā)生[12]。本研究顯示，子癇前期孕婦外周血和胎盤組織中RAGE mRNA相對(duì)表達(dá)量均高于對(duì)照組孕婦(均P<0.05)，重度組孕婦外周血及胎盤組織中RAGE mRNA相對(duì)表達(dá)量>非重度組>對(duì)照組，說明RAGE基因在子癇前期孕婦外周血和胎盤組織中呈高表達(dá)，且與患者病情嚴(yán)重程度有關(guān)，提示隨病情進(jìn)展RAGE表達(dá)愈高。

經(jīng)Pearson相關(guān)分析顯示，子癇前期孕婦外周血及胎盤組織中S100A12 mRNA相對(duì)表達(dá)量與RAGE mRNA相對(duì)表達(dá)量均呈正相關(guān)(r值分別為0.603和0.552，均P<0.05)，說明在子癇前期發(fā)病過程中，S100A12和RAGE相互作用，S100A12和RAGE結(jié)合激活信號(hào)通路可促使核因子κB進(jìn)入細(xì)胞核，在促進(jìn)炎性反應(yīng)的同時(shí)，核因子κB作為RAGE核轉(zhuǎn)錄因子，可促使RAGE基因表達(dá)[13]，同時(shí)，RAGE又可促使S100A12聚集，引發(fā)炎性反應(yīng)瀑布式發(fā)生[14]。

綜上所述，過度炎性反應(yīng)在子癇前期孕婦發(fā)病中具有重要作用，促炎因子S100A12及其受體RAGE在子癇前期孕婦外周血及胎盤組織中呈高表達(dá)，且呈正相關(guān)，可能共同參與了子癇前期孕婦機(jī)體炎性反應(yīng)的發(fā)生，但具體在子癇前期發(fā)生中的作用機(jī)制尚待進(jìn)一步研究明確。

[1]Seely E W, Tsigas E, Rich-Edwards J W.Preeclampsia and future cardiovascular disease in women:how good are the data and how can we manage our patients?[J].Semin Perinatol,2015,39(4):276-283.

[2]Jain A, Schneider H, Aliyev E,etal.Hypoxic treatment of human dual placental perfusion induces a preeclampsia-like inflammatory response[J].Lab Invest,2014,94(8):873-880.

[3]Mihu D, Razvan C, Malutan A,etal.Evaluation of maternal systemic inflammatory response in preeclampsia[J].Taiwan J Obstet Gynecol,2015,54(2):160-166.

[4]Heilmann R M, Grellet A, Allenspach K,etal.Association between fecal S100A12 concentration and histologic, endoscopic, and clinical disease severity in dogs with idiopathic inflammatory bowel disease[J].Vet Immunol Immunopathol,2014,158(3-4):156-166.

[5]Huang S M, Chang Y H, Chao Y C,etal.EGCG-rich green tea extract stimulates sRAGE secretion to inhibit S100A12-RAGE axis through ADAM10-mediated ectodomain shedding of extracellular RAGE in type 2 diabetes[J].Mol Nutr Food Res,2013,57(12):2264-2268.

[6]莊旭,林建華.子癇前期患者24h尿蛋白值與不良妊娠結(jié)局的相關(guān)性[J].中華婦產(chǎn)科雜志,2014,49(7):538-540.

[7]Shah D A, Khalil R A.Bioactive factors in uteroplacental and systemic circulation link placental ischemia to generalized vascular dysfunction in hypertensive pregnancy and preeclampsia[J].Biochem Pharmacol,2015,95(4):211-226.

[8]Liu L, Liu H, Brennecke S,etal.PP023. Soluble Fms-like tyrosine kinase-1 and placental growth factor expression in a rat model of pre-eclampsia[J].Pregnancy Hypertens,2013,3(2):75-76.

[9]Loughran-Fowlds A, Leach S, Lin J,etal.Respiratory disease and early serum S100A12 changes in very premature infants[J].Acta Paediatr,2011,100(12):1538-1543.

[10]Hung K W, Hsu C C, Yu C.Solution structure of human Ca(2+)-bound S100A12[J].J Biomol NMR,2013,57(3):313-318.

[11]Blaauw J, Souwer E T, Coffeng S M,etal.Follow up of intima-media thickness after severe early-onset preeclampsia[J].Acta Obstet Gynecol Scand,2014,93(12):1309-1316.

[12]Myles A, Viswanath V, Singh Y P,etal.Soluble receptor for advanced glycation endproducts is decreased in patients with juvenile idiopathic arthritis(ERA category) and inversely correlates with disease activity and S100A12 levels[J].J Rheumatol,2011,38(9):1994-1999.

[13]Zhu P, Ren M, Yang C,etal.Involvement of RAGE, MAPK and NF-κB pathways in AGEs-induced MMP-9 activation in HaCaT keratinocytes[J].Exp Dermatol,2012,21(2):123-129.

[14]Naruse K, Sado T, Noguchi T,etal.Peripheral RAGE(receptor for advanced glycation endproducts)-ligands in normal pregnancy and preeclampsia: novel markers of inflammatory response[J].J Reprod Immunol,2012,93(2):69-74.

[專業(yè)責(zé)任編輯：李雪蘭]

Expressions of S100A12 and RAGE gene in peripheral blood and placenta tissue of pregnant women with preeclampsia

QIN Li-yun1, LI Jie1, CHEN Hai-ying2

(1.People’s Hospital of Changle County, Shandong Changle 262400, China;2.The Fourth Affiliated Hospital of China Medical University, Liaoning Shenyang 110032, China)

preeclampsia; peripheral blood; placenta tissue; S100 calcium binding protein A12 (S100 A12); receptor for advanced glycation end products (RAGE)

2015-08-08

遼寧省自然科學(xué)基金資助項(xiàng)目(2013021087)

秦麗云(1967-)，女，副主任醫(yī)師，主要從事婦產(chǎn)科臨床工作。

陳海英，副主任醫(yī)師。

10.3969/j.issn.1673-5293.2016.02.011

R714.24

A

1673-5293(2016)02-0180-03