宋麗麗，葉鈞，劉韻，潘瓊，鐘小莉，李?yuàn)檴?，田音，彭志紅，汪榮泉

O型糖鏈合成阻滯對(duì)腸上皮細(xì)胞MUC2表達(dá)及細(xì)菌黏附的抑制作用

宋麗麗，葉鈞，劉韻，潘瓊，鐘小莉，李?yuàn)檴櫍镆?，彭志紅，汪榮泉

目的探討腸上皮細(xì)胞O型糖鏈的合成阻滯對(duì)該細(xì)胞腸分化標(biāo)記物MUC2表達(dá)及細(xì)菌黏附的影響。方法采用O型糖鏈抑制劑benzyl-α-GalNAc抑制結(jié)腸上皮細(xì)胞HT-29及其分化型細(xì)胞(HT-29-Gal)O型糖鏈的合成，經(jīng)benzyl-α-GalNAc處理的HT-29和HT-29-Gal細(xì)胞分別命名為HT-29-OBN和HT-29-Gal-OBN。采用Real-time PCR和Western blotting方法檢測(cè)上述4種細(xì)胞中MUC2基因的轉(zhuǎn)錄和蛋白表達(dá)水平，并將上述細(xì)胞與致病性大腸埃希菌(EPEC)和腸出血性大腸埃希菌(EHEC)O157:H7共培養(yǎng)，采用系列稀釋菌落計(jì)數(shù)法觀(guān)察細(xì)菌在上述細(xì)胞表面的黏附情況。結(jié)果Realtime PCR和Western blotting結(jié)果顯示，經(jīng)benzyl-α-GalNAc處理后，HT-29和HT-29-Gal細(xì)胞MUC2的mRNA和蛋白表達(dá)均明顯減少(P＜0.05)。HT-29-OBN和HT-29-Gal-OBN細(xì)胞與致病性大腸埃希菌EPEC和EHEC O157:H7的黏附明顯少于HT-29和HT-29-Gal細(xì)胞(P＜0.05)。結(jié)論抑制腸上皮細(xì)胞O型糖鏈的合成可阻礙細(xì)菌黏附及MUC2的表達(dá)。

腸黏膜；細(xì)菌黏附；大腸埃希菌

腸道除具有消化吸收及蠕動(dòng)功能外，還有分泌激素、免疫調(diào)節(jié)和黏膜屏障功能，腸黏膜屏障在腸功能維護(hù)中扮演著重要角色[1-2]。腸黏膜表面覆蓋著一層由黏蛋白組成的黏液，目前已經(jīng)發(fā)現(xiàn)的黏蛋白有21種，分為分泌性和跨膜性?xún)煞N。由于腸黏液屏障中黏蛋白分子質(zhì)量的80%是由O-glycans組成，因此黏蛋白的功能與其分子上的O型糖鏈密切相關(guān)[3-4]。近年來(lái)，人們對(duì)病原菌的黏附機(jī)制進(jìn)行了系統(tǒng)深入的研究，發(fā)現(xiàn)黏液黏蛋白為進(jìn)入腸道內(nèi)的細(xì)菌黏附定植提供了受體位點(diǎn)[5]，部分特定的糖鏈成為病原微生物定植黏液層的位點(diǎn)以及聯(lián)結(jié)細(xì)胞與環(huán)境相互交通信號(hào)的生物平臺(tái)[6-7]。研究黏蛋白分子上決定其生物學(xué)功能的O-glycans與病原微生物之間的相互作用，有助于理解宿主與病原微生物之間的關(guān)系。本研究探討腸上皮細(xì)胞O型糖鏈的合成阻滯對(duì)細(xì)胞腸分化標(biāo)記物MUC2表達(dá)及細(xì)菌黏附的影響。

1 材料與方法

1.1 主要材料與儀器 人結(jié)腸癌細(xì)胞株HT-29購(gòu)自中國(guó)科學(xué)院上海細(xì)胞庫(kù)，致病性大腸埃希菌EPEC和腸出血性大腸埃希菌EHEC O157:H7由第三軍醫(yī)大學(xué)微生物免疫教研室毛旭虎教授贈(zèng)送。Macoy's 5A和無(wú)糖Macoy's 5A培養(yǎng)基購(gòu)自Sigma公司，葡萄糖和半乳糖、Trizol試劑購(gòu)自Invitrogen公司，O糖鏈抑制劑benzyl-α-GalNAc購(gòu)自Santa公司，胎牛血清(FBS)購(gòu)自Hyclone公司，反轉(zhuǎn)錄及熒光定量PCR試劑購(gòu)自大連寶生物工程有限公司，CO2恒溫培養(yǎng)箱購(gòu)自美國(guó)Thermo公司，PCR儀和凝膠成像系統(tǒng)均購(gòu)自美國(guó)Bio-Rad公司。MUC2多克隆抗體采用脫糖基化的腸黏蛋白免疫新西蘭兔獲得。

1.2 細(xì)胞培養(yǎng) 將結(jié)腸癌HT-29細(xì)胞用含10% FBS的Macoy's 5A完全培養(yǎng)液進(jìn)行常規(guī)培養(yǎng)；待細(xì)胞生長(zhǎng)融合至60%后再繼續(xù)培養(yǎng)48h；用含10% FBS的Macoy's 5A培養(yǎng)液(以5nmol/L半乳糖取代葡萄糖)培養(yǎng)，誘導(dǎo)得到結(jié)腸癌HT-29細(xì)胞的分化型細(xì)胞HT-29-Gal[8-9]。當(dāng)上述兩種細(xì)胞處于對(duì)數(shù)生長(zhǎng)期時(shí)消化、離心，調(diào)整細(xì)胞濃度至30%，接種至6孔板，接種第2天當(dāng)細(xì)胞生長(zhǎng)融合至50%～60%時(shí)，加入2nmol/L的benzyl-α-GalNAc[10-11]，培養(yǎng)7d，分別獲得O型糖鏈合成抑制的細(xì)胞：HT-29-OBN和HT-29-Gal-OBN。

1.3 細(xì)菌黏附實(shí)驗(yàn) 將處于對(duì)數(shù)生長(zhǎng)期的HT-29、HT-29-Gal、HT-29-OBN、HT-29-Gal-OBN 4種細(xì)胞接種于24孔板直至細(xì)胞生長(zhǎng)融合至70%～80%；將新鮮接種的MacConkey平板上的致病性大腸埃希菌EPEC和EHEC O157:H7，分別挑單菌落于Penassay肉湯培養(yǎng)基中于37℃過(guò)夜培養(yǎng)增殖；細(xì)胞與細(xì)菌共孵育前1h，將含血清的細(xì)胞培養(yǎng)液換成無(wú)血清無(wú)抗生素的Macoy's 5A培養(yǎng)液(含葡萄糖)或無(wú)血清無(wú)抗生素的Macoy's 5A培養(yǎng)液(含半乳糖)。將孵育的EPEC和EHEC O157:H7細(xì)菌分別按106cfu/ml加入24孔板中，37℃共孵育3h；用冷PBS清洗共孵育的細(xì)胞，去除未黏附的EPEC或EHEC O157:H7細(xì)菌并抑制黏附的細(xì)菌；用胰酶消化黏附了細(xì)菌的細(xì)胞，將得到的細(xì)菌按梯度稀釋法涂在MacConkey瓊脂板上，37℃過(guò)夜。用菌落計(jì)數(shù)法定量黏附細(xì)菌數(shù)。

1.4 Real-time PCR檢測(cè)腸分化標(biāo)志物MUC2 mRNA的表達(dá) 按照Trizol試劑說(shuō)明書(shū)提取細(xì)胞總RNA，Real-time PCR引物由大連寶生物工程有限公司合成，退火溫度60℃。按照試劑盒說(shuō)明書(shū)進(jìn)行反轉(zhuǎn)錄，反應(yīng)條件為：37℃ 15min，85℃ 5s。Real-time PCR反應(yīng)體系20μl，反應(yīng)條件為：95℃ 10s；95℃ 5s，60℃ 20s，40個(gè)循環(huán)；55℃ 10s，80個(gè)循環(huán)。MUC2上游引物：5'-GGGGAGTGCTGTAAGAAGTGTGA-3'；下游引物：5'-GTTGGAGACGGACGAGATGAG-3'，擴(kuò)增片段長(zhǎng)度165bp。

1.5 Western blotting檢測(cè)腸分化標(biāo)志物MUC2蛋白的表達(dá) 獲得HT-29、HT-29-Gal、HT-29-OBN和HT-29-Gal-OBN全細(xì)胞裂解液，BCA法定量；取20μg按照4:1比例加入0.5mol/L DTT，煮沸5min變性，上樣、電泳、電轉(zhuǎn)；加入5% BSA室溫封閉2h；加入MUC2一抗(1:1000)，4℃過(guò)夜；洗膜，加入辣根過(guò)氧化物酶標(biāo)記的二抗(1:10 000)，室溫孵育1h；洗膜，化學(xué)發(fā)光顯色，凝膠成像儀成像。采用Quantity-One軟件對(duì)條帶的光密度(A)值進(jìn)行分析，蛋白的相對(duì)定量結(jié)果以實(shí)驗(yàn)條帶A值/β-actin條帶A值表示。

1.6 統(tǒng)計(jì)學(xué)處理 采用SPSS 13.0軟件進(jìn)行數(shù)據(jù)處理。計(jì)量資料以表示，各組間比較采用單因素方差分析，進(jìn)一步兩兩比較采用SNK-q法。P＜0.05表示差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié) 果

2.1 細(xì)菌黏附實(shí)驗(yàn) 對(duì)于同一濃度下的細(xì)菌EPEC和EHEC O157:H7，腸分化細(xì)胞HT-29-Gal的細(xì)菌黏附數(shù)目少于結(jié)腸上皮HT-29細(xì)胞(P＜0.01)；HT-29-OBN細(xì)胞的細(xì)菌黏附數(shù)目少于HT-29細(xì)胞(P＜0.01)；HT-29-Gal-OBN細(xì)胞的細(xì)菌黏附數(shù)目明顯少于HT-29-Gal細(xì)胞(P＜0.05)；HT-29-Gal-OBN細(xì)胞的細(xì)菌黏附數(shù)目少于HT-29-OBN細(xì)胞(P＜0.01，圖1)。

圖1 致病性大腸埃希菌(EPEC和EHEC O157:H7)對(duì)腸上皮細(xì)胞的黏附能力檢測(cè)Fig.1 Adhesion of EPEC and EHEC O157:H7 to intestinal epithelial cells

2.2 腸分化標(biāo)志物MUC2 mRNA的表達(dá)情況Real-time PCR檢測(cè)結(jié)果顯示，HT-29-OBN細(xì)胞中的MUC2 mRNA水平明顯低于結(jié)腸上皮HT-29細(xì)胞(P＜0.05)；HT-29-Gal-OBN細(xì)胞MUC2 mRNA水平明顯低于腸分化的HT-29-Gal細(xì)胞(P＜0.05)；HT-29-Gal細(xì)胞中的MUC2 mRNA表達(dá)明顯高于HT-29細(xì)胞，而HT-29-Gal-OBN細(xì)胞中的MUC2 mRNA表達(dá)高于HT-29-OBN細(xì)胞(P＜0.05，圖2)。

圖2 Real-time PCR檢測(cè)細(xì)胞MUC2 mRNA表達(dá)水平Fig.2 MUC2 mRNA expression detected by real-time PCR

2.3 MUC2蛋白的表達(dá)情況 Western blotting檢測(cè)結(jié)果顯示，HT-29-OBN細(xì)胞MUC2蛋白表達(dá)水平明顯低于HT-29細(xì)胞；HT-29-Gal-OBN細(xì)胞MUC2蛋白表達(dá)水平明顯低于HT-29-Gal細(xì)胞(P＜0.05)；HT-29-Gal細(xì)胞中MUC2蛋白的表達(dá)明顯高于HT-29細(xì)胞(P＜0.05)；而HT-29-Gal-OBN細(xì)胞MUC2蛋白表達(dá)水平高于HT-29-OBN細(xì)胞，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05，圖3)。

3 討 論

腸黏液屏障是組成腸黏膜屏障的重要成分，腸道黏蛋白VNTR內(nèi)的O-glycans含有豐富糖鏈，在腸道黏液層內(nèi)是直接與腸道微生物、病毒、毒素相互作用的一線(xiàn)屏障，并直接成為后者結(jié)合、參與代謝、抑制細(xì)菌生長(zhǎng)的靶點(diǎn)[12-13]。因此，研究黏蛋白分子上決定其生物學(xué)功能的O-glycans與病原微生物之間的相互作用，可更好地理解宿主與病原微生物之間的聯(lián)系。

本研究結(jié)果顯示，相對(duì)于HT-29和HT-29-Gal細(xì)胞，HT-29-OBN和HT-29-Gal-OBN細(xì)胞中的MUC2 mRNA和蛋白質(zhì)表達(dá)水平均下降，考慮原因?yàn)镸UC2轉(zhuǎn)錄、翻譯過(guò)程中所涉及的酶、轉(zhuǎn)錄因子或共激活因子存在O型糖基化。benzyl-α-GalNAC不僅抑制了黏蛋白的O型糖基化，同時(shí)也抑制了培養(yǎng)細(xì)胞中所有蛋白的O型糖基化。benzyl-α-GalNAC通過(guò)與黏蛋白分子O-glycan的核心GalNAC結(jié)構(gòu)(GalNAC-O-Ser/Thr)競(jìng)爭(zhēng)結(jié)合干擾黏蛋白O-glycan的延長(zhǎng)，因此，分化程度高的HT-29-Gal中的MUC2蛋白表達(dá)明顯高于分化程度低的HT-29細(xì)胞。腸杯狀細(xì)胞標(biāo)志物MUC2蛋白表達(dá)在HT-29-OBN和HT-29-Gal-OBN細(xì)胞中低于相應(yīng)的HT-29和HT-29-Gal，提示抑制腸上皮細(xì)胞黏蛋白分子O型糖鏈的合成可抑制細(xì)胞MUC2的表達(dá)。

由于在體內(nèi)評(píng)價(jià)尚存在一定困難，目前多應(yīng)用體外培養(yǎng)腸上皮細(xì)胞和細(xì)菌共孵育的方法來(lái)檢測(cè)候選菌株的黏附力[14]，該方法也可應(yīng)用于細(xì)菌對(duì)腸黏膜上皮的黏附[15]。將HT-29-OBN、HT-29-Gal-OBN、HT-29和HT-29-Gal細(xì)胞與細(xì)菌共孵育發(fā)現(xiàn)，EPEC和EHEC O157:H7在HT-29-Gal細(xì)胞的黏附數(shù)量少于HT-29細(xì)胞；HT-29-Gal-OBN細(xì)胞的細(xì)菌黏附數(shù)量少于HT-29-OBN細(xì)胞，提示分化程度高的細(xì)胞有阻礙細(xì)菌黏附的作用。HT-29-OBN和HT-29-Gal-OBN細(xì)胞的細(xì)菌黏附數(shù)量明顯少于未經(jīng)處理的HT-29和HT-29-Gal細(xì)胞，表明O型糖鏈的合成與細(xì)菌對(duì)腸上皮細(xì)胞的黏附有關(guān)。

綜上所述，我們認(rèn)為O-glycans的合成參與了腸黏液屏障的形成，以及腸道病原體對(duì)腸黏膜上皮細(xì)胞的黏附過(guò)程。

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Inhibitory effect of O-glycosylation inhibition on human intestinal epithelial cells Mucin 2 expression and bacteria adherence

SONG Li-li, YE Jun, LIU Yun, PAN Qiong, ZHONG Xiao-li, LI Shan-shan, TIAN Yin, PENG Zhi-hong, WANG Rongquan*
Institute of Gastroenterology of PLA, Southwest Hospital of Third Military University, Chongqing 400038, China
*

, E-mail: rongquanw@hotmail.com
This work was supported by the National Natural Science Foundation of China (81170340)

ObjectiveTo investigate the effect of O-glycosylation inhibition in intestinal epithelial cells on the expression of Mucin 2 (MUC2) and bacterial adherence.MethodsIntestinal epithelial cells HT-29 and differentiated HT-29 cells (HT-29-Gal) were treated with an inhibitor of O-glycosylation (benzyl-α-GalNAc), and then named as HT-29-OBN and HT-29-Gal-OBN, respectively. The mRNA and protein expression of MUC2 in HT-29, HT29-Gal, HT-29-OBN and HT-29-Gal-OBN were detected by real-time PCR and Western blotting. Then the four kinds of above cells were incubated with enteropathogenicEscherichia coli(EPEC) or enterohemorrhagicEscherichia coliserotype O157:H7 (EHEC O157:H7). The bacteria were quantified by determining the colony forming unit (CFU) following the plating of serial dilutions of the bacteria to evaluate the effect of benzyl-α-GalNAc on bacteria adherence.ResultsThe results of real-time PCR and Western blotting showed that the mRNA and protein expression levels of MUC2 in HT-29-OBN and HT-29-Gal-OBN cells were significantly lower than those in the untreated cells HT-29 and HT-29-Gal (P＜0.05). The bacterial adherence assay showed that the adherence of EPEC and EHEC O157:H7 to HT-29-OBN and HT-29-Gal-OBN cells significantly decreased compared with that to HT-29 and HT-29-Gal cells (P＜0.05). ConclusionInhibition of O-glycosylation in intestinal epithelial cells may reduce the bacteria adherence and MUC2 expression.

intestinal mucosa; bacteria adhesion;Escherichia coli

R34

A

0577-7402(2013)10-0826-04

10.11855/j.issn.0577-7402.2013.10.009

2013-04-27；

2013-08-22)

(責(zé)任編輯：熊曉然)

國(guó)家自然科學(xué)基金(81170340)

宋麗麗，碩士研究生。主要從事腸黏蛋白方面的研究

400038 重慶 第三軍醫(yī)大學(xué)西南醫(yī)院全軍消化病研究所(宋麗麗、葉鈞、劉韻、潘瓊、鐘小莉、李?yuàn)檴?、田音、彭志紅、汪榮泉)

汪榮泉，E-mail: rongquanw@hotmail.com