白細胞介素-33和血管內皮生長因子C在胃癌中的表達及臨床意義*

夏兵祥，李 凡，徐 健，袁振華，鄭蘇文，張業偉

(南京醫科大學附屬腫瘤醫院/江蘇省腫瘤醫院/江蘇省腫瘤防治研究所普外科，南京 210009)

白細胞介素-33和血管內皮生長因子C在胃癌中的表達及臨床意義*

夏兵祥，李 凡，徐 健，袁振華，鄭蘇文，張業偉△

(南京醫科大學附屬腫瘤醫院/江蘇省腫瘤醫院/江蘇省腫瘤防治研究所普外科，南京 210009)

目的 探討白細胞介素-33(IL-33)和血管內皮生長因子C(VEGF-C)在胃癌患者組織標本及血清中表達，以及二者與胃癌淋巴結轉移的關系。方法 分別應用免疫組織化學SP法和酶聯免疫雙抗夾心法(ELISA)檢測98例胃癌患者和36名健康體檢者胃黏膜組織標本及血清中IL-33和VEGF-C水平。結果 胃癌組織中IL-33和VEGF-C的陽性表達率分別為67.35%和74.49%，顯著高于正常胃組織(47.22%和61.11%)，比較差異有統計學意義(P<0.01)。IL-33和VEGF-C表達在不同的腫瘤分化程度、組織浸潤、淋巴結轉移、遠處轉移及臨床分期間比較差異有統計學意義(P<0.05)。胃癌淋巴結轉移患者的IL-33和VEGF-C的表達陽性率高于未發生淋巴結轉移患者(P<0.05)。胃癌患者血清IL-33和VEGF-C水平為(50.24±13.08)pg/mL和(210.73±58.35)pg/mL，高于健康體檢者(P<0.05)；淋巴結轉移患者血清IL-33和VEGF-C水平高于未發生淋巴結轉移患者，比較差異有統計學意義(P<0.05)。結論 胃癌患者血清中高水平IL-33可能誘導VEGF-C分泌，促進胃癌淋巴結轉移，可作為評估胃癌預后的重要指標。

胃腫瘤；血管內皮生長因子C；白細胞介素33；淋巴結轉移

胃癌是消化道最常見的惡性腫瘤，病死率居惡性腫瘤的第2位[1]。胃癌患者死亡的主要原因是腫瘤復發和轉移，其中最常見的轉移途徑為淋巴結轉移。臨床上有超過80%的進展期胃癌患者出現淋巴結轉移，研究證明其是胃癌患者預后不良的獨立危險因素[2]。近年來有關腫瘤侵襲轉移的機制研究發現，血管內皮生長因子C(VEGF-C)與腫瘤的淋巴結轉移密切相關[3-4]，同時白細胞介素-33(IL-33)可刺激并誘導白細胞和肥大細胞VEGF mRNA表達與VEGF蛋白分泌，促進胃癌細胞侵襲和轉移[5]。然而，關于IL-33、VEGF-C與腫瘤淋巴結轉移的研究少有報道。因此，本研究通過檢測胃癌患者組織標本和血清中IL-33和VEGF-C表達情況，分析IL-33和VEGF-C與胃癌淋巴結轉移的關系，探討其在胃癌淋巴結轉移中的作用，以及二者表達水平的相關性及臨床意義。

1 資料與方法

1.1 一般資料 收集2012年1月至2014年12月在本院接受胃癌根治手術的98例胃癌患者(胃癌組)手術切除標本，術前未接受任何放、化療等。其中男50例，女48例；年齡42.0～86.0歲，中位年齡60.8歲；腫瘤直徑1.0～10.0 cm，中位直徑4.6 cm，所有標本經病理學證實為胃癌。根據WHO(2010年)胃癌組織學分類標準[6]，管狀腺癌(高、中分化)35例，低分化腺癌40例，黏液腺癌14例，印戒細胞癌9例；根據國際抗癌聯盟(UICC)胃癌TNM分期標準[6]，Ⅰ期20例，Ⅱ期27例，Ⅲ期38例，Ⅳ期13例；早期胃癌16例，進展期胃癌82例；有淋巴結轉移76例；有遠處轉移16例。另選取同期36名在本院門診體檢的健康人，胃鏡下取正常胃黏膜組織為對照組,均知情同意，自愿參與。本研究經過南京醫科大學倫理委員會批準，批準編號(2011)132號。

納入標準：經病理診斷為胃癌，年齡、性別不限，入院行D2淋巴結清掃的胃切除術患者。排除標準：合并嚴重心腦血管疾??；合并多種腫瘤病史；外科手術前接受放療、化療、靶向藥物治療等其他治療手段；有研究者認為影響預后、評估及難以完成臨床觀察的其他嚴重疾病或殘疾。

1.2 方法 胃癌組患者于術前3 d采集清晨空腹靜脈血2 mL，對照組采集1次空腹靜脈血2 mL，采血后靜置離心(1 000 r/min，10 min)，分離血清，置20 ℃保存待測。所有組織標本均經10%甲醛固定24 h，行常規石蠟包埋，連續切取4 μm厚的切片，采用免疫組織化學SP法染色，具體步驟按說明書進行。每批染色過程中均設磷酸鹽緩沖液(PBS)代替一抗的陰性對照組。以細胞質呈清晰的棕黃色顆粒且其染色強度高于背景非特異染色者為陽性細胞。由兩位經驗豐富的病理科醫生在相同條件下觀察，正常胃組織計數細胞(500)中的陽性細胞數，以百分率表示。無陽性反應細胞或陽性反應細胞數小于10%者為陰性，陽性反應細胞數大于或等于10%者為陽性。酶聯免疫吸附試驗(ELISA)檢測血清IL-33和VEGF-C濃度，免疫組織化學染色SP法檢測IL-33和VEGF-C在胃癌中的表達情況。人IL-33和人VEGF-C ELISA試劑盒均購自上海潤裕生物科技有限公司，兔抗人IL-33和VEGF-C多克隆抗體及S-P試劑盒均購自Dako公司，操作步驟嚴格按照說明書進行。

2 結 果

2.1 IL-33和VEGF-C在胃癌組織及正常胃組織中的表達 光鏡下正常胃黏膜上皮細胞質中IL-33和VEGF-C染色弱或呈陰性。胃癌組織細胞內IL-33和VEGF-C呈彌漫性或簇狀分布的棕黃色顆粒，瘤間質中有時可見IL-33和VEGF-C呈簇狀的淺棕色染色，瘤旁正常胃黏膜中少見IL-33和VEGF-C染色。低分化腺癌中IL-33和VEGF-C染色程度較高、中分化腺癌略深(圖1)。IL-33和VEGF-C在正常胃組織和進展期胃癌及淋巴結陽性組胃癌間的表達有統計學意義(P<0.05)；早期胃癌及淋巴結陰性組胃癌與正常胃組織間表達比較差異無統計學意義(P>0.05)；早期胃癌和進展期胃癌，淋巴結無轉移組和淋巴結轉移組胃癌中IL-33和VEGF-C表達比較差異有統計學意義(P<0.05)，見表1～3。

A～E:SP法(×100)；F：SP法(×400)。

圖1 IL-33與VEGF-C在胃癌組織及正常胃組織中的表達

表1 IL-33和VEGF-C在胃癌組織及正常胃組織中的表達[n(%)]

2.2 IL-33和VEGF-C表達與胃癌臨床病理指標之間的關系 IL-33和VEGF-C表達與胃癌患者性別、年齡、腫瘤位置和大小無相關性(P>0.05)，但與腫瘤分化程度、組織浸潤、淋巴結轉移、遠處轉移及UICC分期有相關性(P<0.05)。腫瘤分化程度越低、組織浸潤越嚴重、UICC分期越晚，IL-33和VEGF-C水平越高(P<0.05)；有淋巴結轉移的胃癌IL-33和VEGF-C水平顯著高于無淋巴結轉移者(P<0.05)；有遠處轉移的胃癌中IL-33和VEGF-C水平明顯高于無遠處轉移者(P<0.05)，見表4。

表2 IL-33和VEGF-C表達與胃癌分期的關系[n(%)]

表3 IL-33和VEGF-C表達與胃癌淋巴結轉移的關系[n(%)]

表4 IL-33和VEGF-C表達與胃癌臨床病理指標之間的關系[n(%)]

2.3 胃癌組織標本中IL-33和VEGF-C表達情況的相關性 98例胃癌組織標本中IL-33和VEGF-C共陽性為54例，66例IL-33陽性患者中VEGF-C陽性率為81.82%,32例IL-33陰性患者中VEGF-C陽性率為59.38%，二者表達水平呈正相關(tau-b等級相關系數Kendall′s tau-b=0.241，P=0.025)。

2.4 胃癌組與對照組間血清IL-33和VEGF-C濃度的比較 98例胃癌患者血清IL-33和VEGF-C水平分別為(50.24±13.08)pg/mL和(210.73±58.35) pg/mL，與對照組血清IL-33和VEGF-C水平[(26.38±9.10)pg/mL和(82.26±35.79)pg/mL]相比，差異有統計學意義(P<0.01)。

2.5 胃癌患者血清IL-33和VEGF-C水平與胃癌淋巴結轉移的關系 本研究98例胃癌患者中，76例患者經組織病理學證實有淋巴結轉移，22例未發生淋巴結轉移。淋巴結轉陰性患者血清IL-33水平和VEGF-C水平分別為(38.37±7.45)pg/mL和(157.97±34.66)pg/mL，較健康人[IL-33：(26.38±9.10)pg/mL，VEGF-C：(82.26±35.79)pg/mL]有升高趨勢，但比較差異無統計學意義(P=0.427、0.734)。淋巴結轉陽性患者血清IL-33和VEGF-C水平分別為(50.24±13.08)pg/mL和(226.00±54.91)pg/mL，較健康人和淋巴結陰性患者明顯升高，比較差異有統計學意義(P<0.05)。

3 討 論

胃癌作為消化道最常見的惡性腫瘤，已嚴重威脅到人類健康。但胃癌的發生與轉移是一個涉及多步驟、多因素的復雜過程，有關其復發轉移機制尚不明確，持續存在的慢性炎癥刺激在胃癌的發生、發展及促進腫瘤復發、轉移過程中發揮重要作用。IL-33具有抑制和(或)促進炎癥反應的雙重生理效應[7-8]，在炎癥、腫瘤等多種疾病中發揮重要的調控作用[9-10]。VEGF-C可誘導腫瘤中心部位新生淋巴管生成[11-12]，并在早期胃癌中均有高表達，與腫瘤浸潤和淋巴結轉移顯著相關[13-15]。

近年來研究結果表明，IL-33可通過致癌抑制因子2-細胞外調解蛋白激酶1/2(ST2-ERK1/2)和c-Jun氨基末端激酶(JNK)信號通路促進胃癌細胞侵襲和轉移[16]，體內IL-33及其受體ST2表達水平的變化對胃癌等惡性腫瘤患者的病情進展、診斷及預后具有重要的參考價值[17-20]。本研究發現胃癌患者血清及組織標本中IL-33表達水平明顯高于健康人，而且其升高程度與胃癌的惡性程度呈正相關，這進一步證實IL-33與胃癌的侵襲和轉移密切相關。

VEGF-C是血管內皮生長因子家族最強的促血管生成因子之一，其主要通過刺激新生血管生成，促進血管內皮細胞增生和遷移，增加微血管通透性及促進細胞外基質的降解等途徑促進腫瘤血管生成、浸潤及轉移[21-22]。諸多研究發現，VEGF-C在胃癌組織中高表達，其表達程度與淋巴結轉移、淋巴管浸潤、靜脈浸潤及腫瘤浸潤生長方式、患者預后呈顯著相關性[23-26]。本研究結果與其一致，VEGF-C在胃癌組織及血清標本中表達水平明顯高于健康人，并且胃癌患者血清VEGF-C濃度與淋巴結轉移、病理分期及分化程度等密切相關，表明VEGF-C可作為反映胃癌侵襲轉移能力的重要指標。

血管生成是許多生理和病理過程的基本事件之一，其中病理性血管生成是腫瘤形成、增殖與轉移的關鍵環節[27-28]。血管生成是一個涉及微血管結構和功能上一系列變化的復雜過程，與多種生長因子相關，如細胞因子、促血管生成因子、黏附分子等[29]。 IL-33能誘導內皮細胞增生、遷移和組織分化，從而促進新生血管生成[30]，同時還與上皮細胞浸潤密切相關，參與腫瘤的發生和發展[31]。

綜上所述，胃癌組織及血清中IL-33和VEGF-C表達水平升高，二者之間存在正相關，并與胃癌的惡性進展、淋巴結轉移等生物學行為有關，提示IL-33參與腫瘤新生血管的形成及腫瘤的浸潤和轉移，可能成為胃癌靶向治療的潛在靶點之一。

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Expression of interleukin-33 and vascular endothelial growth factor-C in gastric cancer and its clinical significance*

XiaBingxiang,LiFan,XuJian,YuanZhenhua,ZhengSuwen,ZhangYewei△

(DepartmentofGeneralSurgery,TumorHospitalAffiliatedtoNanjingMedicalUniversity/JiangsuProvincialCancerHospital/JiangsuProvincialCancerResearchInstitute,Nanjing,Jiangsu210009,China)

Objective To investigate the expression of interleukin-33 (IL-33) and vascular endothelial growth factor C (VEGF-C) in gastric cancer tissues and serum,and to explore the relationship between these two indicators and gastric cancer lymph node metastasis.Methods The levels of IL-33 and VEGF-C in the tissues of gastric mucosa and serum were detected by immunohistochemical SP method and enzyme-linked immunosorbent assay (ELISA) in 98 patients with gastric cancer and 36 healthy subjects.Results The expression rates of IL-33 and VEGF-C in gastric cancer were 67.35% and 74.49%,which were significantly higher than the rates in normal gastric tissue (47.22% and 61.11%).The difference was statistically significant (P<0.01).The expression of IL-33 and VEGF-C was correlated with the degree of tumor differentiation,tissue infiltration,lymph node metastasis,distant metastasis and clinical stage(P<0.05).The positive rates of IL-33 and VEGF-C in gastric cancer lymph node metastasis group were higher than those in non-lymph node metastasis group(P<0.05).The serum concentrations of IL-33 and VEGF-C in patients with gastric cancer were (50.24±13.08)pg/mL and(210.73±58.35)pg/mL,respectively，which were higher than those in healthy control group(P<0.05);the expressions of serum concentration of IL-33 and VEGF-C in the cases with lymph node metastasis were higher than those without lymph node metastasis and the difference was statistically significant(P<0.05).Conclusion High levels of IL-33 in gastric carcinoma patients might induce the secretion of VEGF-C,promote lymph node metastasis,and be applied as an important index of the appraisal to the prognosis of gastric cancer.

stomach neoplasms；vascular endothelial growth factor C；interleukins 33；lymph node metastasis

10.3969/j.issn.1671-8348.2017.15.014

國家自然科學基金面上項目(61371066)； 江蘇省醫學重點人才資助項目(RC2011090)；江蘇省重點研發計劃(社會發展-面上項目)(BE2015720);江蘇省六大高峰人才項目(WSW-041)。 作者簡介：夏兵祥(1988-)，住院醫師，在讀碩士，主要從事普外腫瘤基礎與臨床研究方面研究?！?/p>

，E-mail:zhangyewei@njmu.edu.cn。

R735.2

A

1671-8348(2017)15-2056-04

2016-11-22

2017-01-10)