癌前食管與食管鱗狀細(xì)胞癌表達(dá)CD44及CD44v6的差異

鄭淑妍 楊惠鈿 陳強(qiáng) 蘇少雪 林敏 劉俊斌 楊立業(yè)

?論 著?

癌前食管與食管鱗狀細(xì)胞癌表達(dá)CD44及CD44v6的差異

鄭淑妍 楊惠鈿 陳強(qiáng) 蘇少雪 林敏 劉俊斌 楊立業(yè)★

目的通過免疫組化研究CD44及CD44v6在癌前食管及食管鱗狀細(xì)胞癌中的表達(dá)情況，探討其與食管鱗狀細(xì)胞癌的關(guān)系及臨床意義。方法收集潮州市中心醫(yī)院病理科存檔的食管病例蠟塊（所有病例術(shù)前均未接受放、化療）。采用免疫組化PV-9000二步法（非生物素）檢測(cè)癌前食管63例，食管鱗狀細(xì)胞癌140例。SPSS16.0統(tǒng)計(jì)軟件分析實(shí)驗(yàn)結(jié)果。對(duì)于各組之間表達(dá)情況的數(shù)據(jù)統(tǒng)計(jì)采用多獨(dú)立樣本非參數(shù)檢驗(yàn)：Kruskal-Wallis H檢驗(yàn)；各組中兩兩比較采用兩獨(dú)立樣本非參數(shù)檢驗(yàn)：Kruskal-Wallis H檢驗(yàn)的方法進(jìn)行比較。結(jié)果CD44及CD44v6在正常的鱗狀上皮，只表達(dá)于上皮基底層3～5層細(xì)胞，隨著上皮的增生，表達(dá)的細(xì)胞層數(shù)逐漸增加，在非典型增生的上皮則主要表達(dá)于有異型的上皮，而原位癌則幾乎表達(dá)于上皮的全層。CD44及CD44v6在各種食管癌前病變組織與食管鱗狀細(xì)胞癌中的表達(dá)均存在差異（均P＜0.01），其中從正常食管組織及伴上皮增生但無細(xì)胞異型的食管組織至食管鱗狀細(xì)胞癌之間，其表達(dá)強(qiáng)度逐漸增強(qiáng)，而從非典型增生及原位癌至食管鱗狀細(xì)胞癌之間的表達(dá)強(qiáng)度逐漸減弱。結(jié)論CD44及CD44v6在癌前食管及食管鱗狀細(xì)胞癌中的表達(dá)差異，提示它們可能與食管鱗狀細(xì)胞癌的發(fā)生及發(fā)展有關(guān)，為食管癌干細(xì)胞的進(jìn)一步研究提供線索。

CD44；CD44v6；免疫組化；食管鱗狀細(xì)胞癌

在潮汕地區(qū)，食管癌高發(fā)現(xiàn)象不容忽視，其中，又以汕頭南澳縣最高，其標(biāo)化發(fā)病率在109/10萬，其次是揭陽和潮州的饒平地區(qū)[1]。如果能夠找出其發(fā)病原因，也就意味著能夠在疾病的預(yù)防和治療上爭(zhēng)得先機(jī)。CD44分子是由單一基因編碼的具有高度異質(zhì)性的單鏈細(xì)胞膜表面糖蛋白家族，介導(dǎo)細(xì)胞與細(xì)胞及細(xì)胞外基質(zhì)的粘附作用，從而發(fā)揮廣泛生物學(xué)功能[2]。CD44v6屬于變異型CD44（CD44v）家族成員，其過度表達(dá)使腫瘤細(xì)胞具有轉(zhuǎn)移潛能[3]。我們?cè)?jīng)有實(shí)驗(yàn)證實(shí)胚胎食管粘膜上皮的基底層均有CD44及CD44v6的表達(dá)[4]，而基底層恰恰是上皮的生發(fā)層，因此我們?cè)O(shè)想CD44及CD44v6可能是食管癌干細(xì)胞的標(biāo)志物。本實(shí)驗(yàn)針對(duì)我院收集的食管癌前組織及食管鱗狀細(xì)胞癌病理標(biāo)本及臨床資料，采用免疫組織化學(xué)的方法檢測(cè)CD44及CD44v6的表達(dá)情況，旨在了解其與食管鱗狀細(xì)胞癌發(fā)生、發(fā)展的關(guān)系，進(jìn)一步探索食管鱗狀細(xì)胞癌的發(fā)病機(jī)制，為該病的預(yù)防與臨床治療提供有價(jià)值的信息。

1 材料和方法

1.1 材料

收集我院病理科2003～2006年間收檢的術(shù)前均未接受過放、化療的食管手術(shù)切除標(biāo)本存檔蠟塊，其中正常食管鱗狀上皮23例，伴鱗狀上皮增生19例，鱗狀上皮不同程度非典型增生10例，原位癌11例，鱗狀細(xì)胞癌140例。

1.2 方法

1.2.1 石蠟切片

將收集蠟塊常規(guī)石蠟切片3張，裱于已經(jīng)過APES的硅化玻片上，其中1張用于HE染色，其它2張用于免疫組化染色。

1.2.2 免疫組織化學(xué)染色

采用PV-9000二步法（非生物素）進(jìn)行免疫組化染色。CD44用淋巴結(jié)作陽性對(duì)照；CD44v6用扁桃體作陽性對(duì)照；兩者均用PBS代替一抗作陰性對(duì)照（對(duì)照片均由試劑公司提供），DAB顯色，蘇木素復(fù)染，光鏡觀察。

1.2.3 結(jié)果判斷

CD44定位于細(xì)胞膜及漿，CD44v6陽性定位于細(xì)胞膜，呈顯著棕黃色為陽性，根據(jù)染色強(qiáng)度及陽性細(xì)胞數(shù)量分為：“-”、“+”、“++”、“+++”。細(xì)胞不著色為0分；淺黃色為1分；黃色為2分；棕褐色為3分。陽性細(xì)胞數(shù)＜5%為0分；陽性細(xì)胞數(shù)6%～25%為1分；陽性細(xì)胞數(shù)26%～50%為2分；陽性細(xì)胞數(shù)51%～75%為3分；陽性細(xì)胞數(shù)＞75%為4分。然后著色強(qiáng)度的得分加上著色細(xì)胞數(shù)的得分為總分，總分0～1為“-”、2～3為“+”、4～5為“++”、6～7為“+++”。

1.2.4 統(tǒng)計(jì)學(xué)分析

采用SPSS16.0軟件，對(duì)于各組之間表達(dá)強(qiáng)度的數(shù)據(jù)統(tǒng)計(jì)采用多獨(dú)立樣本非參數(shù)檢驗(yàn)：Kruskal-Wallis H檢驗(yàn)；各組中兩兩比較采用兩獨(dú)立樣本非參數(shù)檢驗(yàn)：Kruskal-Wallis H檢驗(yàn)的方法進(jìn)行比較。

2 結(jié)果

CD44及CD44v6在癌前食管及食管鱗狀細(xì)胞癌中的表達(dá)情況具體見表1。在正常的鱗狀上皮，只表達(dá)于上皮基底層3～5層細(xì)胞，隨著上皮的增生表達(dá)的細(xì)胞層數(shù)逐漸增加，非典型增生的鱗狀上皮則主要在具異型性的上皮細(xì)胞中有表達(dá)，而原位癌則表達(dá)于上皮全層。CD44及CD44v6在食管鱗狀細(xì)胞癌與各種癌前食管組織之間的表達(dá)均存在差異（均P＜0.01），兩者在鱗狀細(xì)胞癌中的表達(dá)均強(qiáng)于正常及增生上皮的表達(dá)，呈遞減趨勢(shì)；而與非典型增生及原位癌比較，兩者的表達(dá)均呈遞減趨勢(shì)（圖1及圖2）。

表1 CD44及CD44v6在癌前食管及鱗狀細(xì)胞癌中的表達(dá)情況Table 1 The expression of CD44 and CD44v6 in different types of esophageal epithelium and squamous cell carcinoma

圖1 癌前食管組織CD44的表達(dá)情況×100Figure 1 The expression of CD44 in different types of esophageal epithelium×100

圖2 癌前食管組織CD44v6的表達(dá)情況×100Figure 2 The expression of CD44v6 in different types of esophageal epithelium×100

3 討論

黏附分子CD44是細(xì)胞膜表面跨膜糖蛋白，它作為透明質(zhì)酸的受體，能與透明質(zhì)酸等多種配體結(jié)合，參與細(xì)胞-細(xì)胞，細(xì)胞-基質(zhì)之間的特異性黏附。由于翻譯后修飾，CD44具有不同亞型：標(biāo)準(zhǔn)型（CD44s）和變異型（CD44v）兩大類。人類CD44基因位于11號(hào)染色體短臂上，有20個(gè)外顯子，分為組成型外顯子和變異型外顯子。其中第6～15外顯子為變異型外顯子，即v區(qū)外顯子（v1～v10），能以變異方式拼接。含有變異型外顯子插入的CD44轉(zhuǎn)錄子統(tǒng)稱為變異型CD44（variance form CD44，CD44v），主要在上皮源性細(xì)胞和腫瘤細(xì)胞中表達(dá)[5]。大量的研究表明CD44的表達(dá)情況與惡性腫瘤的發(fā)生、發(fā)展關(guān)系密切，有關(guān)CD44促進(jìn)腫瘤侵襲和轉(zhuǎn)移越來越受到重視。有報(bào)道認(rèn)為腎細(xì)胞癌[6]中CD44陽性表達(dá)的癌細(xì)胞比陰性的細(xì)胞具有更強(qiáng)的侵襲轉(zhuǎn)移能力，說明CD44在腫瘤侵襲和轉(zhuǎn)移方面發(fā)揮重要的作用，這也是近年來提出的比較新的課題，其研究還處于早期階段，目前大部分研究傾向于把CD44基因作為一種干細(xì)胞標(biāo)記物。CD44v6是含v6變異體的CD44拼接變異體，它通過與透明質(zhì)酸結(jié)合，影響腫瘤細(xì)胞之間以及腫瘤細(xì)胞與基質(zhì)的結(jié)合能力，增強(qiáng)腫瘤細(xì)胞的運(yùn)動(dòng)能力，促進(jìn)腫瘤細(xì)胞的浸潤和轉(zhuǎn)移，為目前比較公認(rèn)的腫瘤轉(zhuǎn)移促進(jìn)基因。它多見于有轉(zhuǎn)移能力的腫瘤細(xì)胞，而正常細(xì)胞和相同組織來源的非轉(zhuǎn)移性腫瘤細(xì)胞表達(dá)較少，CD44v6的表達(dá)可改變腫瘤細(xì)胞表面黏附分子的構(gòu)成和功能，有助于腫瘤細(xì)胞獲得轉(zhuǎn)移潛能。有報(bào)道認(rèn)為CD44v6的過表達(dá)與肺癌的浸潤與轉(zhuǎn)移有關(guān)[7]。

在本組正常食管鱗狀上皮中，CD44及CD44v6大部分表達(dá)于上皮基底層的3～5層細(xì)胞，而隨著上皮的增生、非典型增生到原位癌的演進(jìn)過程，其表達(dá)的細(xì)胞層數(shù)逐漸增多，呈現(xiàn)頗具規(guī)律的變化，這與文獻(xiàn)[8]報(bào)道一致。在原位癌中，CD44及CD44v6幾乎表達(dá)于上皮全層細(xì)胞。食管鱗狀細(xì)胞癌的發(fā)生發(fā)展經(jīng)歷了食管粘膜上皮基底細(xì)胞過度增生、非典型增生和原位癌，這些形態(tài)學(xué)改變是食管鱗狀細(xì)胞癌高易感人群癌變?cè)缙诘闹匾卣鱗9]，并將這些病變稱之為食管的癌前病變[10]。食管癌的早期診斷實(shí)際上就是及時(shí)發(fā)現(xiàn)癌前病變或癌前狀態(tài)患者，尤其是重度非典型增生和原位癌。而CD44及CD44v6在正常鱗狀上皮、增生鱗狀上皮、非典型鱗狀上皮及原位癌中細(xì)胞表達(dá)層次逐漸增加的規(guī)律性變化其演進(jìn)過程存在一定程度的擬合。所以我們認(rèn)為CD44及CD44v6的表達(dá)與腫瘤發(fā)生的早期可能存在一定的關(guān)系，能否作為早期食管癌篩查的一個(gè)指標(biāo)值得我們進(jìn)一步探討。同時(shí)，從正常及增生上皮到鱗狀細(xì)胞癌之間CD44及CD44v6的表達(dá)呈遞增趨勢(shì)，而從非典型增生及原位癌到鱗狀細(xì)胞癌則呈遞減趨勢(shì)，這說明了腫瘤的發(fā)生、發(fā)展是一個(gè)復(fù)雜的過程，仍需我們繼續(xù)探索。

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The difference on expression of CD44 and CD44v6 between esophageal epithelium and squamous cell carcinoma

ZHENG Shuyan, Yang Huitian, CHEN Qiang, SU Shaoxue, LIN Min, LIU Junbin, YANG Liye★
(Department of Pathology, Chaozhou Central Hospital Affiliated to South Medical University, Guangdong, Chaozhou 521000, China)

ObjectiveTo explore the relationship and clinical signi fi cance through investigated the expression of CD44 and CD44v6 in different types of esophageal epithelial hyperplasia and squamous cell carcinoma by immunohistochemical method.MethodsParaf fi n blocks of esophageal carcinoma were collected from department of Pathology, Chaozhou central hospital. The patients were not treated with chemotherapy or radiotherapy before operation. The expression of CD44 and CD44v6 in 63 cases of esophageal epithelial hyperplasia and 140 cases of esophageal squamous cell carcinoma (ESCC) were detected by immunohistochemistry PV-9000 two-steps methods using monoclonal antibodies. The results were analyzed with SPSS 16.0 statistics software. The difference of expression between each sample in one group was analyzed by multi-independent samples nonparameter tests (Kruskal-Wallis H tests). The difference of expression between two samples in one group was analyzed by 2-independent samples nonparameter tests (Kruskal-Wallis H tests).ResultsIn normal squamous epithelium, only 3～5 layers of cells were positive for CD44 and CD44v6, and the expression of cell layers gradually increased with the epithelial hyperplasia. The atypical hyperplasia epithelium were mainly expressed in atypia epithelial, and almost all epithelial layers expressed in carcinoma in situ. There were signi fi cant difference for the expression of CD44 and CD44v6 between normal squamous epithelium and ESCC, the hyperplasia epithelium and ESCC, the atypical hyperplasia epithelium and ESCC, the carcinoma insitu and ESCC (allP＜0.01). The expression of CD44 and CD44v6 in ESCC was greater than normal squamous epithelium and hyperplasia epithelium, but it was weaker than atypical hyperplasia epithelium and carcinoma in situ.ConclusionThe different expression of CD44 and CD44v6 in different types of esophageal epithelium and ESCC implied that CD44 and CD44v6 may be relate to the occurrence and development of ESCC. They may be the stem cells of ESCC.

CD44; CD44v6; immunohistochemistry; Esophageal squamous cell carcinoma

廣東省醫(yī)學(xué)科學(xué)技術(shù)研究基金（B2008179）；廣東省社會(huì)發(fā)展計(jì)劃（2011B031800329）

南方醫(yī)科大學(xué)附屬潮州市中心醫(yī)院病理科，廣東，潮州 521000

★通訊作者：楊立業(yè)，E-mail: yangleeyee@sina.com