趙 帥，黃允寧，趙良玉，盧 林，李智勇，封存志

·論 著·

pN0胃癌淋巴結(jié)微轉(zhuǎn)移臨床意義的研究

趙 帥1，黃允寧2，趙良玉2，盧 林2，李智勇2，封存志1

目的探索聯(lián)合使用RT-PCR及免疫組織化學(xué)染色法(IHC)同時(shí)檢測胃癌淋巴結(jié)微轉(zhuǎn)移(LNM)，分析胃癌淋巴結(jié)微轉(zhuǎn)移對(duì)胃癌臨床診治的影響。方法選取接受手術(shù)的pN0期胃癌患者32例。應(yīng)用IHC和RT-PCR法聯(lián)合檢測術(shù)后淋巴結(jié)CK20的表達(dá)，分析LNM與患者性別、年齡、腫瘤部位、腫瘤大小、浸潤深度、分化程度等臨床病理資料的關(guān)系。結(jié)果經(jīng)IHC法檢測出6例患者、共14個(gè)淋巴結(jié)中CK20陽性，LNM陽性率為21.87%；經(jīng)RT-PCR法檢測出10例患者、共23個(gè)淋巴結(jié)中CK20 mRNA陽性，LNM陽性率為31.25%。兩種方法均為陽性的患者7例，RT-PCR法呈陽性而IHC陰性的患者4例，IHC結(jié)果陽性而RT-PCR法陰性患者1例。此研究共檢出11例患者存在LNM。不同分化程度pN0期胃癌LNM間差異有統(tǒng)計(jì)學(xué)意義(P<0.05)，低分化胃癌較中-高分化胃癌更易出現(xiàn)LNM。結(jié)論胃癌LNM與分化程度有關(guān)，低分化胃癌較中-高分化胃癌更易出現(xiàn)微轉(zhuǎn)移，臨床醫(yī)生給低分化胃癌患者采用縮小手術(shù)治療方式時(shí)需慎重。IHC和RT-PCR法聯(lián)合檢測胃癌INM可提高微轉(zhuǎn)移的檢出率。

胃癌；微轉(zhuǎn)移；CK20；免疫組化法；RT-PCR法

淋巴結(jié)轉(zhuǎn)移的嚴(yán)重程度與胃癌術(shù)后病情發(fā)展呈負(fù)相關(guān)性[1]。臨床醫(yī)生發(fā)現(xiàn)即使接受D2根治手術(shù)，術(shù)后經(jīng)常規(guī)病理染色(HE染色)診斷為pN0期的患者，仍然存在術(shù)后復(fù)發(fā)，甚至因此而死亡。有學(xué)者研究提出淋巴結(jié)微轉(zhuǎn)移(LNM)可能是導(dǎo)致這一現(xiàn)象的一個(gè)主要原因[2]。隨著分子檢測技術(shù)的探索、發(fā)現(xiàn)及應(yīng)用，免疫組織化學(xué)染色技術(shù)和RT-PCR技術(shù)已成為一個(gè)成熟的可進(jìn)行LNM檢測的分子技術(shù)。本研究選取胃腺癌患者術(shù)后經(jīng)常規(guī)病理診斷為pN0淋巴結(jié)蠟塊作為檢測標(biāo)本，采用IHC和RT-PCR法共同檢測其中CK20的表達(dá)，分析胃癌淋巴結(jié)微轉(zhuǎn)移與胃癌患者臨床病理資料的相關(guān)性，探討胃癌LNM對(duì)臨床診治的影響。

1 資料與方法

1.1 研究對(duì)象：選取2013年1月-2016年9月在寧夏人民醫(yī)院接受根治性切除術(shù)，且術(shù)后淋巴結(jié)經(jīng)常規(guī)病理檢測為No期的32例胃癌患者。收集術(shù)后淋巴結(jié)蠟塊共158個(gè)，記錄32例患者的臨床病理資料。術(shù)前或術(shù)中診斷存在其他部位癌癥或存在其他臟器轉(zhuǎn)移癌、胰腺受侵及腹腔種植、卵巢轉(zhuǎn)移等遠(yuǎn)處轉(zhuǎn)移的患者不納入為研究對(duì)象。

1.2 實(shí)驗(yàn)方法

1.2.1 免疫組化實(shí)驗(yàn)方法:主要試劑有CK20抗體(選用武漢三鷹生物有限公司的CK20兔抗人單克隆抗體)、SP9000免疫組化試劑盒、DAB顯色試劑盒。實(shí)驗(yàn)步驟：常規(guī)石蠟切片5 μm,55 ℃烤片2 h，脫蠟水化，枸櫞酸鈉鹽緩沖液高溫4 min，PBS液浸泡5 min，3次；3%去離子雙氧水37 ℃環(huán)境中15 min，5%山羊血清封閉,37 ℃孵育30 min,傾去血清,滴加一抗工作液,4 ℃中過夜。37 ℃溫箱中復(fù)溫1 h，聚合酶輔助劑37 ℃下30 min，PBS洗,5 min× 3次。滴加抗兔IgG二抗,37 ℃孵育30 min，PBS洗，5 min× 3次，DAB顯色2 min，PBS沖洗,蘇木精復(fù)染，封片。陽性對(duì)照采用常規(guī)病理確診的轉(zhuǎn)移陽性淋巴結(jié)組織，陰性對(duì)照采用PBS代替一抗。

1.2.2 RT-PCR實(shí)驗(yàn)步驟

1.2.2.1 引物設(shè)計(jì)與合成：引物序列采用參考文獻(xiàn)[3]中的CK20引物序列并做修改，由上海生物公程有限公司合成，內(nèi)參采用β-actin，引物序列見表1。

表1 引物及內(nèi)參序列

1.2.2.2 實(shí)驗(yàn)耗材：RNA提取試劑盒購于OMEGA公司，逆轉(zhuǎn)錄試劑盒購于美國 Thermo Scientific公司，Marker Ⅱ由天跟生化技術(shù)有限公司合成，β-actin 一抗由武漢博士德生物有限公司合成。

1.2.2.3 RNA提?。?嚴(yán)格按照RNA提取試劑盒說明書步驟進(jìn)行提取，提取出來的RNA在超微量核酸蛋白測定儀上檢測其濃度，放于-80 ℃環(huán)境下保存。

1.2.2.4 擴(kuò)增及逆轉(zhuǎn)錄：逆轉(zhuǎn)錄反應(yīng)體系為20 μl，RevertAid Reverse Transcriptase 1 μl，5x Reaction Buffer 4 μl，RiboLock RNase Inhibitor 1 μl，dNTP Mix 2 μl，Oligo(dT)1 μl；以2 μg為標(biāo)準(zhǔn)加入相應(yīng)體積的RNA提取液，再加入相應(yīng)體積DEPC水，42 ℃×60 min×1個(gè)循環(huán)→70 ℃×5 min×1個(gè)循環(huán)，以mRNA為模板反轉(zhuǎn)錄生成cDNA。擴(kuò)增體系為25 μl，DNA聚合酶復(fù)合物12.5 μl，上游引物1 μl，下游引物1 μl，DEPC水8.5 μl，cDNA 2 μl，94 ℃×2 min×1變性，94 ℃×30 s～56 ℃×30 s～72 ℃×30 s，30個(gè)循環(huán)，72 ℃×2 min延伸。

1.2.2.5 電泳及曝光：配置1.5%瓊脂糖凝膠，120 V電壓，10 mA電流跑膠25 min，凝膠成像系統(tǒng)下分析結(jié)果。

1.3 統(tǒng)計(jì)學(xué)方法：使用SPSS 17.0統(tǒng)計(jì)學(xué)軟件，計(jì)數(shù)資料使用χ2檢驗(yàn)分析，以P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1 實(shí)驗(yàn)結(jié)果：IHC檢測發(fā)現(xiàn)32例患者中有7例術(shù)后LNM陽性，陽性率為21.87%；158個(gè)淋巴結(jié)蠟塊檢檢測發(fā)現(xiàn)有14個(gè)淋巴結(jié)蠟塊出現(xiàn)微轉(zhuǎn)移，檢出率為9%。RT-PCR檢測發(fā)現(xiàn)32例患者中共有10例檢測發(fā)現(xiàn)LNM，陽性率為31.25%；158個(gè)淋巴結(jié)蠟塊中發(fā)現(xiàn)有23個(gè)出現(xiàn)微轉(zhuǎn)移，檢出率為14.93%。

2.2 胃癌淋巴結(jié)微轉(zhuǎn)移與臨床病理資料的關(guān)系：收集32例患者臨床病理資料，包括腫瘤大小、浸潤深度、組織學(xué)類型等信息。分析發(fā)現(xiàn)，胃癌LNM與胃癌患者的年齡、性別、腫瘤部位、腫瘤大小、浸潤深度無相關(guān)性，與分化程度有相關(guān)性。低分化胃癌較中-高分化胃癌容易發(fā)生LNM(P<0.05)，見表2。

表2 胃癌LNM與胃癌病理資料的關(guān)系[n(%)]

臨床病理參數(shù)nIHC陽性P值RT-PCR陽性P值分化程度 中-高分化191(21)<0．053(26)<0．05 低分化136(23)7(38)浸潤深度 未侵犯肌層112(18．2)>0．054(36．4)>0．05 侵犯肌層或超過215(23．8)6(28．6)

3 討論

3.1 胃癌LNM的臨床意義:胃癌LNM與胃癌患者術(shù)后復(fù)發(fā)及存活率的關(guān)系一直是研究的熱點(diǎn)，但是目前兩者的關(guān)系，各學(xué)者還存在一定分歧[4-7]。但是胃癌LNN與術(shù)后胃癌復(fù)發(fā)相關(guān)，存在LNN的患者，術(shù)后復(fù)發(fā)的概率較大，得到諸多研究者的同意。

Fukagawa T等通過研究提出，標(biāo)準(zhǔn)胃癌根治術(shù)加D2淋巴結(jié)清掃術(shù)，對(duì)于pT2N0或pT3N0胃癌患者是一個(gè)恰當(dāng)?shù)氖中g(shù)治療方式[8]。Lee T研究發(fā)現(xiàn)，充分的淋巴結(jié)清掃對(duì)于未分化胃癌十分重要[9]。2011年中國衛(wèi)計(jì)委發(fā)布的《胃癌診療規(guī)范》規(guī)定：ESD和EMR適用于高中分化胃癌，行胃D1切除術(shù)時(shí)，一旦出現(xiàn)淋巴結(jié)轉(zhuǎn)移，應(yīng)當(dāng)施行D2切除術(shù)。本研究收集32例患者臨床病理資料，結(jié)果顯示，胃癌LNM與胃癌分化程度有相關(guān)性；低分化胃癌較中-高分化胃癌容易發(fā)生LNM(P<0.05)。因此，對(duì)低分化胃癌患者采用縮小手術(shù)治療方式時(shí)需慎重。

3.2 IHC與RT-PCR法聯(lián)合檢測胃癌淋巴結(jié)微轉(zhuǎn)移：隨著檢測技術(shù)的發(fā)展，IHC檢測胃癌LNM目前得到廣泛的應(yīng)用，是因?yàn)镮HC具有特異性高、簡便易行的特點(diǎn)[10-11]。逆轉(zhuǎn)錄聚合酶鏈?zhǔn)椒磻?yīng)法(RT-PCR)以其高敏感性的特點(diǎn)，也被廣大學(xué)者用于LNM的檢測。Kubota等探索發(fā)現(xiàn)，RT-PCR法是檢測微轉(zhuǎn)移敏感度最高的方法[12]。陳瑞川等研究發(fā)現(xiàn)，RT-PCR法可從105個(gè)正常組織細(xì)胞中檢出1個(gè)惡性細(xì)胞，具有較高的敏感度[13]。Koshi Kumagai等使用一步核酸擴(kuò)增法(OSNA)來檢測胃癌患者淋巴結(jié)轉(zhuǎn)移，并且可在30 min內(nèi)得出結(jié)果[14]。Yanagita等研究發(fā)現(xiàn)一種用于術(shù)中評(píng)估淋巴結(jié)轉(zhuǎn)移的系統(tǒng)，此系統(tǒng)可以檢驗(yàn)淋巴結(jié)中CEA和CK19的量，在40 min內(nèi)得出結(jié)果[15]。這些研究都為RT-PCR的臨床應(yīng)用提供了便利。

聯(lián)合使用IHC法和RT-PCR法檢測胃癌LNM，可以從細(xì)胞水平及基因水平兩個(gè)方面同時(shí)對(duì)目標(biāo)標(biāo)記物進(jìn)行觀察和分析，兩種方法相互應(yīng)用，可以提高胃癌LNM的檢出率。

[1] Nitti D，Marchet A，Olivieri M，et al.Ratio between metastatic and examined lymph nodes is an Independent prognostic factor after D2 resection for gastric cancer:analysis of a large European monoinstitutional experience[J].Annals of Surgical Oncology，2003，10(9):1077-1085.

[2] Maehara Y，Oshiro T，Endo K，et al.Clinical significance of occult micrometastasis lymph nodes from patients with early gastric cancer who died of recurrence[J].Surgery，1996，119(4):397-402.

[3] Shmica Yoshimasa，Takeuchi H，Sakakura Y，et al.Molecular detection of sentinel node micrometastases in patients with clinical N0 gastric carcinoma with real-time multiplex reverse transcription-polymerase chain reaction assay[J].Annals of Surgical Oncology，2012，19(2):469-477.

[4] Lee CM，Cho JM，Jang YJ，et al.Should lymph node micrometastasis be considered in node staging for gastric cancer[J].Annals of Surgical Oncology，2015，22(3):765-771.

[5] Li Yu，Du Peizhun，Zhou Yangbing，et al.Lymph node micrometastases is a poor prognostic factor for patients in pN0 gastric cancer:a meta-analysis of observational studies[J].The Journal of Surgical Research，2014，191(2):413-422.

[6] Jeuck TL，Wittekind C.Gastric carcinoma:stage migration by immunohistochemically detected lymph node micrometastases[J].Gastric Cancer，2015，18(1):100-108.

[7] Morgagni P，Saragoni L，Scarpi E，et al.Lymph node micrometastases in early gastric cancer and their impact on prognosis[J].World Journal of Surgery，2003，27(5):558-561.

[8] Fukagawa T，Sasako M，Mann GB，et al.Immunohistochemically detected micrometastases of the lymph nodes in patients with gastric carcinoma[J].Cancer，2001，92(4):753-760.

[9] Lee T，Tanaka H，Ohira M，et al.Clinical impact of the extent of lymph node micrometastasis in undifferentiated-type early gastric cancer[J].Oncology，2014，86(4):244-252.

[10] Matsumoto M，Natsugoe S，Ishigami S，et al.Rapid immunohistochemical detection of lymph node micrometastasis during operation for upper gastrointestinal carcinoma[J].The British Journal of Surgery，2003，90(5):563-566.

[11] Kubota K，Nakanishi H，Hiki N，et al.Quantitative detection of micrometastases in the lymph nodes of gastric cancer patients with real-time RT-PCR:a comparative study with immunohistochemistry[J].International Journal of Cancer，2003，105(1):136-143.

[12] 陳瑞川，林嵐，邱達(dá)泰，等.RT-PCR擴(kuò)增MUC1檢測胃癌微轉(zhuǎn)移研究[J].Journal of Fujian Medical University，1999，33(3)：244-247.

[13] Kumagai K，Yamamoto N，Miyashiro I，et al.Multicenter study evaluating the clinical performance of the OSNA assay for the molecular detection of lymph node metastases in gastric cancer patients[J].Gastric Cancer，2014，17(2):273-280.

[14] Yanagita S，Natsugoe S，Uenosono Y，et al.The utility of rapid diagnosis of lymph node metastasis in gastric cancer using a multiplex real-time reverse transcription polymerase chain reaction assay[J].Oncology，2009，77(3/4):205-211.

ClinicalsignificanceoflymphnodemicrometastasisinpN0gastriccancer

ZHAOShuai1,HUANGYunNing2,ZHAOLiangYu2,LULing2,LIZhiYong2,FENGCunzhi1.

1.NingxiaMedicalUniversity,Yinchuan750002,China;2.GastrointestinalSurgery,NingxiaPeople’sHospital,Yinchuan750002,China

Correspondingauthor:HUANGYunNing,Email:nxhyncc@126.com

ObjectiveTo determine the lymph node metastasis of patients who undergo surgery with negative lymph node metastasis by immunohistochemical staining and reverse transcription polymerase chain reaction (RT-PCR) with CK20 as the target marker.To explore the two methods of detecting lymph node micrometastasis and the clinical significance of lymph node micrometastasis in gastric cancer.Methods32 patients with pN0 gastric cancer who underwent radical gastrectomy were selected.158 lymph nodes were collected and all lymph nodes were detected by HE staining.pathology diagnosis.The expression of CK20 in lymph nodes was detected by immunohistochemical staining and RT-PCR.To analyze the relationship between lymph node micrometastasis and sex,age,tumor location,tumor size,depth of invasion,histological type and other clinical pathological parameters of gastric cancer.ResultsThere were seven patients and fourteen lymph nodes were positive of lymph node micrometastasis by immunohistochemically stained.The positive rate of lymph node micrometastasis were 21.87%;A total of 10 patients that were detected by RT-PCR lymph node micrometastasis were positive.The expression of CK20mRNA in 23 lymph nodes was detected by reverse transcription PCR.Lymph node micrometastasis detection rate was 31.25%.Seven patients were positive by both methods.There are 4 patients were positive by RT-PCR and negative by immunohistochemistry.One patient had positive immunohistochemical test and negative RT-PCR.There are 11 patients had positive micrometastasis.There was significant difference between different histological types of pN0 gastric cancer patients (P<0.05).Lymph node micrometastases in patients with poorly differentiated gastric cancer were more likely to occur than patients with moderate to well-differentiated gastric cancer.Lymph node micrometastases were associated with histological types of gastric cancer patients,regardless of age,sex,tumor location,tumor size,and depth of invasion.ConclusionThe lymph node micrometastasis of gastric cancer is related to the histological type of gastric cancer.The patients with poorly differentiated gastric cancer are more likely to undergo micrometastasis than those with moderate to well differentiated gastric cancer.It should be careful to reduced surgical treatment to the patients with poorly differentiated gastric cancer.Immunohistochemical method combine with RT-PCR detection can improve the detection rate of micrometastasis.

Gastriccancer;Micrometastasis;CK20;Immunohistochemistry;RT-PCR

寧夏自然科學(xué)基金項(xiàng)目(NZ14163)

1.寧夏醫(yī)科大學(xué)，寧夏 銀川 750002 2.寧夏人民醫(yī)院胃腸外科，寧夏 銀川 750002

趙帥(1990-)，男，山西籍，在讀碩士研究生，主要從事胃腸外科研究方向。

黃允寧，Email:nxhyncc@ 126.com

http://kns.cnki.net/kcms/detail/64.1008.R.20170814.1453.016.html

10.13621/j.1001-5949.2017.08.0682

R735

A

2017-02-17 [責(zé)任編輯]王凱榮