劉喬飛，牛哲禹，李 媛，王夢(mèng)一，盧朝輝，廖 泉*，趙玉沛*

(中國(guó)醫(yī)學(xué)科學(xué)院 北京協(xié)和醫(yī)院 1.基本外科； 2.病理科，北京 100730)

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研究論文

胰腺癌淋巴結(jié)轉(zhuǎn)移小鼠外周血免疫細(xì)胞群的變化

劉喬飛1，牛哲禹1，李 媛2，王夢(mèng)一1，盧朝輝2，廖 泉1*，趙玉沛1*

(中國(guó)醫(yī)學(xué)科學(xué)院 北京協(xié)和醫(yī)院 1.基本外科； 2.病理科，北京 100730)

目的觀察胰腺癌淋巴結(jié)轉(zhuǎn)移小鼠外周血免疫細(xì)胞群的變化。方法足底注射C57BL/6J小鼠同源Panc02胰腺癌細(xì)胞，流式細(xì)胞計(jì)數(shù)檢測(cè)外周血中13個(gè)免疫細(xì)胞群的變化。 結(jié)果6周后有淋巴結(jié)轉(zhuǎn)移的轉(zhuǎn)移率為65%。與正常對(duì)照小鼠相比，非淋巴結(jié)轉(zhuǎn)移及淋巴結(jié)轉(zhuǎn)移小鼠外周血中B淋巴細(xì)胞(P<0.05，P<0.01)、T淋巴細(xì)胞(P<0.05，P<0.01)均明顯減少；而粒細(xì)胞(P<0.05，P<0.01)、髓系來(lái)源抑制細(xì)胞(MDSC)(P<0.01，P<0.01)以及M2型腫瘤相關(guān)巨噬細(xì)胞(TAM)(P<0.05，P<0.01)均明顯增多。淋巴結(jié)轉(zhuǎn)移小鼠較非淋巴結(jié)轉(zhuǎn)移小鼠上述變化更明顯(P<0.05)。結(jié)論胰腺癌淋巴結(jié)轉(zhuǎn)移可引起強(qiáng)烈的免疫抑制效應(yīng)，其中MDSC以及M2型TAM可能起到關(guān)鍵作用。免疫環(huán)境的重建可能是胰腺癌淋巴結(jié)轉(zhuǎn)移防治的重要靶點(diǎn)。

胰腺癌；淋巴結(jié)轉(zhuǎn)移；髓系來(lái)源抑制細(xì)胞；腫瘤相關(guān)巨噬細(xì)胞；免疫重建

胰腺癌惡性度極高，易發(fā)生淋巴結(jié)轉(zhuǎn)移，預(yù)后極差[1]。胰腺癌淋巴結(jié)轉(zhuǎn)移也是影響胰腺癌預(yù)后的獨(dú)立危險(xiǎn)因素，其機(jī)制不明，有待深入探討。胰腺癌病理組織形態(tài)特殊，腫瘤細(xì)胞少，間質(zhì)細(xì)胞多，且以炎性細(xì)胞為主[2]。炎性細(xì)胞與腫瘤細(xì)胞間的相互作用對(duì)胰腺癌的生物學(xué)行為可產(chǎn)生重要影響。但外周血炎性細(xì)胞在胰腺癌發(fā)生淋巴結(jié)轉(zhuǎn)移過(guò)程中的動(dòng)態(tài)變化情況尚不明確。本研究通過(guò)足底注射C57BL/6J小鼠同源Panc02胰腺癌細(xì)胞誘發(fā)淋巴結(jié)轉(zhuǎn)移，系統(tǒng)分析了正常對(duì)照小鼠，荷瘤未發(fā)生淋巴結(jié)轉(zhuǎn)移小鼠以及淋巴結(jié)轉(zhuǎn)移小鼠外周血中13個(gè)免疫細(xì)胞群的變化。研究結(jié)果提示胰腺癌淋巴結(jié)轉(zhuǎn)移過(guò)程誘發(fā)全身的免疫抑制效應(yīng)，其中髓系來(lái)源抑制細(xì)胞(myeloid derived suppressor cells, MDSC)以及M2型腫瘤相關(guān)巨噬細(xì)胞 (tumor associated macrophages, TAM)可能起關(guān)鍵作用。免疫環(huán)境的重建可能成為胰腺癌淋巴結(jié)轉(zhuǎn)移防治的重要靶點(diǎn)。

1 材料與方法

1.1 實(shí)驗(yàn)動(dòng)物

25只SPF級(jí)雄性C57BL/6J小鼠6～8周齡，體質(zhì)量25 g～30 g。[北京維通利華實(shí)驗(yàn)動(dòng)物技術(shù)有限公司，動(dòng)物生產(chǎn)合格證號(hào) SCKK(京)2002- 2003]其中5只用于正常對(duì)照，剩余20只用于建立胰腺癌淋巴結(jié)轉(zhuǎn)移模型。C57BL/6J小鼠同源胰腺導(dǎo)管腺癌Panc02細(xì)胞系由北京協(xié)和醫(yī)院基本外科實(shí)驗(yàn)室保種傳代。實(shí)驗(yàn)動(dòng)物飼養(yǎng)于SPF級(jí)環(huán)境。動(dòng)物實(shí)驗(yàn)均通過(guò)中國(guó)醫(yī)學(xué)科學(xué)院北京協(xié)和醫(yī)院動(dòng)物倫理委員會(huì)審查。

1.2 試劑及抗體

抗小鼠單克隆或多克隆熒光標(biāo)記抗體(表1)。

1.3 建立淋巴結(jié)轉(zhuǎn)移腫瘤模型

液氮中取出腫瘤細(xì)胞，復(fù)蘇后，培養(yǎng)傳至第三代，待80%～95%匯合時(shí)，消化細(xì)胞，制備細(xì)胞懸液備用(2×107/mL)。將2×106/100 μL Panc02 腫瘤細(xì)胞懸液注入小鼠足底。術(shù)后6周，處死小鼠，觀察腹股溝及腹主動(dòng)脈旁淋巴結(jié)轉(zhuǎn)移情況，留取標(biāo)本常規(guī)行HE染色。處死前1 d，經(jīng)內(nèi)眥取外周血行流式細(xì)胞計(jì)數(shù)。

表1 實(shí)驗(yàn)所用抗體列表Table 1 The list of antibodies

1.4 組織病理染色

獲取腹股溝及腹主動(dòng)脈旁淋巴結(jié)，用10%中性甲醛固定4 d后，常規(guī)制作4 μm石蠟切片。常規(guī)HE染色。

1.5 流式細(xì)胞染色計(jì)數(shù)

毛細(xì)管經(jīng)內(nèi)眥取血后紅細(xì)胞裂解液裂解紅細(xì)胞。常規(guī)流式染色方法染色。利用Accuri C6四通道流式細(xì)胞儀(BD，USA)檢測(cè)各種熒光標(biāo)記表面分子表達(dá)。

1.6 統(tǒng)計(jì)學(xué)分析

2 結(jié)果

2.1 小鼠胰腺癌淋巴轉(zhuǎn)移模型的建立

淋巴結(jié)轉(zhuǎn)移建模6周后，小鼠后肢明顯腫脹。20只荷瘤小鼠中13只病理確認(rèn)淋巴結(jié)轉(zhuǎn)移，轉(zhuǎn)移率65%。典型胰腺癌轉(zhuǎn)移淋巴大體表現(xiàn)為腹股溝及腹主動(dòng)脈旁可見(jiàn)多發(fā)質(zhì)硬腫大淋巴結(jié)。HE染色可見(jiàn)淋巴結(jié)結(jié)構(gòu)破壞，大量低分化癌細(xì)胞侵潤(rùn)(圖1)。

A.the metastatic inguinal lymph node; B.the metastatic paraaortic lymph node; C. HE staining of the metastaic lymph node(×100)

圖1 小鼠胰腺癌淋巴結(jié)轉(zhuǎn)移大體相及病理特征
Fig 1 General figures and pathological characteristics of the metastatic lymph nodeds of pancreatic cancer in mice

2.2 胰腺癌淋巴結(jié)轉(zhuǎn)移小鼠外周血免疫細(xì)胞群的變化

非轉(zhuǎn)移組與對(duì)照組相比，外周血中T淋巴細(xì)胞總數(shù)以及B淋巴細(xì)胞均顯著減少(P<0.05),而粒細(xì)胞(P<0.05)、MDSC(P<0.01)、PMN-MDSC(P<0.01)、MO-MDSC(P<0.05)及M2型TAM(P<0.05)均顯著增多。轉(zhuǎn)移組與對(duì)照組相比，外周血中T淋巴細(xì)胞總數(shù)(P<0.05)、輔助T細(xì)胞(P<0.05)、細(xì)胞毒性T細(xì)胞(P<0.05)以及B淋巴細(xì)胞(P<0.01)均顯著減少，而粒細(xì)胞(P<0.01)、MDSC(P<0.01)、PMN-MDSC(P<0.01)、MO-MDSC(P<0.05)、巨噬細(xì)胞總數(shù)(P<0.05)以及M2型TAM(P<0.01)均增多。轉(zhuǎn)移組與非轉(zhuǎn)移組相比，外周血中粒細(xì)胞(P<0.05)、MDSC(P<0.01)、PMN-MDSC(P<0.01)及M2型TAM (P<0.05)均增多(表 2，圖 2)。

3 討論

胰腺癌易發(fā)生淋巴結(jié)轉(zhuǎn)移預(yù)后極差[3]。胰腺癌腫瘤組織高度纖維化，腫瘤細(xì)胞稀少，富含間質(zhì)細(xì)胞，尤其以各種炎性細(xì)胞為主[4- 5]。不可控炎癥作已經(jīng)成為惡性腫瘤的一個(gè)新的標(biāo)志特征[6]。髓系來(lái)源抑制細(xì)胞(myeloid derived suppressor cells, MDSC)是一群被腫瘤誘導(dǎo)產(chǎn)生的髓系來(lái)源的異質(zhì)性免疫負(fù)向調(diào)節(jié)細(xì)胞，進(jìn)一步分為PMN-MDSC以及MO-MDSC 兩種亞型[7]。巨噬細(xì)胞在腫瘤細(xì)胞的誘導(dǎo)下可由經(jīng)典的M1型向M2型極化，從而促進(jìn)腫瘤進(jìn)展[8]。胰腺癌荷瘤小鼠外周血及腫瘤組織中MDSC與M2型TAM與腫瘤負(fù)荷呈正相關(guān)[4- 5]。胰腺癌、乳腺癌標(biāo)本中粒細(xì)胞數(shù)目、M2型TAM數(shù)目也與患者預(yù)后呈負(fù)相關(guān)[9- 10]。Panc02細(xì)胞胰腺內(nèi)原位注射的方法可建立淋巴結(jié)轉(zhuǎn)移模型，但小鼠大多死于原發(fā)灶快速生長(zhǎng)，淋巴結(jié)轉(zhuǎn)移率低，不利于長(zhǎng)期觀察[11]。P53突變基因工程小鼠可誘導(dǎo)小鼠胰腺癌發(fā)生，但耗時(shí)長(zhǎng)、技術(shù)要求高，淋巴結(jié)轉(zhuǎn)移率低[12]。胰腺癌細(xì)胞足底注射方法簡(jiǎn)便，轉(zhuǎn)移率高達(dá)65%且無(wú)死亡率。轉(zhuǎn)移組及非轉(zhuǎn)移組及對(duì)照組相比，B細(xì)胞、T細(xì)胞均減少，而粒細(xì)胞、MDSC及M2型TAM均顯著升高。而轉(zhuǎn)移組與非轉(zhuǎn)移比，雖然B細(xì)胞、T細(xì)胞均無(wú)顯著差異，但粒細(xì)胞、MDSC及M2型TAM 仍顯著升高。而MDSC又以PMN-MDSC 升高為主。可見(jiàn)胰腺癌淋巴結(jié)轉(zhuǎn)移過(guò)程中，可誘導(dǎo)強(qiáng)烈的全身免疫抑制效應(yīng)，其中MDSC以及M2型TAM可能起到關(guān)鍵作用。以MDSC以及M2型TAM為靶點(diǎn)的免疫重建，可能成為胰腺癌淋巴結(jié)轉(zhuǎn)移防治的新途徑。

表2 各組小鼠外周血免疫細(xì)胞群變化Table 2 The changes of the immune cell populations in peripheral blood[(MEAN±SD)%]

*P<0.05,**P<0.01 compared with the control group;#P<0.05,##P<0.01 compared with non-metastatic group.

Left from upper to lower:total T cell.CD3+, Th.CD3+CD4+, CTL.CD3+CD8+, B cell.CD19+; Right from upper to lower: granulocyte.Gr- 1+, MDSC.CD11b+Gr- 1+, PMN-MDSC/MO-MDSC.CD11b+Ly6ClowLy6G+/ CD11b+Ly6C+Ly6G-(CD11b positive cells were gated); M:F4/80+; M1/M2:F4/80+CD16/32+CD206-/ F4/80+CD16/32+CD206+

圖2 外周血免疫細(xì)胞群變化流式代表圖片
Fig 2 The representative FCM plots of immune cell populations in peripheral blood

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The changes of immune cell populations in peripheralblood of mice with lymph node metastasis of pancreatic cancer

LIU Qiao-fei1, NIU Zhe-yu1, LI Yuan2, WANG Meng-yi1, LU Zhao-hui2, LIAO Quan1*, ZHAO Yu-pei1*

(1.Dept. of General Surgery; 2.Dept. of Pathology, PUMC Hospital, CAMS & PUMC,Beijing 100730，China)

Objective To observe the changes of immune cell populations in peripheral blood of mice with lymph node metastasis of pancreatic cancer. Methods C57BL/6J mouse syngeneic Panc02 pancreatic cancer cells were injected into intra-footpad. The FCM (fluorescence activated cytometry) was adopted to detect 13 immune cell populations in peripheral blood. Results Six weeks after injection, the lymph node metastasis rate was 65%. Compared to that of normal mice, in the peripheral blood of the mice with and without lymph node metastasis, the B lymphocytes(P<0.05，P<0.01) and T lymphocytes (P<0.05，P<0.01)were significantly decreased; however, the granulocytes(P<0.05，P<0.01), myeloid derived suppressor cells (MDSC) (P<0.01，P<0.01)and M2 polarized tumor associated macrophages (TAM) (P<0.05，P<0.01)were significantly elevated. Compared with the tumor bearing mice without lymph node metastasis, in the peripheral blood of the tumor bearing mice with lymph node metastasis, the changes of the above immuen cell populations were more obvious(P<0.05). Conclusions Pancreatic cancer with lmyph node metastasis can induce intensively systemic immunosuppression and the MDSC and M2 polarized TAM may play crucial roles. Immune reconstructions are potential important targets for the prevention and treatment of lymph node metastasis of pancreatic cancer.

pancreatic cancer; lymph node metastasis; myeloid-derived suppressor cells; tumor associated macrophages; immune reconstruction

2015- 06- 02

2015- 07- 17

國(guó)家自然科學(xué)基金(81272573)

1001-6325(2015)11-1471-05

R735.9；R657.5

A

*通信作者(corresponding author)：lqpumc@126.com; zhao8028@263.net