虞先濬 劉辰 徐近 龍江 倪泉興

復(fù)旦大學(xué)附屬腫瘤醫(yī)院胰腺肝膽外科，復(fù)旦大學(xué)上海醫(yī)學(xué)院腫瘤學(xué)系，上海 200032

CD24+CD44+胰腺癌細(xì)胞的獲取及其干細(xì)胞特性的初步鑒定

虞先濬 劉辰 徐近 龍江 倪泉興

復(fù)旦大學(xué)附屬腫瘤醫(yī)院胰腺肝膽外科，復(fù)旦大學(xué)上海醫(yī)學(xué)院腫瘤學(xué)系，上海 200032

背景與目的：近年研究發(fā)現(xiàn)，腫瘤內(nèi)存在極小一部分細(xì)胞，決定腫瘤的惡性表型及生物學(xué)特性，被稱為“腫瘤干細(xì)胞”。本研究擬從人原發(fā)胰腺癌組織中找到具有干細(xì)胞特性的胰腺癌細(xì)胞，為今后靶向干預(yù)胰腺癌干細(xì)胞，解決胰腺癌治療的臨床難題提供研究基礎(chǔ)。方法：將人原發(fā)胰腺癌組織種植于NOD/SCID小鼠成瘤，對(duì)移植瘤細(xì)胞進(jìn)行流式細(xì)胞分選；將流式細(xì)胞儀分選獲得的細(xì)胞用無(wú)血清、含有生長(zhǎng)因子的DMEM/F12培養(yǎng)基培養(yǎng)，觀察細(xì)胞的生長(zhǎng)、傳代及體外干細(xì)胞球形成能力；將細(xì)胞接種于非肥胖型糖尿病/重癥聯(lián)合免疫缺陷型 (Nonobese diabetic/severe combined immunodeficiency，NOD/SCID)小鼠，觀察、比較成瘤率，免疫組化法檢測(cè)干細(xì)胞移植瘤腫瘤分化相關(guān)抗原Ki67、CK7及甲基化調(diào)控因子MBD1的表達(dá)；免疫熒光檢測(cè)細(xì)胞Hedgehog、BMI-1的表達(dá)。結(jié)果：人原發(fā)胰腺癌組織種植于NOD/SCID小鼠可部分成瘤，對(duì)移植瘤細(xì)胞進(jìn)行流式細(xì)胞分選，獲得CD24+CD44+細(xì)胞(獲得率0.6%～1.8%)；將CD24+CD44+細(xì)胞用無(wú)血清、含有生長(zhǎng)因子的DMEM/F12培養(yǎng)基培養(yǎng)，細(xì)胞呈懸浮球狀生長(zhǎng)，可連續(xù)傳代并能再次形成干細(xì)胞球；將102個(gè)CD24+CD44+細(xì)胞接種于NOD/SCID小鼠，成瘤率12.5%(1/8小鼠成瘤)，將103個(gè)CD24+CD44+細(xì)胞接種于NOD/SCID小鼠，4周后100%成瘤，而同樣數(shù)量的CD24-CD44-胰腺癌細(xì)胞則無(wú)法成瘤(P＜0.05)，免疫組化法檢測(cè)干細(xì)胞移植瘤中部分細(xì)胞表達(dá)Ki67、CK7及MBD1陽(yáng)性；免疫熒光檢測(cè)證實(shí)CD24+CD44+細(xì)胞陽(yáng)性表達(dá)Hedgehog、BMI-1，表達(dá)量分別高于CD24-CD44-細(xì)胞9.95倍和2.74倍(P＜0.05)。結(jié)論：所獲得的CD24+CD44+細(xì)胞具有胰腺癌干細(xì)胞特性，可作為今后胰腺癌干細(xì)胞研究的實(shí)驗(yàn)基礎(chǔ)。

胰腺癌； 干細(xì)胞； Hedgehog； BMI-1

近年來(lái)，胰腺癌的發(fā)病率明顯上升。胰腺癌惡性程度高，生物學(xué)行為特異，容易發(fā)生早期轉(zhuǎn)移，且對(duì)放、化療不敏感，手術(shù)切除率低，發(fā)病率接近死亡率，預(yù)后很差［1］。因此，深入研究胰腺癌的特性，探求胰腺癌發(fā)生、發(fā)展的根源，具有重要的意義。研究表明，多數(shù)惡性腫瘤中存在極少部分的細(xì)胞群體，能夠無(wú)限生長(zhǎng)、永久生存，并具備自我更新、多向分化的能力，是惡性腫瘤失控生長(zhǎng)、轉(zhuǎn)移、耐藥和預(yù)后差的根源［2］。本研究擬從人原發(fā)胰腺癌組織中找到具有干細(xì)胞特性的胰腺癌細(xì)胞，為今后靶向干預(yù)胰腺癌干細(xì)胞，解決胰腺癌治療的臨床難題提供研究基礎(chǔ)。

1 資料和方法

1.1 人原發(fā)胰腺癌組織的異種種植

取手術(shù)切除的新鮮人原發(fā)胰腺癌組織10例，其中6例男性，4例女性，平均年齡(54.7±10.3)歲(41～70歲)，TNM分期(UICC，2010)均為Ⅲ/Ⅵ期，病理證實(shí)為導(dǎo)管腺癌，術(shù)前均未行化療。將腫瘤組織在無(wú)菌RPMI 1640培養(yǎng)基內(nèi)制成2 mm×2 mm大小的腫瘤組織條，用無(wú)血清的PBS溶液沖洗，種植于8周齡的非肥胖型糖尿病/重癥聯(lián)合免疫缺陷型 (Nonobese diabetic/severe combined immunodeficiency，NOD/SCID) 雌性小鼠(8～10周齡，SPF級(jí)環(huán)境飼養(yǎng))背部皮下，成瘤后取出移植瘤，分別用膠原酶消化后制成單細(xì)胞懸液。

1.2 流式細(xì)胞分選

取對(duì)數(shù)生長(zhǎng)期的細(xì)胞(1×106/100 μL)，用胰酶消化2～3 min，加入HBSS/2%FBS溶液沖洗，重懸細(xì)胞，DAPI(濃度為1 μg/mL)染色。加入PE標(biāo)記的抗人CD44，F(xiàn)ITC標(biāo)記的抗人CD24(美國(guó)PharMingen公司，濃度均為1∶40)，冰上溫育20 min。用流式細(xì)胞儀(美國(guó)BD公司)進(jìn)行分選。細(xì)胞分選重復(fù)2次，分選后各亞群細(xì)胞純度＞95%。

1.3 CD24+CD44+細(xì)胞的體外培養(yǎng)

收集對(duì)數(shù)生長(zhǎng)期的細(xì)胞，用無(wú)血清、含生長(zhǎng)因子EGF、FGF10的DMEM-F12培養(yǎng)基重懸、培養(yǎng)，收集細(xì)胞制成單細(xì)胞懸液，接種于無(wú)血清DMEM-F12培養(yǎng)基傳代。

1.4 CD24+CD44+細(xì)胞體內(nèi)成瘤實(shí)驗(yàn)

取8周齡NOD/SCID雌性小鼠24只，飼養(yǎng)于SPF級(jí)環(huán)境。分為3組，每組8只，分別接種102、103、104數(shù)量級(jí)的細(xì)胞。將CD24+CD44+細(xì)胞接種于小鼠右側(cè)背部， CD24-CD44-細(xì)胞接種于小鼠左側(cè)背部，每周觀察成瘤情況及腫瘤大小，12周后處死小鼠。移植瘤取出后切片，進(jìn)行HE及免疫組化染色。

1.5 免疫組化

采用二步法免疫組化染色(EnVision System)，Ki67(購(gòu)自美國(guó)DAKO公司)、CK7、MBD1(均購(gòu)自美國(guó)Santa Cruz公司)一抗的工作濃度均為1∶100。常規(guī)脫蠟、水化、抗原修復(fù)，一抗和二抗分別溫育、DAB顯色、蘇木精復(fù)染，中性樹脂封片觀察。用PBS代替一抗作為陰性對(duì)照。

1.6 免疫熒光

將細(xì)胞分為CD24+CD44+和CD24-CD44-2組。取對(duì)數(shù)生長(zhǎng)細(xì)胞，制備細(xì)胞涂片，固定、通透、封閉后加入一抗(抗人Hedgehog、抗人BMI-1，均購(gòu)自美國(guó)Santa Cruz公司，濃度為1∶100)溫育1 h，PBS沖洗，加入熒光標(biāo)記的二抗(濃度為1∶50)，沖洗、封片，熒光顯微鏡觀察。MoticFluo1.0圖像分析系統(tǒng)進(jìn)行圖像分析，根據(jù)熒光顯示的亮度，測(cè)定其灰度值，代表蛋白的相對(duì)表達(dá)量。

1.7 統(tǒng)計(jì)學(xué)處理

采用SPSS 19.0統(tǒng)計(jì)軟件進(jìn)行分析，計(jì)數(shù)資料用χ2檢驗(yàn)或Fisher’s確切概率檢驗(yàn)，計(jì)量資料的比較用t檢驗(yàn)。P＜0.05為有統(tǒng)計(jì)學(xué)意義。

2 結(jié) 果

2.1 CD24+CD44+胰腺癌細(xì)胞的獲取

10例人原發(fā)胰腺癌組織異種種植2周后開始成瘤，4周后共有3例成瘤，瘤體大小(995.8±46.8) mm3，未見明顯轉(zhuǎn)移灶，移植瘤經(jīng)HE染色證實(shí)為導(dǎo)管腺癌。將3例移植瘤分別進(jìn)行流式細(xì)胞分選，CD24+細(xì)胞的比例為2.5%～26.8%，CD44+細(xì)胞的比例為2.8%～9.5%，CD24+CD44+細(xì)胞的比例為0.6%～1.8%(圖1)。

2.2 CD24+CD44+細(xì)胞的體外成球能力

將CD24+CD44+細(xì)胞用無(wú)血清的含生長(zhǎng)因子的DMEM/F12培養(yǎng)基培養(yǎng)，4～5 d后細(xì)胞呈懸浮球狀生長(zhǎng)(圖2)，12 d左右將細(xì)胞傳代，傳代細(xì)胞仍能懸浮球狀生長(zhǎng)。

2.3 CD24+CD44+細(xì)胞的體內(nèi)成瘤能力

將CD24+CD44+細(xì)胞接種于NOD/SCID小鼠右側(cè)背部，CD24-CD44-細(xì)胞接種于小鼠左側(cè)背部，觀察小鼠成瘤率。102組：CD24-CD44-細(xì)胞均未成瘤(0/8)，CD24+CD44+細(xì)胞有1例成瘤(1/8)；103組：CD24-CD44-細(xì)胞均未成瘤(0/8)，CD24+CD44+細(xì)胞均成瘤(8/8)(P＜0.05)；104組：CD24-CD44-細(xì)胞有2例成瘤(2/8)，CD24+CD44+細(xì)胞均成瘤(8/8)。移植瘤取出后切片，進(jìn)行HE染色及Ki67、CK7、MBD1免疫組化染色(圖3)。

2.4 腫瘤干細(xì)胞“自我更新”相關(guān)信號(hào)通路因子的表達(dá)

免疫熒光檢測(cè)CD24+CD44+和CD24-CD44-2組細(xì)胞Hedgehog、BMI-1的表達(dá)，發(fā)現(xiàn)CD24+CD44+細(xì)胞Hedgehog、BMI-1的表達(dá)較CD24-CD44-細(xì)胞分別上調(diào)了9.95倍和2.74倍(P＜0.05，圖4)。

3 討 論

胰腺癌作為一個(gè)日趨多見的消化道惡性腫瘤，有其特有的生物學(xué)特性：容易發(fā)生早期轉(zhuǎn)移；對(duì)化、放療耐藥；進(jìn)展迅速、預(yù)后極差。因此，深入研究胰腺癌的特性，探求胰腺癌發(fā)生、發(fā)展的根源，具有重要的意義。

2007年，Simeone等首次報(bào)道發(fā)現(xiàn)了胰腺癌干細(xì)胞，這些細(xì)胞僅占癌細(xì)胞總數(shù)的0.2%～0.8%，具有自我更新及多向分化潛能，并能不斷適應(yīng)各種惡劣環(huán)境的變化而永久生存，被認(rèn)為是胰腺癌無(wú)限增殖、早期轉(zhuǎn)移和化、放療耐藥的根源。100個(gè)具有干細(xì)胞特性的腫瘤細(xì)胞就可使半數(shù)以上的小鼠成瘤，其致癌性是普通腫瘤細(xì)胞的100倍［3］。有理由相信，如果能夠靶向干預(yù)胰腺癌的干細(xì)胞特性，就有可能在這惡性頑疾的治療上獲得重大的突破。

對(duì)于胰腺癌干細(xì)胞的表面標(biāo)志物，目前尚無(wú)一致的定論。2007年Li等［4］用流式細(xì)胞儀分離出CD44、CD24、ESA三陽(yáng)性的胰腺癌干細(xì)胞。同年，Hermann等［5］報(bào)道CD133+的胰腺癌細(xì)胞具有強(qiáng)大的致瘤、耐藥和轉(zhuǎn)移等干細(xì)胞特性。

甲基化結(jié)合域蛋白1 (methyl-CpG binding domain protein 1，MBD1)是一個(gè)重要的轉(zhuǎn)錄調(diào)控因子，它通過(guò)特異地結(jié)合DNA上甲基化的CpG位點(diǎn)而發(fā)揮活躍的轉(zhuǎn)錄抑制作用。我們前期的研究發(fā)現(xiàn)，MBD1在胰腺癌中存在高表達(dá)，并與胰腺癌的生長(zhǎng)增殖、淋巴轉(zhuǎn)移以及上皮-間質(zhì)轉(zhuǎn)化過(guò)程密切相關(guān)［6-9］。本研究在胰腺癌干細(xì)胞移植瘤中檢測(cè)發(fā)現(xiàn)MBD1表達(dá)陽(yáng)性，提示胰腺癌干細(xì)胞特性可能與甲基化調(diào)控相關(guān)，為今后深入研究甲基化調(diào)控與胰腺癌干細(xì)胞特性之間的聯(lián)系及相關(guān)機(jī)制提供啟示。

胰腺癌干細(xì)胞的核心本質(zhì)為自我更新及無(wú)限生長(zhǎng)能力，多種信號(hào)傳導(dǎo)通路的異常激活參與了胰腺癌干細(xì)胞的自我更新特性，如Hedgehog、Notch、BMI-1、PTEN等［10-13］。研究發(fā)現(xiàn)，Hedgehog與BMI-1的活化可使腫瘤干細(xì)胞球數(shù)量增多、體積增大，增強(qiáng)干細(xì)胞的多向分化及傳代后再次形成干細(xì)胞球的能力，在體內(nèi)，下調(diào)Hedgehog與BMI-1表達(dá)可抑制NOD/SCID小鼠成瘤［14］。Li等［4］發(fā)現(xiàn)，胰腺癌干細(xì)胞中Hedgehog的表達(dá)高于普通胰腺癌細(xì)胞近10倍。異常表達(dá)的Hedgehog信號(hào)可促使正常胰腺導(dǎo)管發(fā)生癌前病變，并向胰腺癌逐漸發(fā)展，活化的Hedgehog還可維持胰腺癌的惡性表型，而阻斷Hedgehog表達(dá)則會(huì)減緩胰腺癌增殖、促進(jìn)細(xì)胞凋亡，進(jìn)而抑制腫瘤轉(zhuǎn)移、改善腫瘤耐藥［15-16］。BMI-1同樣與胰腺癌的增殖、轉(zhuǎn)移、預(yù)后有關(guān)［17］，本研究中，具有腫瘤干細(xì)胞特性的CD24+CD44+細(xì)胞表達(dá)Hedgehog、BMI-1均明顯高于普通胰腺癌細(xì)胞，證實(shí)Hedgehog和BMI-1的表達(dá)與胰腺癌干細(xì)胞特性相關(guān)。

本研究將人原發(fā)胰腺癌組織種植于NOD/SCID小鼠成瘤，對(duì)移植瘤細(xì)胞進(jìn)行流式細(xì)胞分選，獲得CD24+CD44+細(xì)胞(獲得率0.6%～1.8%)； 將CD24+CD44+細(xì)胞用無(wú)血清、含有生長(zhǎng)因子的DMEM/F12培養(yǎng)基培養(yǎng)，細(xì)胞呈懸浮球狀生長(zhǎng)，可連續(xù)傳代并能再次形成干細(xì)胞球；將103個(gè)CD24+CD44+細(xì)胞接種NOD/SCID小鼠，4周后100%成瘤，而同樣數(shù)量CD24-CD44-胰腺癌細(xì)胞則無(wú)法成瘤；免疫熒光檢測(cè)證實(shí)CD24+CD44+細(xì)胞陽(yáng)性表達(dá)Hedgehog、BMI-1，表達(dá)量顯著高于CD24-CD44-細(xì)胞。證明我們所獲得的CD24+CD44+細(xì)胞具有胰腺癌干細(xì)胞特性，為今后靶向干預(yù)胰腺癌干細(xì)胞，解決胰腺癌治療的臨床難題提供研究基礎(chǔ)。

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The isolation and functional verification of the cells with CD24CD44 double positive expression from primary human pancreatic adenocarcinomas

YU Xian-jun,LIU Chen,XU Jin,LONG Jiang,NI Quan-xing(Department of Pancreatic & Hepatobiliary Surgery, Fudan University Shanghai Cancer Center; and Department of Oncology, Shanghai Medical College, Fudan University,Shanghai 200032, China)

NI Quan-xing E-mail：blurlc@hotmail.com

Background and purpose：Emerging evidence has shown that cancer stem cells, which is a few subset of cells in tumor, could determine the malignant phenotype and biological behavior of the tumor, This study aimed to isolate a highly tumorigenic subpopulation of cells from primary human pancreatic adenocarcinomas and verify the biological function of the cells.Methods：Primary human pancreatic adenocarcinomas was planted in immunocompromised (NOD-SCID) mice, and then fl ow cytometry was used to sort the cells into several subpopulation.The sorted cells were cultured in a serum-free DMEM/F12 medium with EGF and FGF to observe the proliferation,passage and spheroid forming capacity of the cells, the cells with those unique features were injected s.c. into NOD/SCID mice to determine the tumorigenicity. The expression of differentiation markers like Ki67, CK7 and DNA methylation regulating factor MBD1 in xenograft tumors was examined by immunohistochemistry, and the expression of Hedgehog and BMI-1 was observed by immunof l uorescence method.Results：Through the processes, a highly tumorigenic subpopulation of pancreatic cancer cells were isolated and had been shown positive expression of CD44 and CD24 of cell surface markers. CD24+CD44+cells could form tumor spheroids in nonadherent culture conditions,and the spheroids also had CD24+CD44+features and could be passaged multiple times without loss of tumor spheroids forming capability. In vivo study, the tumorigenesis was dependent on the number of the cells that implanted into mice.Some of CD24+CD44+tumor cells had been observed to also express the differentiation markers Ki67, CK7 and DNA methylation regulating factor MBD1, and the expression of Hedgehog and BMI-1 were significantly up-regulated about 9.95 fold and 2.74 fold, respectively, in CD24+CD44+cells compared with CD24-CD44-cells.Conclusion：The CD24+CD44+cells are highly tumorigenic subpopulation of pancreatic cancer cells and might be the stem cells of pancreatic adenocarcinomas.

Pancreatic carcinoma; Stem cell; Hedgehog; BMI-1

10.3969/j.issn.1007-3969.2011.02.002

R735.9；R73-35

A

1007-3639(2011)02-0086-05

國(guó)家自然科學(xué)基金資助項(xiàng)目(No:81001058)。

倪泉興 E-mail:blurlc@hotmail.com

2010-08-25

2010-10-23）