經(jīng)卡介苗活化的負(fù)載PANC1裂解產(chǎn)物的樹突狀細(xì)胞疫苗體外抗胰腺癌作用

楊德紅 劉文佳 陳敏 吳育美 鄒曉平

·論著·

經(jīng)卡介苗活化的負(fù)載PANC1裂解產(chǎn)物的樹突狀細(xì)胞疫苗體外抗胰腺癌作用

楊德紅 劉文佳 陳敏 吳育美 鄒曉平

目的探討負(fù)載胰腺癌細(xì)胞株P(guān)ANC1裂解產(chǎn)物(tumor lysate,TL)的樹突狀細(xì)胞(dendritic cells, DC)經(jīng)卡介苗(CGB)活化后誘導(dǎo)的自體淋巴細(xì)胞體外抗胰腺癌效應(yīng)。方法應(yīng)用rhGM-CSF和rhIL-4從健康人外周血單個(gè)核細(xì)胞誘導(dǎo)培養(yǎng)DC,用TL負(fù)載DC，并用CGB促其成熟。倒置相差顯微鏡觀察DC形態(tài)，流式細(xì)胞儀檢測細(xì)胞CD1a、CD83、CD86及HLA-DR表型， ELISA法檢測培養(yǎng)上清液中TNF-α和IL-12p70含量。CCK-8檢測DC誘導(dǎo)自體混合淋巴細(xì)胞的增殖率以及活化淋巴細(xì)胞對PANC1、PaTu8988及SCG7901細(xì)胞的殺傷率。結(jié)果CGB活化負(fù)載TL的DC后，CD83和CD86陽性率分別從(3.7±0.3)%和(38.6±5.0)%顯著增加至(16.5±0.6)%和(76.5±2.5)%(P<0.05)；DC分泌的IL-12p70和TNF-α量分別從(20.18±2.06)pg/ml和(61.38±1.19)pg/ml顯著增加至(62.48±3.80)pg/ml和(592.53±17.96)pg/ml(P<0.01)；DC與自體混合淋巴細(xì)胞以1∶100、1∶50、1∶10、1∶5的比例共培養(yǎng)，淋巴細(xì)胞增殖率分別從(3.90±1.41)%、(4.07±0.40)%、(3.39±1.05)%、(2.82±0.39)%顯著增加至(55.38±3.58)%、(75.00±2.54)%、(77.07±3.40)%、(99.07±2.40)%(P<0.01)；DC活化淋巴細(xì)胞對PANC1細(xì)胞的殺傷率在效靶比為1∶5、1∶10、1∶20、1∶50時(shí)分別可達(dá)(71.73±0.46)%、(49.44±0.98)%、(31.76±2.77)%、(19.03±3.04)%，但對PaTu8988和SCG7901細(xì)胞的殺傷率顯著降低。結(jié)論經(jīng)CGB活化的胰腺癌DC疫苗成熟度增加，體外表現(xiàn)出高效特異的抗胰腺癌細(xì)胞的效應(yīng)。

胰腺腫瘤； 樹突細(xì)胞； 卡介苗

隨著腫瘤免疫治療的研究進(jìn)展，承載腫瘤抗原的樹突狀細(xì)胞(dendritic cells, DC)疫苗成為治療惡性腫瘤的重要手段。聯(lián)合應(yīng)用IL-4和GM-CSF培養(yǎng)外周血單個(gè)核細(xì)胞(peripheral blood mononuclear cells,PBMC)可誘導(dǎo)出足夠數(shù)量的DC用于臨床治療[1]。DC疫苗治療腫瘤關(guān)鍵在于保證DC攝取腫瘤抗原后能夠充分成熟，這樣DC回輸體內(nèi)才能有效激活機(jī)體的抗腫瘤免疫反應(yīng)。為促進(jìn)DC成熟，可使用免疫佐劑[2]。卡介苗(Calmette-Guerin bacillus，CGB)是其中一種應(yīng)用較廣的微生物免疫佐劑。本實(shí)驗(yàn)用人胰腺癌細(xì)胞株P(guān)ANC1裂解產(chǎn)物制備DC疫苗，并用CGB進(jìn)一步促其成熟，觀察DC成熟情況及用DC刺激的自體淋巴細(xì)胞對PANC1細(xì)胞的殺傷效應(yīng)。

材料和方法

一、PANC1細(xì)胞裂解產(chǎn)物(TL)制備

PANC1胰腺癌細(xì)胞株購自上海生命科學(xué)院。常規(guī)培養(yǎng)后制成1×107/ml細(xì)胞懸液，經(jīng)-80～37℃反復(fù)凍融5次，每次10 min后高速離心收集上清液，BCA法測定蛋白含量，-80℃保存?zhèn)溆谩?/p>

二、DC體外誘導(dǎo)及自體混合淋巴細(xì)胞獲取

健康人外周新鮮白細(xì)胞濃縮液(南京市中心血站)用PBS等容積稀釋，采用密度梯度離心法獲取PBMC，進(jìn)一步用貼壁分離法獲取DC前體細(xì)胞和自體混合淋巴細(xì)胞。混合淋巴細(xì)胞用含rhIL-2(800 μ/ml)的RPMI 1640液培養(yǎng)維持活力。DC前體細(xì)胞采用含rhGM-CSF(20 ng/ml)及rhIL-4(10 ng/ml)的RPMI 1640液(DC培養(yǎng)液)培養(yǎng)，第5天的DC為未成熟的DC(imDC)。將imDC分為4組，分別用CGB、TL、CGB+TL及DC培養(yǎng)液(陰性對照)刺激2 d，繼續(xù)用DC培養(yǎng)液培養(yǎng)。為確定CGB最佳刺激劑量，先分別用30、60、90、300、500 μg的CGB刺激相同數(shù)量imDC，2 d后加入FITC標(biāo)記人CD86單抗孵育，流式細(xì)胞儀檢測CD86表型最多的DC組的CGB劑量為最佳刺激劑量。DC功能檢測包括：(1)觀察培養(yǎng)第5、7天DC大小、形態(tài)及集落生長情況；(2)將培養(yǎng)第7天各組DC分別與FITC標(biāo)記人CD86、CD83、HLA-DR單抗以及PE標(biāo)記人CD1a單抗共孵后,采用流式細(xì)胞儀檢測DC的CD1a、CD83、CD86及HLA-DR表型(重復(fù)5次)；(3)ELISA法檢測培養(yǎng)第6天收集的培養(yǎng)上清液中TNF-α、IL-12p70濃度(重復(fù)7次)。

三、DC刺激自體混合淋巴細(xì)胞的增殖及其對胰腺癌細(xì)胞的殺傷效應(yīng)

在96孔板，將培養(yǎng)第7天各組DC與自體混合淋巴細(xì)胞按1∶5、1∶10、1∶50、1∶100的比例共培養(yǎng)4 d，同時(shí)設(shè)淋巴細(xì)胞及DC對照組。應(yīng)用CCK-8測淋巴細(xì)胞活力，按說明書操作，最后測各孔細(xì)胞在450 nm波長處A值。按公式計(jì)算淋巴細(xì)胞增殖率：增殖率=[(實(shí)驗(yàn)組-DC對照組)-淋巴細(xì)胞對照組-空白孔]/(淋巴細(xì)胞對照組- 空白孔)×100%。

以1∶10共培養(yǎng)的混合淋巴細(xì)胞為活化淋巴細(xì)胞(效應(yīng)細(xì)胞，E)，以PANC1為靶細(xì)胞(T)。按效靶比50∶1、20∶1、10∶1和5∶1共培養(yǎng)于96孔板，同時(shí)設(shè)單純混合淋巴細(xì)胞為對照。培養(yǎng)3 d后應(yīng)用CCK-8測腫瘤細(xì)胞活力，計(jì)算淋巴細(xì)胞對腫瘤細(xì)胞的殺傷率。殺傷率={[靶細(xì)胞對照組-(實(shí)驗(yàn)組-效應(yīng)細(xì)胞對照組)]-空白孔}/(靶細(xì)胞對照組-空白孔)×100%。同樣以CGB+TL組DC刺激的淋巴細(xì)胞與PANC1、PaTu8988、SCG7901細(xì)胞不同比例共培養(yǎng)后檢測淋巴細(xì)胞對治療細(xì)胞的殺傷率。以上實(shí)驗(yàn)均重復(fù)6次。

四、統(tǒng)計(jì)學(xué)方法

結(jié) 果

一、DC形態(tài)變化和DC表型測定

DC培養(yǎng)第5天形成明顯的簇狀細(xì)胞集落,懸浮或半懸浮。加入各組刺激后培養(yǎng)至第7天，可見CGB及CGB+TL組DC集落逐步散開，呈均勻散在分布，胞體明顯增大,形狀不規(guī)則,胞質(zhì)豐富、透亮，表面有粗大樹突狀突起，為典型DC形態(tài)，而TL及陰性對照組DC形態(tài)較培養(yǎng)至第5天變化不明顯(圖1)。CGB的刺激劑量以90 μg時(shí)最佳(圖2)。與陰性對照組比較，單純TL刺激不增加CD83表型數(shù)量，甚至減少CD86表型數(shù)(P<0.05)。但CGB及CGB+TL組CD83及CD86表型數(shù)較TL組顯著增加(P<0.01)，且CGB+TL組的增加較CGB組更顯著(P<0.05)。而各組CD1a及HLA-DR表型無顯著差異(表1)。

圖1CGB刺激2 d后培養(yǎng)第5天(a, ×200)和第7天的CGB組(b)、CGB+TL組(c)、TL組(d)和陰性對照組(e)的DC形態(tài)(b～e， ×400)

組別例數(shù)CD1aCD83CD86HLA-DR陰性對照組529.7±1.23.7±0.338.6±5.098.1±0.4TL組529.9±1.25.2±0.2a32.9±2.2a98.2±0.4CGB組528.8±0.811.5±1.1abc76.6±2.4ab98.5±0.2TL+CGB組528.3±0.716.5±0.6abc76.5±2.5ab98.6±0.4

注：與陰性對照組比較，aP<0.05；與TL組比較，bP<0.01；與CGB組比較，cP<0.05

二、DC的IL-12p70和TNF-α分泌量的變化

陰性對照組、TL組、CGB組和CGB+TL組培養(yǎng)上清中IL-12p70含量分別為(20.18±2.06)pg/ml、(19.47±4.63)pg/ml、(59.18±2.88)pg/ml、(62.48±3.80)pg/ml；TNF-α含量為(61.38±1.19)pg/ml、(63.32±1.77)pg/ml、(498.23±13.29)pg/ml、(592.53±17.96)pg/ml。TL組與陰性對照組間無顯著差異。但CGB組和CGB+TL組培養(yǎng)上清的IL-12p70和TNF-α含量均較TL組明顯增加(P<0.01)，且CGB+TL組的TNF-α含量又較CGB組增加(P<0.05)。

三、DC對自體混合淋巴細(xì)胞增殖的影響

與陰性對照組比較，TL組的DC可顯著增加淋巴細(xì)胞增殖率(P<0.01)，CGB組及CGB+TL組DC對自體混合淋巴細(xì)胞增殖率的影響又顯著高于TL組DC(圖3，P<0.01)。

四、DC活化的淋巴細(xì)胞對腫瘤細(xì)胞的殺傷效應(yīng)

未活化淋巴細(xì)胞(L)、陰性對照組(陰性對照-DC-L)及TL組DC活化的淋巴細(xì)胞(TL-DC-L)幾乎不能殺傷PANC1細(xì)胞，CGB組DC活化的淋巴細(xì)胞(CGB-DC-L)較前3組有明顯殺傷率，CGB+TL組DC活化淋巴細(xì)胞(CGB+TL-DC-L)則具有更高的殺傷率(表2，P值均<0.01)。雖然CGB+TL-DC-L對PANC1細(xì)胞有很高的殺傷率，但對PaTu8988和SCG7901殺傷率顯著降低(表2，P<0.01)。

圖3各組DC與自體混合淋巴細(xì)胞以不同比例共培養(yǎng)后的淋巴細(xì)胞增殖率

討 論

DC是體內(nèi)功能最強(qiáng)的專職APC,而腫瘤患者體內(nèi)腫瘤細(xì)胞常能逃脫機(jī)體免疫監(jiān)視，其中主要一個(gè)原因是DC數(shù)量明顯減少，且處于免疫耐受狀態(tài)[3]。因此DC疫苗治療腫瘤需解決兩個(gè)關(guān)鍵問題：一是能夠獲取足夠數(shù)量的DC；二是獲取的DC能激活抗腫瘤免疫反應(yīng)。本實(shí)驗(yàn)用經(jīng)典細(xì)胞因子誘導(dǎo)法培養(yǎng)的DC，CD1a陽性率近30%，并呈典型樹突形態(tài)，表明細(xì)胞因子誘導(dǎo)可獲得數(shù)量可觀的DC。因未成熟DC(imDC) 低表達(dá)MHC分子及共刺激分子，可誘導(dǎo)免疫耐受，而成熟DC(mDC)高表達(dá)MHC分子及共刺激分子，誘導(dǎo)免疫激活[4]，所以DC的成熟度是影響DC效能的關(guān)鍵因素。單純腫瘤抗原并不能促進(jìn)DC成熟，本實(shí)驗(yàn)結(jié)果也如此，故需要免疫佐劑。

CGB是一種應(yīng)用較廣的微生物免疫佐劑。Kim等[5]研究證實(shí)，CGB活菌與imDC相互作用后能顯著上調(diào)CD83、CD80、CD86及HLA-DR的表達(dá)。本結(jié)果也顯示，經(jīng)CGB進(jìn)一步修飾后CD83及CD86表型的DC顯著增加，表明DC明顯成熟。

表2 各組DC活化的淋巴細(xì)胞對腫瘤細(xì)胞株的殺傷率

注：與TL-DC-L組比較，aP<0.01；與PANC1細(xì)胞組比較，bP<0.01

近年來的研究發(fā)現(xiàn)，TNF-α在DC成熟過程中有重要作用。用GM-CSF和IL-4刺激培養(yǎng)人外周血PBMC，于第7天加入TNF-α繼續(xù)培養(yǎng)至第14天，與未加入TNF-α比較，DC表面CD83、CD80和CD86表達(dá)增強(qiáng)[6]。去血清培養(yǎng)mDC可使其產(chǎn)生大量活性氧中間產(chǎn)物而誘導(dǎo)自身凋亡，TNF-α可阻斷這一過程[7]。本實(shí)驗(yàn)結(jié)果顯示，CGB促進(jìn)DC分泌大量TNF-α，提示CGB促進(jìn)DC表型成熟可能與自身分泌TNF-α有關(guān)。但Kim等[5]發(fā)現(xiàn)中和CGB刺激的DC培養(yǎng)上清中的TNF-α，不能使CD83表達(dá)下降，得出CGB直接促DC成熟。因此CGB促進(jìn)DC成熟是否與TNF-α分泌有關(guān)還需進(jìn)一步實(shí)驗(yàn)驗(yàn)證。

抗腫瘤免疫反應(yīng)主要為細(xì)胞免疫，主要效應(yīng)細(xì)胞是CD8+細(xì)胞毒T淋巴細(xì)胞(cytotoxic T lymphocyte，CTL)，其發(fā)揮免疫殺傷功能需CD4+輔助性T細(xì)胞(T helper，Th)的調(diào)節(jié)。Th細(xì)胞可分為Th0、Th1、Th2三種類型，其中Th1激活并增強(qiáng)CTL的殺傷功能，形成激活型Th1反應(yīng)；相反Th2抑制CTL活性，形成抑制型Th2反應(yīng)。Th1、Th2由Th0 分化而來[8-9]。DC作用T淋巴細(xì)胞時(shí)分泌的細(xì)胞因子，即構(gòu)成Th0細(xì)胞所在細(xì)胞因子微環(huán)境，可顯著影響Th0細(xì)胞分化方向,其中IL-12p70使Th0顯著向Th1分化，成為產(chǎn)生Th1優(yōu)勢的關(guān)鍵細(xì)胞因子[10]。本實(shí)驗(yàn)結(jié)果顯示，CGB+TL能明顯提高DC上清IL-12p70的濃度，從而增加向Th1的分化。

此外，本實(shí)驗(yàn)還發(fā)現(xiàn)：(1)單獨(dú)CGB也能促進(jìn)DC成熟，該組DC活化的淋巴細(xì)胞對PANC1細(xì)胞也有一定殺傷能力。考慮其原因?yàn)镈C攝取CGB非特異性抗原后激活混合淋巴細(xì)胞中的NK細(xì)胞，可非特異殺傷腫瘤細(xì)胞[11]；CGB可誘導(dǎo)DC表達(dá)TRAIL[12]，后者與胰腺癌細(xì)胞表面的TRAIL受體結(jié)合后可導(dǎo)致腫瘤細(xì)胞凋亡[13]。(2)單純TL致敏DC不能促進(jìn)DC成熟，但可以輕度刺激淋巴細(xì)胞增殖，然而誘導(dǎo)的淋巴細(xì)胞對腫瘤細(xì)胞幾乎無殺傷力。這可能是PANC1裂解產(chǎn)物中的自身抗原成分作用DC，使其誘導(dǎo)調(diào)節(jié)性T細(xì)胞及無反應(yīng)性T細(xì)胞增生，從而引起免疫耐受[14]，導(dǎo)致不能殺傷腫瘤細(xì)胞。(3)負(fù)載PANC1 TL的DC僅對PANC1細(xì)胞具有高的殺傷率，顯示其抗腫瘤細(xì)胞的特異性。

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2010-03-25)

(本文編輯：呂芳萍)

Invitroanti-tumoreffectofautologousmixedlymphocyteprimedbyBCGactivateddendriticcellsbasedPANC1lysate

YANGDe-hong,LIUWen-jia,CHENMin,WUYu-mei,ZOUXiao-ping.

DepartmentofGastroenterology,DrumTowerHospital,MedicalSchool,NanjingUniversity,Nanjing210008,China

ZOUXiao-ping,Email：zouxiaoping795@hotmail.com

ObjectiveTo evaluate in vitro anti tumor effect of host lymphocyte primed by Calmette-Guerin bacillus (CGB) activated dendritic cells (DC) based PANC1 lysate.MethodsDCs were obtained from peripheral blood mononuclear cells of healthy volunteer and cuitured by rhGM CSF and rhIL 4. DC vaccines for pancreatic cancer were loaded with PANC1 tumor lysate (TL) and were further maturated by CGB. CD1a, CD83, CD86and HLA-DR phenotype was characterized by flow cytometer, and IL-12p70 and TNF-α concentration in DC culture supernatant were measured by ELISA. Autologous mixed lymphocyte proliferation and the cytotoxicity of cytotoxic T lymphocytes primed by activated DCs to PANC1, PaTu8988 and SCG7901 tumor cells was measured by CCK 8 test.ResultsWhen DCs based PANC1 lysate were activated by CGB, the expression rates of CD83and CD86were increased from (3.7±0.3)% and (38.6±5.0)% to (16.5±0.6)% and (76.6±2.5)% (P<0.05). The concentrations of cytokines ILp12p70 and TNF-α were increased from (20.18±2.06)pg/ml and (61.38±1.19)pg/ml to (62.48±3.80)pg/ml and (592.53±17.96)pg/ml (P<0.01). When co-cultured with CGB activated DCs based PANC1 lysate in proportion of 1∶100,1∶50,1∶10 and 1∶5, the proliferation rate of the lymphocytes from (3.90±1.41)%,(4.07±0.40)%, (3.39±1.05)%, (2.82±0.39)% significantly increased to (55.38±3.58)%, (75.0±2.54)%, (77.07±3.4)%, (99.07±2.4)% (P<0.01), respectively. The killing effects of lymphocytes stimulated by DCs at E/T of 1∶5, 1∶10, 1∶20, 1∶50 were (71.73±0.46)%, (49.44±0.98)%, (31.76±2.77)%, (19.03±3.04)%; but the killing effects on PaTu8988 and SCG7901 were significantly decreased.ConclusionsDC vaccines for pancreatic cancer could be more maturated when activated by CGB, and could show a high capability of anti-tumor in vitro.

Pancreatic neoplasms; Dendritic cells; CGB vaccine

10.3760/cma.j.issn.1674-1935.2010.04.014

210008 南京，南京大學(xué)醫(yī)學(xué)院附屬鼓樓醫(yī)院消化科

鄒曉平，Email：zouxiaoping795@hotmail.com