龐 沖 張騰月 王長利△

腫瘤干細(xì)胞來源的DC-CIK對(duì)同源腫瘤細(xì)胞的殺傷作用

龐 沖1張騰月2王長利1△

目的 觀察干細(xì)胞負(fù)載樹突細(xì)胞誘導(dǎo)的殺傷細(xì)胞（CSC-DC-CIK）作為效應(yīng)細(xì)胞對(duì)同源腫瘤細(xì)胞的殺傷作用，探討CSC抗原參與腫瘤殺傷作用的可行性。方法培養(yǎng)腎癌細(xì)胞株A498和肺癌細(xì)胞株A549，用流式分選術(shù)分離純化CD133+細(xì)胞，分別作為腎癌干細(xì)胞（KSC）和肺癌干細(xì)胞（LSC），凍融法制備抗原。提取健康產(chǎn)婦臍帶血的單個(gè)核細(xì)胞，體外擴(kuò)增誘導(dǎo)生成DC和CIK細(xì)胞。分別用上述CSC抗原負(fù)載DC，與CIK共培養(yǎng)（CSC-DC-CIK），流式細(xì)胞術(shù)分析DC和CIK細(xì)胞免疫表型，ELISA法檢測細(xì)胞因子分泌水平，用乳酸脫氫酶（LDH）釋放法檢測CSCDC-CIK對(duì)同源腫瘤細(xì)胞的殺傷效率。結(jié)果CSC-DC的DC免疫表型CD40+、CD80+、CD86+及HLA-DR+的表達(dá)均高于單純DC的相應(yīng)免疫表型的表達(dá)（P＜0.01）；DC、CSC-DC與CIK共培養(yǎng)后的DC免疫表型CD40+、CD80+、CD86+及HLA-DR+的表達(dá)均高于共培養(yǎng)前（P＜0.01）；CSC-DC與CIK共培養(yǎng)后的DC免疫表型CD40+、CD80+、CD86+及HLA-DR+的表達(dá)高于DC與CIK共培養(yǎng)后的相應(yīng)免疫表型（P＜0.01）；DC、CSC-DC與CIK共培養(yǎng)后的CIK免疫表型CD3+、CD8+、CD56+的表達(dá)高于共培養(yǎng)前（P＜0.01）；CSC-DC與CIK共培養(yǎng)后的CIK免疫表型CD3+、CD8+、CD56+的表達(dá)高于DC與CIK共培養(yǎng)后的CIK相應(yīng)免疫表型（P＜0.01）；DC、CSC-DC與CIK共培養(yǎng)后的IFN-γ、TNF-α和IL-2分泌水平高于共培養(yǎng)前（P＜0.01）；CSC-DC與CIK共培養(yǎng)后的IFN-γ、TNF-α和IL-2分泌水平高于DC與CIK共培養(yǎng)后的相應(yīng)細(xì)胞因子表達(dá)（P＜0.01）；KSC-DC-CIK組和LSC-DC-CIK組對(duì)靶細(xì)胞的殺傷率為（50.21±4.24）％和（49.32±3.89）％，明顯高于DC-CIK組的（30.25±3.11）％（F=89.157，P＜0.01）。結(jié)論CSC抗原負(fù)載DC活化CIK （CSC-DC-CIK）對(duì)同源腫瘤細(xì)胞有更好的殺傷作用，對(duì)其作用機(jī)制和臨床應(yīng)用的可能性尚需深入研究。

腫瘤干細(xì)胞；樹突細(xì)胞；免疫療法；腎腫瘤；肺腫瘤；細(xì)胞因子誘導(dǎo)的殺傷細(xì)胞

近年來有關(guān)腫瘤生物免疫治療領(lǐng)域的研究極為活躍，成為繼手術(shù)、化療、放療之外不可忽視的治療手段，包括細(xì)胞因子、單克隆抗體及各種免疫細(xì)胞等，并且取得了顯著效果。其中樹突狀細(xì)胞（DC）聯(lián)合細(xì)胞因子誘導(dǎo)的殺傷細(xì)胞（DC-CIK）過繼免疫治療具有很好的抗腫瘤作用，DC-CIK共同培養(yǎng)可明顯提高CIK的增殖活性和細(xì)胞毒作用，提高患者自身免疫力，其安全性及療效已經(jīng)在轉(zhuǎn)移性黑素瘤[1]、晚期非小細(xì)胞肺癌[2]、白血病[3]的治療中得到了驗(yàn)證。腫瘤干細(xì)胞(cancer stem cell,CSC)，具有無限自我更新及分化的潛能，是腫瘤發(fā)生發(fā)展、轉(zhuǎn)移復(fù)發(fā)和放化療抗性等產(chǎn)生的根源，成為惡性腫瘤研究的熱點(diǎn)[4]。本研究從腎癌細(xì)胞A498和肺癌細(xì)胞A549分離腫瘤干細(xì)胞制備抗原負(fù)載DC，活化CIK，觀察CSC-DC-CIK作為效應(yīng)細(xì)胞對(duì)同源腫瘤細(xì)胞的殺傷作用，探討CSC抗原參與腫瘤殺傷作用的可行性。

1 材料與方法

1.1 細(xì)胞株、試劑及儀器 腎癌細(xì)胞株A498和肺癌細(xì)胞株A549，本室保存。RPMI-1640培養(yǎng)基（賽默飛世爾生物化學(xué)制品有限公司）；新生胎牛血清（FCS）、淋巴細(xì)胞分離液（天津血液研究所試劑公司）；異硫氰酸熒光素（FITC）-CD40、APC-HLA-DR、PE-CD80、PE-CD86，葉綠素蛋白（Percp）-cy5.5-CD56、PE-CD8、FITC-CD3（美國BD Bioscience公司）,重組人干細(xì)胞生長因子(rhSCF)、重組人FMS樣酪氨酸激酶3(FLT3)配體、重組人粒細(xì)胞巨噬細(xì)胞刺激因子（rhGMCSF）、重組人白介素-4（rhIL-4）、重組腫瘤壞死因子-α（rhTNF-α）、重組人白介素-2（rhIL-2）、重組干擾素（rhINF）-γ(英國Peprotech公司)；抗人CD3單抗（武漢生物制品研究所）。流式分析儀（美國BD公司），CO2孵箱（Thermo Forma公司）。

1.2 方法

1.2.1 腫瘤干細(xì)胞凍融抗原的制備 流式分析儀分別收集腎癌細(xì)胞A498和肺癌細(xì)胞A549的CD133+細(xì)胞，前者作為腎癌腫瘤干細(xì)胞（KSC），后者作為肺癌腫瘤干細(xì)胞（LSC），調(diào)整細(xì)胞濃度為1×107∕mL，加入500 μL生理鹽水中，置液氮中5 min，迅速放入37℃水浴中，反復(fù)3次，離心、微孔濾膜過濾除菌，4℃保存?zhèn)溆谩?/p>

1.2.2 細(xì)胞活率的測定 用Hanks液配制0.1％臺(tái)盼藍(lán)溶液；用0.5％胰蛋白酶∶0.2％EDTA=1∶1混合液來消化培養(yǎng)的貼壁細(xì)胞；加入適量Hanks液制成細(xì)胞懸液；每0.1 mL細(xì)胞懸液約加新鮮配制的染液一小滴，室溫下染3～5 min；染色過的細(xì)胞材料，取一滴細(xì)胞懸液置玻片上，加蓋玻片后放高倍鏡下觀察；計(jì)數(shù)1 000個(gè)細(xì)胞中的活細(xì)胞和死細(xì)胞數(shù)目；統(tǒng)計(jì)未染色細(xì)胞。細(xì)胞活率（％）=未染色的細(xì)胞數(shù)∕觀察的細(xì)胞總數(shù)×100％。

1.2.3 單個(gè)核細(xì)胞的收集和DC體外誘導(dǎo)培養(yǎng) 經(jīng)本人知情同意，采用健康產(chǎn)婦新鮮的臍帶血，使用淋巴細(xì)胞分離液梯度離心法獲取單個(gè)核細(xì)胞。將單個(gè)核細(xì)胞置于含10％新生牛血清的RPMI-1640完全培養(yǎng)基中，收集黏附細(xì)胞，加培養(yǎng)液調(diào)細(xì)胞濃度為1×106∕mL，加入rhGM-CSF和rhIL-4，培養(yǎng)至第5天時(shí)，用上述制備的凍融抗原以1∶5的比例進(jìn)行沖擊，即為CSC-DC（分別命名為KSC-DC或LSC-DC），繼續(xù)培養(yǎng)2 d，與未沖擊的DC均于第7天加入腫瘤壞死因子（TNF）-α。培養(yǎng)結(jié)束后用熒光倒置顯微鏡觀察細(xì)胞形態(tài)，并流式細(xì)胞術(shù)進(jìn)行CSC-DC和DC的表型檢測。

1.2.4 CIK細(xì)胞的分離及培養(yǎng) 收集單個(gè)核細(xì)胞中的非黏附細(xì)胞，用RPMI-1640完全培養(yǎng)基調(diào)細(xì)胞數(shù)為3×106∕mL，加入rhINF-γ，刺激24 h后，加入抗CD3單抗及rhIL-2；培養(yǎng)至第7天將CIK細(xì)胞分為4組，分別是凍融抗原沖擊的DC 與CIK共培養(yǎng)組（KSC-DC-CIK組和LSC-DC-CIK組）、未沖擊的DC與CIK共培養(yǎng)組（DC-CIK組）及CIK對(duì)照組，DC 與CIK細(xì)胞按照1∶10比例進(jìn)行共培養(yǎng)。第10天后收集上述各組細(xì)胞，采用流式細(xì)胞術(shù)進(jìn)行免疫表型檢測；倒置顯微鏡觀察細(xì)胞形態(tài)。

1.2.5 細(xì)胞因子的檢測 取上述各組細(xì)胞上清液用酶聯(lián)免疫吸附測定（ELISA）試劑盒進(jìn)行干擾素（IFN）-γ、TNF-α和白細(xì)胞介素（IL）-2水平的檢測，具體操作按試劑盒說明書進(jìn)行。

1.2.6 對(duì)同源腫瘤細(xì)胞的殺傷作用 采用乳酸脫氫酶（LDH）釋放法測定上述各組細(xì)胞對(duì)同源腫瘤細(xì)胞的殺傷作用，將CSC-DC-CIK作為效應(yīng)細(xì)胞，并調(diào)整細(xì)胞濃度為1× 106∕mL，同源腫瘤細(xì)胞作為靶細(xì)胞，調(diào)細(xì)胞濃度為1×105∕mL。LDH釋放法分別測定各實(shí)驗(yàn)組在效靶比20∶1的殺傷活性。設(shè)3個(gè)復(fù)孔，用酶標(biāo)儀在490 nm波長下檢測其光密度（OD）值。按以下公式計(jì)算殺傷率：殺傷率（％）=（實(shí)驗(yàn)組OD值-效應(yīng)細(xì)胞自然釋放OD值-靶細(xì)胞自然釋放OD值）∕（靶細(xì)胞最大釋放OD值-靶細(xì)胞自然釋放OD值）×100％。

1.3 統(tǒng)計(jì)學(xué)方法 采用SPSS 18.0進(jìn)行統(tǒng)計(jì)處理，計(jì)量數(shù)據(jù)用均數(shù)±標(biāo)準(zhǔn)差（±s）表示，組間比較采用方差分析，多重比較采用LSD-t法。組內(nèi)共培養(yǎng)前后比較采用配對(duì)t檢驗(yàn)，P＜0.05為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1 腫瘤干細(xì)胞凍融抗原 倒置顯微鏡下KSC和LSC細(xì)胞形態(tài)一致，均為圓形，胞體較小，形似淋巴細(xì)胞，見圖1。臺(tái)盼藍(lán)染色顯示其細(xì)胞活率＞95％。

Fig.1 Microscopic morphologies of KSC and LSC圖1 顯微鏡下KSC和LSC細(xì)胞形態(tài)

2.2 CSC-DC的培養(yǎng)與形態(tài) 新分離的臍帶血單個(gè)核細(xì)胞呈球形散在分布，表面光滑；加入細(xì)胞因子組合培養(yǎng)2 d后，倒置顯微鏡下可見部分細(xì)胞懸浮并發(fā)生聚集樣增生；培養(yǎng)第5天，出現(xiàn)細(xì)胞體積增大，并出現(xiàn)貼壁細(xì)胞，數(shù)量增多，多形性細(xì)胞增多。第5天用凍融抗原進(jìn)行沖擊后大部分細(xì)胞聚集成團(tuán)，胞體較大，培養(yǎng)7 d后具有樹突狀突起的細(xì)胞增多且突起更為明顯，見圖2。

Fig.2 Microscopic morphology of dendritic cells induced from mononuclear cells isolated from cord blood after been pulsed with KSC and LSC frozen-thawed antigen(×400)圖2 凍融抗原從臍帶血單個(gè)核細(xì)胞誘導(dǎo)出的樹突狀細(xì)胞(×400)

2.3 CIK細(xì)胞的培養(yǎng)與鑒定 CIK細(xì)胞培養(yǎng)第4天開始數(shù)量倍增，體積開始增大，細(xì)胞核大而圓，部分細(xì)胞聚集成簇。培養(yǎng)10 d后CIK成熟表型檢測結(jié)果為CD3+細(xì)胞占（68.321±3.891）％，CD3+CD8+細(xì)胞占（36.792±2.822）％，CD3+CD56+細(xì)胞占（25.334± 2.652）％，提示細(xì)胞達(dá)到成熟程度，見圖3。

2.4 免疫表型表達(dá)的測定結(jié)果 KSC-DC組和LSC-DC組CD40+、CD80+、CD86+及HLA-DR+的表達(dá)均高于DC的相應(yīng)免疫表型的表達(dá)（P＜0.01）；DC、 CSC-DC與CIK共培養(yǎng)后的DC免疫表型CD40+、CD80+、CD86+及HLA-DR+的表達(dá)均高于共培養(yǎng)前（P＜0.01）；KSC-DC和LSC-DC與CIK共培養(yǎng)后的DC免疫表型CD40+、CD80+、CD86+及HLA-DR+的表達(dá)高于DC與CIK共培養(yǎng)后的相應(yīng)免疫表型（P＜0.01），見表1。DC-CIK、KSC-DC-CIK和LSC-DCCIK組的CIK免疫表型CD3+、CD3+CD8+、CD3+CD56+的表達(dá)高于CIK組（P＜0.01）；KSC-DC-CIK和LSC-DC-CIK組的CIK免疫表型CD3+、CD8+、CD56+的表達(dá)高于DC-CIK組（P＜0.01）；KSC-DC-CIK組和LSC-DC-CIK組間差異無統(tǒng)計(jì)學(xué)意義（P＞0.05），見表2。

2.5 細(xì)胞因子分泌水平的測定結(jié)果 DC-CIK、KSC-DC-CIK和LSC-DC-CIK組的IFN-γ、TNF-α 和IL-2表達(dá)高于CIK組（P＜0.01）；KSC-DC-CIK 和LSC-DC-CIK組的IFN-γ、TNF-α和IL-2表達(dá)高于DC-CIK組（P＜0.01）；KSC-DC-CIK組和LSCDC-CIK組TNF-α和IL-2表達(dá)的差異無統(tǒng)計(jì)學(xué)意義（P＞0.05），見表3。

2.6 CSC-DC-CIK對(duì)同源腫瘤細(xì)胞的殺傷作用 KSC-DC-CIK組和LSC-DC-CIK組對(duì)靶細(xì)胞的殺傷率為（50.21±4.24）％和（49.32±3.89）％，明顯高于DC-CIK組的（30.25±3.11）％（F=89.157，P＜0.01），KSC-DC-CIK組和LSC-DC-CIK組對(duì)腎癌和肺癌細(xì)胞的殺傷作用差異無統(tǒng)計(jì)學(xué)意義（P＞0.05）。

Fig.3 Phenotypes of CIK analyzed by flow cytometry圖3 CIK細(xì)胞表型的流式細(xì)胞術(shù)檢測分析圖

Tab.1 Phenotype analysis of CIK before and after co-culture表1 與CIK共培養(yǎng)前后DC表型分析 （n=10，％，±s）

Tab.1 Phenotype analysis of CIK before and after co-culture表1 與CIK共培養(yǎng)前后DC表型分析 （n=10，％，±s）

＊＊P＜0.01；a與（1）組比較，b與（2）組比較，P＜0.01；表2同

組別DC(1) KSC-DC(2) LSC-DC(3) F組別DC(1) KSC-DC(2) LSC-DC(3) F CD40+共培養(yǎng)前23.86±3.83 52.43±4.08a52.36±4.34a162.552＊＊CD86+共培養(yǎng)前28.94±3.68 62.89±4.38a60.05±5.28a175.691＊＊共培養(yǎng)后35.36±3.46 78.08±4.45a83.32±2.99ab509.780＊＊t t 7.186＊＊12.914＊＊20.675＊＊共培養(yǎng)后52.39±4.16 83.50±4.49a82.38±3.87a178.040＊＊10.067＊＊17.675＊＊14.870＊＊共培養(yǎng)后53.44±4.47 85.93±5.53a84.62±7.01a101.715＊＊t t 12.031＊＊12.165＊＊9.214＊＊CD80+共培養(yǎng)前36.18±2.90 53.82±3.84a55.84±5.59a64.526＊＊HLA-DR+共培養(yǎng)前32.61±3.97 53.56±7.06a55.04±8.03a36.280＊＊共培養(yǎng)后53.31±4.11 88.84±4.16a87.61±5.40a192.328＊＊11.471＊＊14.798＊＊9.425＊＊

Tab.2 Phenotype analysis of CIK before and after been co-cultured with DC表2 與DC共培養(yǎng)結(jié)束后CIK表型分析（n=10，％，±s）

Tab.2 Phenotype analysis of CIK before and after been co-cultured with DC表2 與DC共培養(yǎng)結(jié)束后CIK表型分析（n=10，％，±s）

?

Tab.3 Cytokines levels in cell supernatant after co-culture表3 共培養(yǎng)后4組細(xì)胞上清中細(xì)胞因子的分泌水平（n=10，ng∕L，±s）

Tab.3 Cytokines levels in cell supernatant after co-culture表3 共培養(yǎng)后4組細(xì)胞上清中細(xì)胞因子的分泌水平（n=10，ng∕L，±s）

＊＊P＜0.01；a與（1）組比較，b與（2）組比較，c與（3）組比較，P＜0.01

組別CIK組(1) DC-CIK組(2) KSC-DC-CIK組(3) LSC-DC-CIK組(4) F IFN-γ 871.05±82.46 1 532.51±60.23a3 238.14±246.50ab3 511.28±354.44abc337.212＊＊TNF-α 326.67±55.89 498.31±43.07a986.67±73.00ab1 034.53±77.51ab305.320＊＊IL-2 108.20±17.97 214.18±24.76a439.33±37.39ab418.90±31.84ab309.024＊＊

3 討論

DC是功能最強(qiáng)的抗原遞呈細(xì)胞，能激活初始T細(xì)胞，激發(fā)特異性免疫應(yīng)答和促進(jìn)非特異性細(xì)胞毒殺傷，是抗腫瘤免疫的重要載體。DC誘導(dǎo)培養(yǎng)CIK可以使CIK細(xì)胞中的免疫抑制T細(xì)胞降低，達(dá)到增強(qiáng)CIK細(xì)胞的殺傷作用[5]，而且體外擴(kuò)增速度快，殺瘤譜廣，對(duì)耐藥腫瘤細(xì)胞敏感[6]，是一種比較理想的具有細(xì)胞毒作用的免疫活性細(xì)胞。DC-CIK共同培養(yǎng)可明顯提高CIK的增殖活性和細(xì)胞毒作用，體內(nèi)外研究均表明DC-CIK過繼免疫治療具有很好的抗乳腺癌[7-8]、結(jié)腸癌[9]、肝癌作用[10]。譚潔等[11]報(bào)道DC-CIK細(xì)胞體外能有效殺傷腎癌細(xì)胞786-0和慢性粒細(xì)胞白血病細(xì)胞K562，輸注后可提升患者的免疫水平，可作為晚期腫瘤患者的輔助治療。王賽男等[12]也發(fā)現(xiàn)DC-CIK免疫治療能改善腎癌患者的免疫抑制狀態(tài)，提高機(jī)體的抗腫瘤免疫效應(yīng)，有較好的近期療效。陳詩萍等[13-14]等發(fā)現(xiàn)樹突細(xì)胞誘導(dǎo)的CIK細(xì)胞能夠有效抑制肺癌移植瘤生長，有望成為非小細(xì)胞肺癌有效的過繼免疫治療方法。本文未進(jìn)行DC-CIK對(duì)比CIK的體外殺傷腫瘤實(shí)驗(yàn)，但通過對(duì)比DC與CIK共同培養(yǎng)前后DC免疫表型、CIK免疫表型及細(xì)胞因子分泌水平，可以推測出DC-CIK共同培養(yǎng)可以明顯提高抗腫瘤效果，和前述文獻(xiàn)結(jié)果一致。

CSC是影響腫瘤治療效果和轉(zhuǎn)歸的重要因素之一，鑒于其對(duì)放化療的抗性，故免疫治療具有獨(dú)特的優(yōu)勢[15]。理論上推測，CSC應(yīng)具有一般腫瘤細(xì)胞抗原和CSC特有的抗原，以CSC抗原負(fù)載DC激活CIK，有望達(dá)到對(duì)一般腫瘤細(xì)胞和CSC的特異性殺傷作用。唐家平等[16]用負(fù)載胃癌干細(xì)胞抗原的DCCIK對(duì)胃癌細(xì)胞的殺傷作用明顯高于未負(fù)載組，在效靶比為20∶1時(shí)對(duì)胃癌細(xì)胞的殺傷率為80.6％，而未負(fù)載組為49.7％(P＜0.05)。本研究以CD133+作為篩選腎癌和肺癌CSC的表面標(biāo)志[4]，凍融法獲取CSC抗原，制備CSC-DC-CIK細(xì)胞，確實(shí)能促進(jìn)免疫細(xì)胞的成熟和細(xì)胞因子的分泌，當(dāng)效靶比為40∶1時(shí)對(duì)同源腫瘤細(xì)胞的殺傷率達(dá)50％左右。本研究結(jié)果初步證實(shí)了DC-CIK負(fù)載KSC及LSC能夠有效提高對(duì)同源腫瘤細(xì)胞的殺傷率，不同CSC抗原參與的同源腫瘤殺傷作用方面雖然LSC-DC與CIK共培養(yǎng)后的DC免疫表型CD40+與KSC-DC組有差異，KSC-DC-CIK組和LSC-DC-CIK組的IFN-γ表達(dá)有差異，但是在CD80+等其他表型及TNF-α和IL-2表達(dá)方面沒有明顯差異，因此筆者認(rèn)為在殺傷其同源腫瘤細(xì)胞方面還不能說兩種CSC抗原哪種更有特異性。對(duì)于是否存在某種CSC抗原能夠較KSC和LSC抗原更有效地殺傷其同源腫瘤細(xì)胞仍需日后增加腫瘤干細(xì)胞種類以進(jìn)一步深入探索。

[1]Bernatchez C,Radvanyi LG,Hwu P.Advances in the treatment of metastatic melanoma:Adoptive T-cell therapy[J].Semin Oncol, 2012,(2):215-226.doi:10.1053∕j.seminoncol.2012.01.006.

[2]Shi SB,Ma TH,Li CH,et al.Effect of maintenance therapy with dendritic cells:cytokine-induced killer cells in patients with advanced non-small cell lung cancer[J].Tumori,2012,98(3):314-319.doi:10.1700∕1125.12398.

[3]Pan Y,Tao Q,Wang H,et al.Dendritic cells decreased the concomitant expanded Tregs and Tregs related IL-35 in cytokine-induced killer cells and increased their cytotoxicity against leukemia cells [J]. PLoS One, 2014,9(4):e93591. doi: 10.1371∕journal. pone.0093591.eCollection 2014.

[4]Carsten R,Christian S,Adrian F,et al.From''magic bullet''s to specific cancerimmunotherapy[J].Swiss Medical Weekly,2013,143: w13734.doi:10.4414∕smw.2013.13734.

[5]Schmidt J,Eisold S,Buchler MW,et al.Dendritic cells reduce number and function of CD4+CD25+in cytokine-induced killer cells derived from patients with pancreatic carcinoma[J].Cancer Immunology immunotherapy,2004,53(11):1018-1026.doi:10.1007∕s00262-004-0554-4.

[6]Niu Q,Wang W,Li Y,et al.Cord blood-derived cytokine-induced killer cells biotherapy combined with second-line chemotherapy in the treatment of advanced solid malignancies[J].Int Immunopharmacol,2011,(04):449-456.doi:10.1016∕j.intimp.2010.12.014.

[7]Yue LL,Zhang LL,Chai Y,et al.Effect of cytokine-induced killer cells co-cultured with dendritic cells pulsed with antigens on cytotoxicity against multidrug resistant breast cancer cells[J].Chin J Cancer Biother,2012,19(4):437-441.[岳玲玲，張連生，柴曄,等.負(fù)載抗原的DC與CIK共培養(yǎng)對(duì)耐藥乳腺癌細(xì)胞的殺傷作用[J].中國腫瘤生物治療雜志,2012,19(4):437-441.]doi:10.3872∕j. issn.1007-385X.2012.04.017.

[8]Lyu Y,Pang H,Pang CM,et al.Study on the Kill Activity of the Whole Tumor Cell Antigen Pulsed DC Co-Culture with CIK for Breast Cancer Circulating Tumor Cells[J].Tianjin Med J,2013,41 (12):1173-1176.[呂艷，龐華，龐春淼,等.負(fù)載抗原DC-CIK共培養(yǎng)對(duì)富集培養(yǎng)乳腺癌CTCs殺傷作用研究[J].天津醫(yī)藥,2013,41 (12):1173-1176.]doi:10.3969∕j.issn.0253-9896.2013.12.010.

[9]Wang B,Liu YX,He CY,et al.Compare study of killing activity of DC and CIK on colorectal cancer SW1116[J].Chin J Lab Diagn, 2013,17(9):1571-1573.[王斌，劉玉俠，何春瑩,等.抗原負(fù)載的DC及CIK對(duì)結(jié)腸癌細(xì)胞SW1116殺傷活性的比較研究[J].中國實(shí)驗(yàn)診斷學(xué),2013,17(9):1571-1573.]doi:10.3969∕j.issn.1007-4287.2013.09.007.

[10]Zhou B,Zhang DC.Killing activity of antigen-pulsed dendritic cells combined with cytokine induced killer cells against hepatocarcinoma cells[J].Journal of Chongqing Medical University,2012,37 (8):703-706.[周兵，張德重.抗原致敏的樹突狀細(xì)胞聯(lián)合細(xì)胞因子誘導(dǎo)的殺傷細(xì)胞對(duì)肝癌細(xì)胞殺傷活性的研究[J].重慶醫(yī)科大學(xué) 學(xué) 報(bào),2012,37(8):703-706.]doi: 10.3969∕j.issn.0253-3626.2012.08.012.

[11]Tan J,Wang B,He ZJ,et al.Curative effect of autologous dendritic cell stimulated by cytokine-induced killer cells on metastatic renal carcinoma[J].Chin J Cancer Biother,2013,20(6):711-716.[譚潔，王彬，何志潔，等.自體DC-CIK細(xì)胞治療晚期腎細(xì)胞癌的療效[J].中國腫瘤生物治療雜志,2013,20(6):711-716.]doi:10.3872∕j. issn.1007-385X.2013.06.014.

[12]Wang SN,Gao HY,Pan X,et al.Impact of DC-CIK treatmeat on lymphocyte subsets of patients with renal cell carcinoma[J].Chin J Cancer Biother,2012,19(3):309-312.[王賽男，高海燕，潘欣，等. DC-CIK治療對(duì)腎癌患者淋巴細(xì)胞亞群的影響[J].中國腫瘤生物治 療 雜 志,2012,19(3):309-312.]doi: 10.3872∕j.issn.1007-385X.2012.03.016.

[13]Chen SP,Mai SJ,Yu XJ,et al.Inhibitory effect of cytokine-induced killer(CIK)cells induced by dendritic cells on lung cancer xenograft in nude mice[J].Chin Med Biotechnol,2012,7(2):125-129.[陳詩萍，買世娟，佘杏娟，等.樹突狀細(xì)胞誘導(dǎo)的CIK細(xì)胞對(duì)肺癌生長的抑制作用[J].中國醫(yī)藥生物技術(shù),2012,7(2):125-129.]doi: 10.3969∕cmba.j.issn.1673-713X.2012.02.008.

[14]Cai JX,Tan J,Wang B,et al.Short-term curative efficacy of DCCIK cell-therapy on patients with advanced non-small cell lung cancer[J].Modern Oncology,2014,22(1):67-70.[蔡俊霞，譚潔，王彬，等.DC-CIK治療43例晚期非小細(xì)胞肺癌的近期療效觀察[J].現(xiàn)代腫瘤雜志,2014,22(1):67-70.]doi:10.3969∕j.issn.1672-4992.2014.01.20.

[15]Yang L,Ren B,Li H,et al.Enhanced antitumor effects of DC-activated CIKs to chemotherapy treatment in a single cohort of advanced non-small cell lung cancer patients[J].Cancer Immunol Immunother,2013,62:65-73.doi:10.1007∕s00262-012-1311-8.

[16]Tang JP,Li JY.A study of the immune function of DC-CIK loaded with gastric cancer stem cell antigen against the activity of gastric cancer[J].China Medical Engineering,2013,21(3)1-5.[唐家平，李錦毅.負(fù)載胃癌干細(xì)胞抗原的樹突細(xì)胞對(duì)胃癌細(xì)胞免疫作用的研究[J].中國醫(yī)學(xué)工程,2013,21(3):1-5.]

(2014-09-11收稿 2014-09-18修回)

(本文編輯 魏杰)

Effect of Tumor Stem Cell Derived CSC-DC-CIK on Destructing Homologous Tumor Cells

PANG Chong1,ZHANG Tengyue2,WANG Changli1△
1 Tianjin Medical University Cancer Institute and Hospital,National Clinical Research Center for Cancer,Key Laboratory of Cancer Prevention and Therapy,Tianjin，Tianjin 300060,China;2 Tianjin Eye Hospital△

E-mail:E-mail：wangchangli@medmail.com.cn

ObjectiveTo investigate the destructive effect of CSC-DC-CIK who were induced by cytokine induced killer(CIK)cells co-cultured with dendritic cells(DCs)on homologous tumor cells and to explore the possibility of CSC antigen involving in killing tumor.MethodsKidney cancer stem cells(KSCs)and lung cancer stem cells(LSCs)were isolated through FACS using CD133+as a selection marker from cultured kidney cancer cell line A498 and lung cancer cell line A549 respectively.Freeze-thaw method was used to obtain the cancer stem cells（CSCs）antigens.DC cells and CIK cells were collected by in vitro expansion and inducted from the mononuclear cells isolated from human cord blood.The CIK cells were co-cultured with the DCs which were pulsed with the CSCs antigens（CSC-DC-CIK）mentioned above.Immunophenotypes of DC and CIK were analyzed by flow cytometry;cytokines levels were detected by ELISA kits and the destructive effects of two kinds of CSC-DC-CIKs were tested by lactate dehydrogenase(LDH)release assay.ResultsThe expression of phenotypes CD40+,CD80+,CD86+and HLA-DR+were higher in CSC-DC than in CD（P＜0.01）;the expression of phenotypes CD40+,CD80+,CD86+and HLA-DR+of DC and CSC-DC were higher after co-culture than those before co-culture（P＜0.01）;the expression of phenotypes CD40+,CD80+,CD86+and HLA-DR+of CSC-DC after been co-cultured with CIK were higher than those of DC after been co-cultured with CIK（P＜0.01）.The CIK phenotypes:CD3+,CD8+,CD56+were increased in CIK co-cultured with both CSC-DC and DC than those before co-culture(P＜0.01);the expression of phenotypes CD3+,CD8+,CD56+were higher in CSC-DC co-cultured with CIK than in DC co-cultured with CIK.DC-CIK and CSC-DC-CIK groups were more capable to express IFN-γ,TNF-α,IL-2 than they were before co-cultured with CIK(P＜0.01).CSC-DC-CIK group can secrete more above cytokines than DC-CIK group does（P＜0.01）.The destructive rates of KSC-DC-CIK and LSC-DC-CIK on target cells were(50.21±4.24)％and(49.32±3.89)％respectively which were muchhigher than that in DC-CIK（30.25±3.11）％（F=89.157，P＜0.01）.ConclusionCSC-DC-CIKs have destructive effects on homologous tumor cells.More researches are needed to explore the mechanism and to evaluate the clinical applications.

neoplastic stem cells;dendritic cells;immunotherapy;kidney neoplasms;lung neoplasms;cytokine induced killer

R730.3

A

10.3969∕j.issn.0253-9896.2014.10.004

1天津醫(yī)科大學(xué)腫瘤醫(yī)院，國家腫瘤臨床醫(yī)學(xué)研究中心，天津市“腫瘤防治”重點(diǎn)實(shí)驗(yàn)室（郵編300060）；2天津市眼科醫(yī)院

△通訊作者 E-mail：wangchangli@medmail.com.cn