顺铂和紫杉醇对CIK 细胞杀伤食管癌细胞活性的影响及其分子机制研究*

目的:研究紫杉醇、顺铂对人食管癌EC9706细胞NKG 2D 配体表达及CIK 细胞杀伤活性的影响,探讨相关分子机制。方法:MTT 法测定紫杉醇、顺铂对EC9706细胞的24h 半数抑制浓度（IC 50）。 流式细胞仪检测1/ 2 IC 50浓度紫杉醇、顺铂作用前、后EC9706细胞NKG 2D 配体的表达。乳酸脱氢酶释放法检测效靶比2 0 :1、3 0 :1 时,CIK 细胞对1/ 2 IC 50浓度紫杉醇、顺铂作用前、后EC9706细胞的杀伤活性。荧光定量PCR 法检测1/ 2 IC 50浓度紫杉醇、顺铂作用EC9706细胞24h 前、后DNA 损伤修复基因（ATM 、ATR 、CHK 1、CHK 2、P 53）表达的变化。结果:紫杉醇、顺铂的24h 半数抑制浓度分别为10、5 μ g/mL 。1/ 2 IC 50浓度紫杉醇作用24h后,EC9706细胞MICB、ULBP2、ULBP3 表达均明显增强（P < 0.05）,MICA、ULBP1 表达无显著性变化（P > 0.05）;1/ 2 IC 50浓度顺铂作用24h 后,EC9706细胞 MICA、MICB、ULBP2、ULBP3 表达均明显增强（P < 0.05）,ULBP1 表达无显著性变化（P > 0.05）。效靶比 20 :1、30 :1 时,CIK 细胞对 1/ 2 IC 50浓度紫杉醇、顺铂作用后的 EC9706细胞的杀伤活性均明显增强（P < 0.05）。1/ 2 IC 50浓度紫杉醇作用 24h后,DNA 损伤修复基因表达均无显著性变化（P > 0.05）;1/ 2 IC 50浓度顺铂作用24h 后,ATM 、ATR 、CHK 1、CHK 2 基因表达均明显增加（P < 0.05）,P 53基因表达无显著性变化（P > 0.05）。 结论:顺铂、紫杉醇均可增强CIK 细胞的杀伤活性,其分子机制可能与激活DNA 损伤修复基因,进而增加NKG 2D 配体表达有关。      

顺铂与CIK细胞对A549细胞杀伤协同作用机制的初步研究

目的:探讨不同浓度顺铂作用人肺腺癌A549细胞24h后,NKG2D配体表达的改变及细胞因子诱导的杀伤细胞(CIK)杀伤活性的变化。方法:MTT法测定顺铂作用A549细胞24h的50%抑制浓度(IC50);流式细胞仪检测IC50、1/2IC50和1/4IC50浓度顺铂分别作用A549细胞24h后,A549细胞表面NKG2D配体表达的变化,配体包括MHC-I类链相关分子MICA和MICB以及人巨细胞病毒糖蛋白UL16结合蛋白ULBP1、ULBP2和ULBP3;乳酸脱氢酶释放法检测不同效靶比时,CIK细胞对IC50浓度的顺铂作用前及作用24h后A549细胞的杀伤活性。结果:不同浓度顺铂作用24h后,A549细胞表面MICA(F=12.490,P=0.002)、MICB(F=41.492,P<0.001)、ULBP2(F=375.773,P<0.001)和ULBP3(F=59.594,P<0.001)表达均显著升高;ULBP1表达降低,F=55.693,P<0.001。效靶比10∶1时,IC50浓度顺铂作用24h前、后的A549细胞的杀伤活性分别为(11.88±1.57)%和(17.64±1.44)%,F=21.770,P=0.010;20∶1时分别为(35.56±1.98)%和(46.39±3.51)%,F=21.653,P=0.010;30∶1时分别为(45.03±1.74)%和(73.81±1.62)%,F=439.578,P<0.001。结论:顺铂提高A549细胞NKG2D配体MICA、MICB、ULBP2和ULBP3的表达,增强A549细胞对CIK细胞杀伤的敏感性。     

紫杉醇增强肺癌细胞NKG2D配体表达和CIK细胞杀伤活性

目的研究不同浓度紫杉醇作用人肺腺癌A549细胞48小时后NKG2D配体表达的改变及CIK细胞杀伤活性的变化。方法 采用MTT法测定紫杉醇对A549细胞24小时的50%抑制率(IC50);流式细胞术检测IC50、1/2 IC50、1/4 IC50浓度紫杉醇作用A549细胞48小时后A549细胞表面NKG2D配体(MICA、MICB、ULBP1、ULBP2、ULBP3)表达的变化;乳酸脱氢酶释放法检测不同效靶比时,CIK细胞对IC50浓度的紫杉醇作用前及作用48小时后A549细胞的杀伤活性。结果 不同浓度紫杉醇作用48小时后A549细胞表面MICA、MICB、ULBP2、ULBP3表达显著升高,ULBP1表达降低(P<0.05)。效靶比10∶1、20∶1、30∶1时,CIK细胞对A549细胞的杀伤活性分别为(11.08±1.22)%、(36.22±0.91)%、(45.73±2.00)%;CIK细胞对IC50浓度紫杉醇作用48小时后的A549细胞杀伤活性分别为(20.79±3.33)%,(53.47±1.62)%、(66.39±0.77)%,与作用前比较均明显增强(P<0.05)。结论 紫杉醇作用后能提高A549细胞NKG2D配体(MICA、MICB、ULBP2、ULBP3)的表达,从而增强A549细胞对CIK细胞杀伤的敏感性。     

穿膜肽介导的NY-ESO-1155-163诱导CTL对黑素瘤A-375细胞的杀伤活性

目的：探讨穿膜肽（cell penetrating peptide, CPP）Tat49-57携带NY-ESO-1155-163抗原肽致敏DC后诱导获得抗原特异性细胞毒性T淋巴细胞（cytotoxic lymphocyte, CTL),并体外检测其对黑素瘤细胞株A-375的杀伤效果。 方法：采集健康志愿者外周血50 ml,用淋巴细胞分离液分离外周血单个核细胞,经细胞因子诱导,获得DC和T淋巴细胞。实验组用Tat49-57-NY-ESO-1155-163致敏DC,待DC成熟后与T淋巴细胞混合诱导产生CTL,并设PBS组和NY-ESO-1155-163组作为对照。流式细胞术检测致敏前后DC的表型,乳酸脱氢酶（LDH）释放法检测抗原肽疫苗致敏DC后诱导的CTL对黑素瘤细胞株A-375的体外杀伤活性,同时与对人肺癌A549细胞株、人白血病K562细胞的杀伤作用进行比较。结果：Tat显著提升NY-ESO-1155-163进入DC的穿膜能力。与NY-ESO-1155-163致敏的DC相比,Tat49-57-NY-ESO-1155-163致敏后DC的CD80/CD86\[(54.9 ±3.3 )% vs (43.8±5.7)%,P<0.05\]和CD40\[(42.1±1.9)% vs (23.7±2.8)%,P<0.05\]的表达率显著升高。Tat49-57-NY-ESO-1155-163刺激DC后诱导培养的T细胞亚群主要以MHC-Ⅰ类分子介导的CD3+CD8+细胞为主。Tat49-57-NY-ESO-1155-163组CTL对A-375细胞的杀伤能力显著高于NY-ESO-1155-163组,且随着效靶比的增加,杀伤活性逐渐增强（P<0.05）。A-375细胞高表达NY-ESO-1,Tat49-57-NY-ESO-1155-163组CTL对A-375细胞具有特异性杀伤作用,杀伤作用显著强于A549和K562细胞（均P<0.05）。结论：Tat49-57可以增强NY-ESO-1155-163抗原多肽的免疫原性,Tat49-57-NY-ESO-1155-163多肽致敏DC能有效诱导CTL抗黑素瘤细胞A-375的特异性免疫应答。     

顺铂对鼻咽癌细胞NKG2D配体表达和NK细胞杀伤活性的增强作用

 目的 研究顺铂(Cisplatin, DDP)作用前后人鼻咽癌细胞CNE2 NKG2D配体和HLA-Ⅰ类分子表达的改变及NK细胞杀伤活性的变化。 方法 MTT法测定DDP对CNE2细胞的50%抑制量（IC₅₀）;以此浓度DDP作用CNE2细胞24h,乳酸脱氢酶释放法检测效靶比20∶1时,NK细胞对DDP作用前后的CNE2细胞的杀伤活性;流式细胞仪检测DDP作用前后的CNE2细胞表面NKG2D配体(MICA/B、ULBP1、ULBP2、 ULBP3)和HLA-Ⅰ类分子表达的变化。 结果 DDP对CNE2细胞的IC₅₀为5mg/L。效靶比20∶1时,NK细胞对5mg/L DDP作用前后的CNE2细胞的杀伤活性分别为 （38.11±1.41）%,（47.71±1.53）%,差异有统计学意义（P＜0.05）,DDP作用后CNE2细胞表面MICA/B、ULBP1、 ULBP3表达显著升高,与作用前相比差异有统计学意义（P＜0.05）。ULBP2、HLA-Ⅰ类分子无明显变化(P＞0.05)。 结论 DDP能提高CNE2细胞NKG2D配体(MICA/B、ULBP1、ULBP3)的表达,从而增强CNE2细胞对NK细胞杀伤的敏感度。     

体外培养不同时间后冻存对脐血来源CIK细胞生物学活性的影响

目的： 观察体外培养不同时间后冻存对人脐血来源细胞因子诱导杀伤（cytokine induced killer,CIK）细胞增殖、免疫表型、细胞毒活性以及细胞因子分泌的影响。 方法： CIK细胞在体外培养6 、9 、12 d后冻存1个月,复苏后培养至72 h,其间每隔24 h检测细胞增殖情况,并采用流式细胞术检测复苏后细胞免疫表型、CCK-8法检测复苏后细胞对A549细胞的杀伤活性、ELISA方法检测复苏后CIK细胞分泌细胞因子IFN-γ和TNF-α的变化。 结果： 体外培养不同时间后冻存的CIK细胞在复苏后仍然显示出较好的增殖活性,其中,体外培养6 d后冻存组CIK细胞增殖活性明显高于体外培养9 d和12 d后冻存组CIK细胞,复苏72 h时,6、9、12 d冻存组CIK细胞数分别为(35.90±1.67)×106、(18.98±2.13)×106和(11.76±2.12)×106个（P<0.01）。 体外培养6、9、12 d后冻存组CIK细胞复苏24 h后,各冻存组CD3+CD56+、CD3+CD8+细胞比例逐渐增加,且复苏72 h后6 d冻存组CIK细胞中CD3+CD56+和CD3+CD8+细胞比例最低。体外培养后冻存组CIK细胞在复苏24 h内对A549细胞的杀伤活性较低,但24 h后细胞毒活性有较大幅度的增加,且体外培养12 d后冻存组CIK细胞对A549细胞的杀伤活性高于6 d和9 d冻存组CIK细胞,如复苏后72 h,12、6、9 d冻存组CIK细胞对A549细胞的抑瘤率分别为（0.81±009）%、（0.59±0.06）%、（0.42±0.08）%（P<0.01）。ELISA检测结果显示,随着复苏时间的延长,体外培养不同时间后冻存的CIK细胞分泌IFN-γ和TNF-α的水平也逐渐上升;复苏72 h时,体外培养6 d后冻存组CIK细胞分泌IFN-γ的量要高于其他组,而体外培养12 d后冻存组TNF-α的分泌量则明显高于其他组。 结论： 体外培养不同时间后冻存对CIK细胞的生物学活性有一定影响,但复苏后经短时间培养后基本恢复,提示CIK细胞可以在培养至12 d后进行冻存。     

舒尼替尼促进人肝癌细胞HepG2的凋亡及其分子机制

目的：探讨苹果酸舒尼替尼对人肝癌HepG2细胞凋亡的作用及其机制。方法：常规体外培养HepG2细胞,利用MTT法检测舒尼替尼杀伤HepG2细胞的IC50,Western blotting 检测药物处理前后HepG2细胞分子靶点蛋白表达,膜联蛋白(Annexin-V)/碘化丙啶(PI)双标法和TUNEL染色法检测舒尼替尼处理前后HepG2细胞凋亡情况,实时荧光定量PCR检测药物处理前后HepG2细胞凋亡基因 mRNA的表达情况。结果：舒尼替尼杀伤HepG2细胞的IC50值为（3.22±0.50）μmol/L。以对HepG2细胞无明显抑制作用的剂量1 μmol/L舒尼替尼处理HepG2细胞后,细胞内VEGFR1、VEGFR2、PDGFRα、Kit、FLT3蛋白表达均有不同水平下降（均P<0.05）,HepG2细胞的凋亡率\[(15.18±1.28)% vs (5.90±0.45)%,P<0.05\]、凋亡指数（AI）\[(23.54±4.73) vs (4.17±0.64), P<0.05\]均显著升高。舒尼替尼处理HepG2细胞后,上调促凋亡基因Bax、NOXA、PUMA、P53 mRNA表达水平（均P<0.05),降低抑凋亡基因Bcl-2、X-IAP mRNA表达水平（均P<0.05）。结论： 舒尼替尼能够诱导肝癌HepG2细胞凋亡,其机制可能是通过上调促凋亡基因的表达及降低抑凋亡基因表达水平来实现的。     

舒尼替尼促进HepG2细胞NKG2DLs表达从而增强NK细胞杀伤活性

目的：探讨舒尼替尼促进肝癌细胞自然杀伤细胞2族成员D配体（natural killer group 2 member D ligands,NKG2DLs）表达及提高NK细胞杀伤肿瘤细胞的作用。方法：常规体外培养HepG2细胞,免疫磁珠法从健康志愿者外周静脉血中分选NK细胞,流式细胞技术检测NK细胞纯度及1 μmol/L舒尼替尼孵育24 h前后肝癌细胞NKG2DLs表达率;LDH释放测定法检测NK细胞对药物处理前后靶细胞的杀伤活性,实时荧光定量PCR检测药物处理前后HepG2细胞NKG2DLs mRNA的表达情况。结果：分选后NK细胞(CD3-CD16+CD56+细胞)的纯度达78%以上;经舒尼替尼处理后靶细胞MHC-Ⅰ类链相关分子A或B（MHC class Ⅰ-related chain molecules A/B, MICA/MICB）、人巨细胞病毒糖蛋白UL16结合蛋白（UL16-binding proteins,ULBPs）的表达率均有升高,以MICA、MICB和ULBP2升高最明显（F=17.73,P=0.000）。当效靶比为10∶1、20∶1时,NK细胞对舒尼替尼处理前后HepG2细胞的杀伤活性分别从(9.47±1.11)%、(20.45±1.94)%上升到(28.88±123)%、(44.93±1.57)%,舒尼替尼处理前后NK细胞对靶细胞杀伤活性的差异有明显统计学意义(P<0.05)。舒尼替尼处理HepG2细胞后,MICA、MICB和ULBP2 mRNA表达水平明显升高（F=62.628,P=0.000）。结论：舒尼替尼能选择性诱导肿瘤细胞高表达NKG2DLs(MICA/B和ULBP2),并增强NK细胞杀伤活性。     

重组腺病毒rAd-E1A-CDglyTK对人卵巢癌SKOV3细胞的抑制作用

目的: 探讨含腺病毒基因E1A、融合自杀基因CDglyTK及报告基因GFP的重组腺病毒rAdE1ACDglyTK与酶前体药物5FC和(或)GCV联合应用对人卵巢癌SKOV3细胞的抑制作用。方法: 将重组腺病毒质粒prAdE1ACDglyTK经脂质体介导转染293细胞，进行病毒的扩增、纯化与PCR鉴定，以紫外分光光度法与TCID50法测定纯化病毒的颗粒浓度与感染性滴度，计算病毒比活性；在SKOV3细胞中观察重组病毒的复制能力及感染效率，确定感染率达90%以上的最小MOI。用MTT法观察感染重组病毒的SKOV3细胞对5FC或GCV的敏感性，并计算IC50；观察一定MOI的rAdE1ACDglyTK或重组复制缺陷型腺病毒rAdCDglyTK与IC50的5FC和(或)GCV联合应用对SKOV3细胞生长的抑制作用。结果: 经PCR和琼脂糖凝胶电泳证实，纯化重组腺病毒中存在E1A和CDglyTK两个基因片段，其病毒颗粒浓度和感染性滴度分别为9.47×1011VP/ml和3.16×1010IU/ml，病毒比活性为3.34%。重组病毒可在SKOV3细胞中复制，对SKOV3细胞的感染效率随MOI的增加而增加，可致细胞感染率达90%以上的最小MOI为50 IU/cell。5FC或GCV明显抑制感染细胞的生长，且存在明显的剂量依赖效应。两种重组腺病毒/前药系统的抑瘤作用比较显示，rAdE1ACDglyTK组细胞生长抑制率\[(16.57±1.59)%\]显著高于rAdCDglyTK组\[(10.44±1.80)%，P<0.01\]；与IC50的5FC和GCV双药联合应用，其抑制率达(71.68±2.63)%，显著高于rAdCDglyTK/5FC+GCV组的(63.64±2.91)%（P<0.01）。结论: 重组腺病毒rAdE1ACDglyTK与酶前体药物联合应用对卵巢癌SKOV3细胞具有显著的抑制作用，其效果优于重组复制缺陷型腺病毒rAdCDglyTK。     

紫杉醇和顺铂对EGFR野生型与突变型肺腺癌细胞的杀伤作用比较及其分子机制

目的 探讨化疗相关基因表达水平与EGFR突变状态的关系及其对化疗疗效的影响。方法 采用MTT法分别检测紫杉醇、顺铂对A549与HCC827细胞的24 h半数抑制浓度（IC50）;比较紫杉醇、顺铂单独及联合作用于A549与HCC827细胞杀伤作用的差异;荧光定量PCR法检测紫杉醇、顺铂作用前后A549与HCC827细胞化疗相关基因（BRCA1、ERCC1、TUBB3）的表达水平。结果 紫杉醇对A549与HCC827细胞24 h的IC50分别为100和70 μg/ml,顺铂对两种细胞的IC50分别为40和50 μg/ml。单独作用时,紫杉醇对HCC827细胞的杀伤作用明显高于A549细胞（P<0.05）;顺铂对A549细胞的杀伤作用明显高于HCC827细胞（P<0.05）;紫杉醇和顺铂联合作用对A549和HCC827细胞的杀伤作用差异无统计学意义（P>0.05）,且均表现为单纯相加作用（Q=1.03, 1.06）。HCC827细胞中BRCA1基因表达明显高于A549细胞（P<0.05）;顺铂作用于A549细胞后,其ERCC1基因表达明显升高（P<0.05）。结论 紫杉醇、顺铂单独作用对EGFR野生型与突变型肺腺癌细胞杀伤作用有明显差异,而两者联合无明显差异,可能与BRCA1基因的表达有关;EGFR野生型比突变型肺腺癌细胞更容易对顺铂出现耐药。     

CIK、DC-CIK细胞对神经母细胞瘤细胞杀伤作用的研究

目的：研究细胞因子诱导的杀伤细胞（ CIK）与树突状细胞（ DC）共培养后对神经母细胞瘤（ neuro-blastoma,NB）细胞株的杀伤作用。方法：取健康人和肿瘤患者外周血单个核细胞（ PBMC）,加入不同的细胞因子分别诱导出DC和CIK细胞,用流式细胞术测定诱导培养前后DC和CIK细胞的表型,MTT法测定不同组CIK细胞对NB细胞株的杀伤活性。结果：流式细胞仪检测健康人PBMC培养后CD3＋CD56＋淋巴细胞百分比以及对NB细胞株的杀伤活性均显著高于肿瘤患者（ P〈0.05）。此外,与单纯CIK细胞相比,DC-CIK细胞具有更强的杀伤NB细胞株的活性（ P〈0.05）。结论：DC-CIK细胞是一种细胞毒作用高于单纯CIK细胞的免疫活性细胞。健康人和肿瘤患者的PBMC经诱导培养获得的CIK细胞有显著差别,为临床进一步提高CIK细胞的治疗效果提供了实验依据。     

Differences in inducibility of morphological differentiation of parental cells, selected variant cells and cells from spontaneous metastases of mouse neuroblastoma cells

The in vitro sensitivities to differentiating agents of a murine neuroblastoma cell line (N18) and a selected variant cell line (N18-LM5) were examined. In addition, the sensitivities to differentiating agents of cells from spontaneous metastases produced by N18 cells were examined. When N18 cells (1 X 10(5) cells/mouse) were injected into the lateral tail vein of syngeneic A/J mice, only a few metastatic nodules formed in the liver and lung, while similar injection of N18-LM5 cells produced larger numbers of metastatic nodules. Exposure of N18 cells to differentiating agents, such as dibutyryl cyclic 3':5'-AMP (db-cAMP), prostaglandin E1, and dexamethasone, resulted in induction of differentiation in terms of neurite extension. N18-LM5 cells responded to differentiating agents to a greater extent than N18 cells, and most of the cells extended neurites when they were exposed to 1 mM db-cAMP for 3 days. On the other hand, not all cell lines from spontaneous metastases produced by N18 cells responded to db-cAMP. These results suggest that the colonizing potential of neuroblastoma cells is not necessarily correlated with loss of responsiveness to differentiating agents and that various spontaneous metastases show heterogeneity in responsiveness to differentiating agents.     

Melanoma cells interfere with the interaction of dendritic cells with NK/LAK cells

Dendritic cells (DCs) and natural killer (NK) cells are key players at the interface between innate resistance and acquired immunity. NK cells can induce DC maturation, a differentiation process whereby DCs respond to a environmental stimulus and acquire the ability of eliciting adaptive immunity. Conversely, maturing DCs promote NK functions in vivo and in vitro. This interplay has important consequences on the immune response to pathogens and possibly to neoplastic cells. Here, we show that B16 melanoma cells actively modulate the interaction between DCs derived from bone marrow precursors and NK/LAK cells propagated from the spleen of C57BL/6 mice. DCs increased in a dose-dependent manner the ability of NK/LAK cells to kill melanoma cells and to produce cytokines. This activatory cross-talk entailed the production of IL-18 by DCs and of IFN-gamma by NK/LAK cells. Melanoma cells were not a passive target of NK activity; they regulated the outcome of the interaction between DCs and NK/LAK cells, inhibiting the in vitro production of cytokines as effectively as the genetic deletion of IL-18 or IFN-gamma. Interference with the NK/DC interaction possibly represents a mechanism used by growing tumors to evade the immune response.     

Immunogenic reactivity of CTLs induced by electrofusion cells of human dendritic cells and gastric cancer cells

In tumor immunotherapy, there were several reports of attempts to induce anti-tumor immunity by fusion hybrid cells generated with dendritic and tumor cells. One of them reported that vaccination of hybrid cells resulted in a remarkable reduction of tumor cells in a lab mouse experiment. In our study, fusion cells were generated successfully with human matured dendritic and human gastric cancer cells by electrofusion technique and employed to induce CTLs. The evaluated fusion rate was 47.8% by FACS analysis. We tried to induce CTLs by co culture of effector and stimulator cells in the presence of IL-2, IL-7 and IL-12 for 4 weeks. Although it was not statistically significant in tumor cytotoxic assay, effector cells induced by the fusion cells as stimulator cells showed a few cytotoxic responses in an immunological tumor specific manner. Our data suggest that fusion hybrid cells may facilitate stimulation and expansion of tumor-specific T cells, but further investigation is required for clinical application of fusion cells in adoptive immunotherapy.     

Marker profile of mesothelial cells versus ovarian carcinoma cells

We investigated the marker profile of human ascitic and cultured mesothelial cells, and compared it to that of ovarian carcinoma cells which are related in terms of their histogenesis, unrelated colon carcinomas being used as reference. Mesothelial and ovarian carcinoma cells could not be distinguished by (intermediate) filament typing, using monoclonal antibodies (MAbs) to keratins, vimentins and desmins. Colon carcinomas differed from mesothelial cells and ovarian carcinomas by the absence of keratin-7 filaments. The epithelial marker BW 495/36 was completely negative on mesothelial cells and positive on all ovarian and colon carcinoma cells. While CEA was found on about 85% of all colon carcinomas, CEA expression on mesothelial cells and ovarian carcinoma cells was below 20%. The ovarian carcinoma markers (OV-TL 3, OV-TL 10, OC 125, MOV 18) were strongly positive on ovarian carcinomas and negative on colon carcinomas (or limited to traces of immunofluorescence on some samples). Although the mesothelial cells showed weak or negative reactivity with these markers, OC 125 antigen was found by immunoelectron microscopy on the surface of cultured mesothelial cells, and was shed in the culture supernatant at concentrations of 50, 28, and 25 CA 125 U/ml/10(4) positive cells. This suggests that mesothelial cells may be responsible for the synthesis of CA 125 in ascitic fluid. The data indicate that ovarian carcinomas, mesothelial cells and colon carcinomas can be distinguished using a combination of anti-keratin antibodies with BW 495/36 and anti-ovarian carcinoma markers.     

Radial glia cells are candidate stem cells of ependymoma

Tumors of the same histologic type often comprise clinically and molecularly distinct subgroups; however, the etiology of these subgroups is unknown. Here, we report that histologically identical, but genetically distinct, ependymomas exhibit patterns of gene expression that recapitulate those of radial glia cells in the corresponding region of the central nervous system. Cancer stem cells isolated from ependymomas displayed a radial glia phenotype and formed tumors when orthotopically transplanted in mice. These findings identify restricted populations of radial glia cells as candidate stem cells of the different subgroups of ependymoma, and they support a general hypothesis that subgroups of the same histologic tumor type are generated by different populations of progenitor cells in the tissues of origin.     

Can cancer cells transform normal host cells into malignant cells?

A human prostate tumour cell line, LNCaP C4-2, when injected into athymic male nude mice, produced tumours containing: (1) only human cancer cells similar to those injected; (2) only murine stromal cells containing abnormal chromosome constitutions; or (3) both human prostate cancer cells similar to those injected and the transformed murine stromal cells with altered chromosome constitutions. Karyotypic analysis of murine metaphases from all the host-derived tumours showed mostly pseudodiploid chromosome constitutions, with multiple copies (amplification) of mouse chromosome 15 and the absence of a typical Y chromosome. Fluorescence in situ hybridization analysis of these murine cells, using a biotin-labelled total human DNA painting probe, further demonstrated the absence of human DNA and the presence of only mouse metaphase and interphase cells in these transformed stromal cells. These results suggest that cancer cells are capable of inducing neoplastic transformation in stromal cells of the host organ by some, as yet unknown, epigenetic mechanism(s).     

α-半乳糖神经酰胺负载DC促进CIK细胞对肝癌细胞的杀伤效应

目的:研究负载α-半乳糖神经酰胺(alpha-galactosylceramide,α-GalCer)的DC功能与成熟度的改变情况,探讨α-GalCer-DC与CIK共培养对CIK细胞表型、增殖活性及杀伤肝癌细胞效率的影响.方法:采用密度梯度离心法从人外周血中分离出单个核细胞,悬浮细胞诱导培养CIK细胞,贴壁细胞诱导培养DC;流式细胞仪检测α-GalCer负载DC的表型,Real-time PCR检测DC相关基因的mRNA表达改变.将α-GalCer-DC与CIK细胞共培养,流式细胞仪检测DC与CIK细胞表面标志物;锥虫蓝染色法检测CIK细胞的增殖倍数;Real-time PCR检测CIK细胞功能相关基因的表达情况;CCK-8试剂盒检测α-GalCer-DC对CIK杀伤HepG2细胞的影响.结果:经过多种细胞因子诱导,可获得CIK细胞和成熟的DC;α-GalCer负载可促进DC成熟,DC表面标志物CD80、CD86、CD83和CD11c的阳性率均升高(P＜0.05或P＜0.01),表面趋化因子受体CCR-7、IL-12、IL-10的mRNA水平升高(P＜0.05).α-GalCer-DC与CIK共培养,可显著提高CIK细胞CD3+ CD56+的表达和增殖活性(P＜0.05或P＜0.01),并显著提高INF-γ、IL-12、穿孔素和颗粒酶素B的mRNA表达水平(P＜0.05或P＜0.01);CIK、DC-CIK、α-GalCer-DC-CIK细胞对HepG2细胞的杀伤作用随效靶比的升高而增强,在同一效靶比时α-GalCer-DC-CIK细胞对靶细胞的杀伤作用最强(P＜0.05).结论:α-GalCer负载可促进DC成熟,α-GalCer-DC与CIK共培养能促进后者增殖和成熟,能显著增强其对肝癌细胞的杀伤活性,为DC-CIK在肿瘤免疫治疗中的应用提供了实验依据.     

Chemoresistant colorectal cancer cells and cancer stem cells mediate growth and survival of bystander cells

Background:

Recent studies suggest that cancer stem cells (CSCs) mediate chemoresistance, but interestingly, only a small percentage of cells in a resistant tumour are CSCs; this suggests that non-CSCs survive by other means. We hypothesised that chemoresistant colorectal cancer (CRC) cells generate soluble factors that enhance survival of chemonaive tumour cells.

Methods:

Chemoresistant CRC cells were generated by serial passage in oxaliplatin (Ox cells). Conditioned media (CM) was collected from parental and oxaliplatin-resistant (OxR) cells. CRC cells were treated with CM and growth and survival were assessed. Tumour growth rates were determined in nude mice after cells were treated with CM. Mass spectrometry (MS) identified proteins in CM. Reverse phase protein microarray assays determined signalling effects of CM in parental cells.

Results:

Oxaliplatin-resistant CM increased survival of chemo-naive cells. CSC CM also increased growth of parental cells. Parental and OxR mixed tumours grew larger than tumours composed of parental or OxR cells alone. Mass spectrometry detected unique survival-promoting factors in OxR CM compared with parental CM. Cells treated with OxR CM demonstrated early phosphorylation of EGFR and MEK1, with later upregulation of total Akt .We identified progranulin as a potential mediator of chemoresistance.

Conclusion:

Chemoresistant tumour cells and CSCs may promote resistance through soluble factors that mediate survival in otherwise chemosensitive tumour cells.