负载抗原的DC与CIK共培养对耐药乳腺癌细胞的杀伤作用

目的:观察负载抗原的树突状细胞(dendritic cell,DC)与细胞因子诱导的杀伤细胞(cytokine-induced killer cell,CIK)对高表达P-糖蛋白(P-glycoprotein,P-gp)的多药耐药(multidrug resistance,MDR)人乳腺癌MCF-7/ADR细胞的杀伤作用。方法:提取健康人外周血单个核细胞,常规诱导出CIK及DC;制备MCF-7/ADR细胞冻融抗原后冲击DC,并与CIK共培养作为实验组(冻融物DC-CIK组),未负载抗原的DC与CIK共培养作为对照组(DC-CIK组),同时设单独培养的CIK组或DC组作为空白对照组。流式细胞术分析细胞的表型及P-gp表达,ELISA法测定细胞上清中IL-12、IFN-γ水平,MTT法检测对MCF-7/ADR及MCF-7细胞株的杀伤活性。结果:冻融物DC-CIK组、DC-CIK组细胞增殖活性均大于CIK组(P<0.05)。冻融物DC-CIK组对MCF-7/ADR、MCF-7细胞的杀伤活性在效靶比10∶1、20∶1、40∶1时分别为(44.29±1.39)%、(58.24±3.52)%、(68.9±2.83)%和(33.51±2.18)、(40.43±2.3)%、(44.62±1.19)%,均高于DC-CIK组及CIK组(P<0.05);冻融物DC-CIK组对MCF-7/ADR耐药细胞株的杀伤活性高于非耐药细胞株MCF7(P<0.05);而对于非耐药株MCF-7细胞的杀伤活性,冻融物DC-CIK组和DC-CIK组之间差异无统计学意义(P>0.05)。结论:DC与CIK共培养细胞增殖活性和细胞毒活性均强于CIK,经冻融抗原冲击的DC与CIK共培养可以显著提高对MCF-7/ADR耐药细胞株的杀伤活性。     

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。     

TIL与脐血LAK细胞对肿瘤细胞的杀伤作用

肿瘤浸润性淋巴细胞（TIL）和脐血淋巴因子激活杀伤（LAK）细胞在含重组白细胞介素2（rlL－2）的完全培养基中可大量扩增培养，体外培养肿瘤组织的TIL及癌性腹水的淋巴细胞对自体瘤细胞（黑色素瘤，卵巢囊腺癌，大网膜转移性腺癌及癌性腹水）有明显的杀伤作用，胎儿脐血中分离培养的LAK细胞对肿瘤细胞也有较强的杀伤作用，在效靶比例分别为20：1和40：1时，对肿瘤细胞的杀伤作用后者均明显高于前者（P=0.025和P=0．01），且TIL对自体瘤细胞的杀伤作用明显高于脐血LAK细胞（P＜0．01），为临床应用TIL和脐血细胞提供实验依据。     

脐血和合并乙型肝炎病毒感染肝癌患者来源CIK细胞的增殖及杀伤活性

目的：观察脐血和乙型肝炎病毒感染肝癌患者外周血诱导的CIK细胞的体外增殖能力及杀伤活性的特点。方法： 分别采集解放军第105医院妇产科住院的健康产妇剖宫产胎儿的脐带血5份（A组）、感染科住院的合并乙型肝炎病毒感染的原发性肝癌患者自体外周血15份（B组）、无乙型肝炎病毒感染的原发性肝癌患者自体外周血5份（C组）,采用Ficoll两步分离法分离出单个核细胞,细胞因子诱导培养成细胞因子诱导的杀伤(ClK)细胞,流式细胞仪检测CIK细胞的免疫表型CD3＼CD8＼CD16＼CD56,MIT法测定其对白血病K562细胞的杀伤活性。结果：体外培养15 d时, C组CIK细胞体外增殖倍数与A组相似\[（577.24±202.4）vs（600.93±249.1）倍, P >0.05\],该两组均显著高于B组\[（385.16±117.3）倍, P <0.05\];A组CIK细胞杀伤活性显著高于B组和C组\[（77.3±5.1）% vs （44.1±3.4）%、（54.5±3.7）%, P <0.01\]。结论： 脐血来源的CIK细胞体外增殖快、杀伤活性强;合并有乙型肝炎病毒感染的肝癌患者CIK细胞体外增殖能力和杀伤能力明显降低,不宜接受自体CIK细胞移植。     

脐带血和乳腺癌患者外周血来源CIK细胞PD-1表达及对MCF-7细胞杀伤

目的：探讨脐带血和乳腺癌患者外周静脉血来源的CIK细胞程序性死亡分子-1（programmed cell death-1,PD-1）的表达及其对乳腺癌MCF-7细胞的杀伤作用。方法: 采集2015年6月至2015年12月在解放军第105医院住院的健康产妇脐带血和乳腺癌患者外周静脉血各5例,分离PBMC,体外培养、扩增CIK细胞。流式细胞术检测不同时间节点两种来源的CIK细胞PD-1表达情况,分别取培养第7、14、21、28天的CIK细胞与MCF-7细胞共培养,细胞计数（CCK-8）法测定CIK细胞对MCF-7细胞的杀伤率, 吖啶橙-溴化乙啶双染(AOEB)法观察共培养后CIK细胞凋亡变化,流式细胞术检测CIK细胞凋亡率。结果: 随着体外培养时间的延长,脐带血和乳腺癌患者静脉血来源的CIK细胞PD-1表达率均逐渐上升,第14天时脐带血组CIK细胞PD-1表达率低于乳腺癌组\[（38.42±4.76）% vs （50.54±3.50）%,P<0.05\],至第21天后两组PD-1表达率均升高,但差异无统计学意义（P>0.05）。培养第7、14、21、28天两组CIK细胞对MCF-7细胞的杀伤率分别为(18.54±354）%和（21.74±427）%、（71.86±16.86）% 和（58.78±24.25）%、（44.32±26.87）%和（43.96±26.04）%、（43.24± 24.27）%和（40.28±23.69）%,以培养第14天的脐带血来源的CIK细胞的杀伤活性最强（P<0.05）。分析发现,两种来源的CIK细胞PD-1表达水平与CIK细胞的凋亡率呈正相关(r=0.971,r=0.900, 均P<0.01）,与杀伤率呈负相关（r=-0.865,r=-0885, 均P<0.01）。结论: 活化的CIK细胞表面高表达PD-1,脐带血较乳腺癌患者静脉血来源的CIK细胞表面PD-1水平低;培养第14天的CIK细胞凋亡率均较低,其杀伤能力更强。     

抗EGFR-抗CD3双功能抗体介导CIK细胞、LAK细胞或PBLS细胞对胃癌荷瘤裸鼠治疗效果的比较

 目的　比较抗EGFR-抗CD3双功能抗体在体内条件下介导CIK细胞、LAK细胞及人类PBLS细胞三类不同的免疫效应细胞对胃癌细胞的杀伤能力,为临床应用该抗体治疗胃癌的细胞选择提供实验指导。方法　采用化学耦联法合成的抗EGFR-抗CD3双功能抗体分别与CIK细胞、LAK细胞及人类PBLS细胞联合经尾静脉注入SGC7901胃癌细胞移植瘤小鼠体内,同时以等量0.9 % NaCl溶液尾静脉注射建立对照,治疗后检测在体情况下4组对胃癌细胞的杀伤能力,进行组间比较。结果　抗EGFR-抗CD3双功能抗体介导CIK细胞治疗组小鼠肿瘤抑制率为（64.9±7.7）%,显著高于抗EGFR-抗CD3双功能抗体介导LAK细胞及人类PBLS细胞组的（43.5±8.2）%和（39.7±6.5）%（均P＜0.05）,治疗结束时平均肿瘤质量为（473.9±37.7）mg,显著低于其他两组的（764.6±88.3）mg和（829.1±104.4）mg（均P＜0.05）。结论　初步的裸鼠模型治疗实验显示由抗EGFR-抗CD3双功能抗体介导的CIK细胞在体内条件下对胃癌治疗作用优于其他常用的免疫效应细胞。     

CIK细胞联合顺铂对卵巢癌耐药细胞SKOV3/CDDP的杀伤作用

目的：探讨细胞因子诱导的杀伤（cytokine-induced killer,CIK）细胞联合顺铂对卵巢癌顺铂耐药细胞SKOV3/CD-DP小鼠腹腔移植模型的体内外杀伤作用。方法：取正常人的外周血单个核细胞（PBMC）,加入IFN-γ、IL-2、CD3McAb和IL-1,体外诱导成CIK细胞。用MTT法测定CIK细胞、顺铂及两者联合对人卵巢癌细胞株SKOV3/CDDP细胞的杀伤活性。在SCID小鼠腹腔内接种卵巢癌SKOV3/CDDP细胞,观察CIK细胞对荷瘤鼠的体内抑瘤作用,并用RIA法检测各组血清中CA125的含量。结果：顺铂对SKOV3/CDDP细胞的IC50为315.5μg/ml,较其对SKOV3细胞的IC50（85μg/ml）增加了4倍;CIK细胞对SKOV3与SKOV3/CDDP细胞的杀伤活性均在48h最高,且随效靶比的增高而增强,其对SKOV3/CDDP细胞的杀伤活性明显高于SKOV3（P〈0.05）。仅25μg/ml的顺铂即可使效靶比为12.5∶1组中的联合杀伤率比单纯顺铂组增加5.8倍,比单纯加CIK（相同效靶比）细胞杀伤率增加2.3倍,且随效靶细胞比值和顺铂浓度的升高呈依赖性增加。与对照组相比,CIK组与CIK＋顺铂组均能显著抑制癌性结节的生长,抑瘤率达100%,且3组SCID鼠血中CA125值有显著性差异（P〈0.05）。结论：正常人CIK细胞联合顺铂对清除晚期卵巢癌表达耐药蛋白的腹腔种植转移病灶可能会有较好的效果,并有希望成为前景良好的综合治疗方案。     

顺铂和氟尿嘧啶增强食管癌细胞NKG2D配体的表达及CIK细胞的杀伤活性

目的研究顺铂(Cisplatin,DDP)和氟尿嘧啶（5-fluorouracil,5-Fu）对人食管癌EC9706 细胞NKG2D配体表达及CIK细胞杀伤活性的影响。方法MTT法测定DDP、5-Fu的50%抑制浓度（IC50）;以1/2 IC₅₀浓度DDP、5-Fu作用EC9706细胞72 h,RT-PCR检测DNA损伤修复系统相关信号分子的表达。流式细胞仪检测DDP、5-Fu作用前、后EC9706细胞NKG2D配体的表达。乳酸脱氢酶释放法检测效靶比20∶1时,CIK细胞对DDP、5-Fu作用前、后EC9706细胞的杀伤活性。结果DDP、5-Fu的IC₅₀分别为5 μg/ml、10 μg/ml。DDP、5-Fu可上调DNA损伤修复系统相关信号分子mRNA的表达。DDP与 EC9706细胞共孵育72 h后,EC9706细胞MICA、MICB、ULBP2、ULBP3表达较DDP作用前明显增强（P<0.05）,ULBP1无明显变化（P>0.05）。5-Fu与 EC9706细胞共孵育72 h后,EC9706细胞MICA、ULBP2、ULBP3表达明显增强（P<0.05）,MICB、ULBP1无明显变化（P>0.05）。效靶比20∶1时,CIK细胞对EC9706细胞的杀伤活性为（37.08±0.62）%,CIK细胞对1/2 IC₅₀ DDP、1/2 IC₅₀ 5-Fu作用后的EC9706细胞杀伤活性分别为（59.33±2.10）%、（52.44±0.97）%,与作用前相比差异均有统计学意义（P<0.05）。结论DDP、5-Fu通过激活DNA损伤修复系统相关信号分子,提高EC9706细胞NKG2D配体的表达,从而增强EC9706细胞对CIK细胞杀伤的敏感度。     

IL-15上调NKG2D表达对CIK细胞杀伤活性的增强效应

目的观察IL-15对细胞因子诱导的杀伤细胞( Cytokine-induced killer cells,CIK)NKG2D受体表达及其对食管癌EC9706细胞杀伤活性的影响。方法体外分离外周血单个核细胞,分为两组。对照组：干扰素-γ、白细胞介素-2、CD3单抗诱导培养CIK细胞。IL-15组：加用IL-15培养。流式细胞仪检测细胞免疫表型及CD3+细胞、CD56+细胞表面NKG2D的表达,LDH法测定第14天细胞在效靶比20∶1、30∶1时对EC9706细胞的杀伤活性;效靶比30∶1时,观察NKG2D单抗封闭细胞表面NKG2D分子后对两组细胞杀伤活性的影响。结果随着培养时间的延长,CIK群体细胞及CD56+细胞表面NKG2D表达逐渐增强,IL-15组与对照组相比差异有统计学意义(P＜0.05);效靶比20∶1、30∶1时,IL-15组细胞对EC9706细胞的杀伤活性均较对照组明显增强,差异均有统计学意义(P＜0.05);效靶比30∶1时NKG2D单抗封闭CIK细胞表面NKG2D分子后,对照组细胞、IL-15组细胞对EC9706细胞的杀伤活性均较阻断前明显下降,差异均有统计学意义 (P＜0.05)。结论IL-15上调CIK细胞表面NKG2D分子表达,增强CIK细胞对EC9706细胞的杀伤活性,CIK细胞通过NKG2D发挥作用。     

负载肺癌干细胞膜微粒的 DC －CIK 对耐药肺癌细胞的靶向杀伤作用及对肺癌干细胞凋亡的影响

目的：探讨负载肺癌干细胞膜微粒的 DC －CIK 细胞对 EGFR －TKI 耐药肺癌细胞的杀伤作用及对肺癌干细胞凋亡的影响机制。方法：无血清悬浮细胞培养法富集 EGFR －TKI 耐药肺癌细胞 A549、H292干细胞样细胞,RT －PCR 检测干细胞标志物,裸鼠成瘤实验鉴定致瘤性。超滤和差速离心法获取肺癌干细胞膜微粒。流式细胞术分别测定共孵育组和常规培养组 DC 成熟标志 CD86和 CD83,测定两组 DC －CIK 细胞表型CD3＋、CD3＋CD8＋、CD3＋CD56＋、CD3＋CD4＋;形态观察及 MTT 法分别测定不同效靶比两组 DC －CIK 对A549、H292的杀伤效应;ELISA 法分别检测两组 DC －CIK 上清中 IL －2、IFN －γ、TNF －α分泌水平;流式细胞术分别测定两组 DC －CIK 对肺癌干细胞凋亡的影响。结果：富集培养获得的 EGFR －TKI 耐药肺癌干细胞样细胞高表达干细胞标志物 Sox2和 Oct4,并具较强裸鼠致瘤性。负载膜微粒的 DC 成熟标志 CD86和 CD83较常规 DC 表达显著升高;负载膜微粒的 DC －CIK 较常规 DC －CIK 细胞表型 CD3＋、CD3＋CD8＋、CD3＋CD56＋、CD3＋CD4＋升高,对 EGFR －TKI 耐药肺癌细胞杀伤效应高于常规 DC －CIK,并具有显著提高的靶向趋向性;负载膜微粒的 DC －CIK 分泌因子对 EGFR －TKI 耐药肺癌干细胞样细胞凋亡的影响与常规 DC －CIK 不同。结论：与常规培养 DC －CIK 相比,负载膜微粒的 DC －CIK 活性提高,对 EGFR －TKI 耐药肺癌细胞的体外特异靶向杀伤效应显著提高。细胞分泌因子可显著上调耐药肺癌干细胞的细胞凋亡率。     

两种来源的树突状细胞体外培养及免疫学特征比较

目的对脐血(UCB)和外周血(PB)体外诱导培养成树突状细胞(DC),并进行免疫学特征的比较,探索更好的DC来源。方法UCB和正常成人PB各10例为UCB组和PB组,分离出单个核细胞后,分别加入粒细胞巨细胞集落刺激因子(GM-CSF),白介素4(IL-4)和肿瘤坏死因子(TNF-α)培养基中,培养12d,每天用光镜观察细胞形态,于第3、6d和10d,用流式细胞仪进行CD11c、CD80、CD86及HLA-DR表型分析,于第10d透射电镜和扫描电镜观察细胞形态,并在第12d与淋巴细胞混合培养,对比两种源性DC激发淋巴细胞增殖能力。结果两者均诱导出具有典型形态的DC,UCB组诱导的DC细胞数量明显多于外周血诱导的细胞数(P<0.01)。激发淋巴细胞增殖能力,两者无区别(P>0.05)。结论UCB和PB中的MNs均可在体外诱导为成熟DC,UCB诱导的DC细胞数高于PB,两者激发淋巴细胞增殖能力相同,故UCB有可能成为免疫治疗DC的丰富来源。     

Flow cytometric identification of cell surface markers on cultured human colonic cell lines using monoclonal antibodies

Background. The expression of tumor-associated cell surface antigens is a reflection of the state of cell differentiation of tumor cells in culture. Method. Monoclonal antibodies (MoAbs) against the tumor-associated antigens carcinoembryonic antigen (CEA) and CA19-9 and the extracellular matrix protein CD44 were used to label the cell surface of human colonic cells in culture. The binding of each antibody to its respective antigen was measured by fluorescence-activated flow cytometry and expressed as a percentage of positive cells. Results. The human colon adenocarcinoma cell (HCAC) line, LS-180, showed strong binding with CEA (81%), CA 19-9 (87%), and CD44 (83%). LS-174t cells, a trypsinized variant of LS-180 cells, showed less binding with CEA (66%) and CA 19-9 (49%), but no binding with CD44. With cells from HCAC line HT-29, antigen expression was highly variable for CEA (13% ± 18) and CD44 (31% ± 35) but was consistently positive for CA19-9 (33% ± 13). The expression of CEA in the Caco-2 cell line was weak (24%), whereas there was no expression of CA19-9 and CD44. Normal human colon fibroblast cells (CCD-18Co) did not recognize the monoclonal antibodies to CEA or CA 19-9, but were strongly positive with the CD44 antibody (97%). Conclusions. These results support the concept that the expression of the tumor associated markers CEA and CA19-9 and the cell surface marker CD44 on human colonic cell lines varies with the degree of cellular differentiation. Carcinoembryonic antigen and/or CA19-9 were expressed in all four human colon adenocarcinoma cell lines, but not in the normal colon fibroblast cells (CCD-18Co). Using these two MoAbs appeared to be a more reliable measure of the state of differentiation of human colon adenocarcinoma cells. Cancer 1995; 75:195–200.     

脐血来源的DC增强NK和NKT细胞对白血病细胞的杀伤作用

目的:探讨脐血来源的树突状细胞(DC)增强自然杀伤(NK)细胞和自然杀伤性T(NKT)细胞对白血病细胞杀伤作用的影响机制.方法:提取脐带血单个核细胞(PBMC),体外诱导培养DC、NK和NKT淋巴细胞.以人髓性白血病K-562细胞作为靶细胞,检测NK、NKT、NK+DC和NKT+DC对靶细胞的杀伤效应.检测共培养上清液...     

Phase I clinical study applying autologous immunological effector cells transfected with the interleukin-2 gene in patients with metastatic renal cancer, colorectal cancer and lymphoma

Natural killer-like T lymphocytes termed cytokine-induced killer (CIK) cells have been shown to eradicate established tumours in a severe combined immune deficient (SCID) mouse/human lymphoma model. Recently, we demonstrated that CIK cells transfected with cytokine genes possess an improved proliferation rate and a significantly higher cytotoxic activity as compared to non-transfected cells. Here, in a phase I clinical protocol, autologous CIK cells were generated from peripheral blood obtained by leukapheresis in patients with metastatic renal cell carcinoma, colorectal carcinoma and lymphoma. CIK cells were transfected with a plasmid containing the interleukin-2 (IL-2) gene via electroporation. Transfected cells generated IL-2 in the range of 330-1800 pg 10(-6) cells 24 h(-1) with a mean of 836 pg 10(-6) cells 24 h(-1). Ten patients received 1-5 intravenous infusions of IL-2-transfected CIK cells; five infusions with transfected CIK cells were given. In addition, the same patients received five infusions with untransfected CIK cells for control reasons. In three patients, WHO grade 2 fever was observed. Based on polymerase chain reaction of peripheral blood transfected cells could be detected for up to 2 weeks after infusion. There was a significant increase in serum levels of interferon gamma (IFN-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor beta (TGF-beta) during treatment. Interestingly, there was also an increase in CD3+ lymphocytes in the blood of patients during therapy. In accordance, a partial increase in cytotoxic activity in peripheral blood lymphocytes (PBLs) was documented when patient samples before and after therapy were compared. Concerning clinical outcome, six patients remained in progressive disease, three patients showed no change by treatment, and one patient with lymphoma developed a complete response. In conclusion, we were able to demonstrate that CIK cells transfected with the IL-2 gene can be administered without major side-effects and are promising for future therapeutic trials.     

肺癌患者外周血血小板活化标志物水平及其临床意义

Objective： To investigate the relationship between the activation markers of platelets and the lung cancer. Methods： Based on international stages of lung cancer in 1997, lung cancer patients of 120 cases diagnosed by pathology as well as with operation indication were selected as the experimental group. During the process of experiment, 60 cases concluded as healthy in the physical examination were chosen as control group. The activation markers of platelets were detected by FCM method. The experimental result would be processed by SPSS 11.5. Results： The level of activation markers of platelets in peripheral blood of lung cancer patients was significantly higher than those healthy people （P 〈 0.01）. The level of activation markers of platelets in peripheral blood of lung cancer patients on the seventh postoperative day was significantly lower than that before operation and on the first postoperative day （P 〈 0.01）. The level of activation markers of platelets in peripheral blood of lung cancer patients was closely related to the size of the primary tumor, lymph node status and stages, but not to the grade of cell differentiation, type of tumor, age, sex of the patients （P 〉 0.05）. Conclusion： Elevation of the level of activation markers of platelets in peripheral blood exists in lung cancer patients and the levels of activation marker of platelets plays an important role in tumor growth and lymphatic metastasis. The levels of activation markers of platelets maybe a predictor for prognosis.     

石斛多糖增强脐带血和肿瘤病人外周血LAK细胞体外杀伤作用的研究

目的：探讨石斛多糖（ｄｅｎｄｒｏｂｉｕｍ ｃａｎｄｉｄｕｍ ｐｏｌｙｓａｃｃｈａｒｉｄｅ，ＤＣＰ）与ｒＩＬ－２联合应用对脐带血ＬＡＫ细胞（ＣＢ－ＬＡＫ）和肿瘤病人外周血ＬＡＫ细胞（ＰＢ－ＬＡＫ）体外杀伤肿瘤细胞作用的影响。方法：应用＾３Ｈ－ＴｄＲ释放法测定ＣＢ－ＬＡＫ和ＰＢ－ＬＡＫ细胞的杀伤活性。结果：①ｒＩＬ－２（５００ｕ／ｍｌ）能明显提高ＣＢ－ＬＡＫ对Ｒａｊｉ细胞的杀伤活性，０ｈ的ＣＢ－ＬＡＫ活性为１３．６３％，ｒＩＬ－２（５００ｕ／ｍｌ）诱导４８、７２、９６、１２０ｈ后，ＣＢ－ＬＡＫ活性分别提高至２２．２８％、２６．２３％、２８．５５％和２５．７６％（Ｐ〈０．０１）；②ＤＣＰ（１００ｍｇ／Ｌ或４００ｍｇ／Ｌ）与ｒＩＬ－２（５００ｕ／ｍｌ）联合诱导ＣＢ－ＬＡＫ的活性，比单纯ｒＩＬ－２（５００ｕ／ｍｌ）的作用     

舒尼替尼诱导耐药鼻咽癌CNE2/DDP细胞表达NKG2DLs的机制

目的：初步探讨舒尼替尼诱导高、低表达ABCG2(ATPbinding cassette superfamily G member 2)分子的耐药鼻咽癌CNE2/DDP细胞（简称ABCG2highCNE2/DDP细胞、ABCG2lowCNE2/DDP细胞）中NKG2D配体（natural killer group 2 member D ligands,NKG2DLs）表达的分子机制。方法：Caspase8活化试剂盒和线粒体膜电位法分别检测NK细胞处理后ABCG2highCNE2/DDP细胞和ABCG2lowCNE2/DDP细胞caspase8活化水平和线粒体膜电位。RTPCR检测舒尼替尼处理前后两种CNE2/DDP细胞DNA损伤修复系统相关信号分子mRNA的表达。结果：CNE2/DDP细胞+NK细胞组中两种CNE2/DDP细胞caspase8活性均明显增强;舒尼替尼处理后的ABCG2low CNE2/DDP细胞+NK细胞组和ABCG2highCNE2/DDP细胞＋NK细胞组中caspase8的活性是处理前的2～2.5倍（P<0.01）。舒尼替尼预处理后,CNE2/DDP细胞和NK细胞共培养体系中两种CNE2/DDP细胞的线粒体膜电位分别为(76.58±2.32)%和(73.11±1.93)%,较舒尼替尼处理前明显降低（P<0.05）。舒尼替尼可上调两种CNE2/DDP细胞中ATR、CHK1和CHK2 mRNA的表达,并诱导P53和NFκB mRNA的表达。结论：舒尼替尼可能通过激活DNA损伤修复系统相关信号分子和NFκB的表达,诱导耐药鼻咽癌CNE2/DDP细胞NKG2DLs的表达,同时经由死亡受体信号通路和线粒体信号通路增强NK细胞诱导的肿瘤细胞凋亡。     

DC瘤苗联合CIK姑息治疗广泛期小细胞肺癌的临床分析

目的:探讨负载同源肿瘤抗原(antigen,Ag)的树突状细胞(denritic cells,DC)联合细胞因子诱导的杀伤细胞(cytokine-induced killer,CIK)对广泛期小细胞肺癌(small cell lung cancer,SCLC)患者的临床姑息疗效.方法:选择120例诊断明确的广泛期SCLC患者(不再适宜行手术或放化疗),随机分为2组:对照组60例,接受内科姑息治疗;免疫组60例,除接受相同治疗外,还给予Ag-DC-CIK过继免疫治疗,记录2组患者的生活质量和疗效.结果:与对照组相比,细胞组患者的免疫功能增强,生活质量改善,临床疗效提高(P＜0.05).结论:Ag-DC-CIK过继免疫治疗对广泛期SCLC患者有较好的姑息治疗价值且安全可行.     

Therapy of recurrent high grade gliomas with surgery,and autologous mitogen activated IL-2 stimulated killer (MAK) Lymphocytes: I. Enhancement of MAK lytic activity and cytokine production by PHA and clinical use of PHA

Nineteen patients with recurrent high grade gliomas were treated in a phase I/II trial with aggressive debulking of the tumor, mitogen activated IL-2 stimulated peripheral blood lymphocytes (MAK cells), and rIL-2. Phytohemagglutin (PHA) was introduced into the tumor site in 16 patients prior to implanting MAK cells and IL-2 in an attempt to trigger more effective lysis of the tumorin vivo. In vitro both TNF bioactivity and cytolytic activity of long term cultured MAK (LMAK) cells were dramatically enhanced by adding PHA to the cultures of these activated PBL. Three of eleven patients (27%) had a decrease in size of the enhancing lesion on CT and/or MRI. Seven (37%) patients clinically improved. Median survival after therapy was 30 weeks. PHA was shown to be safein vivo and more effective than IL-2 triggering enhanced effector functionin vitro.