胃癌抗原特异性树突状细胞疫苗的抗瘤效应

我们利用液氮冻融提取胃癌细胞抗原刺激小鼠骨髓来源树突状细胞(DCs)制备肿瘤疫苗,进而诱导小鼠脾脏淋巴细胞制备肿瘤抗原特异细胞毒性淋巴细胞(CTLs) ,应用于胃癌裸小鼠皮下模型,探讨其多方面抗胃癌效应。一、材料与方法1.BALB/c小鼠骨髓DCs肿瘤疫苗制备:依据文献[1] ,分离BALB/c小鼠股、胫骨骨髓细胞,经1g/L浓度SCG790 1肿瘤可融性抗原孵育过夜后,终浓度5 0 μg/L的粒细胞 巨噬细胞集落刺激因子(GM CSF)及10 0 μg/L的白细胞介素(IL) 4(PeproTech公司)诱导培养7d获得DCs肿瘤疫苗。抗CD40 FITC、抗CD80 FITC直标荧光…     

癌细胞裂解物修饰的DC疫苗体外诱导T细胞特异性抗胰癌免疫

目的　观察经胰癌细胞裂解物修饰的树突状细胞 (DC )疫苗 ,在体外诱导抗胰癌的特异性T细胞免疫应答的效果。方法　从胰腺癌患者外周血中分化、增殖出DC ,经胰癌细胞裂解物修饰 ,并与T细胞体外共培养 (分为致敏DC组、未致敏DC组、肿瘤裂解物组、对照组 ) ,检测了各组上清液中细胞因子IL 12与IFN γ的浓度和T细胞的反应性增殖 ,并评估激活的T细胞对胰腺癌肿瘤细胞的特异性细胞毒作用。结果　致敏DC组中IL 12与IFN γ的浓度 [(1161± 2 39) pg/ml和 (10 44± 312pg/ml) ]与未致敏DC组及肿瘤裂解物组相比差异有极显著性 (P <0 .0 5 ,P <0 .0 1) ;在致敏DC组中T细胞有明显的增殖 ;激活的T细胞对胰腺癌细胞有高效、特异的细胞毒作用。结论　癌细胞裂解物致敏的DC可高效的诱发机体T细胞 (Th1和CTL )多重的特异性抗肿瘤免疫应答     

转染肿瘤mRNA的树突状细胞疫苗诱导抗肝癌免疫研究

目的探讨转染原发性肝癌(HCC)mRNA的树突状细胞(DC)能否诱导抗肿瘤特异性细胞毒性T淋巴细胞(CTL)。方法采用HCC患者外周血单核细胞(PBMC)体外刺激分化为DC细胞；从人肝癌HepG-2细胞和3例HCC患者的肝癌组织中体外扩增mRNA。以mRNA转染DC细胞，并与PBMC混合培养诱导扩增CTL。流式细胞计数仪检测培养细胞中CD3^ 、CD4^ 、CD8^ 细胞的比例。^51Cr释放法测定CTL的杀瘤活性。结果经扩增人肝癌HepG-2mRNA和2例AFP( )患者的AFP( )HCCmRNA诱导3周后，CD3^ 、CD8^ 细胞占淋巴细胞总数由诱导前的27．8％、26．5％、29．6％升高至89．3％、73．6％、86．8％；而经扩增AFP(-)HCCmRNA诱导3周后，CD3^ 、CD8^ 细胞占淋巴细胞总数由诱导前的25．4％升高至53．6％。转染HepG-2细胞和AFP( )的患者HCCmRNA的DC诱导的CTL对HepG-2细胞杀瘤活性明显高于AFP(-)的患者，其杀瘤特性由MHC-I限制的CD8^ T细胞所介导。结论HCCmRNA体外转染DC能诱导肿瘤特异性CTL，可为肝癌的免疫治疗提供新的有效手段。     

胃癌肿瘤裂解物修饰的树突状细胞体外诱导抗原特异性细胞毒T淋巴细胞

目的　利用树突状细胞呈递肿瘤抗原的特性提高细胞毒T淋巴细胞 (CTLs)对胃癌细胞的杀伤活性。方法　胃细胞癌患者外周血来源的有核细胞体外经GM -CSF和IL -4诱导产生树突状细胞 ,负载肿瘤裂解物后诱导自体CTLs产生。用细胞毒试验检测CTLs杀伤活性 ,和用ELISA测定细胞因子的分泌。结果　胃癌患者自体来源的DC裂解物能诱导产生的CTLs对自体胃癌细胞具有高杀伤率 ,可达 83 % ;致敏的DC组中IL -12与TNF -α的浓度 ( 1161± 2 3 9pg/ml,10 44± 3 12 pg/ml)显著高于未致敏的DC组 ( P <0 .0 5 )。结论　DC能呈递胃癌裂解物 ,诱导产生抗原特异性CTLs。     

新型肿瘤抗原gp96在肝癌小鼠体内的抗瘤效应

目的　观察从肿瘤细胞中提取的热休克蛋白gp96在小鼠体内的特异抗瘤效应。 方法　BALB/C小鼠肝脏成功接种肿瘤后 ,分成 4组。第Ⅰ组单纯切除肿瘤 ,不做免疫治疗 ;第Ⅱ组切除肿瘤 ,用gp96进行免疫治疗 ;第Ⅲ组不切除肿瘤 ,用 gp96进行免疫治疗 ;第Ⅳ组为对照组。第2 8天检测血清中白细胞介素 (IL) 10、干扰素 (IFN ) γ水平 ;CD4、CD8和IFN γ、IL 10双阳性细胞比例。用51Cr释放法测定各组小鼠脾细胞对亲本H2 2 肝癌细胞的杀伤活性。结果　第Ⅱ组血清IFN γ为 13 .3 3ng/L ,CD8+ IFNγ+ 和CD4+ IFNγ+ 细胞比例分别为 5 .79%和 2 .91% ,较其余组升高 (P <0 .0 1) ;血清IL 10为 5 .2 8ng/L ,CD8+ IL 10 + 和CD4+ IL 10 + 细胞比例分别为 4.5 4%和0 .75 % ,低于其他各组 (P <0 .0 1)。效靶比为 10 0∶1时 ,Ⅱ组小鼠脾细胞体外杀伤亲本H 2 2癌细胞的杀伤率为 3 9.3 % ,显著高于其他各组 (P <0 .0 5 )。结论　gp96可激发特异性细胞介导的免疫反应 ,改善抗肿瘤免疫反应。     

负载肾癌肿瘤裂解物的树突状细胞诱导产生抗原特异性细胞毒T淋巴细胞

目的　利用树突状细胞呈递肿瘤抗原的特性提高细胞毒T淋巴细胞 (CTLs)对肾癌细胞的杀伤活性。　方法　肾细胞癌患者骨髓来源的有核细胞体外经GM CSF和IL 4诱导产生树突状细胞 ,负载肿瘤裂解物后诱导自体CTLs产生。用细胞毒试验和ELISA测定CTLs杀伤活性和细胞因子的分泌。　结果　肾癌患者自体来源的DC Tuly能 (1)增加CTLs增殖 ,16d时使T细胞增殖达 4 3倍 ;(2 )上调CTLs中CD3 和CD8 T细胞群 ;(3)诱导产生的CTLs对自体肾癌细胞具有高杀伤率 ,显著高于异体肾癌和异种肿瘤细胞 (P <0 .0 5 ) ;(4)上调TNF α分泌。　结论　DCs能呈递Tuly抗原。诱导产生抗原特异性CTLs,提示DC Tuly具有为肾癌患者制作疫苗和进行特异性CTLs过继免疫治疗的临床应用前景     

树突状细胞疫苗与肝癌免疫治疗进展

以肿瘤疫苗为基础的主动免疫治疗和以T细胞介导的抗肿瘤免疫应答成为肿瘤治疗新的热点,近来发现,肝癌患者癌灶缺乏成熟而有活性的CD8+树突状细胞(dendriticcells,DC),表明肝癌发生过程中有活性DC的浸润作用[1]。DC是机体免疫系统中功能最强的专职性抗原提成细胞(Antigen-presentingcells,APC),其最大特点是能激活初始型T细胞(CD4+Th细胞和CD8+CTL细胞),生成辅助性T细胞和杀伤性T细胞,DC能识别、加工、提呈抗原,表达高水平MHC类分子,共刺激分子,粘附分子,同时分泌高水平的IL-12,从而主导初始型T细胞生成Th1型应答,Th1型应答在…     

肝癌病人树突状细胞诱导高效而特异的抗肝癌免疫

目的 以肝癌病人树突状细胞（ＤＣ）体外诱导抗肝癌免疫。方法 自肝癌病人外周血中分离出单个核细胞（ＰＢＭＣ０;以人肝癌细胞系ＨｅｐＧ２肿瘤细胞的肿瘤相关抗原（ＴＡＡ）激活ＤＣ;以粒／巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）及白介素４（ＩＬ－４）联合刺激ＰＢＭＣ中ＤＣ;ＤＣ诱导自体Ｔ淋巴细胞增殖,分化为细胞毒性Ｔ细胞（ＣＴＬ）;检测ＣＴＬ及其上清液对ＨｅｐＧ２,ＢＥＬ－７４０２,ＬＯＶＯｅｙ ＨＯＳ－８６     

肿瘤坏死因子α基因转导的人肝癌细胞疫苗对小鼠肝癌移植瘤的影响

目的　用肿瘤坏死因子α (TNF α)基因转导的人肝癌细胞作为疫苗接种小鼠 ,研究其对小鼠肝癌移植瘤的免疫排斥作用。方法　取昆明种小鼠 40只 ,分为 5组 ,每组 8只。分别采用经60 Co照射的TNF α基因转导的人肝癌细胞 (BEL 740 4 TNF Co组 )、未经60 Co照射的TNF α基因转导的人肝癌细胞 (BEL 740 4 TNF组 )、经60 Co照射的人肝癌细胞 (BEL 740 4 Co组 )以及未经60 Co照射的人肝癌细胞 (BEL 740 4组 )接种小鼠 ,生理盐水对照组仅注射生理盐水。然后将小鼠肝癌细胞株H2 2移植于上述 5组小鼠 ,观察肝癌移植瘤生长情况 ,并应用脱氧核糖核酸末端转移酶介导的缺口末端标记 (TUNEL)法观察肿瘤细胞凋亡指数。结果　BEL 740 4 TNF Co组小鼠接种经60 Co照射的TNF α基因转导的人肝癌细胞疫苗后 ,肝癌移植瘤成瘤率下降 ,瘤体生长受抑制 ,细胞凋亡指数增高 ,与BEL 740 4 Co组、BEL 740 4组及生理盐水对照组相比 ,差异有显著性 (P <0 .0 1) ,但与BEL 740 4 TNF组相比 ,差异则无统计学意义 (P>0 .0 5 )。结论　TNF α基因转导的人肝癌细胞疫苗对小鼠肝癌移植瘤有较强的免疫排斥作用。     

白细胞介素-18与癌细胞裂解物修饰的树突状细胞疫苗对胰腺癌的免疫治疗作用

目的　观察经白细胞介素 18(IL 18)和胰腺癌细胞裂解物修饰的树突状细胞 (DC)疫苗对胰腺癌荷瘤小鼠的免疫治疗作用。方法　用药物诱导制成BALB/c小鼠的胰腺癌模型。通过IL 18和癌细胞裂解物修饰小鼠DC(MTSC4) ,制成DC疫苗 ,检测各组血清中细胞因子IL 18、干扰素γ(IFN γ)的浓度 (分为DC IL18 裂解物组 ,DC 裂解物组 ,DC IL 18组 ,DC组 ,PBS组 ) ,研究了其对小鼠胰腺癌的免疫治疗作用。结果　DC IL18 裂解物组中IL 18与IFN γ的浓度 ( 2 161± 43 9)μg/L和 ( 4 3 5± 72 ) μg/L ,与其余各组比较差异有显著性 (P <0 .0 5 ,P <0 .0 1)。DC IL18 裂解物组接种胰腺癌细胞后 5 0d均未见移植肿瘤形成 ,与其余各组比较差异有非常显著性 (P <0 .0 1)。对胰腺癌细胞的细胞毒作用以DC IL18 裂解物组最强 ,DC 裂解物组次之 ,DC IL18组较弱 ,其余 2组则缺乏 (P <0 .0 1)。DC IL18 裂解物组的小鼠的生存期比他各组明显延长 ,且肿瘤的重量比其他各组明显减轻 (P <0 .0 1,P <0 .0 5 )。结论　IL 18和胰腺癌细胞裂解物修饰的DC疫苗对胰腺癌荷瘤小鼠有明显的免疫治疗作用。     

负载冻融抗原的树突状细胞诱导特异性抗膀胱癌作用的研究

目的研究负载膀胱癌冻融抗原的树突状细胞（DC）诱导的对膀胱癌细胞的特异性杀伤效应。方法反复冻融法获得BIU-87细胞抗原;人单个核细胞在含重组人GM—CSF、IL-4和TNF-a的RPMI1640培养基中体外诱导出人DC并负载肿瘤抗原;9d后成熟DC与自体T细胞共孵育诱导膀胱癌抗原特异性CTLs; 用MTT法检测其对BIU-87体外杀伤效应;用ELISA法检测DC分泌IL-12的能力。结果负载膀胱癌抗原的DC诱导的特异性CTLs可明显杀伤BIU-87细胞,在效靶比为40：1时杀伤率为（65．5±6．4）％,显著高于各对照组（P〈0．01）;负载抗原DCs较未负载抗原DCs有更强的分泌IL-12能力（P〈0．05）,而且不同时期的DC分泌的量不同。结论负载膀胱癌抗原的DC在体外可诱导出高效而特异的抗膀胱癌效应,提示以DC为中心的肿瘤生物治疗有望提高膀胱癌综合治疗水平。     

癌细胞裂解物修饰的DC疫苗对胰腺癌荷瘤小鼠的免疫治疗作用

目的　观察经胰腺癌细胞裂解物修饰的DC疫苗对胰腺癌荷瘤小鼠的免疫治疗作用。方法　通过胰腺癌细胞裂解物修饰小鼠DC制成DC疫苗 ,研究其对小鼠抗胰腺癌的免疫保护作用 ,并评估了其对胰腺癌荷瘤小鼠的免疫治疗效果。结果　DC疫苗组的小鼠接种胰腺癌细胞 2 5d后未见移植肿瘤形成 ,其余各组在 3～ 9d后可见有移植肿瘤的生长。DC疫苗组的CTL对胰腺癌细胞有明显的细胞毒作用 ,但其余各组的CTL则缺乏明显的细胞毒作用。DC疫苗组小鼠的生存期 (5 6± 9)d比其余各组明显延长 ,且肿瘤重量 (1 4± 0 8)g明显低于其余各组 (P <0 0 1,P <0 0 5 )。结论　癌细胞裂解物致敏的DC可诱导荷瘤小鼠产生高效的抗胰腺癌免疫应答反应。     

自体负载肿瘤抗原树突状细胞治疗大鼠甲状腺癌

目的 观察自体负载肿瘤抗原树突状细胞(DC)对大鼠甲状腺癌的治疗作用.方法 建立Wistar荷瘤大鼠动物模型,随机分为治疗组和对照组,治疗组静脉输入负载肿瘤抗原DC、对照组输入生理盐水,检测分析2组血清中自细胞介素(IL)-12、IL-8表达水平,以及瘤重、瘤体体积及大鼠存活.结果 治疗组与对照组瘤重、瘤体体积、存活期、IL-12及IL-8表达水平分别为:(3.56±0.79)g比(4.97±0.56)g、(3.98±0.84)cm3比(5.06±0.58)cm3、(36.0±3.0)d比(28.0±3.2)d、(368.9±21.7)ng/L比(227.7±13.4)ng/L和(218.9±19.3)ng/L比(371.2±24.1)ng/L(P值分别<0.01、<0.01、<0.05、<0.01、<0.01).结论 自体负载肿瘤抗原DC对甲状腺癌具有较好的抑瘤效应,可延长荷瘤鼠的生存期.     

自体负载肿瘤抗原树突状细胞治疗大鼠甲状腺癌

Objective To investigate the immunotherapeutic effects of auto dendritic cells (DC) loaded with tumor lysates on rat transitional thyroid carcinoma. Methods After the thyroid tumor models in Wistar rats were successfully established, all rats were divided randomly into the treatment group and the control group. DC loaded with tumor lysates were injected into the tail vein of the rats in the treatment group, and the rats in control group, normal saline was injected into the tail vein. The levels of interleukin (IL)-12 and IL-8 in serum, the tumor weight and volume, and the life span of the rats were measured. Results The average tumor weight and volume, the life span, the levels of IL-12 and IL-8 in the treatment group and the control group were respectively (3. 56±0. 79) g vs (4. 97±0. 56) g, (3. 98±0. 84) cm3 vs (5.06±0.58) cm3, (36.0±3.0) days vs (28.0±3.2) days, (368.9±21.7) ng/L vs (227.7±13.4) ng/L, (218.9±19.3) ng/L vs (371.2±24. 1) ng/L. P values <0.01, <0.01, <0.05,<0.01, <0.01, respectively. Conclusion Auto DC loaded with tumor lysates can inhibit tumor growth and prolong the survival time of Wistar rats with thyroid carcinoma.     

自体负载肿瘤抗原树突状细胞治疗大鼠甲状腺癌

Objective To investigate the immunotherapeutic effects of auto dendritic cells (DC) loaded with tumor lysates on rat transitional thyroid carcinoma. Methods After the thyroid tumor models in Wistar rats were successfully established, all rats were divided randomly into the treatment group and the control group. DC loaded with tumor lysates were injected into the tail vein of the rats in the treatment group, and the rats in control group, normal saline was injected into the tail vein. The levels of interleukin (IL)-12 and IL-8 in serum, the tumor weight and volume, and the life span of the rats were measured. Results The average tumor weight and volume, the life span, the levels of IL-12 and IL-8 in the treatment group and the control group were respectively (3. 56±0. 79) g vs (4. 97±0. 56) g, (3. 98±0. 84) cm3 vs (5.06±0.58) cm3, (36.0±3.0) days vs (28.0±3.2) days, (368.9±21.7) ng/L vs (227.7±13.4) ng/L, (218.9±19.3) ng/L vs (371.2±24. 1) ng/L. P values <0.01, <0.01, <0.05,<0.01, <0.01, respectively. Conclusion Auto DC loaded with tumor lysates can inhibit tumor growth and prolong the survival time of Wistar rats with thyroid carcinoma.     

肿瘤靶向性细胞穿膜肽介导小分子干扰RNA对肝癌细胞的抑制作用

目的 观察以基质金属蛋白酶为靶点的肿瘤靶向性细胞穿膜肽介导小干扰RNA(siRNA)进入肝癌并对肝癌细胞SMMC-7721起一定抑制作用.方法 以体外培养的肝癌细胞SMMC-7721为研究对象,将细胞分为实验组和对照组,实验组加入siRNA质粒/穿膜肽复合物,对照组加入空质粒/穿膜肽复合物,培养48 h后收集两组细胞,流式细胞仪检测各组细胞的细胞周期,逆转录-聚合酶链反应(RT-PCR)检测各组细胞人端粒酶逆转录酶(hTERT)基因的表达水平,噻唑蓝(MTT)法检测SiRNA质粒/穿膜肽复合物对细胞增殖的抑制作用.结果 实验组与对照组比较G1期细胞所占比例增多(实验组75.14％、对照组62.55％),G2期细胞比例减少(实验组11.39％、对照组12.14％)、S期细胞比例减少(实验组13.47％、对照组25.31％),说明经穿膜肽质粒复合物处理后细胞被阻滞在G1期,实验组细胞hTERT mRNA的表达量约为未对照组的74％,即实验组mRNA表达水平下调约26％.肝癌细胞经穿膜肽复合物处理48 h后MTT法测细胞增殖抑制率为34.82％.结论 肿瘤靶向性细胞穿膜肽可介导siRNA进入肝癌细胞并可对肝癌细胞起一定的抑制作用.     

The effects of different types of pulsed electromagnetic fields on fibroblast cell cultures

The aim of this study is to determine which is the most effective pulsed electromagnetic field (PEMF) on a fibroblastic culture cells. MATERIAL AND METHODS: Five different types of PEMF are generated by a pulsed generator created by the author. The electric signal, received by a reference coil is pulse bursts type. The growth rate of a fibroblastic culture cells is measured with and without different types of PEMF. RESULTS: Comparative results showed the the most effective type of PEMF is characterized by a 15 mV positive amplitude of the signal and a 15 Hz frequency of the bursts. The intermittent stimulation (8 hours/day) was more effective than continuous stimulation.     

Enhanced anti-tumor effects of recombinant human tumor necrosis factor plus VP-16 on metastatic renal cell carcinoma in a xenograft model

A nude mouse renal subcapsular and subcutaneous implantation xenograft model utilizing the SN12C human renal carcinoma cell line was investigated. In the absence of treatment, renal subcapsular implantation of SN12C resulted in metastatic spread (lung, liver and lymph nodes) and death of all animals. Radical nephrectomy of the tumor-bearing kidney after various periods of tumor implantation demonstrated that surgery alone after 18 days of tumor growth resulted in no statistically significant increase in survival with 100% of the nephrectomized animals succumbing to local recurrence and distant metastases. Recombinant human tumor necrosis factor (rTNF) and VP16 (etoposide), both well known cytotoxic and cytostatic anticancer agents, were tested singly and in combination against this metastatic model of human renal adenocarcinoma. Single agent rTNF or VP16 therapy after radical nephrectomy demonstrated only minimal efficacy with no significant decrease in local recurrence and distant metastases as compared to nephrectomy only control animals. In contrast, the combination of rTNF plus VP16 when given after nephrectomy resulted in a significant decrease in local recurrence and no gross evidence of metastasis in any animal. Subcutaneously growing SN12C tumor nodules were also treated with the same rTNF, VP16 and combination regimens. Regression in tumor size was noted only in the combination treatment group. rTNF or VP16, as single agents, demonstrated only slight growth inhibition that was not statistically significant. These results suggest that by combining TNF plus VP16, a synergistic enhancement of antineoplastic activity against local as well as metastatic human renal cell carcinoma can be produced.