骨髓间充质干细胞表达外源性IL-12对胶质瘤C6细胞增殖的影响

目的:探讨以骨髓间充质干细胞（mesenchymal stem cells,MSCs） 为基因治疗载体表达外源性IL12对胶质瘤C6细胞增殖的影响。方法：分离培养大鼠MSCs, 腺病毒介导IL12基因转染大鼠MSCs(AdIL12MSCs),RTPCR 及Western Blotting检测AdIL12MSCs中IL12基因mRNA及蛋白表达。MTT法检测AdIL12MSCs分泌的外源性IL12对C6胶质瘤细胞增殖活性 的影响,光镜下观察外源性IL12对C6细胞形态的影响。结果：腺病毒介导IL12基因成功转染MSCs形成AdIL12MSC,其IL12基因在mRNA及蛋白水平均有明显表达。AdIL12MSC分泌的外源性IL12显著抑制胶质瘤C6细胞的增殖（P<0.05）。结论：转染IL12的MSCs(AdIL12MSC)能够在mRNA及蛋白水平表达外源性IL12基因,显著抑制胶质瘤C6细胞的增殖。     

IL-24对C6细胞JNK及caspase-3表达的影响

目的:白细胞介素24(interleukin 24,IL-24)是由黑素瘤分化相关基因-7(melanoma differentiation -associated gene-7,mda-7)编码的一种分泌蛋白.以转入外源性IL-24基因的C6/IL-24细胞、空载体C6/pLXSN细胞以及亲代C6细胞为研究对象,探讨外源性IL-24体外抑制胶质瘤细胞增殖的相关机制.方法:应用MTT法体外观察IL-24对胶质瘤细胞增长的抑制作用.Western blot方法检测c-Jun氨基末端蛋白激酶(c-Jun NH2-terminal kinases,JNK)和半胱氨酸蛋白酶-3(caspase-3)蛋白的表达.探讨外源性IL-24体外抑制胶质瘤细胞增殖的相关机制.结果:C6/IL-24细胞与C6/pLXSN和亲代C6细胞相比,其吸光值降低,细胞生长曲线显示其体外增殖能力降低.与C6/pLXSN及C6细胞比较,C6/IL-24细胞的JNK和caspase-3蛋白表达增高(P＜0.05).C6/pLXSN与C6细胞比较无显著性差异(P＞0.05).结论:外源性IL-24基因可抑制C6胶质瘤细胞增殖.外源性IL-24基因对胶质瘤细胞诱导凋亡作用,与其上调并激活JNK及caspase-3表达有关.     

IL-18基因增强肿瘤抗原致敏DC诱导的CTL特异性杀伤肝癌细胞

目的: 探讨腺病毒介导的白细胞介素-18(IL-18)基因转染能否使肿瘤抗原冲击的树突状细胞(dentritic cell,DC) 在体外诱导出更强的抗肝癌免疫反应.方法:携IL-18 的重组腺病毒载体感染经肝癌细胞株HepG2冻融抗原致敏的DC(AdIL-18-HepG2/DC), FACS分析AdIL-18-HepG2/DC表面分子的表达, ELISA法检测IL-18的分泌水平, 3H-TdR掺入法检测T淋巴细胞增殖能力, MTT法检测细胞毒性T 淋巴细胞杀伤效应.结果:AdIL-18-HepG2/DC较未转染DC能高水平地表达CD1a、CD11c、CD80、CD86以及HLA-DR;较未经IL-18转染的DC分泌较高水平的IL-18.AdIL-18-HepG2/DC 能非常有效地刺激自体T 细胞增殖(CPM 值为228 018±1 079),其刺激强度显著强于AdIL-18DC、HepG2/DC、AdlzcZ/DC及DC(均P＜0.05).当靶细胞为HepG2时,AdIL-18-HepG2/DC诱导的CTL杀伤活性显著高于其他各组(均P＜0.05),并且其杀伤能力与效应细胞数量成正比.结论:IL-18 基因转染且肝癌抗原致敏的DC可以显著增强DC的特异性抗肝癌效应.     

腺病毒介导的大鼠增殖抑制基因抑制大鼠胶质瘤的生长

目的:探讨腺病毒介导的大鼠增殖抑制基因(rat hyperplasia suppressor gene,rHSG)对大鼠胶质瘤生长的抑制作用.方法:建立大鼠C6胶质瘤动物模型,分别在接种C6胶质瘤细胞后第4和11天,于肿瘤原位注射0.9％氯化钠溶液(对照组)、重组腺病毒Adv-rHSG-GFP和腺病毒Adv-GFP,并于接种C6胶质瘤细胞后第9、15和21天取脑胶质瘤观察肿瘤的生长情况,免疫组织化学法检测增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)和rHSG蛋白的表达以及微血管密度(microvessel density,MVD),RT-PCR和蛋白质印迹法检测rHSG mRNA及蛋白的表达.结果:Adv-rHSG-GFP组肿瘤体积明显小于对照组和Adv-GFP组(P＜0.01).Adv-rHSG-GFP组PCNA标记指数和MVD均低于对照组和Adv-GFP组.Adv-rHSG-GFP组rHSG mRNA和蛋白的相对表达量均明显高于对照组和Adv-GFP组(P＜0.01).结论:Adv-rHSG-GFP能够抑制胶质瘤血管生成和肿瘤细胞增殖,对恶性胶质瘤的生长具有明显的抑制作用.     

外源性IL-18基因对C6胶质瘤细胞周期及相关基因表达的影响

目的:本研究拟从分子水平探讨IL-18基因转染对C6胶质瘤细胞周期及其相关基因表达的影响。方法:用流式细胞仪检测C6/IL-18细胞周期、细胞增殖指数的变化;以MTT法检测细胞的增殖活性及细胞抑制率,用RT-PCR、Western blot分析C6/IL-18细胞PCNAc、yclin D1c、yclin B1、p21 mRNA及蛋白的表达。结果:与C6/LXSN细胞及亲代C6细胞相比,C6/IL-18细胞周期有所改变,表现为G0/G1期细胞增多、G2/M期细胞减少,细胞增殖指数(PI)与细胞增殖活性降低。C6/IL-18细胞的PCNAc、yclin D1c、yclin B1 mRNA及蛋白表达降低,p21 mRNA及蛋白表达增高。结论:外源性IL-18基因表达可降低C6细胞的增殖活性,下调PCNA,cyclin D1,cyclin B1表达,上调p21基因表达,其作用机制有待进一步研究。     

外源性白介素24促进C6细胞凋亡的体外研究

目的 探讨外源性白细胞介素24(interleukin 24,IL-24)体外抑制胶质瘤细胞增殖及促凋亡的相关机制.方法 应用MTT体外观察转染IL-24对胶质瘤C6细胞增殖的抑制作用.应用Hoechst 33258荧光染色体外观察IL-24对C6细胞的促凋亡作用.Western blot法检测c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)和caspase-3蛋白的表达变化情况.结果 IL-24能使C6细胞的体外增殖能力降低,转入IL-24的C6细胞(C6/IL-24)增殖能力下降.经Hochest 33258染色,转入IL-24的C6细胞凋亡后出现核固缩、染色质凝集呈块或碎裂状改变.与转入空载体的C6细胞(C6/pLXSN)及C6细胞比较,C6/IL-24细胞的JNK和caspase-3蛋白表达均增高(均P＜0.05).C6/pLXSN与C6细胞比较差异无统计学意义(P＞0.05).结论 外源性IL-24基因可抑制C6胶质瘤细胞的增殖并诱导其凋亡,该诱导凋亡的作用可能与其上调并激活JNK及caspase-3表达有关.     

重组腺病毒介导survivin基因转染树突状细胞对肾细胞癌的免疫效应

目的:研究重组腺病毒介导survivin基因转染的树突状细胞(DCs)体外诱导特异性抗肾细胞癌的免疫效应.方法:以携带survivin基因的重组腺病毒(Ad-svv)感染DCs, Western bloting检测转染DCs的survivin的表达,流式细胞术检测DCs表面分子CD83、MHCⅡ、CD80、CD86的表达,ELISA法检测DCs培养上清中IL-12 的含量;混合淋巴细胞反应(MLR)测定DCs刺激同种异体淋巴细胞增殖的能力, ELISA法检测DCs刺激淋巴细胞后上清中IFN-γ含量,MTT法检测其诱导的特异的细胞毒性T淋巴细胞(CTLs)免疫效应.结果:各组Ad-svv转染DCs后均表现出成熟的DCs表型特征,均可见survivin蛋白表达;转染Ad-svv或转染空白腺病毒载体(Ad-CMV)的DCs上清中IL-12均高于对照组(P<0.01),转染Ad-svv的DCs上清中IL-12高于转染Ad-CMV-DCs组(P<0.01).MLR中,转染或未转染腺病毒的DCs均能刺激同种异体T淋巴细胞的增殖,Ad-svv-DC刺激T淋巴细胞增殖的能力最强(P<0.01);MLR后,转染或未转染重组腺病毒的DCs均能刺激T淋巴细胞IFN-γ的分泌,Ad-svv-DC组IFN-γ的分泌能力最强(P<0.01).Ad-svv-DCs组诱导的CTL对survivin表达阳性的786-0肾癌细胞的杀伤率明显高于无survivin表达的L-02肝细胞(P<0.01),Ad-CMV-DCs、空白DC对786-0肾癌细胞无杀伤作用.结论:重组腺病毒介导survivin基因转染能显著提高DCs对肾癌细胞抗原的提呈能力、激活特异性细胞毒性T淋巴细胞、诱导针对survivin的特异性抗癌免疫效应.     

连接蛋白43基因治疗鼠C6脑胶质瘤的体内实验研究

目的 研究连接蛋白（Cx）43基因抑制脑胶质瘤生长的作用。方法 将Cx43基因表达缺失的大鼠C6胶质瘤细胞（对照组）和转染Cx43 cDNA的C6细胞（转染组）种植于SD大鼠右侧尾状核,荷载C6脑胶质瘤鼠用Cx 43 cDNA原位治疗（治疗组）,并以空载体治疗作为对照（空载组）,每组10只,观察各组大鼠一般情况、生存期、MRI动态变化及病理改变,通过原位杂交及免疫组化染色检测肿瘤Cx43 mRNA及蛋白表达,以AgNOR平均计数检测增殖活性,以Tunel法检测细胞凋亡。结果 对照组和空载组大鼠均于3周内死亡;转染组6只大鼠和治疗组8只大鼠观察120 d内无自然死亡。除治疗组1只尚有残瘤外,余瘤体消失,转染组和治疗组肿瘤细胞均有Cx43 mRNA及蛋白表达,且增殖治疗组1只尚有残瘤外,余瘤体消失。转染组和治疗组肿瘤细胞均有Cx43 mRNA及蛋白表达,且增殖活性下降,但凋亡不增加。结论 转染Cx43基因后的C6胶质瘤细胞致瘤性显著降低,且对荷载脑胶质瘤鼠具有明显的治疗作用,有可能成为恶性胶质瘤基因治疗的优先靶的之一。     

大鼠增殖抑制基因表达变化与恶性脑胶质瘤生长的关系

目的:探讨大鼠增殖抑制基因(rat hyperplasia suppressor gene,rHSG)表达变化与恶性脑胶质瘤生长之间的关系,为外源性rHSG治疗提供实验依据.方法:采用立体定向法,于尾状核注射C6细胞建立SD大鼠脑胶质瘤动物模型;采用SABC免疫组织化学法检测注射C6细胞后第9、15和21天,脑胶质瘤中增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)、rHSG及微血管度(microvessel density,MVD)的表达;RT-PCR法检测不同生长期脑胶质瘤中rHSG mRNA的表达.结果:第9、15和21天脑胶质瘤中PCNA标记指数和MVD高于对照组,rHSG蛋白的表达低于对照组,差异均有统计学意义(P<0.05);实验组大鼠胶质瘤rHSG mRNA相对表达量分别为(13.53±2.11)%、(10.39±2.71)%和(8.86±1.91)%,低于对照组的(19.94±3.23)%.差异有统计学意义(P<0.05).结论:rHSG表达下调可能与大鼠脑恶性胶质瘤的发生、发展及血管增生有关,rHSG的高表达可能会抑制肿瘤的生长.     

IL-24基因对大鼠胶质瘤细胞生长状况的影响

目的　探讨IL 2 4基因对C6大鼠胶质瘤细胞生长状况的影响。方法　应用逆转录病毒载体 ,将IL 2 4基因导入C6细胞 ,经G4 18筛选后获得表达IL 2 4分子的阳性细胞克隆C6 /IL 2 4 ;用RT PCR方法检测目的基因表达 ;四甲基偶氮唑蓝 (MTT)法检测细胞体外增殖状况 ,流式细胞技术检测细胞的增殖活性 ,并制作荷瘤动物模型 ,观察C6 /IL 2 4和C6细胞的体内致瘤性。结果　RT PCR检测表明 ,外源IL 2 4基因于mRNA水平在C6 /IL 2 4细胞已获得稳定表达。C6 /IL 2 4细胞系的体外增殖性较亲代C6细胞明显下降 ,流式细胞术检测其细胞增殖指数 (PI)为 (2 9.71± 0 .89) %。 9只接种C6 /IL 2 4细胞的实验组大鼠中 ,6只颅内成瘤 ,肿瘤体积为 (14 .0 8± 9.81)mm3 ,明显小于接种C6细胞大鼠的肿瘤体积 (P <0 .0 5 )。结论　外源性IL 2 4基因可部分抑制胶质瘤细胞异常增殖的肿瘤特性。     

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.