自体肿瘤抗原致敏的树突状细胞联合细胞因子诱导的杀伤细胞治疗晚期肾癌的疗效观察

目的探讨负载自体肿瘤抗原的树突状细胞(DC)联合细胞因子诱导的杀伤细胞(CIK细胞)在晚期肾癌治疗中的临床疗效,并监测患者的免疫状态及不良反应。方法回顾分析2011-01/2013-12期间在我科治疗的82例晚期肾癌患者,单采其外周血单个核细胞(PBMC),其中贴壁细胞经体外诱导产生DC,并负载自体肿瘤细胞裂解物(Ag)制备Ag-DC;T淋巴细胞经体外诱导产生CIK细胞,将Ag-DC与CIK细胞共培养制备Ag-DC-CIK瘤苗,检测DC表面分子的表达及IL-12的分泌,监测CIK细胞增殖情况,并将Ag-DC-CIK分次回输给其中41名患者,以41例单纯的CIK细胞治疗作为对照,治疗2疗程后,检测患者外周血细胞因子水平及T细胞亚群变化,结合影像学等检查综合评价疗效,同时观察不良反应。结果负载肿瘤细胞冻融抗原的DC,高表达CD11c、CD83、CD86、HLA-DR表面分子,IL-12的分泌显著增加。Ag-DC与CIK细胞共培养后,可明显刺激CIK细胞的增殖,同时CD3+CD8+细胞及CD3+CD56+细胞的含量明显增加,此外,Ag-DC-CIK瘤苗的临床疗效明显高于单纯CIK细胞治疗组,2组均未发生严重的不良反应。结论晚期肾癌PBMC来源的DC负载自身肿瘤细胞冻融抗原能提高晚期肾癌患者的免疫状态。     

自体细胞因子诱导的杀伤细胞降低恶黑患者外周血髓样抑制性细胞的数量研究

目的 探讨恶性黑色素瘤(MM)患者外周血中髓样抑制性细胞(MDSCs)的数量及自体细胞因子诱导的杀伤细胞(CIK)对MDSCs数量的影响.方法 采集20例MM患者CIK细胞治疗前后和18例健康志愿者外周血,用anti-CD33-mAb和anti-CD11b-mAb标记,应用流式细胞仪检测MDSCs比例.结果 MM患者外周血中MDSCs水平高于健康对照组,差异具有统计学意义(P＜0.05).MM患者经CIK治疗后,其MDSCs水平低于治疗前,差异有统计学意义(P＜0.05).结论 自体CIK可降低MM患者外周血MDSCs水平,这一结果揭示了CIK抗肿瘤作用的新机制.     

细胞因子诱导的杀伤细胞的研究进展

正常人或患者外周血、骨髓单个核细胞中定期加入IFN-γ、IL-1、IL-2、CD3mAb经2-4W的诱导可获得大量的新型肿瘤杀伤细胞—细胞因子诱导的杀伤细胞CIK,此细胞增殖旺盛杀伤活性高。CIK在体内和体外都具有非MHC限制抗肿瘤活性,在临床上已经用于治疗多种恶性肿瘤表现出强大的杀瘤活性,是一种很好的免疫治疗方法。     

细胞因子诱导的杀伤细胞联合白细胞介素2治疗恶性胸腔积液

目的 观察应用细胞因子诱导的杀伤（CIK）细胞联合白细胞介素2（IL-2）治疗恶性胸腔积液的疗效.方法 收集2009年7月至2013年7月于本院肿瘤科就诊的恶性胸腔积液患者50例,随机数字表法分为治疗组（n=24）和对照组（n=26）.胸腔积液引流净后,治疗组胸腔灌注CIK细胞联合IL-2,连续3d;对照组灌注IL-2联合顺铂,隔日1次,连续3次.治疗4周后评价疗效和不良反应.结果 治疗组有效率87.50％（21/24）,完全缓解率54.17％（13/24）;对照组有效率57.69％（15/26）,完全缓解率42.31％（11/26）.与对照组比较,治疗组的完全缓解率、有效率均增加（均P＜0.05）.不良反应比较显示,治疗组发热的发生率较高,而对照组胸痛、胃肠道反应、骨髓抑制的发生率较高（均P＜ 0.05）.与治疗前比较,治疗后两组患者的Karnofsky评分均增加（均P＜0.05）.结论 CIK细胞联合IL-2胸腔灌注治疗恶性胸腔积液不良反应小,疗效确切.     

细胞因子诱导杀伤（CIK）细胞的大容量扩增与杀伤活性观察

建立大容量细胞因子诱导杀伤(Cytokine-inducedkiller,CIK)细胞培养方法,观察CIK细胞回输后对患者细胞免疫功能的影响。采用1000ml培养袋大量扩增患者自体CIK细胞,用MTT法检测CIK细胞杀伤活性,比较回输前后患者外周血单个核细胞(PBMNC)对靶细胞的杀伤活性及其毒副作用。结果表明大容量培养法使自体CIK细胞扩增总量达1.6×1010以上,回输CIK细胞使患者PBMNC的杀伤活性明显增加,未出现毒副作用。因此CIK细胞大容量扩增方法在治疗肿瘤微小残留病变上有着良好的临床应用前景。     

制备细胞因子诱导的杀伤细胞的方法比较

生物治疗是目前国内外治疗恶性肿瘤常规方法即手术治疗、化疗和放疗之外的一种新型疗法.过继性细胞免疫治疗(adaptive cellular immunotherapy,ACI)是生物治疗的一种,它是一种通过将具有抗肿瘤活性的、能直接或间接介导抗肿瘤效应的免疫细胞输注肿瘤宿主体内进行肿瘤治疗的方法[1].对于促进患者免疫系统的重建、消除残留病变及骨髓净化都具有良好效果[2].细胞因子诱导的杀伤细胞(cytokine-induced killer cells,CIK细胞)是将人外周血单个核细胞在体外用多种细胞因子共同培养一段时间后获得的一群异质细胞,具有增殖速度快、杀瘤活性高、杀瘤谱广等优点[3-4],已广泛应用于临床,但疗效差异很大,原因可能与制备CIK细胞的方法、所使用细胞因子的种类和剂量密切相关.本研究拟采用3种方法制备CIK细胞,并比较用这3种方法制备的CIK细胞的生物学特性及其杀瘤活性,为制备高活性的CIK细胞提供方法,以提高CIK细胞临床应用的疗效.     

-80℃冰箱直接冻存对细胞因子诱导的杀伤细胞杀伤活性的影响

目的:研究-80℃冰箱直接冻存对细胞因子诱导的杀伤细胞(CIK)杀伤活性的影响。方法采用非程序降温方法-80℃冰箱冻存CIK,于冻存后6、12、18周复苏,通过LDH释放法分别测定其对K562细胞的杀伤活性,与新鲜未经冻存的CIK的杀伤活性进行比较。结果:未冻存的CIK在培养至第11天时,有较多贴壁成团细胞,而冻存后复苏培养的CIK贴壁成团细胞较少。比较各组细胞杀伤活性,冻存6、12周后复苏的CIK与未冻存的CIK,对K562细胞的杀伤活性无显著性差异(P>0.05),冻存18周后复苏的CIK的杀伤活性与未冻存的CIK对K562细胞的杀伤活性有显著性差异(P<0.05)。结论:采用-80℃冰箱直接冻存CIK,在12周内复苏应用于临床治疗较好。     

恩替卡韦联合细胞因子诱导的杀伤细胞序贯治疗慢性乙型肝炎患者树突状细胞相关功能观察

观察恩替卡韦联合细胞因子诱导的杀伤细胞(cytokine-induced kill cells,CIK)序贯治疗慢性乙型肝炎患者DC功能变化,为指导CIK联合核苷(类)药物抗病毒治疗提供理论依据。以15例接受恩替卡韦联合CIK序贯治疗的慢性乙型肝炎患者为观察对象,单独接受CIK治疗患者为对照,分别在治疗前、HBVDNA500IU/ml及CIK治疗2周后流式细胞技术测定DC表面共刺激分子CD1a、CD80、CD83及HLA-DR表达水平,同时淋巴细胞增殖实验评估DC细胞功能。结果显示15例恩替卡韦联合CIK序贯治疗患者同治疗前相比在接受恩替卡韦治疗并达到HBVDNA500IU/ml时仅有HLA-DR表达水平高于治疗前,其它共刺激分子标志物及淋巴细胞增殖能力没有显著变化。CIK序贯治疗后DC共刺激分子标志物和HLA-DR水平明显升高,并且淋巴细胞增殖能力也明显升高。单独CIK治疗患者同治疗前比较DC表面分子标志物水平及淋巴细胞增殖能力均无变化,同联合治疗组相比DC标志物水平和淋巴细胞增殖能力均低于联合治疗组。以上结果提示恩替卡韦联合CIK序贯治疗明显提高慢性乙型肝炎患者DC共刺激分子及HLA-DR表达,并诱导免疫细胞应答功能,恩替卡韦联合CIK序贯治疗可能通过增强DC相关功能提高慢性乙肝患者的抗病毒疗效。     

细胞因子诱导杀伤细胞免疫治疗晚期乳腺癌老年患者的临床研究

目的：观察在晚期乳腺癌老年患者的治疗中,采用细胞因子诱导的免疫胞进行肿瘤杀伤的临床相关研究进而探究其治疗方法。方法选择2012年2月~9月在我院治疗的晚期老年乳腺癌患者51例。将患者随机分为观察组和对照组,对照组采用该病的常规治疗措施进行治疗,而观察组则在常规的传统治疗的基础上进行细胞因子诱导性杀伤细胞的免疫治疗方法,在患者治疗两个疗程后对患者的治疗效果、机体免疫状态及药物毒副反应情况进行比较分析。结果对照组和观察组患者的临床治疗有效率分别为36.0%和69.2%,统计结果显示,该疾病治疗的有效率总体较低,但观察组明显有与对照组(P<0.05);两组患者的主要免疫细胞也有显著差异,观察组的免疫细胞数量明显高于对照组,差异具有统计学意义(P<0.05);观察组对患者的毒副作用也明显低于对照组(P<0.05)。结论对细胞因子诱导性的杀伤细胞进行免疫治疗能够显著提高老年晚期乳腺癌患者的治疗效果,同时在不提升患者毒副反应的前提下,改善患者的免疫状态,其临床效果具有安全、可靠性,值得在临床广为使用。     

黏附分子整合素CD11c对细胞因子诱导的杀伤细胞治疗胃癌疗效的影响

探讨胃癌组织中黏附分子CD11c的表达与患者预后的关系,并分析CD11c的表达对细胞因子诱导的杀伤细胞(CIK)治疗胃癌患者复发和死亡风险的影响。采用免疫组织化学染色法确定CD11c表达;应用COX多因素模型分析化疗联合CIK治疗组(下称联合组)与CD11c表达状态联合影响胃癌复发和死亡的风险。单因素统计学分析显示:联合组+CD11c高表达、联合组+CD11c低表达、化疗组+CD11c高表达和化疗组+CD11c低表达患者的中位无瘤生存时间及中位生存时间差异有统计学意义(均P<0.01)。在调整了性别、年龄、胃癌部位、肿瘤大小、分期、转移和治疗方式等混杂因素后,与联合组+CD11c高表达组相比,化疗组+CD11c低表达组复发和死亡风险显著增加(HR=5.76,95%CI=2.42~13.70;HR=5.00,95%CI=2.22~11.12),且联合组+CD11c高表达、联合组+CD11c低表达、化疗组+CD11c高表达和化疗组+CD11c低表达患者复发和死亡的HR呈上升趋势(趋势检验,均P<0.01)。肿瘤组织中CD11c高表达的胃癌患者采用化疗联合CIK细胞治疗可显著延长患者的生存时间,CD11c高表达可作为CIK细胞治疗的纳入标准。     

Human T cells with a type-2 cytokine profile are resistant to apoptosis induced by primary activation: consequences for immunopathogenesis

The mechanisms leading to a relative dominance of T cells producing type 2 cytokines in certain human immune disorders are still unclear. We investigated the relative susceptibility to apoptosis induced by primary in vitro activation of human type 1 (producing interferon-gamma (IFN-gamma)) or type 2 (producing IL-4) T cells. Peripheral blood lymphocytes were isolated from patients with immune disorders characterized by expansion of type 2 cells (four with AIDS and hyper-IgE/hypereosinophilia, one with Churg-Strauss syndrome, and one with idiopathic hypereosinophilic syndrome) or from individuals with normal cytokine balances. Cells were stimulated for 16 h with ionomycin and phorbol ester, and apoptosis of cytokine-producing cells was assessed by flow cytometry. T cells with a type-2 cytokine profile, i.e. producing IL-4 alone, were significantly more resistant to activation-induced apoptosis than those producing IFN-gamma alone. This was observed in AIDS patients, whose type 2 cells were mostly CD8+, as well as in the patients with Churg-Strauss and with hypereosinophilic syndrome. CD4+ and CD8+ IL-4-producing cells were equally resistant to apoptosis. Lower susceptibility to apoptosis of type-2 T cells was also observed in subjects with normal cytokine balances. Bcl-2 expression was high in type-2 cells and in viable type-1 cells, whereas it was low in apoptotic type-1 cells. Resistance to activation-induced apoptosis may explain the expansion of cells producing type-2 cytokines in certain immune disorders.     

Serum cytokine detection in the clinical follow up of patients with cystic echinococcosis

The relation of serum cytokine levels and outcome of chemotherapy was evaluated in 15 patients with cystic echinococcosis. Serum IL-4, IL-10 and interferon-gamma (IFN-gamma) concentrations were determined by ELISA before and after a 3-month course of albendazole treatment: at least one serum sample per patient from 13 patients (87%) contained measurable amounts of IL-4; samples from five patients (33%) measurable amounts of IL-10 and samples from only two patients (13%) measurable amounts of IFN-gamma. Clinical assessment at 1 year after the end of therapy showed that 11 of the 15 patients had responded clinically. Seven of these patients had lower IL-4 serum concentrations, two had unchanged and two undetectable amounts (pre- versus post-therapy, n = 11, P = 0.008). Conversely, of the patients who did not respond, three had higher and one patient unchanged serum IL-4 concentrations. Serum IL-10 10 levels also decreased in all patients who responded (3/5) and increased in all patients who did not (2/5). No association was found between cytokine concentrations and cyst characteristics or antibody levels. Overall these data suggest that serum IL-4 detection may be useful in the follow up of patients with cystic echinococcosis.     

中药对溴隐亭致流产大鼠保胎机理的实验研究

目的：探讨补肾益气中药对流产大鼠保胎的作用及其机理。方法：采用孕6、7、8d皮下注射溴隐亭0.125mg/d，诱发SD大鼠流产模型，以放免法测血清PRL、P的动态变化；RT-PCR法测孕12d蜕膜Th1/Th2型细胞因子的表达，观察中药治疗对妊娠结局及以上指标的影响。结果：溴隐亭致流产模型血清PRL、P水平下降，母胎界面Th1/Th2型细胞因子平衡偏向Th1型，补肾益气中药可使其血清PRL及P水平升高，母胎界面Th1/Th2型细胞因子平衡恢复正常。结论：改良溴隐亭致流产模型具备免疫内分泌网络失调的特征，中药可通过对母胎界面免疫内分泌网络的调控维持妊娠。     

自体外周血造血干细胞移植后序贯CIK细胞治疗急性髓系白血病

背景：细胞因子诱导的杀伤细胞作为一种有效的过继免疫治疗方法,成为治疗急性髓系白血病的一种新的手段,目前关于急性髓系白血病自体移植后序贯细胞因子诱导的杀伤细胞治疗的报道尚少,值得进一步研究。 目的：观察急性髓系白血病M2患者自体外周血造血干细胞移植后序贯细胞因子诱导的杀伤细胞治疗的临床疗效和不良反应。 方法：入选45例低、中危急性髓系白血病M2患者,其中19例在自体外周血造血干细胞移植后序贯了细胞因子诱导的杀伤细胞治疗,另26例未序贯细胞因子诱导的杀伤细胞治疗,比较两组患者的复发率、无病生存率、总生存率,观察细胞因子诱导的杀伤细胞治疗的安全性。 结果与结论：①序贯细胞因子诱导的杀伤细胞治疗组的复发率低于未序贯细胞因子诱导的杀伤细胞治疗组(21.05%,38.46%,P < 0.05);序贯细胞因子诱导的杀伤细胞治疗组患者的2年无病生存率和2年总生存率均高于未序贯细胞因子诱导的杀伤细胞治疗组,差异均有显著性意义(P < 0.05)。②19例接受细胞因子诱导的杀伤细胞治疗的患者均顺利完成治疗方案,治疗过程中除4例出现寒战、发热外,无其他不良反应。结果显示低、中危急性髓系白血病M2患者自体外周血造血干细胞移植后序贯细胞因子诱导的杀伤细胞治疗可降低原发病的复发率,提高患者的无病生存率及总生存率,且无明显不良反应,是一种安全、有效、可行的治疗方法。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

T cell proliferation and cytokine secretion to T cell epitopes of Asp f 2 in ABPA patients

The pathogenesis of allergic bronchopulmonary aspergillosis (ABPA) involves specific cytokines secreted by lymphocytes in response to Aspergillus fumigatus (Af) allergens. To gain information about the lymphoproliferative response and cytokine production against a major Af allergen, Asp f 2, we studied Asp-f-2-specific T cell clones (TCCs) from ABPA patients. TCCs were stimulated with rAsp f 2, its deletion mutants, and synthetic peptides to identify the T cell epitope(s) and to understand cytokine production. PBMCs from four of five ABPA patients showed proliferation in response to Asp f 2. Three TCCs from one patient showed higher IL-5 secretion compared to IL-4 and IFN-gamma. Two TCCs from the second patient showed a mixed Th1/Th2 response, as evidenced by production of IL-4, IL-5, and IFN-gamma. An epitope from the N-terminal region of Asp f 2 induced only IL-5 secretion. High IL-5 secretion might explain the marked eosinophilia observed in ABPA patients.     

Transcatheter arterial injection of autologous lymphokine-activated killer (LAK) cells into patients with liver cancers

We studied the effect of transcatheter arterial injection of lymphokine-activated killer (LAK) cells in conjunction with interleukin-2 on 13 patients with liver cancers, 9 primary hepatocellular carcinomas (HCC), and 4 metastatic liver cancers. Based on the fact that the LAK-cell phenomenon was observed to exist equally in these patients as in healthy controls, an injection of autologous LAK cells into these patients was performed. Of 13 patients receiving LAK cells plus interleukin-2, one had a partial response and three had minor responses. The response duration of this partial-response patient was around 6 months after the LAK-cell injection. Simultaneously, a reduction of serum AFP level was found in six of eight HCC patients. One patient having a minor response after receiving this therapy had severe side effects, such as dyspnea and hypotension. The others had only a transient fever which was easy to control by medication. Thus the present study indicates that a measurable tumor regression and a reduction of serum AFP level are observed in some liver cancers after undergoing this therapy.     

Kinetics and functional implications of Th1 and Th2 cytokine production following activation of peripheral blood mononuclear cells in primary culture

The importance of cytokine production in some disease processes is now widely recognized. To investigate temporal relationships between cytokines, we stimulated peripheral blood mononuclear cells (PBMC) in vitro using the T cell mitogen phytohemagglutinin (PHA) and various antigens chosen to induce predominantly Th1 (streptokinase: streptodornase or purified protein derivative) or Th2 (Dermatophagoides pteronyssinus, bee or wasp venom: allergens in sensitive subjects) responses. Cytokine production was measured by sensitive bioassays or enzyme-linked immunosorbent assays. Of the 30 subjects studied, 10 were normal and 20 individuals were allergic to either D. pteronyssinus (n = 10) or bee venom (n = 10) (examined before specific allergen immunotherapy). We examined the temporal profiles of a panel of cytokines produced in prmary culture. In PHA-driven cultures, cytokines were found to be sequentially produced in the order interleukin (IL)-2, IL-4, IL-5, IL-3, interferon (IFN)-γ, IL-10, IL-6, IL-12 and tumor necrosis factor (TNF)-α. The response to allergen in allergic patients was predominantly Th2 in nature, with the production of IL-4, IL-5, IL-6 and IL-10, but little or no IFN-γ. IL-2, IL-3, TNF-α and IL-12 were also produced in low amounts. The response of both atopic and normal subjects to recall bacterial antigens was predominantly Th1, with high levels of IFN-γ, IL-2 and TNF-α. The relevance of the order, amount and speed of production, characteristic kinetics (production, consumption, homeostatic regulation) and the cell source of the cytokines are discussed.     

Th1/Th2相关细胞因子基因检测方法的建立

目的：建立检测Ｔｈ１／Ｔｈ２相关细胞因子的方法。方法：设计６对用于ＩＬ－２、ＩＬ－４、ＩＬ－１０、ＩＬ－１３、ＩＦＮγ和β－ａｃｔｉｎ基因扩增的引物,建立可用于Ｔｈ１／Ｔｈ２相关细胞因子检测的ＲＴ－ＰＣＲ技术,同时 口平移法制备６种细胞因子的ｃＤＮＡ探针,建立ＲＮＡ斑点杂交技术。结果：用ＲＴ－ＰＣＲ和ＲＮＡ斑点杂交技术检测２０种肿瘤细胞株,发现１１／２０为Ｔｈ２型细胞因子表达,８／２０为Ｔｈ０型,