人外周血NK细胞的纯化及体外扩增

ＮＫ细胞是机体防御体系的第一道防线,近年来对它的研究不断深入,本文概述了ＮＫ细胞的纯化手段和扩增技术,并简单介绍了其应用情况。     

高聚金葡素对中晚期肝癌患者多药抗药性基因表达及NK细胞活性的影响

研究高聚金葡素对中晚期肝癌患者多药抗药性（ＭＤＲ）基因表达及ＮＫ细胞活性的影响。结果显示化疗联合高聚金葡素治疗组（５０例）较单纯化疗组（３０例）可明显抑制ＭＤＲ基因过度表达，升高ＮＫ细胞活性，临床疗效显著优于对照组，副反应发生率明显降低     

金米冲剂对原发性肝癌患者多药抗药性基因表达及NK细胞活性的影响

以肝动脉插管化疗栓塞（ＴＡＣＥ）是当前临床治疗原发性肝癌的主要手段。但由于肿瘤细胞的多药抗药性（ＭＤＲ）,常常影响治疗效果。为此我们研究了金米冲剂对原发性肝癌ＭＤＲ基因表达及ＮＫ细胞活性的影响,以期提高肝动脉插管化疗栓塞（ＴＡＣＥ）疗效。 将经ＣＴ、ＡＦＰ等检查证实的原发性肝癌的患者随机分成治疗组,对照组,各组各３０例。 治疗组于ＴＡＣＥ前一周即开始以金米冲剂（慧苡仁、云苓、炙鸡金、炒白术、昆布、海带、山慈姑、白英、丹参、白茅根、仙鹤草、八月扎、陈皮诸药为原料经科学提炼加工而成）每次１０克,每日 …     

口腔癌患者外周血NK细胞活性的临床意义

我们采用四甲基偶氮唑蓝（ＭＴＴ）法测定３３例口腔癌患者外周血ＮＫ细胞活性，并以正常人群作对照，观察不同病理类型、分化程度及分期的癌症患者ＮＫ细胞活性水平，探讨ＮＫ细胞活性的检测对口腔癌辅助诊断价值及临床应用前景。１资料与方法１．１检测对象实验组：口腔...     

热疗对荷瘤小鼠NK细胞活性的影响

为研究热化疗综合治疗癌症对机体免疫功能的影响，采用ＹＡＣ－１细胞￣３Ｈ－ＴｄＲ掺入抑制的ＮＫ细胞活性检测法，测定了经不同方法治疗的荷瘤鼠脾脏ＮＫ细胞的变化情况。ＢＡＬＢ／Ｃ小鼠６８只，设５组，为正常对照组，实验组分为不治疗组、平阳霉素治疗组、热疗组及热疗联合平阳霉素组。实验组小鼠于右后足底支下接种Ｓ＿（１８０）细胞２×１０￣７／０．１ｍｌ．治疗两次，间隔７天后处死取脾脏进行ＮＫ细胞活性检测。结果表明不治疗组与平阳霉素组ＮＫ细胞活性无差异（Ｐ＞０．０５）；热化疗组与正常对照组无差异。证实单纯化疗组其ＮＫ效应细胞功能受到严重损害，不能发挥应有的免疫作用；而联合热疗则可恢复其正常ＮＫ细胞活性，增强免疫功能。     

冷冻影响肝癌细胞HLA和B7分子表达的实验研究

目的：探讨冷冻治疗后机体免疫功能提高的机制。方法：应用流式细胞仪检测人肝癌细胞经0℃或－20℃冷冻后，HLA和B7分子表达率和平均荧光强度的变化。结果：0℃组与对照组比较，HLA－Ⅰ分子和B7－2分子的表达率和平均荧光强度均无明显变化；HLA－Ⅱ分子和B7－1分子的表达率增高，平均荧光强度增强。－20℃组与对照组比较，HLA－Ⅰ、HLA-Ⅱ、B7-1和B7－2分子的表达率均增高，平均荧光强度均增强。－20℃组和0℃组比较，HLA－Ⅱ和B7－1分子的表达率和平均荧光强度均无显著性差异；HLA－Ⅰ和B7－2分子表达率均增高，平均荧光强度均增强。结论：0℃和－20℃冷冻可增强人肝癌细胞HLA和B7分子的表达，这可能是冷冻治疗提高机体免疫功能的机制之一。     

全反式维甲酸对大鼠肝癌前状态NK细胞活性的影响

本文采用乳酸脱氢酶释放法测定了全反式维甲酸对二乙基亚硝胺诱导的鼠肝癌前状态自然杀伤细胞活性。结果表明二乙基亚硝胺诱癌１２周可明显导致大鼠ＮＫ细胞活性下降，而全反式维甲酸可预防二乙基亚硝胺的这种作用。因此，我们认为全反式维甲酸提高机体自然杀伤细胞活性可能为它预防肿瘤发生的机制之一。     

肿瘤逃逸NK细胞攻击的分子机制及逆转作用

通过分析NK细胞活化性受体、抑制性受体以及它们相应的配体，概括了NK细胞的天然肿瘤免疫监视功能和肿瘤细胞逃逸NK细胞杀伤的分子机制，在此基础上提出阻断NK细胞的抑制性信号，激发活化性信号，可以提高NK细胞的杀瘤效应，为肿瘤生物治疗探索新路。     

高聚金葡素对中晚期肝癌患者多药抗药性基因表达及NK细胞活性 …

研究高聚金葡素对中晚期肝癌患者多药抗药性（ＭＤＲ）基因表达及ＮＫ细胞活性的影响，结果显示化疗联合高聚金葡素治疗（５０例）较单纯化疗组（３０例）可明显抑制ＭＤＲ基因过度表达，升高ＮＫ细胞活性，临床疗效显著优于对照组，副反应发生率明显降低。     

DC-CIK共培养细胞联合索拉菲尼对肝癌细胞体内外的杀伤效应

目的：观察DC、CIK共培养细胞（DC-CIK）联合索拉菲尼(sorafenib)对肝癌细胞BEL-7402的体内外杀伤效应。方法：取健康人外周血单个核细胞,加入不同细胞因子促进DC及CIK细胞成熟并混合共培养。CCK8试剂盒检测DC-CIK共培养细胞联合索拉菲尼对BEL-7402细胞的体外杀伤效应,Annexin V-FITC试剂盒检测两者联合对肝癌细胞凋亡率的影响。用肝癌细胞BEL-7402建立裸鼠皮下移植瘤模型,分为生理盐水对照组、索拉菲尼组、DC-CIK组、DC-CIK+索拉菲尼组,观察它们对裸鼠移植瘤生长的抑制作用。结果：联合组对肝癌细胞的杀伤率及诱导凋亡率均明显高于各单独治疗组,联合组杀伤率高达（75.24±1.91）%,是DC-CIK组的1.8倍,是索拉菲尼单药组的2.1倍（P<0.01）;联合组诱导肝癌细胞凋亡率达（78.32±2.54）%,与单独治疗组相比差异有统计学意义（P<0.05）。体内实验表明,DC-CIK+索拉菲尼组可明显抑制裸鼠BEL-7402移植瘤的生长,抑制率为（83.37±0.16）%,与单独治疗组相比差异有显著统计学意义（P<0.01）。结论：DC-CIK共培养细胞联合索拉菲尼在体内、外可显著抑制肝癌细胞的生长,分子靶向治疗联合细胞免疫治疗可能成为肝癌综合治疗的方法之一。     

GM-CSF gene or B7-1 gene modified murine EL-4 cells vaccine

Since 1990, gene transduced tumor vaccine has been studied. Many articles reported that tumor cells transduced with some cytokine or costimulatory molecule could induce system antitumor immunity[1,2] In this study, EL-4 lymphoma was transduced with recombinant retrovirus containing the murine GM-CSF gene and B7-1 gene, respectively. The effect of gene transduction on antitumor immunity was investigated.MATERIALS AND METHODSMice and Cell Lines Female C57BL/6 mice were bought from …     

Direct effect of dasatinib on proliferation and cytotoxicity of natural killer cells in in vitro study

Lymphocytosis predominantly due to natural killer (NK) cells has been reported in nearly a half of chronic myelogenous leukemia (CML) patients who were being treated with dasatinib. Besides, dasatinib‐treated patients with lymphocytosis have a better prognosis than patients without lymphocytosis. In order to elucidate the effects of dasatinib on the proliferation of lymphocyte subset, dasatinib was added to the culture of peripheral blood mononuclear cells with IL‐2 (lymphokine‐activated killer culture) or a low dose of IL‐2 with zoledronate (γδ T‐cell culture). In both culture conditions, NK cells were increased in both percentage and absolute number in the culture with dasatinib compared with control culture without dasatinib. The increase of NK cells was dose dependent of dasatinib in the range of 2–25 nM. NK cell cytotoxicity of cultured cells with dasatinib was demonstrated to be superior to control cells without dasatinib in cytotoxicity assay using EGFP‐transfected K562 cells as target cells. The present study suggested that lymphocytosis in dasatinib‐treated CML patients is at least partly associated with a direct effect of dasatinib to stimulate the proliferation of NK cells. Favourable prognosis in patients with dasatinib‐induced lymphocytosis might be associated with the effects of dasatinib to potentiate NK cytotoxicity in vivo. Copyright © 2012 John Wiley & Sons, Ltd.     

KIR2DS1受体对人类NK细胞杀伤白血病细胞的作用研究

目的 探讨活化性杀伤细胞免疫球蛋白样受体（KIR）家族成员KIR2DS1在自然杀伤（NK）细胞杀伤白血病细胞中的作用.方法 采集健康人群外周血单个核细胞,采用NK细胞分选试剂盒富集NK细胞,将分选的NK细胞进行体外扩增培养,以作为效应细胞.取初治确诊的急性髓细胞白血病（AML）患者新鲜骨髓液,分离后的白血病细胞作为靶细胞.采用CCK8比色法检测NK细胞对白血病细胞的杀伤率.采用聚合酶链反应和顺序特异性引物（PCR-SSP）基因分型法分别检测NK细胞及靶细胞的KIR基因和HLA-Cw;根据KIR2DS1表达与否将NK细胞分为KIR2DS1阳性NK细胞与KIR2DS1阴性NK细胞;用抗-KIR2DS1抗体封闭NK细胞的KIR2DS1受体,用CCK8比色法检测封闭前后KIR2DS1阳性组NK细胞对靶细胞的杀伤率.根据HLA-Cw将NK细胞和靶细胞分别分为C1纯合子组（表达HLA-Cw 01、03、07、08、12、14、16）、C2纯合子组（表达HLA-Cw02、04、05、06、15、17、18）和C1/C2杂合子组（既表达C1组的等位基因又表达C2组的等位基因）.结果 经过富集后NK细胞的纯度为（91.2±5.94）％;不同效靶比下,KIR2DS1阳性NK细胞的杀伤率明显高于阴性组;当效靶比为10：1时,KIR2DS1阳性NK细胞组对白血病细胞的杀伤率（42.82±6.81）％高于KIR2DS1阴性组（28.61±5.14）％;抗-KIR2DS1封闭后NK细胞对白血病细胞杀伤作用明显减弱（t=-3.00,P＜0.05）;KIR2DS1阳性NK细胞C1纯合子组对靶细胞C2纯合子组的杀伤率（54.39±3.46）％明显高于靶细胞C1组（41.22±3.68）％（t=8.33,P＜0.05）和C1/C2组（41.32±5.09）％（t=6.37,P＜ 0.05）.结论 KIR2DS1阳性NK细胞对白血病细胞的杀伤效力高于KIR2DS1阴性NK细胞,即KIR2DS1可以有效介导NK细胞杀伤白血病细胞;HLA-C1纯合子的KIR2DS1阳性NK细胞对HLA-C2纯合子的靶细胞杀伤作用强.     

同种异体NK细胞KIR2DL1表达差异对其杀伤KG1A细胞活性的影响

目的:研究同种异体自然杀伤(natural killer,NK)细胞对人急性髓系白血病细胞株KG1A的杀伤率,探讨供者NK细胞表面杀伤细胞免疫球蛋白样受体2DL1(killer immunoglobulin-like receptor 2DL1,KIR2DL1)表达差异对NK细胞杀伤KG1A细胞活性的影响。方法:自9名健康志愿供者分离外周血NK细胞,流式细胞术检测NK细胞表面KIR2DL1的表达率,LDH释放法测定NK细胞在效靶比为20∶1时对KG1A细胞的杀伤活性。结果:NK细胞表面KIR2DL1的表达率存在个体差异,9名供者的表达范围为(33.20±1.95)%～(92.69±1.47)%;9名健康供者NK细胞对KG1A细胞的杀伤活性不同,杀伤率范围为(44.40±1.97)%～(93.70±1.12)%。不同个体NK细胞KIR2DL1表达率与其对KG1A细胞的杀伤率呈负相关(r=-1.00,P=0.00)。结论:不同个体NK细胞其表面KIR2DL1表达率与NK细胞对KG1A细胞的杀伤率呈负相关。     

自然杀伤T细胞对小鼠肝癌移植瘤的抑制作用

Objective:The aim of this study was to investigate the inhibition effect of natural killer T(NKT) cells on transplantation hepatocellular carcinoma in mice.Methods:α-galactosylceramide(α-GalCer)-pulsed DC and Hep S were prepared as stimulus.Hepatoma xenograft model was established and mice were randomly divided into 4 groups(n=13 each group):(1) control group,intravenous injection of the same volume of saline.(2) mature DC group,intravenous injection of mature DC cells(4×106 cells).(3) α-GalCer-pulsed HepS ...     

In vitro natural killer and lymphokine-activated killer activity in patients with bronchogenic carcinoma.

The authors examined peripheral blood mononuclear cells from 45 patients with bronchogenic carcinoma to determine natural killer (NK) and lymphokine-activated killer (LAK) activity after in vitro incubation with media alone or media plus interferon gamma (IFN, 200 U/ml) and/or interleukin-2 (IL-2, 100 U/ml). Our results show that lymphocytes from patients with bronchogenic carcinoma can acquire LAK activity, but the level of activity acquired was significantly lower compared with lymphocytes from 25 control subjects when IL-2 cultures were supplemented with 10% autologous human serum (AHS) (15.6% +/- 2.1% specific release versus 26.0% +/- 2.9% specific release, P = 0.004). The LAK activity, defined as cytotoxicity of an NK-resistant cell line, of the patients' lymphocytes was augmented when cells were cultured with both IL-2 and IFN compared with IL-2 alone (P = 0.0001, paired t-test). Control subjects were unchanged (P = 0.09). There was no significant difference between groups of patients with different histologic types of tumor or different stages of disease. The NK activity, defined as killing of NK-sensitive K-562 target cells, of the patients' lymphocytes was not significantly different from that of the controls' lymphocytes (42.8% +/- 3.0% specific release versus 49.3% +/- 3.3% specific release, P = 0.16). These studies indicate the feasibility of IL-2 and IFN therapy in patients with bronchogenic carcinoma.     

二甲双胍体外诱导人肝癌Huh-7细胞凋亡及其作用机制

目的 探讨二甲双胍对人肝癌Huh-7细胞增殖、凋亡的影响及其可能的作用机制.方法 二甲双胍干预Huh-7细胞后,四甲基偶氮唑蓝(MTT)法检测其对细胞活力及生长抑制的影响;Western blot检测凋亡相关蛋白及CD133蛋白的变化;流式细胞术检测细胞凋亡及肝癌干细胞相关标志物CD133的表达;无血清悬浮培养观察二甲双胍对肿瘤干细胞球形成的影响;逆转录聚合酶链反应(RT-PCR)检测其对肿瘤干细胞相关基因CD133、β-catenin、ABCG2 mRNA表达的影响.以加入与实验组相同的培养液但未给予药物处理的组为对照组.结果 MTT检测结果显示,与对照组比较,随着二甲双胍浓度的增高,肝癌Huh-7细胞的增殖率下降,差异均有统计学意义(均P＜0.05).10 mmol/L二甲双胍干预48 h后肝癌Huh-7细胞的早期凋亡率为(22.29±0.80)％,对照组的早期凋亡率为(6.70±0.50)％,差异有统计学意义(P ＜0.05);10 mmol/L二甲双胍干预48 h后肝癌Huh-7细胞的晚期凋亡率为(13.87±1.20)％,对照组的晚期凋亡率为(1.10±0.02)％,差异有统计学意义(P＜0.05).25 mmol/L二甲双胍干预48 h后细胞的早期凋亡率为(15.28±2.10)％,晚期凋亡率为(25.89±2.30)％,均高于对照组(均P＜0.05).Western blot检测结果显示,25 mmol/L二甲双胍干预Huh-7细胞48 h后,p-Akt、Bcl-2/Bax比值表达下调,PTEN表达上调.随着二甲双胍浓度的增加,CD133蛋白的表达下调.流式细胞仪检测结果显示,25 mmol/L二甲双胍干预Huh-7细胞48 h前后,Huh-7细胞中CD133+细胞的表达率分别为(40.7±2.1)％和(14.6±1.85)％ (P ＜0.05).在低黏附培养皿、无血清培养液的生长环境中,对照组细胞球体积较大,中心厚重,周围清亮,折光度强;10mmol/L二甲双胍组细胞球体积较小,球体折光度较弱,细胞球数量少于对照组.RT-PCR检测结果显示,肿瘤干细胞相关基因CD133、β-catenin、ABCG2 mRNA表达水平均下调.结论 二甲双胍能显著抑制人肝癌Huh-7细胞增殖,促进凋亡.其机制可能与抑制肝癌PI3 K/Akt信号通路和CD133+肿瘤干细胞的表达有关.     

Involvement of interaction between Fractalkine and CX3CR1 in cytotoxicity of natural killer cells against tumor cells

Many chemokine receptors are typically found on natural killer cells, including CX3CR1, the receptor for the chemokine fractalkine (FKN). This study explored whether interaction between CX3CR1 and FKN is relevant for NK cell functions in cytotoxicity against tumors. FKN expression was examined by polymerase chain reaction and CX3CR1 expression in NK cells was analyzed by flow cytometry. NK cell cytotoxicity was examined by 4-h 51Cr-release assay. FKN was expressed in a variety of tumor cell lines such as K562 cells, an NK-sensitive cell line. Approximately 90% of peripheral blood NK cells and almost all of the NK cell line, NK-92 cells, expressed CX3CR1. Anti-CX3CR1 antibody strongly neutralized the cytotoxicity of NK cells against K562 cells, and pretreatment of NK cells with recombinant soluble FKN improved the cytolytic function on tumor cells. This study demonstrates that an interaction between CX3CR1 on NK cells and FKN on tumor cells is involved in the natural cytotoxicity of NK cells against tumors.