同种异体NK细胞对CD+34早期急性髓系白血病细胞的杀伤活性

目的探讨同种异体NK细胞对CD3+4早期急性髓系白血病(AML)细胞的体外杀伤活性。方法免疫磁珠法分离5例健康个体NK细胞,以NK杀伤敏感细胞株K562作为对照,乳酸脱氢酶(LDH)释放法测定不同效靶比时NK细胞对CD3+4早期AML细胞KG1a的杀伤活性。结果AML细胞株KG1a中CD34抗原表达率为98.0%±1.1%,分选后的NK细胞纯度为93.2%±3.7%。不同效靶比时NK细胞对KG1a细胞均有杀伤活性,且随着效靶比的增高,其杀伤活性增高(P<0.05)。结论同种异体NK细胞对CD34+早期AML细胞具有一定的杀伤活性。     

CIK对耐药肿瘤细胞的杀伤作用

目的探讨细胞因子活化的杀伤细胞(CIK细胞)对耐药肿瘤细胞的杀伤作用。方法选用人食管癌细胞株Ecap-109作为实验细胞,用顺铂(cDDP)反复刺激建立人食管癌细胞耐药细胞模型,检测cDDP对Ecap-109的抑制率和药物半数抑制浓度(IC50)确定诱导耐药效果,之后用CIK细胞对耐药的Ecap-109细胞进行体外杀伤实验,并与未诱导的Ecap-109细胞杀伤活性进行比较。结果经长期反复诱导后,检测诱导的Ecap-109细胞IC50为70.869μmol/L,而亲本Ecap-109细胞为20.561μmol/L,两者相比有统计学意义(P0.01)。用此细胞做耐药细胞,同时用亲本Ecap-109细胞做对照,检测杀伤活性。CIK细胞对耐药细胞Ecap-109和亲本细胞Ecap-109的杀伤活性分别为83.01%和80.43%,两者比较没有显著差异。结论 CIK细胞对耐药肿瘤细胞Ecap-109的杀伤作用与对正常Ecap-109细胞的杀伤作用没有明显差异,证明CIK细胞可以杀伤耐药肿瘤细胞。     

不同细胞因子联合诱导K562细胞向杀伤细胞转化的研究

目的 探讨肿瘤细胞系K562向杀伤细胞转化的可能性。方法 通过rhIL-2、rhIL-15和A23187的不同组合诱导K562细胞,动态监测细胞形态学、免疫表型及杀伤功能变化。结果 经过40d的诱导,K562细胞在各细胞因子组合下均可以被诱导成具有杀伤功能的CD3^-CD36^＋NK细胞和CD3^＋CD56^＋NKT细胞,以IL-2＋IL-15组的诱导率最高,杀伤功能最强;IL-2＋A23187组的免疫标记出现最早。结论 IL-2可将K562细胞诱导成杀伤细胞dL-15可提高K562细胞向杀伤细胞转化的诱导率;A23187可能加速K562细胞向杀伤细胞的转化。     

家蝇抗菌肽体外抗弓形虫的效果观察

目的 观察家蝇幼虫和蛹抗菌肽对弓形虫速殖子的抑制作用. 方法 采用损伤加感染的方法,诱导家蝇幼虫和蛹大量产生抗菌肽,通过研磨、低温高速离心等方法获得抗菌肽粗提物,分别用磷酸缓冲液(PBS)调整为4 mg/ml、2 mg/ml和1 mg/ml并作用于弓形虫速殖子,24 h后,通过血细胞计数法和扫描电镜观察两种粗提物对弓形虫速殖子的抑制作用. 结果 幼虫抗菌肽组和蛹抗菌肽组速殖子存活率均低于对照组,差异有统计学意义(P<0.05),其抗弓形虫作用随着抗菌肽浓度的减小而减小;扫描电镜观察,对照组弓形虫速殖子外型完整,表面平滑,抗菌肽组速殖子则虫体膨胀.表面凹凸不平. 结论 从家蝇幼虫和蛹中提取的抗菌肽都有抗弓形虫作用,幼虫抗菌肽效果更好.     

EphB4介导K562细胞株对伊马替尼耐药性的产生

目的:探讨EphB4受体在慢性髓细胞白血病细胞株产生伊马替尼(IM)耐药性中的作用。方法:IM诱导K562-W(野生型)产生耐药的K562-R细胞株。同时采用RNA干扰技术,利用K562-R细胞株构建稳定低表达EphB4的K562-R-EphB4-sh细胞株。MTT法分别检测3种细胞对IM耐药性的变化(IC50)。结果:对IM耐受的K562-R细胞株显著高表达EphB4mRNA和蛋白,而构建的K562-R-EphB4-sh细胞株EphB4表达显著低于K562-W与K562-R细胞株(P0.01)。耐药性检测表明,与K562-R细胞比较,K562-R-EphB4-sh细胞株对IM的敏感性明显加强(IC50为0.93mg/L∶5.45mg/L,P0.01)。结论:K562-R细胞株低表达EphB4后,细胞对IM的敏感性显著恢复,提示EphB4可能是慢性髓细胞白血病细胞株中产生对IM耐药的一个新作用因子。     

CD4+ T细胞克隆对自体非霍奇金淋巴瘤具有特异的肿瘤杀伤作用

目的:研究CD4 T细胞的抗肿瘤能力。方法:利用1例滤泡B淋巴细胞淋巴瘤患者的外周血淋巴细胞,将缺乏人类白细胞抗原Ⅰ(HLA-Ⅰ)的恶性CH1细胞株作为惟一的刺激源,以梯度稀释法,得到3种自体的、细胞毒性的、具有肿瘤杀伤特异性的CD4 T细胞克隆。结果:3种T细胞克隆可以杀伤来自于新鲜恶性B细胞的自体HLA-Ⅰ分子缺乏的淋巴瘤细胞株,但却对EB病毒(EBV)阳性的正常B细胞或K562细胞无杀伤溶解作用。3种肿瘤特异杀伤的CD4 T细胞均为TCR Vβ17-Dβ1-Jβ1.2,并具有同样的CDR3。结论:研究表明,CD4 T细胞克隆能够被扩增,其能够特异性地杀伤从自体淋巴瘤细胞衍生的细胞株。通过产生CD4 T细胞来杀伤肿瘤细胞及其相应的过继转移,特别是在肿瘤逃避CD8 T细胞介导的免疫杀伤作用时,CD4 T细胞能起到替代作用。     

树突细胞负载骨髓瘤抗原介导的免疫反应

目的 研究负载肿瘤抗原的树突细胞能否诱导特异性细胞霉T淋巴细胞反应。方法 用多发性骨髓瘤患者骨髓CD34^ 细胞诱生树突细胞，并将骨髓瘤冻融物冲击致敏树突细胞。采用MTT法检测骨髓瘤抗原致敏及未致敏树突细胞诱导的自身T细胞对不同靶细胞(患者骨髓瘤细胞、K562细胞株)的杀伤率。结果 骨髓瘤冻融物致敏树突细胞诱导的自身T细胞对患者骨髓瘤细胞的杀伤远大于对K562细胞的杀伤。结论 患者骨髓瘤冻融物致敏的树突细胞能有效诱导自身T细胞发生特异性抗肿瘤免疫。     

抗菌肽抗肿瘤作用机制的研究进展

抗菌肽(antimicrobal peptides,AMPs)是天然免疫系统中的一种小分子多肽,一些具有抗肿瘤活性,毒副反应小,不易产生耐药性.近年来,研究已经明确了抗菌肽对肿瘤细胞的杀伤抑制作用及其作用机制,其中作用机制包括以下5方面:(1)在肿瘤细胞膜上形成穿膜孔道,使肿瘤细胞死亡;(2)作用于细胞骨架使其结构紊乱;(3)抑制DNA合成,影响细胞增殖;(4)作用于线粒体,引起肿瘤细胞凋亡;(5)影响免疫效应进而杀伤肿瘤细胞.该文就抗菌肽抗肿瘤细胞的作用机制作一综述.     

脐血CIK细胞对耐药白血病细胞体外杀伤效应及其机制的研究

目的:探索干预或克服白血病细胞耐药的新策略.方法:采用抗CD3McAb联合多种细胞因子诱导的脐血杀伤细胞(CB-CIK)体外作用于K562耐药细胞株(K562/A02),用MTT比色法检测其杀伤效应;用免疫组化染色法检测杀伤前后K562/A02细胞表面多药耐药基因mdr1表达产物P170水平;采用DNA凝胶电泳进行CB-CIK细胞诱导白血病细胞凋亡的检测.结果:①CB-CIK细胞杀伤K562/A02细胞活性(73.647±5.72)与杀伤K562细胞活性(75.124±4.36)相比差异无统计学意义,P＞0.05;其杀瘤活性显著高于CB-LAK细胞、CB-CD3AK细胞,P<0.05,与成人CB-CIK细胞相比差异无统计学意义,P＞0.05.②经CIK细胞杀伤后,K562/A02细胞表面mdr1表达产物P170含量明显减少.③K562/A02细胞在CIK细胞作用20 h后,其DNA结构断裂,在DNA凝胶电泳上呈现凋亡特有的梯形图谱(DNA Ladder).结论:CB-CIK细胞对K562细胞及其耐药株K562/A02细胞均有较强的杀伤作用,其杀伤机制可能与CIK细胞能下调白血病细胞表面多药耐药基因mdr1表达产物P170水平,以及诱导白血病细胞凋亡有关.提示脐血CIK细胞在抗白血病多药耐药性方面具有独特的优势,若与化疗联合使用,有可能成为克服白血病细胞多药耐药性的一种可供选择的新策略,因脐血的来源较广,采制相对简便,该研究方法可能具有广泛的应用前景.     

脐带血DCs对CIK细胞杀伤活性的影响研究

目的：探讨相同来源脐带血DCs对CIK细胞杀伤活性的影响作用。方法：采用相同来源的脐带血单个核细胞分别制备树突状细胞，CIK细胞，用流式细胞术检测免疫表型，MTT法检测CIK和CIK＋DC对K562，HL－60两种肿瘤细胞株的杀伤活性。结果：脐带血DC高表达CD1a,HLA-DR,CD80,CD86等抗原，CIK细胞是以CD3^ T细胞为主的异质性细胞群体，随培养时间延长CD3^ CD56^ 细胞比例明显升高，DC不改变CIK的免疫表型，但能明显促进其对K562和HL－60的杀伤活性（P＜0．05），结论：适量DC能增强CIK的杀伤活性，为DC的免疫治疗提供了新的思路。     

Target cell-induced apoptosis of interleukin-2-activated human natural killer cells: roles of cell surface molecules and intracellular events

We previously reported that natural killer (NK)-sensitive target cells, K562, kill interleukin-2-stimulated (lymphokine-activated killer [LAK]) but not unstimulated NK cells. We have now investigated the molecular basis of this phenomenon. Soluble monoclonal antibody (MoAb) to CD18 inhibited 75% of K562-induced DNA fragmentation and membrane disruption, whereas blocking MoAb to Fas partially inhibited only the DNA fragmentation. MoAbs to CD2, CD11a, CD11b, B7, or CD16 had limited or no effect on K562-induced death of LAK cells. Receptor ligation with either immobilized MoAb to CD18 or Fas induced membrane disruption and DNA degradation in LAK cells independently of K562, and MoAb to CD18, CD11a, or CD11b enhanced DNA fragmentation induced by anti-Fas. Fas-L- transfected Raji cells also killed LAK cells, but only if Fas-L expression was amplified. K562 cells rapidly triggered protein phosphorylation in LAK cells, and the tyrosine kinase inhibitor, Herbimycin A, inhibited DNA fragmentation and membrane disruption. Protease inhibitors strongly suppressed K562-mediated DNA fragmentation of LAK cells, but not membrane disruption. In conclusion, (1) K562- induced death of LAK cells involves primarily CD18, although other molecules, such as Fas, may also be involved; (2) K562-mediated apoptosis of LAK cells requires tyrosine phosphorylation and protease activity; (3) engagement of Fas by immobilized MoAb or Fas-L on target cells can also kill LAK cells; and (4) Fas-immobilized MoAb synergizes with coimmobilized MoAb to CD11a, CD11b, or CD18 for LAK cell killing. Activation-induced death of NK cells may represent a mechanism for NK cell regulation.     

齐墩果酸对K562细胞系VEGF表达影响的实验研究

目的观察齐墩果酸(Oleanolic Acid,OA)对慢性粒细胞白血病系K562细胞中血管内皮生长因子(VEGF)表达的影响。方法采用MTT实验观察OA对体外培养K562细胞增殖抑制作用,用RT-PCR以及Western印迹法研究OA处理后K562细胞中VEGF基因表达的变化。并用ELISA方法检测培养液中VEGF的表达。结果OA能抑制K562细胞生长,呈一定的量效特征;并能抑制K562细胞中VEGF的表达。结论OA通过下调VEGF表达,抑制白血病细胞的增殖。     

外体负载特异性抗原促进小鼠抗肿瘤效应的研究

目的:验证人树突状细胞(DC)分泌的外体(exosomes,EXO)负载NY-ESO-1抗原促进转基因小鼠特异性细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL)扩增及增强肿瘤杀伤效应。方法:应用人类白细胞抗原(human leukocyte antigen,HLA)-A2阳性健康成人的外周血分离诱导外周血单个核细胞(peripheral blood mononuclear cells,PBMC)来源的成熟DC,利用一系列不同速度的离心结合膜超滤的方法,从其上清中提取成熟DC分泌的EXO。人工负载肿瘤睾丸抗原NY-ESO-1于EXO之上(EXO/Ag),与聚肌胞(PolyI-C)共同免疫健康HLA-A2转基因C57小鼠,并在体系中加入EXO/Ag继续体外培养脾细胞。使用以上方法获得的脾细胞行四聚体实验,并对K562细胞及黑色素瘤细胞(对照组)行杀伤实验。结果:脾细胞中NY-ESO-1特异的CTL有所扩增,对K562肿瘤细胞有一定杀伤效应。体外实验显示NY-ESO-1特异的CTL较前大量扩增,对K562肿瘤细胞杀伤效应大大增加。结论:EXO/Ag结合PolyI-C作为新一代抗肿瘤疫苗具有良好的应用前景。     

Hormonal effects on cell proliferation in a human erythroleukemia cell line (K562)

We have investigated the hormonal responsiveness of K562 cells using a serum-substituted in vitro clonogenic assay. Dexamethasone inhibited colony formation by the K562 cells, and the inhibitory effect could be reversed by progesterone (10(-6) M). Fluoxymesterone caused a prominent enhancement of K562 colony growth, whereas estriol had no effect. Stimulation by triiodothyronine was maximal at 10(-7) M, and the thyroid effect could be abrogated by the beta 2-adrenergic antagonist butoxamine in equimolar concentrations. Using standard tissue culture conditions, the beta-adrenergic agent isoproterenol, but not the alpha catecholamine phenylephrine, enhanced the proliferation of K562 cells. When K562 cells were grown under hormone-depleted conditions, they developed responsiveness to phenylephrine and were no longer stimulated by isoproterenol. DbcAMP and prostaglandins of the E series also caused K562 colony enhancement. Prostaglandin F2 alpha had no effect on cell proliferation. Insulin was an effective stimulant of colony formation of K562 cells, as were human growth hormone and ovine prolacin. Bovine growth hormone had no effect. Our results are consistent with the identificaiton of K562 as an erythroid line, and they indicate that K562 cells respond to endocrine hormones in a manner analogous to normal erythroid progenitors.     

Mannan and oligomers of N-acetylglucosamine protect intestinal mucosa of celiac patients with active disease from in vitro toxicity of gliadin peptides

Wheat flour and other cereals toxic for celiac patients contain an alcohol-soluble protein fraction that, under experimental conditions simulating in vivo protein digestion, yields peptides that agglutinate undifferentiated K 562(S) cells. In contrast, cereals well tolerated in celiac disease (i.e., rice and maize) do not. Furthermore, purified A-gliadin peptides that damage in vitro-cultured flat celiac mucosa are powerful agglutinins for K 562(S) cells, whereas A-gliadin peptides that do not show any adverse in vitro effect on celiac intestine lack agglutinating activity. Mannan, acetylglucosamine, and its oligomers (N,N'-diacetylchitobiose and N,N',N"-triacetylchitotriose) were able to prevent and reverse cell agglutination induced by peptides from all the toxic cereals. Moreover, mannan and N,N',N"-triacetylchitotriose exhibited a protective effect on intestinal mucosa specimens of patients with active celiac disease cultured with wheat protein-derived peptides. These data are consistent with the hypothesis that the agglutinating and toxic peptides are bound by carbohydrates.     

Enhanced CML stem cell elimination in vitro by bryostatin priming with imatinib mesylate

OBJECTIVE: In chronic myeloid leukemia (CML), imatinib mesylate (IM; Gleevec, Glivec) induces a G0/G1 cell-cycle block in total CD34(+) cells without causing significant apoptosis. Bryostatin-1 (bryo), a protein kinase C (PKC) modulator, was investigated for its ability to increase IM-mediated apoptosis either through induction of cycling of G0/G1 Ph(+) cells or antagonism of the IM-induced cell-cycle block. METHODS: The Ph(+) K562 cell line and primary CD34(+) CML cells were studied for cell-cycle progression (PI staining), proliferation ((3)H thymidine uptake), and survival (dye exclusion). RESULTS: Following 48 hours exposure to IM, on average more than 80% of surviving K562 cells were in G0/G1 as compared to approximately 50% for untreated control cultures (p < 0.001). After accounting for IM-induced cell kill, the absolute number of viable G0/G1 cells was significantly increased, confirming its anti-proliferative effect. However, pretreatment for 24 hours with bryo both increased K562 total cell kill and normalized the percentage of cells recovered in G0/G1, thus reducing their absolute number. For primary CML CD34(+) cells, pretreatment with bryo prior to IM significantly enhanced cell death of both total and, critically, G0/G1 populations. CONCLUSION: These results suggest that carefully scheduled drug combinations that include an agent to antagonize the anti-proliferative effect of IM may prove more efficacious within the Ph(+) stem cell compartment than IM monotherapy.     

Identification of Functional Fragments in gMYL6 and the Mechanism of Improving NK Cell-Mediated Cytotoxicity

Background: In a previous study, the unrecognized role of gMYL6 in the up-regulation of human NK cells development and cytotoxicity was reported. Objective: To further elucidate the mechanism of action of small recombinant fragments of gMYL6 enhancing the NK cells activity. Methods: Mononuclear cells were isolated from umbilical cord blood (UCB) by density-gradient centrifugation and NK cells were propagated and cultured. The small peptides from the gMYL6, with the ability to enhance the cytotoxicity of NK cells were screened by CCK-8 method and one of the most powerful peptides was identified for the next study. Flow cytometry was used to assess the proliferation and apoptosis of K562 cells, as well as the cell cycle arrest. The apoptosis of target cells was observed by AO/EB fluorescence staining, and the degree of apoptosis was assessed by flow cytometry. Protein imprinting method was also used to explore the pathway of small peptides to enhance the NK cells' activity. On the other hand, Real-time Quantitative PCR Detecting System was used to verify the mechanism of K562 cells suppression. Results: Small D peptide significantly increased NK cells cytotoxicity and induced both cell cycle arrest at G2/M and apoptosis of K562 cells. Conclusion: Small D peptide could be a novel promising peptide for cancer immunotherapy since it was shown to promote the cytotoxicity of cord blood-derived NK cells.     

Expansion of autorecognitive cytotoxic effectors in human cancer by T cell growth factor (Interleukin 2)1

Conditions for the production of supernates from mitogen stimulated human lymphocytes with the capacity to induce proliferation in long term MLC or PHA stimulated cultures which no longer respond to PHA have been investigated. These supernates can be used to maintain lymphocytes in continuous growth with a doubling time of approximately 48 hours. Cells grown from MLC stimulated cultures show less than 80% SRBC rosetting cells, are Fc-, do not show ADCC activity and induce reduced lysis of K562 compared with freshly isolated effectors. Cultured T cells (CTC) show high lytic activity against the inducing PHA blasts but not autologous or third part targets. Similar experiments have been performed with lymphocytes from the blood, lymph node, spleen and tumour of cancer patients and CTC tested for cytotoxicity against autologous and allogeneic tumour cells and the K562 compared with freshly isolated effectors. Cultured T cells (CTC) show high lytic activity against the inducing PHA blasts but not autologous or third part targets. Similar experiments have been performed with lymphocytes from the blood, lymph node, spleen and tumour of cancer patients and CTC tested for cytotoxicity against autologous and allogeneic tumour cells and the K562 cell line. Cytotoxicity for autologous tumour was found in all samples. This was accompanied by killing of allogeneic cells in most instances but killing of K562 was only rarely demonstrable. These data would be consistent with a polyclonal expansion of cytotoxic effectors in the samples. The finding of autologous reactivity suggests the presence of autorecognitive cytotoxic T cells in cancer patients with specificity for tumour. Cloning experiments are currently in progress to investigate this possibility further.