IL-15基因修饰的NK细胞对原代卵巢癌细胞的杀伤作用

目的:探讨γ射线照射后的IL-15基因修饰的NK细胞(简称NK-ustc细胞)对原代卵巢癌细胞的体内外杀伤活性。方法:分离卵巢癌患者腹水原代卵巢癌细胞。不同剂量γ射线(0、1、2、4、8、16 Gy)照射NK-ustc细胞,3H-TdR掺入法检测照射后NK-ustc细胞的增殖情况,51Cr释放法检测NK-ustc细胞对K562和原代卵巢癌细胞的杀伤活性。建立人裸鼠荷卵巢癌模型,随机分为NK-ustc治疗组(模型鼠腹腔注射辐照后的NK-ustc细胞)和培养基对照组,同时设空白对照组(正常裸鼠腹腔注射辐照后的NK-ustc细胞),观察各组裸鼠体重、腹围及生存期。结果:1、2、4、8、16 Gy辐照后NK-ustc细胞的增殖率分别为(62.1±9.8)%、(41.3±8.7)%、(14.6±4.1)%、(0.1±0.03)%和(0.2±0.04)%。当效靶比为10∶1时,0、8 Gy辐照后NK-ustc细胞对K562的杀伤率分别为(45.4±8.9)%和(43.1±6.4)%,对原代卵巢癌细胞的杀伤率分别为(54.6±6.4)%和(48.3±5.8)%,说明辐照不影响NK-ustc细胞的杀伤活性(P>0.05)。辐...     

IL-15联合GM-CSF对急性白血病NK细胞作用的研究

目的选择细胞因子IL-15和GM—CSF作为刺激物，观察其对急性白血病自然杀伤细胞(NK细胞)的激活作用。方法分离健康成人及化疗缓解后的白血病患者外周血单个核细胞(PBMC)，分别加入IL-2、IL-15、GM—CSF培养，MTr法检测PBMC及活化后的PBMC对白血病K562的细胞毒作用。流式细胞仪检测NK细胞并计数NK／PBMC比例。结果正常人及化疗缓解组PBMC经过IL-15、IL-15 GM—CSF分别作用后，细胞均呈现不同程度的增殖。化疗后缓解的增殖程度均小于正常人。正常人及化疗缓解组PB—MC联合应用IL-15和GM—CSF后对k562杀伤活性优于单用IL-15。缓解组对k562的杀伤活性明显低于正常对照组，化疗缓解后病人的NK／PBMC比例低于正常人。经联合应用IL-15、GM—CSF孵育后NK／PB—MC高于正常人。结论正常人及白血病患者PBMC与IL-15和GM—CSF联合孵育，能有效刺激PBMC增殖，提高NK／PBMC的比例。通过正常人及白血病患者的PBMC对K562的杀伤能力，间接反应IL-15和GM—CSF能有效激活外周血NK细胞，增加杀伤活性。     

CD137单抗联合IL-15体外扩增NK细胞及其对肺癌细胞的杀伤活性

目的:建立CD137单抗联合IL-15体外扩增NK细胞的方案,研究扩增后NK细胞对肺癌细胞的杀伤活性。方法:分离健康人外周血单个核细胞,经免疫磁珠阴性分选获取CD3-CD56+NK细胞,将NK细胞分成对照组、IL-2组、IL-2+CD137单抗组、IL-2+IL-15组、IL-2+CD137单抗+IL-15组进行培养,经锥虫蓝染色法计算NK细胞的扩增倍数,流式细胞术检测NK细胞的表型,LDH酶释放法检测NK细胞对肺癌细胞株A549的杀伤活性,ELISA法检测扩增后NK细胞培养液上清中IFN-γ的分泌量。结果:经磁珠分选后NK细胞纯度为(93.28±3.21)%。IL-2+CD137单抗+IL-15组NK细胞扩增倍数明显高于IL-2+CD137单抗组、IL-2+IL-15组、IL-2组及对照组[(86.20±5.00)vs(60.01±5.00)、(49.06±4.39)、(17.04±1.49)、(3.95+0.23)倍,P<0.01]。IL-2+CD137单抗+IL-15组NK细胞对A549细胞的杀伤效率明显高于其他各组[(93.14±3.27)%vs(83.15±4.03)%、(71.25±3.24)%、(62.27±3.01)%、(49.38±2.35)%,P<0.01]。IL-2+CD137单抗+IL-15组培养液上清中的IFN-γ的分泌水平明显高于IL-2+CD137单抗组、IL-2+IL-15组、IL-2组及对照组[(296.25±9.79)vs(260.47±11.55)、(201.13±6.36)、(138.36±6.09)、(38.42±3.56)pg/ml,P<0.01]。结论:CD137单抗联合IL-15能高效扩增NK细胞,扩增的NK细胞高效杀伤A549肺癌细胞。     

IL-27联合IL-15通过磷酸化STATs途径调控NK92细胞的抗肿瘤作用

目的:探讨IL-27联合IL-15对NK92细胞抗肿瘤作用的影响及其分子和信号通路机制.方法:将高表达IL-15的NK92 (IL-15-NK92)细胞分别置于不同质量浓度的IL-27(0、10、20、30及60 ng/ml)下培养24 h,ELISA法检测IL-27对IL-15-NK92细胞分泌IL-15的影响,Tr...     

IL-2+IL-15组合培养方案对乳腺癌患者外周血中NK细胞体外扩增的效果

目的：观察IL-2+IL-15组合体外培养方案对于NK、NKT和T细胞亚群的比例、表型、杀伤肿瘤细胞活性与黏附活性的影响,并初步探讨其作用机制。方法：采集天津医科大学附属肿瘤医院生物治疗科2012年5月至2012年7月期间收治的5例乳腺癌患者的外周血单个核细胞（peripheral blood mononuclear cell,PBMC）,用IL-2+IL-15 联合培养方案进行培养,观察培养15 d后细胞的扩增倍数和淋巴细胞亚群比例变化,流式细胞术检测细胞免疫表型、细胞表面受体的表达,LDH释放法和CD107a释放法检测不同细胞亚群对于HLA匹配或不匹配的靶肿瘤细胞系的杀伤活性,活细胞工作站检测总扩增产物对于不同靶肿瘤细胞系的黏附作用。结果：与扩增前相比,IL-2+IL-15培养方案扩增15 d后,NK细胞\[（36.74±1710）% vs （16.34±3.12）%,P<0.05\]、NKT细胞\[（24.88±12.34）% vs （4.06± 2.05）%,P<0.05\]比例显著增加,CD4+ T细胞和Treg细胞比例显著降低（P<0.05）,CD8+ T细胞比例显著升高（P<0.05）;NK细胞表面活化受体NKp30\[（ 92.38±7.19）% vs（32.14±17.64）%,P<0.05\]、NKp44\[（74.26±16.28）% vs（1.94±1.60）%,P＜0.05\]、NKG2D \[（ 98.58±128）% vs（6650±24.84）%,P<0.05\] 的表达率均显著升高,CD16表达率显著降低\[（ 85.12±7.66）% vs（95.60±253）%,P<0.05\]; NKT细胞、T细胞表面活化受体DNAM-1和NKG2D明显升高（P<0.05）。总扩增产物、NK细胞和NKT细胞对HLA不匹配的靶肿瘤细胞的杀伤率均显著高于HLA匹配靶细胞系的杀伤（P<0.05）;在共孵育至84 min时,与HLA匹配的靶肿瘤细胞系相比,总扩增产物细胞与HLA不匹配的靶细胞系黏附结合数目显著增多\[（ 4.80±0.53） vs （2.25±035）个,P<0.05\]。结论：IL-2+IL-15组合方案在扩增NK细胞的同时,也能够有效扩增NKT细胞,即可以扩增CD56+细胞群。并且,扩增产物主要以不受HLA限制的NK细胞杀伤活性为主来杀伤肿瘤细胞。     

MICA基因多态性与乳腺癌细胞对NK细胞杀伤的敏感性

目的：探讨MHC-Ⅰ类链相关分子A（MHC class I chain-related molecule A,MICA）多态性与乳腺癌细胞对NK细胞杀伤敏感性的关系。方法：测序分析乳腺癌细胞系MCF-7、MDA-MB-231、MDA-MB-435S和SK-BR-3 的MICA等位基因,用Western blotting 和流式细胞术检测MICA 重组表达载体转染293T 细胞（分别命名为pMCFA5.1、pMCFA4、p231A5R、p231A9 和p435A5P）MICA蛋白的表达水平,用LDH法检测NK细胞对转染MICA的293T细胞的杀伤活性,酶联免疫斑点法检测NK细胞穿孔素、颗粒酶B分泌水平。结果：MCF-7 细胞表达MICA*008/A5.1 和MICA*001/A4,MDA-MB-231 和SK-BR-3 细胞均表达MICA*019/A5 和MICA*002/A9,MDA-MB-435S 细胞表达MICA*010/A5。转染MICA 后,pMCFA5.1 组293T 细胞MICA蛋白的表达水平最低（P<0.05）,p435A5P组次之（P<0.05）,pMCFA4 组、p231A5R组和p231A9 组表达水平较高（均P<0.05）。NK细胞对转染MICA的293T细胞杀伤活性及穿孔素、颗粒酶B分泌：p435A5P组对NK细胞杀伤的敏感性最低（P<0.05）,穿孔素、颗粒酶B分泌水平最低（均P<0.05）;pMCFA5.1、pMCFA4、p231A5R和p231A9 各组之间比较差异无统计学意义（P>0.05）。结论：MICA基因多态性与乳腺癌细胞对NK细胞杀伤的敏感性密切相关。     

IL-2、IL-15增强免疫编辑后NK细胞NKG2D的表达及其对鼻咽癌CNE2细胞的杀伤活性

目的：观察IL2、IL15对免疫编辑后NK细胞NKG2D的表达及其对鼻咽癌CNE2细胞杀伤活性的影响。方法：抗CD56磁珠纯化NK细胞后分为4组：（1）编辑前NK细胞组：加入100 U/ml IL2;（2）单纯编辑组：NK细胞与CNE2细胞10∶1混合,加入100U/ml IL2;（3）IL2再培养组：纯化编辑后的NK细胞,加入1 000 U/ml IL2;（4）IL15再培养组：纯化编辑后的NK细胞,加入10 ng/ml IL15。24 h后,流式细胞仪检测各组NK细胞表面NKG2D的表达;LDH释放测定法测定效靶比20∶1时各组NK细胞对CNE2细胞的杀伤活性。结果：编辑前NK细胞组、单纯编辑组、IL2再培养组、IL15再培养组NK细胞表面NKG2D的表达率分别为(97.63±0.83)%、(53.50±1.25)%、(94.47±1.00)%、(98.07±0.21)%。IL2、IL15再培养组NK细胞 NKG2D的表达分别比单纯编辑组有显著的增加（P<0.01),该4组NK细胞对CNE2细胞的杀伤活性分别为(35.90±3.27)%、(4.70±2.30)%、(31.70±3.56)%、(40.18±2.94)%,IL2再培养组、IL15再培养组明显提高编辑后NK细胞对CNE2细胞的杀伤活性（P<0.01）, IL15的作用强于IL2。结论：高剂量IL2、IL15可以上调免疫编辑后NK细胞表面NKG2D的表达,恢复编辑后NK细胞对鼻咽癌细胞CNE2的杀伤活性,IL15的作用强于IL2。     

异体NK细胞,LAK细胞对新鲜急性粒细胞白血病的杀伤作用

异体ＮＫ细胞、ＬＡＫ细胞对新鲜急性粒细胞白血病的杀伤作用浙江医科大学附属第一医院（３１０００３）陈湖光在血液肿瘤领域，自然杀伤（ｎａｔｕｒａｌｋｉｌｌｅｒ，ＮＫ）细胞被认为与一些白血病的发生、发展及转归有关。有人观察到，白血病（包括急性粒细胞白血病ａ...     

Recombinant IL-2-activated NK cells mediate LAK activity against ovarian cancer

We investigated the ability of endogenous and recombinant interleukin-2 (IL-2)-stimulated NK cells from normal donors and ovarian cancer patients to mediate lysis of ovarian tumors, and found that peripheral blood (PB) mononuclear cells of normal donors or cancer patients did not display tumoricidal activity against ovarian cell line or fresh ovarian tumors. In addition, ovarian cancer patients displayed a defect in NK-cell activity against the highly NK-sensitive target, K-562. However, lytic activity against ovarian tumors (including cultured and primary tumors) was induced and that against K-562 was potentiated in PB of normal donors and PB and peritoneal cavity of ovarian cancer patients after enrichment of LGL on Percoll density gradient or after stimulation of effector cells with recombinant IL-2 in vitro. The IL-2-generated cytotoxic cells in PB were characterized as NK cells displaying CD16+, NKH1 (Leu-19)+, CD3- and CD5- phenotype, while those in the peritoneal cavity were predominantly CD16-, NKH1+, CD3- and CD5-. Studies on the mechanism of IL-2-dependent generation of cytotoxicity showed that such an effect was mediated via recruitment of tumor-binding cells as well as by an increase in the frequency of cytotoxic cells.     

IL-2- or IL-15-activated NK cells enhance Cetuximab-mediated activity against triple-negative breast cancer in xenografts and in breast cancer patients

Triple-negative breast cancer (TNBC) patients do not benefit from target-specific treatments and is associated with a high relapse rate. Epidermal growth factor receptor is frequently expressed in TNBC and is a candidate for new therapies. In this work, we studied Cetuximab-mediated immune activity by NK cells. Thirteen activating/inhibitory receptors were examined on peripheral blood and tumor infiltrating NK cells. NK-cell functionality was evaluated using as effectors tumor-modulated NK cells and NK cells from patients. We evaluated the treatment with Cetuximab plus IL-2 or IL-15 in vivo in TNBC xenografts. Tumor NK-cells receptor profile showed upregulation of inhibitory receptors and downregulation of activating ones. Tumor-modulated NK cells were less cytotoxic. They could perform antibody-dependent cellular cytotoxicity (ADCC) triggered by Cetuximab, although impaired, it could still be restored by stimulation with IL-2 or IL-15. Patients with advanced disease displayed diminished levels of ADCC compared to healthy volunteers. ADCC was restored and potentiated with both cytokines, which were also effective in enhancing the therapeutic activity of Cetuximab in vivo. The combination of Cetuximab with IL-15 and IL-2 may be considered an attractive therapeutic approach to enhance the clinical efficacy of Cetuximab in TNBC.     

Molecular analysis of the retinoblastoma gene in primary ovarian cancer cells

Loss of heterozygosity (LOH) at the site of the retinoblastoma (RBI) gene, at 13ql4, has been shown to occur in a high proportion of ovarian cancer patients. Based on this, RBI gene mactivation was studied in primary tumor cells from 15 patients with ovarian cancer. Structural changes as well as expression of the RBI gene were investigated. One patient had a noncisiunctional deletion at the RBI locus, and the duplicated smaining allele carried a deletion of exon 21. In another patient, without detectable structural changes of the RBI gene. no RB protein was detected. Allelic losses at the RBI locus were observed in 8/13 informative cases (61%). Our results indicate that inactivation of the RBI gene plays a role in tumor development in a minority of ovarian cancer patients. Another gene(s) on 13q also seem(s) to influence malignant transformation in patients with ovarian cancer.     

Cytotoxic activity of autologous lymphocytes against lung cancer cells; correlation of prognosis and recurrence pattern

Cytotoxic activity of lymphocytes cultured in IL-2 against autologous primary lung cancer cells was studied in relation to curativity, prognosis and relapse rate. A total of 51 patients, 44 males and 7 females, consisting of those with adenocarcinoma (n = 27), squamous cell carcinoma (n = 19), large cell carcinoma (n = 2), small cell carcinoma (n = 1), lung sarcoma (n = 1), and carcinoid (n = 1), were evaluated. Pathological stages of the patients were stage I (n = 16), stage II (n = 1), stage III (n = 28), and stage IV (n = 6). Thirteen patients (25.5%) underwent curative surgery, 23 patients (45.1%) received relative curative surgery and 15 patients (29.4%) received non-curative surgery. The mean value of cytotoxic activity in the patients who received curative surgery was 34.7 +/- 15.3%, relative curative surgery 26.5 +/- 18.9%, and non-curative surgery 42.8 +/- 22.3%. Among the patients who underwent curative and relative curative surgery, 23 patients survived more than 2 years and 13 patients died of cancer recurrence. Mean value of cytotoxic activity in the former (36.7 +/- 15.9%) was significantly (p less than 0.01) higher than that in the latter (17.1 +/- 14.7%). Positive rate (percentage of patients whose CA exceeded 15%) of the former (86.9%) was also higher than that of the latter (46.1%). Comparison between the survival curves of the positive cases (CA 15.0%) and negative cases (CA less than 15.0%) revealed a significantly better prognosis for the former (generalized Wilcoxon test: W/square root VarW = 2.198). The mean cytotoxic activity in the cases of local recurrence (25.7 +/- 16.6%, n = 7) was higher (p less than 0.10) than that in the cases with distant metastases (9.3 +/- 6.3%).     

来源于未缓解白血病患者的DC-CIK细胞对白血病细胞的杀伤活性

目的：体外诱导培养缓解期及未缓解期急性白血病患者来源的树突状细胞（dendritic cell, DC)联合细胞因子诱导的杀伤(cytokine-induced killer, CIK)细胞,比较两者的增殖能力、免疫表型及对白血病细胞的杀伤活性。方法: 提取2013年4月25日至2014年1月17日河北医科大学第二医院血液科收治的复发难治未缓解期白血病患者（7例）和缓解期患者（7例）的外周血单个核细胞,分别按常规方法诱导产生DC-CIK细胞,在培养过程中检测细胞增殖倍数,流式细胞术检测CD3+CD8+、CD3+CD56+细胞比例,细胞涂片法和流式细胞术检测培养前后CD34+白血病细胞比例,CCK-8法检测两组DC-CIK细胞对K562细胞及患者单个核细胞的杀伤活性。结果: 缓解组和未缓解组来源的DC-CIK细胞均以相似的速度快速增殖,CD3+CD8+和CD3+CD56+细胞比例均随培养时间延长显著升高（ P <0.05）,但两组间差异没有统计学意义（ P >0.05）。培养第15天时,非缓解组DC-CIK细胞涂片检测未见CD34+原始白血病细胞,流式细胞术检测显示CD34+细胞比例较培养前显著降低\[（0.1±0.05）% vs (8.3±3.1)%, P <0.05\]。效靶比在5 ∶1~20 ∶1范围内时,缓解组与未缓解组DC-CIK细胞对K562细胞的杀伤率差异没有统计学意义（ P >0.05）,但未缓解组DC-CIK对患者单个核细胞的杀伤率显著高于缓解组（ P <0.05）,并且显著高于未缓解组对K562细胞的杀伤率（ P <0.05）。结论:未缓解期白血病患者来源的DC-CIK细胞在增殖活性、CD3+CD8+和CD3+CD56+表达水平和对K562细胞的非特异性杀伤活性方面与缓解期患者来源的DC-CIK细胞均相似,但对患者单个核细胞的杀伤具有更强的特异性。     

Cytotoxic activity of azelaic acid against human melanoma primary cultures and established cell lines

The in vitro cytotoxic activity of azelaic acid was studied with 25 human melanoma primary cultures and with 5 established cell lines characterized by different contents of melanotic pigment. A dose-dependent antiproliferative effect was observed in both the experimental systems, even though cell lines displayed a slightly greater susceptibility to the compound, with ID50 values generally lower than those for fresh human tumors. Our results do not demonstrate a clear difference between melanotic and non-melanotic melanomas in sensitivity to azelaic acid. The early interference of azelaic acid on nucleic acid metabolism was investigated additionally with 15 human melanoma primary cultures. There were significant inhibitions of RNA and DNA synthesis in a remarkable percentage of tumors, at the highest concentrations of the compound. Moreover, cell proliferation of tumors that showed these antimetabolic effects was always significantly depressed by lower drug concentration as well as by the highest.     

靶向MUC16的嵌合抗原受体修饰的NK-92细胞对卵巢癌的抗肿瘤活性

目的:探索靶向MUC16的嵌合抗原受体修饰的NK-92(CARNK-92)细胞对卵巢癌的抗肿瘤活性.方法:通过免疫组化法及流式细胞术检测庆阳市中医医院妇产科卵巢癌外科手术15例患者的肿瘤组织及4种卵巢癌细胞系中MUC16的表达情况.通过全基因合成的方法合成MUC CAR序列并构建重组慢病毒表达载体,利用慢病毒感染的方式...     

ANTITUMOR EFFECTS OF HUMAN IL-15 GENE MODIFIED LUNG CANCER CELL LINE

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