嵌合抗原受体修饰的NK-92MI细胞对 HER2 阳性乳腺癌细胞的杀伤

目的:探讨用特异性靶向HER2抗原的嵌合抗原受体(HER2-CAR)修饰的NK-92MI细胞对HER2阳性乳腺癌细胞的杀伤效率.方法:通过PCR获得HER2-CAR片段,运用常规分子克隆技术,将其构建到慢病毒载体上,利用包装的慢病毒颗粒对NK-92MI细胞进行转染;应用流式细胞术检测细胞转染效率以及转染前后NK-92MI的IFN-γ和颗粒酶分泌差异;使用7-AAD和流式细胞术体外检测HER2-CAR-NK92MI细胞对乳腺癌细胞的杀伤效率;构建小鼠乳腺癌原位肿瘤模型进行体内细胞毒性的检测.结果:用HER2-CAR修饰的NK-92MI细胞对HER2阳性乳腺癌肿瘤细胞MCF-7和T47D的杀伤效率明显高于未被基因修饰的NK-92MI细胞[分别为(62.4±1.3)％ vs (22.1±1.2)％和(50.0±1.9)％vs(16.9±0.6)％,P＜0.01];而两者对HER2阴性的乳腺癌细胞MDA-MB-468的杀伤效率没有明显差异[(13.6±1.4)％vs(12.7±0.8)％,P＞0.05].同时,经HER2-CAR修饰的NK-92MI细胞相比未被基因修饰的NK-92MI细胞,IFN-γ的分泌明显升高(P＜0.01).结论:经HER2-CAR修饰的NK-92MI细胞对HER2阳性乳腺癌细胞的杀伤效率明显提高,且具有特异性靶向,此为治疗乳腺癌等恶性肿瘤提供了临床依据.     

IL-15、IL-21和4-1BBL增强NK-92细胞增殖和细胞毒性的实验研究

目的:探讨IL-15、IL-21和4-1BBL组成的不同培养体系,对体外扩增的NK-92细胞毒活性的影响。方法:以三质粒系统包被携带IL-15、IL-21和4-1BBL基因的慢病毒,用慢病毒感染的方式制备2组饲养层细胞;筛选后经MMC处理分5组体外扩增NK-92细胞（NK-92组:NK-92细胞;K562组:K562细胞+NK-92细胞;IL-15组:表达IL-15和4-1BBL的K562细胞+NK-92细胞;IL-21组:表达IL-21和4-1BBL的K562细胞+NK-92细胞;IL-15+IL-21组:表达IL-15和4-1BBL的K562细胞、表达IL-21和4-1BBL的K562细胞+NK-92细胞）;通过CCK-8法,乳酸脱氢酶释放法检测体外扩增后NK-92细胞的细胞毒性;ELISA检测NK-92细胞上清中IFN-γ的水平,以评估NK-92细胞的抗肿瘤效应。结果:两组转染后的HEK293FT细胞以及慢病毒感染的K562细胞携带绿色荧光,两组饲养层细胞成功表达目的基因;各组饲养层细胞都能刺激NK-92细胞大量增殖;随着效靶比的逐渐增加,IL-15组扩增后的NK-92细胞毒性显著高于其余各组（P<0.05）;效靶比为5∶1和10∶1时,IL-15组NK细胞IFN-γ的释放量显著高于其余各组（P<0.05）。结论:膜结合型IL-15、IL-21与4-1BBL联合都能引起NK-92细胞的大量增殖,且IL-15与4-1BBL联合运用扩增的NK-92细胞有更强的杀伤肿瘤细胞的能力。     

抗CD19 嵌合受体修饰的NK-92 细胞的构建及其对CD19 阳性非霍奇金淋巴瘤细胞的杀伤作用

目的：构建表达CD19 的二代CAR-NK-92 细胞系,探讨其对CD19 阳性非霍奇金淋巴瘤细胞的特异性杀伤作用。方法：构建表达CD19-CAR基因的载体并包装慢病毒颗粒,感染NK-92 细胞,流式细胞术检测感染率,并进一步分选阳性细胞,Western blotting 检测CD19-CAR 在NK-92 细胞中的表达;以CD19 阴性骨髓瘤细胞U-266、CD19 阳性的非霍奇金淋巴瘤细胞ARH-77 和HS-Sultan 细胞为靶细胞,以CD19CAR-NK-92 为效应细胞,流式细胞术检测细胞杀伤实验中存活肿瘤细胞的绝对数量并计算杀伤率。结果: 成功构建慢病毒载体pLVX-CD19-CAR并包装病毒颗粒,感染NK-92 细胞后CD19CAR-NK-92 细胞纯度高于90%;Western blotting 分析显示CD19-CAR已经成功表达在NK-92 中。CD19CAR-NK-92 对ARH-77[ （70.10±1.86）% vs （1.95±0.63）%,P<0.01]和HS-Sultan[ （74.98±1.60）% vs（0.58±1.49）%,P<0.01]细胞的杀伤率显著高于空载体对照组ZsGreen-NK-92 细胞,对U266 的杀伤率无显著差别（P>0.05）。结论：成功构建了表达CD19CAR的NK-92 细胞系,其对CD19 阳性的非霍奇金淋巴瘤细胞有特异性杀伤作用。     

靶向CD19 抗原的CAR-NK-92MI和CAR-CD19-T 细胞对套细胞淋巴瘤的体外杀伤

[摘要] 目的：探讨靶向CD19 抗原的CAR-NK-92MI和CAR-CD19-T 细胞对套细胞淋巴瘤（mantle cell lymphoma,MCL）的体外杀伤作用。方法：利用近年来在B细胞急性淋巴细胞白血病（B-lineage acute lymphoblastic leukemia, B-ALL）临床试验中获得的成功嵌合抗原受体基因修饰的T（CAR-T 细胞）技术,针对MCL高表达CD19 抗原的情况,分别构建了靶向CD19 抗原的CAR-CD19-T和CAR-NK-92MI细胞,运用LDH释放法检测两者对MCL细胞的体外杀伤作用,另外通过流式细胞术对其杀伤作用进行了验证。结果：相对于对照,无论是CAR-NK-92MI细胞还是CAR-CD19-T 细胞,都对MCL细胞具有极高的杀伤能力（均P <0.01）,都对K562-CD19 细胞具有非常高的毒性（均P <0.01）,而对K562 细胞基本不起作用。CAR-NK-92MI细胞组MCL细胞的死亡率比对照组高30%~40%,CAR-CD19-T 细胞组MCL细胞的死亡率比对照组高40%~50%。结论：CAR-NK-92MI和CARCD19-T细胞在体外对MCL细胞具有强的特异性杀伤作用。     

靶向CD19的CAR修饰的NK细胞对B细胞淋巴瘤的杀伤

目的:利用鼠杂交瘤技术筛选和制备治疗性CD19单克隆抗体,探讨以CD19 scFv序列构建CD19-CAR(pCD19-CAR)修饰的NK细胞对CD19+B细胞淋巴瘤细胞的杀伤作用.方法:用偶联多肽免疫小鼠制备CD19单克隆抗体,然后用基因测序法获得抗体的序列.分析抗体序列,并通过基因合成和分子克隆技术构建pCD19-CAR片段,然后将其克隆到慢病毒载体上,病毒包装制备后,转染NK-92MI细胞.最后,用流式细胞术检测不同的pCD9-CAR-NK-92MI细胞对CD19+细胞的杀伤率.结果:(1)成功筛出特异性强的CD19单克隆抗体-pCD19;(2)抗体检测结果显示CD19+的Ramos细胞的阳性率为84.3％,Raji为85.6％,与商业化抗体结果相似;(3)被pCD19-CAR修饰的CD19-CAR阳性率为28.72％的NK-92MI细胞对CD19+的Ramos和Raji细胞的杀伤效率明显高于未被修饰的NK-92MI细胞株对Ramos和Raji细胞的杀伤率[(47.1±1.7)％vs(24.7±6.2)％和(51.8±7.9)％vs(27.6±9.6)％,均P＜0.05];对CD19-细胞Jurkat,不论是未被pCD19-CAR修饰的NK-92MI或是被修饰的NK-92MI细胞,几乎都不存在特异性杀伤作用[(16.1±0.7)％vs(17.7±2.9)％,P＞0.05].结论:成功构建pCD19-CAR,被pCD19-CAR修饰的NK-92MI细胞能特异性的识别CD19抗原并杀伤CD19+B细胞淋巴瘤细胞.     

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...     

联合应用IL-2和IL-7对增强T细胞体外增殖及功能的作用

应用体外诱导扩增的抗原特异性T细胞治疗恶性肿瘤及某些病毒性疾病已成为一种有效的临床免疫治疗方法，这一方法常规需要用特异性抗原(灭活的肿瘤细胞或提纯抗原)以及抗原递呈细胞作周期性刺激诱导，并在含特定细胞因子，如IL-2的培养系统中扩增以获得具有足够治疗剂量的功能性T细胞。     

IL-2短期活化的HLA半相合的外周血单个核细胞治疗晚期恶性实体瘤初步观察

目的:初步探讨IL-2短期活化的HLA半相合的异体外周血单个核细胞(allo-mPBMCs)治疗晚期转移性/化疗抗性实体瘤的可行性。方法:11例晚期转移性/化疗抗性恶性实体瘤患者,供者为与患者HLA半相合直系亲属。供者经10μg/kg·dG-CSF动员3天后采集PBMC,体外用大剂量rhIL-2培养后回输患者。观察治疗后临床反应,采用流式细胞仪、LDH和ELISA试剂盒检测动员、活化前后表型,IL-2短期活化前后的非特异性杀瘤活性以及患者回输前后血清中各种细胞因子的含量变化。结果:11例患者回输的HLA半相合allo-mPBMCs总数达(2.5～4.3)×1010。体外经G-CSF动员后T细胞,尤其是CD4+T细胞显著下降,而NK细胞比例显著上升;经大剂量IL-2短期活化后,各种细胞亚群比例未见改变,但活化细胞(包括CD69+和CD25+细胞)亚群比例显著升高。体外实验中可观察到IL-2短期活化的allo-mPBMCs具有类似LAK样的较高的非特异性杀瘤活性。经过治疗,11例患者血清中Th1类细胞因子,如IL-2、IL-12、IFN-γ、TNF-α含量明显升高,而Th2类细胞因子,如TGF-β含量明显下降。治疗后8例观察到临床症状的缓解,或肿瘤标志物的降低,或影像学资料的改善,其中1例治疗后4个月达到部分缓解,而3例出现进展,其中2例死亡。结论:IL-2短期活化的HLA半相合的allo-mPBMCs具有明显的抗肿瘤效应,为治疗晚期转移性/化疗抗性实体瘤提供了新的希望。     

鼻咽癌患者外周血单个核细胞中P-gp和LRP的表达及其临床意义

目的通过检测鼻咽癌患者外周血单个核细胞中P-糖蛋白(P-gp)和肺耐药相关蛋白(LRP)的表达,探讨两者在鼻咽癌多药耐药中的作用及意义.方法采用流式细胞术对54例鼻咽癌患者化疗前后外周血单个核细胞中P-gp、LRP进行检测,同期选择鼻咽部非恶性肿瘤患者10例,正常健康人10例为对照组.结果 P-gp在正常人、非恶性肿瘤及鼻咽癌初治组中的表达差异无显著性,但在复发组的表达明显高于其他组,差异有显著性.LRP在初治及复发组中的表达明显高于对照组,且化疗前后差异有显著性.P-gp和LRP的表达与年龄、性别、临床分期、病理类型、淋巴结转移无显著相关.P-gp、LRP的表达与化疗疗效密切相关.结论 P-gp、LRP的表达在鼻咽癌多药耐药的产生中具有重要意义,对临床制定化疗方案具有潜在的指导意义.     

IL-21对慢性粒细胞白血病患者调节性T细胞的影响

目的:观察白细胞介素21(interleukin-21,IL-21)对慢性粒细胞白血病(chronic myeloid leukemia,CML)患者CD4+CD25+调节性T细胞(regulatory T cell,Treg)的作用,为CML的免疫治疗提供新思路.方法:收集兰州大学第二医院血液科初诊CML患者外周血单个核细胞(peripheral blood mononuclear cells,PBMCs),免疫磁珠法分选出CD4+T细胞.分别加入50、100、200 ng/ml IL-21与CD4+T细胞共培养72 h后,流式细胞术检测Treg的比例,并设生理盐水空白对照组.Real-time PCR法检测Treg细胞中Foxp3 mRNA的表达,ELISA法检测各组细胞上清液中IL-10、TGF-β的水平.结果:与空白对照组相比,50、100、200 ng/ml IL-21组CD4+ CD25+ Treg占CD4+T细胞的比例均降低[(3.42±0.76)％、(6.81±0.33)％、(7.98±0.76)％vs(12.09±0.91)％,P＜0.05];且3组的Foxp3 mRNA均降低[(0.05±0.02)、(0.16±0.02)、(0.25±0.02)vs 1,P＜ 0.05];Treg分泌IL-10量减少[(26.13 ±7.28)、(44.88 ±3.72)、(79.77±3.94) vs(133.00±12.32) pg/ml,P＜0.05],分泌TGF-β量也减少[(9.25 ±0.84)、(16.70±1.00)、(20.47±1.60)vs (26.05±1.81) pg/ml,P＜0.05].结论:IL-21在体外可减少CML患者外周血中Treg的比例,并抑制其功能.     

MicroRNA-150对NK/T细胞淋巴瘤组织和NK-92细胞辐射敏感性的影响

目的:探讨microRNA-150 (miR-150)对NK/T细胞淋巴瘤组织和NK-92细胞辐射敏感性的影响.方法:选取2010年1月至2016年1月在南方医科大学珠江医院收治的NK/T细胞淋巴瘤患者36例为研究对象,并收集患者的组织标本.所有患者均接受放疗并采用淋巴瘤国际工作组标准((IWG)评价患者的近期疗效,根据疗效分为完全缓解(CR)组和未完全缓解组(Non-CR).实时荧光定量PCR检测患者肿瘤组织及NK/T细胞淋巴瘤细胞系中miR-150的表达量,向淋巴瘤NK-92细胞转染miR-150 mimics,利用MTT法和集落形成实验分析过表达miR-150对NK-92细胞辐射敏感性的影响,流式细胞术检测转染过表达miR-150对NK-92细胞辐射致凋亡的影响,Western blotting实验检测miR-150对凋亡相关蛋白Caspase3和PARP表达的影响.结果:根据IWG标准,12例患者CR,24例患者为Non-CR;与CR组相比,Non-CR组患者miR-150表达量普遍下调(P＜0.05).与正常对照sCD3-CD56+ NK细胞相比,NK/T细胞淋巴瘤组织(9.10±0.19 vs4.01±0.22,P＜0.01)和5种NK/T细胞淋巴瘤细胞系中miR-150呈明显低表达(P＜0.05).过表达miR-150可明显降低辐射后NK-92细胞的增殖能力(P＜0.01)和集落形成能力(P＜0.01),明显提高NK-92细胞的辐射增敏比(10 Gy时辐增敏比为5.375),显著促进辐射诱导的NK-92细胞的凋亡[(37.3±1.24)％vs(28.3±2.34)％,P＜0.05],可促进激活的Caspase 3和PARP蛋白表达.结论:miR-150在NK/T细胞淋巴瘤组织标本及体外培养细胞系中的表达呈显著低水平,miR-150表达量低的患者放疗后缓解率低;转染miR-150 mimics能够增强辐射对NK-92细胞增殖的抑制作用和促进辐射诱导致凋亡作用,对NK/T细胞淋巴瘤有辐射增敏作用.     

Effect of hyperthermia on the survival of normal human peripheral blood mononuclear cells

Human peripheral blood mononuclear cells from normal healthy volunteers were exposed to elevated temperatures of 41-43 degrees for up to 6 hr. Thereafter, the cells were stimulated with phytohemagglutinin in vitro in order to measure indirectly the surviving fraction. DNA replication in heated cells in response to phytohemagglutinin was found to be a sensitive indicator of thermal injury. Exposure to even 40 degrees for 2 hr lowered thymidine incorporation at early time points after phytohemagglutinin stimulation, but the cells were able to recover from thermal injury after exposure for up to 4 hr at 42 degrees. At 43 degrees, exposure for even 1 to 2 hr caused irreversible damage. The changes in thymidine incorporation were not due to changes in endogenous nucleotide pools since parallel changes were observed in DNA polymerase activity. Thus, the heat sensitivity of normal human lymphocytes could be a limiting factor for use of hyperthermia as an adjunct to radiotherapy and chemotherapy of human cancer.     

Reduced numbers of IL-7 receptor (CD127) expressing immune cells and IL-7-signaling defects in peripheral blood from patients with breast cancer

Interleukin-7-receptor-signaling plays a pivotal role in T-cell development and maintenance of T-cell memory. We studied IL-7Ralpha (CD127) expression in PBMCs obtained from patients with breast cancer and examined IL-7 receptor-mediated downstream effects defined by STAT5 phosphorylation (p-STAT5). Reduced numbers of IL-7Ralpha-positive cells were identified in CD4+ T-cells as well as in a CD8+ T-cell subset defined by CD8alpha/alpha homodimer expression in patients with breast cancer. PBMCs obtained from healthy donors (n = 19) and from patients with breast cancer (n = 19) exhibited constitutive p-STAT5 expression in the range of 0-6.4% in CD4+ T-cells and 0-4% in CD8+ T-cells. Stimulation with recombinant human IL-7 for 15 min increased p-STAT5 expression up to 36-97% in CD4+T-cells and to 26-90% in CD8+T-cells obtained from healthy control donors (n = 19). In contrast, PBMCs obtained from 13/19 patients with breast cancer did not respond to IL-7 as defined by STAT5 phosphorylation, despite expression of IL-7Ralpha on T-lymphocytes. T-cells were further characterized for IL- 2 and IFN-gamma production induced by PMA/Ionomycin. PBMCs from 9/19 patients with breast cancer showed decreased IL-2 and IFN-gamma production combined with IL-7-signaling defects; PBMCs from 4 patients with breast cancer exhibited deficient IL-7-signaling, yet intact cytokine production. Reduced numbers of IL-7Ralpha-positive cells and nonresponsiveness to IL-7, defined by lack of STAT5 phosphorylation, characterizes the immunological profile in T-cells from patients with breast cancer.     

电离辐射对人外周血线粒体编码基因mRNA表达的影响

目的： 观察γ射线对人外周血线粒体编码基因mRNA表达水平的影响,探讨线粒体编码基因用于辐射生物剂量估算的可行性。方法：用不同剂量水平 (0~5 Gy)的60Co γ射线照射3名正常人离体外周血样本,照射6 h后,提取总RNA,应用实时荧光定量PCR技术检测ND1,ND6,细胞色素C氧化酶亚基 (cytochrome C oxidase subunit,COX)I、II、III,三磷酸腺苷酶6 (adenosine triphosphatase,ATPase6),三磷酸腺苷酶8(adenosine triphosphatase,ATPase8) mRNA表达水平的变化,采用Origin 7.5拟合照射剂量与基因表达水平的剂量-效应关系曲线。结果：γ射线照射人外周血后,线粒体编码基因COXI及 ATPase6的相对表达水平在0~5 Gy剂量范围内表达先增加,3 Gy剂量点之后显现下降趋势,对这两个基因在0~3 Gy剂量范围内分别进行拟合,得到COXI 剂量-效应关系方程为Y=0.625 9+0.256 1D (r2=0.916 3,P<0.01); ATPase6基因表达剂量-效应关系方程为Y=0.218 9+ 0.806 0 D (r2=0.912 6,P<0.01),其他5个基因的相对表达水平与照射剂量间不存在明显的剂量-效应关系 (P>0.05)。结论：γ射线对人外周血线粒体编码基因COXI 及 ATPase6的mRNA表达水平具有明显的影响,这两个基因的表达水平与γ射线的照射剂量存在线性关系,有望用于辐射生物剂量的估算。     

In vitro drug sensitivity of normal peripheral blood lymphocytes and childhood leukaemic cells from bone marrow and peripheral blood

In vitro drug sensitivity of leukaemic cells might be influenced by the contamination of such a sample with non-malignant cells and the sample source. To study this, sensitivity of normal peripheral blood (PB) lymphocytes to a number of cytostatic drugs was assessed with the MTT assay. We compared this sensitivity with the drug sensitivity of leukaemic cells of 38 children with acute lymphoblastic leukaemia. We also studied a possible differential sensitivity of leukaemic cells from bone marrow (BM) and PB. The following drugs were used: Prednisolone, dexamethasone, 6-mercaptopurine, 6-thioguanine, cytosine arabinoside, vincristine, vindesine, daunorubicin, doxorubicin, mafosfamide (Maf), 4-hydroperoxy-ifosfamide, teniposide, mitoxantrone, L-asparaginase, methotrexate and mustine. Normal PB lymphocytes were significantly more resistant to all drugs tested, except to Maf. Leukaemic BM and PB cells from 38 patients (unpaired samples) showed no significant differences in sensitivity to any of the drugs. Moreover, in 11 of 12 children with acute leukaemia of whom we investigated simultaneously obtained BM and PB (paired samples), their leukaemic BM and PB cells showed comparable drug sensitivity profiles. In one patient the BM cells were more sensitive to most drugs than those from the PB, but the actual differences in sensitivity were small. We conclude that the contamination of a leukaemic sample with normal PB lymphocytes will influence the results of the MTT assay. The source of the leukaemic sample, BM or PB, does not significantly influence the assay results.     

Generation of anti-p53 cytotoxic T lymphocytes from human peripheral blood using autologous dendritic cells.

CTLs recognizing the HLA-A2.1-restricted, wild-type sequence p53 epitopes p53(149-157) and p53(264-272) were generated from CD8-enriched populations of nonadherent peripheral blood lymphocytes (PBLs) obtained from healthy donors. The PBLs were restimulated in vitro with peptide-pulsed granulocyte macrophage colony-stimulating factor- and interleukin (IL)-4-induced autologous dendritic cells in the presence of IL-6 and IL-12 and subsequently cultivated with IL-1alpha, IL-2, IL-4, IL-6, and IL-7. Bulk anti-p53(264-272) CTL populations were generated from PBLs obtained from two of five donors. Both CTL populations were cytotoxic against peptide-pulsed HLA-A2+ target cells, but not against untreated target cells. A CD8+ anti-p53 CTL clone designated p264#2 was isolated from one of the bulk populations. It was found to have an intermediate affinity of approximately 10(-9) M for the epitope and to mediate cytotoxicity against several human tumor cell lines, including the squamous cell carcinoma of the head and neck cell line SCC-9, which is known to present the wild-type sequence p53(264-272) epitope. In addition, CTLs reactive against p53(149-157)-pulsed targets as well as a HLA-A2+ tumor cell line were cloned from a bulk population of antitumor CTLs obtained from one of the five normal PBLs restimulated with this epitope. The results indicate that CTLs recognizing wild-type sequence epitopes can be generated from precursors present in PBLs obtained from some normal individuals using autologous dendritic cells as antigen-presenting cells and suggest that vaccine strategies targeting these epitopes can lead to antitumor CTL generation, thereby emphasizing the therapeutic potential of p53-based cancer vaccines.