新城鸡瘟病毒激活巨噬细胞释放一氧化氮及对肝癌瘤株细胞毒活性的研究

目的:分析新城鸡瘟病毒Lossta系(NDV-L)对小鼠腹腔巨噬细胞是否具有激活作用并对其机制进行探讨.方法:采用电镜、氚标记的胸腺嘧啶核苷(3H-TdR)和Griess方法.结果:①NDV-L与小鼠腹腔巨噬细胞(PEM)作用 24h后,光镜和扫描镜均显示细胞明显增大和伪足伸出;②被NDV-L作用的PEMф与3H-标记的H22靶细胞作用 24h后,发现具有明显的细胞毒杀伤效应,且该效应与阳性对照组[卡介苗、细菌内毒素联合激活的PEMф(BCG-LPS-PEMф)]的杀伤效应接近;③收集NDV-L与PEMф作用 24h上清进行检测,发现一氧化氮(NO)分泌量增高.结论:被NDV-L激活的PEMф对肿瘤细胞的细胞毒效应与其分泌NO有关.     

程控冻存对NK细胞杀伤作用影响的研究

以~3H-TdR释放法和效靶细胞结合试验检测程控冻存前后单个核细胞(MNC_(?))中NK细胞杀伤活性。结果当效靶细胞比例为20∶1、60∶1、100∶1时,程控冻存后即刻、冻存7天和冻存30天的MNC(?)中NK细胞杀伤活性与冻存前无显著性差异,冻存复苏后早期效靶细胞结合率降低,在预培养12小时左右恢复至冻存前水平。以间接免疫荧光法检测冻存前后MNC_(?)中heullb、leul9阳性细胞亚群百分率无显著差异。     

氚标记的脱氧胸苷诱发人胚肺成纤维细胞恶性转化

本文用氚标记的脱氧胸苷(~3H-TdR)诱发人胚肺成纤维细胞(2 BS)的恶性转化,并对转化细胞进行了形态学和生物学的初步鉴定。 首先用含有0.5～2.0μCi/ml ~3H-TdR的培养液培养人胚肺成纤维细胞(1×10~6/瓶)48小时,吸出含~3H-TdR的培养液,用磷酸缓冲液生理盐水(PBS)洗细胞2次,再加入新鲜培养液于37℃继续培养5天。然后对各瓶细胞进行计数,每3瓶为1组,取平均值。对照组不加~3H-TdR,观察~3H-TdR剂量对细胞     

口腔粘膜鳞状细胞癌的细胞动力学研究

本实验用体外~3H-TdR两次标记、放射自显影术对9例口腔粘膜鳞状细胞癌进行了细胞动力学研究。结果表明:该肿瘤的LI高于正常口腔粘膜,Tc短于正常口腔粘膜,Ts延长。经放射治疗后,残留的变性癌细胞仍被标记。可能与复发有关。     

药物标记抗体体外导向治疗系统性红斑狼疮所致血小板减少

目的：探讨药物标记抗体导向治疗系统性红斑狼疮（SLE）所致血小板减少的可行性及效果。 方法：将静脉注射用免疫球蛋白（IVIG）与免疫抑制剂MTX交联制备成直接和间接标记抗体,使之对单核-巨噬细胞产生特异的杀伤作用,用免疫荧光法检测Fc段结合活性,以Fc受体阳性的小鼠巨噬细胞和人单核细胞白血病U937为靶细胞,用MTT法检测标记前后MTX杀伤活性及标记抗体的特异性杀伤活性。 结果：标记抗体的杀伤活性明显大于游离MTX,标记抗体对Fc受体阳性的U937细胞的杀伤活性明显大于Fc受体阴性细胞,标记抗体能抑制巨噬细胞的吞噬功能,间接标记抗体IVIG-HSA-MTX杀伤活性明显大于直接标记抗体IVIG-MTX。 结论：标记抗体在体外对单核、巨噬细胞显示了相对特异的杀伤活性。     

体外培养人癌细胞在细胞周期不同时相形态结构变化的研究

本文应用过量胸腺嘧啶核苷阻断法使细胞同步化,应用~3H-TdR标记放射自显影及液体闪烁计数法检测,分别获取G_1及S期细胞。利用扫描电子显微镜、荧光显微镜及细胞化学等技术系统地观察了在细胞周期不同时相两种体外培养人癌细胞的形态、表面结构及细胞化学的变化。     

应用~(125)I—UdR释放法检测LAK细胞的杀伤作用

本文利用~(125)I-UdR标记K562、Raji靶细胞技术,建立体外测定LAK细胞的细胞毒方法。Raji细胞与K562细胞的标记条件相似,即在标记时间为4h、5×10~(-5)SMFUdR条件下,标记1×10~6细胞/ml。~(125)I-UdR最适用量为2—3μCi,效靶细胞孵育最佳时间为16-18h。~(125)I-UdR释放法检测的结果符合LAK细胞的一般特性。     

氚—胸腺嘧啶核苷掺入法测定T淋巴细胞转化试验

本文介绍了应用氚—胸腺嘧啶核苷(~3H-TdR)掺入法测定体外淋巴细胞培养在PHA刺激下DNA合成的条件。实验结果表明,细胞培养72小时后,加~3H-TdR掺入24小时和PPO、POPOP、萘、二氧六(?)配制的闪烁液测得放射性为最灵敏。加~3H-TdR剂量3.0微居是可取的。     

人食管癌ECa109球体生长的放射自显影观察

前言为解决实体瘤的治疗问题,目前国外对体外培养动物细胞球体研究较多。使用~3H-胸腺嘧啶核苷(~3H-TdR)标记放射自显影方法对其生长情况、治疗后变化的观察已见报道,有的用球体切片法,有的把球体消化成细胞悬液,涂于载片观察,但缺     

高胆固醇血症对鹌鹑在体动脉内皮细胞损伤的定量研究

为定量分析高胆固醇血症对动脉内皮细胞的损伤,并观察调脂通脉灵对此损伤可能存在的保护作用,采用内皮细胞铺片法和放射自显影技术,对鹌鹑在体动脉内皮细胞再生进行了定量分析。研究发现,高胆固醇组动脉内皮细胞排列不规则、密度增加,~3H-TdR标记率为2.05±4.85‰,与对照组相比(~3H-TdR标记率为0.81±0.87‰)差异有显著性。经调脂通脉灵治疗后,血脂浓度明显降低,~3H-TdR标记率随之下降。提示:通过对内皮细胞再生的定量分析,可对内皮细胞的损伤程度作出定量分析。     

Induction of cytotoxic T lymphocytes from the peripheral blood of a hepatocell ular carcinoma patient using melanoma antigen- 1 (MAGE- 1) peptide

Objective To investigate the possibility of using melanoma antigen- 1 (MAGE- 1) peptide as a tumor vaccine to treat hepatocellular carcinoma (HCC). Methods The expressions of MAGE- 1 in 8 HCC cell lines and in liver cancer tissue from a patient were detected using RT- PCR. The type of human leucocyte antigen Ⅰ (HLAⅠ) of both 8 HCC cell lines and peripheral blood mononuclear cells of the patient was detected using a microcytotoxicity method to screen out target cell lines for the cytotoxicity assay. Peripheral blood mononuclear cells from the HCC patient pulsed with an MAGE- 1 peptide (NYKCRFPEI) were used as antigen presenting cells. Autogenous peripheral blood mononuclear cells were stimulated with antigen presenting cells every 7 days for 4 times to elicit cytotoxic T lymphocytes. The phenotype of effector cells was analyzed using flow cytometry. The cytotoxicity of effector cells was detected with a lactate dehydrogenase releasing assay. Results The expressions of both MAGE- 1 and HLA- A24 were detected in BEL7405 cell line which were used as the positive target cell line in the cytotoxicity assay. The expression of MAGE- 1 alone was detected in HLE, BEL7402, BEL7404, QGY7703 and SMMC7721 cell lines, and the expression of neither MAGE- 1 nor HLA- A24 was shown in QGY 7701 and HpG2 cell lines. The last 7 cell lines could be used as negative target cell lines in the cytotoxicity assay. Peripheral blood mononuclear cells expanded 32 folds during 28- day culture. The ratio of CD3[ + T cells in creased by 16% (from 54% to 70%), and the ratio of CD8[ + T cells increased by 20% (from 36% to 56%) during 28- day culture. When the ratio of effector cells to target cells was 10∶1,effector cells exhibited 62. 5% cytotoxicity against autogenous lymphoblasts pulsed with the peptide (NYKCRFPEI) of MAGE- 1 antigen, 40. 25% cytotoxicity against BEL7405 cells, compared with 17. 88% cytolysis observed against autogenous lymphoblasts, 19. 55% against HLE cells, and 1. 6% against QGY7701 cells. When the ratio of effector cells to target cells was 3. 3∶1, the cytotoxicity of effector cells against the peptide pulsed autogenous lymphoblasts was 53. 6%, which was much higher against autogenous lymphoblasts, HLE cells and QGY7701 cells at 15. 6%, 13% and 1%, respectively. Conclusion The results demonstrate that cytotoxic T lymphocytes with the ability to specifically lyse target cells expressing both MAGE- 1 and HLA- A24 could be successfully induced by the MAGE- 1 peptide NYKCRFPEI in vitro. This indicates that a good result might be anticipated if this peptide is used as a tumor vaccine to treat HLA- A24 HCC patients.     

正常鼠和免疫鼠CD3AK细胞和LAK细胞杀伤活性的比较研究

实验取正常鼠和Ｐ８１５肿瘤细胞致敏的免疫鼠脾细胞，经抗ＣＤ３抗体与低剂量ＩＬ－２或高剂量ＩＬ－２分别诱生为ＣＤ３ＡＫ细胞和ＬＡＫ细胞，用改良的乳酸脱氢酶释放法以Ｐ８１５细胞和ＹＡＣ－１细胞为靶细胞，测定ＣＤ３ＡＫ和ＬＡＫ细胞的杀伤活性。结果表明：正常鼠ＣＤ３ＡＫ细胞对Ｐ８１５细胞的杀伤活性高于ＬＡＫ细胞，对ＹＡＣ－１细胞的杀伤活性低于ＬＡＫ细胞。免疫鼠ＬＡＫ细胞对Ｐ８１５细胞的杀伤活性明显增高，高     

CD3AK细胞抗肿瘤作用与MHC限制性关系

目的 研究抗CD3单克隆抗体激活的杀伤细胞(anti-CD3McAb activated killer cells,CD3AK细胞)杀伤肿瘤细胞作用与MHC限制性的关系。方法 采用微量淋巴细胞毒实验方法测定CD3AK细胞及肿瘤细胞MHC表型，用MTT法测定CD3AK细胞杀伤活性。结果 (1)不同人来源的CD3AK细胞，其MHC表型不同，但对同种肿瘤传代细胞都有很强的杀伤作用，且不同人来源的CD3AK细胞间杀伤作用差异无显著性(P＞0．05)；(2)同一来源CD3AK细胞对不同肿瘤传代细胞株(MHC表型不同)的杀伤作用差异无显著性(P＞0．05)；(3)人外周血诱导的CD3AK细胞对鼠肿瘤细胞株(Yac—1)与对人肿瘤传代细胞株(K—562，Raji)一样具有很强的杀伤作用。结论 CD3AK细胞杀伤肿瘤的作用是非MHC限制的，在临床应用中可用正常人外周血诱导得到的CD3AK细胞体外培养增殖后输给肿瘤病人进行治疗。该效应细胞来源方便，可用于肿瘤术后清除残留肿瘤细胞，具有重要应用价值。     

rIL_（－2）对慢性HBV感染者NK细胞功能调节及影响因素

本文采用４小时、１６小时微量释放细胞毒法监测慢性ＨＢＶ感染者ＮＫ细胞对Ｋ５６２细胞及ＰＬＣ／ＰＲＦ／５细胞杀伤活性及某些影响因素。实验提示慢性活动型肝炎（ＣＡＨ）、慢性无症状携带者（ＡＳＣ）ＮＫ细胞杀伤活性均高于健康人。ｒＩＬ－２可明显增加健康人ＮＫ细胞活性，但对ＨＢＶ感染者ＮＫ细胞增强作用不明显。ＨＢｓＡｇ、ｒＨＢｅＡｇ和ｒＨＢｃＡｇ均可明显抑制ＮＫ细胞活性，并有剂量依赖性，加入外源性ｒＩＬ－２仍不能纠正。提示ＨＢｓＡｇ、ｒＨＢｅＡｇ和ｒＨＢｃＡｇ可通过某种机制阻抑ｒＩＬ－２与ＮＫ细胞结合发挥作用。     

THE KINETICS OF CYTOPLASMIC GRANULE SECRETION IN NATURAL KILLER CYTOTOXICITY

Antisexum against purified cytoplasmic granules from rat LGL tumor cells, and protein A-gold inmmnoelec-tron microscopy were used to study the secretory events in lysis of YAC-1 tumor cells by rat LGL tumor cells or by isolated LGL from normal rats. After 30 min incubation of effector and target cells together, gold-labeled cyto-plasmic granules were often seen concentrated in the area of the LGL adjacent to the ~ YAC-1 Within 60min,the grantees were observed to move to the cell border near the conjugazed site. At this point, fine granules were fused with file cell membrane, and subsequently released file gold-labeled contents into the junction between the LGL and the target cell. Gold particles could be seen at the B-T interface, on the surface,or sometimes on the target cell surface.These data provide direct evidence for the hypothesis that under conditions of active cytotoxicity,natural killer cells secrete their cytoplasmic granule contents leading to the deposition of granule material on the target cell surface and the eventual lysis of the cell.     

慢性活动性肝炎中单核性细胞超微形态研究

经电镜观察,慢活肝中主要为淋巴细胞及单核巨噬细胞浸润。淋巴细胞分3型,通过4种方式与肝细胞接触,可能代表杀伤肝细胞的动态过程。与肝细胞接触的主要为起细胞毒作用的T_8~+Ⅱ型淋巴细胞及单核巨噬细胞。多数T_4~+淋巴细胞为Ⅰ型淋巴细胞,可能为T辅助细胞。在肝细胞损伤中,淋巴细胞及单核巨噬细胞均起重要作用。     

Preparation and cytotoxicity of McAb-HSA-MTX conjugates

In the present study, methotrexate (MTX), was conjugated with a murine monoclonal antibody (79) to human common acute lymphoblastic leukemia antigen (CALLA), with human serum albumin (HSA) as an intermediary. The highest molar ratio of McAb 79:HSA:MTX in the conjugates was 1:2. 63:117. The conjugates obtained retained both antibody binding and drug activities. Although there was some loss of drug activity in binding to antibody, the toxicity of McAb79-HSA-MTX was entirely specific for the target cell, and the cytotoxicity of McAb79-HSA-MTX against CALLA+ cells was greater than that of control S13 (Anti-human urokinase)-HSA-MTX. The ratio of 79-HSA-MTX cytotoxicity to the target and non-target cells was 66:1, whereas there was no cytotoxicity to target cells when McAb79 was used only. There was no cytotoxic difference between 79-HSA-MTX and S13-HSA-MTX against CALLA- SB cells. These results suggest that the cytotoxicity of 79-HSA-MTX against CALLA leukemia cells is specific and this specificity is mediated by McAb79.     

IL-2激活的TIL对自体肿瘤细胞的杀伤活性及其与LAK细胞活性的比较

肿瘤浸润性淋巴细胞(TIL)经白细胞介素2(IL-2)体外培养后具有很强的体内外抗肿瘤作用,且有一定的靶细胞特异性,其抗肿瘤效果强于淋巴因子激活的杀伤细胞即LAK细胞(P<0.01)。从瘤体中新鲜分离到的TIL对自体肿瘤细胞的杀伤活性极低,经IL-2体外培养后,其杀伤活性逐渐增高,以培养至7～25d的杀伤活性最强,这与IL-2使TIL分泌3种抗癌淋巴因子包括IL-2、IFN-γ、淋巴毒素(LT)增加有关。体外培养25d后,TIL的抗肿瘤活性下降,实验表明这与培养过程中TIL的Lyt-2~+细胞(Tc)减少而L3T4~+细胞(T_H)增多有关。TIL经冻存复苏和IL-2体外培养后仍保持很强的抗肿瘤活性,冻存前后比较未见显著差异(P>0.05),这为间断地运用TIL治疗复发性、晚期肿瘤提供了一条可行的途径。     

MAGE-A3抗原肽负载树突状细胞诱导乳腺癌患者免疫应答的研究

目的研究MAGE-A3抗原肽负载对乳腺癌患者树突状细胞(DC)的功能的影响,探讨其是否可以在体外诱导特异性细胞毒性T淋巴细胞(CTL)应答.方法体外培养HLA-A2乳腺癌患者外周血来源的DC,并经孵育携带MAGE-A3抗原肽,用以刺激CTL,用3H-TdR渗入法检测抗原肽负载前后DC刺激同种淋巴细胞增殖能力(MLR),并用乳酸脱氢酶(LDH)释放法检测CTL对靶细胞的杀伤效应.结果DC:T为1:10时,单纯DC组刺激能力显著高于抗原肽负载组DC(DC-P)(P＜0.05),但当DC:T低于1:20时后DC-P组刺激能力显著强于单纯DC组(P＜0.05).DC-P组和单纯DC组所激发的CTL对细胞株MCF-7,Sk-Br-3,MDA-MB-435s的杀伤活性有显著性差异,而对细胞株Raji无显著性差异,DC-P组对细胞株MCF-7,Sk-Br-3,MDA-MB-435s的杀伤活性明显高于对细胞株Raji的杀伤活性.结论负载MAGE-A3抗原肽的DC在体外可以诱导乳腺癌患者特异性的CTL免疫应答,为临床以DC为基础的过继免疫治疗的应用提供理论基础.     

Antibody-dependent cell-mediated cytotoxicity and spontaneous cell-mediated cytotoxicity against cells infected with porcine transmissible gastroenteritis virus

The objective of this study was to determine whether porcine peripheral blood leukocytes and intestinal intraepithelial leukocytes can mediate antibody-dependent cell-mediated cytotoxicity and spontaneous cell-mediated cytotoxicity against target cells infected with transmissible gastroenteritis virus. Peripheral blood leukocytes collected from six young adult pigs and intraepithelial leukocytes from a further five pigs were used as effector cells in chromium release assays against PK-15 cells persistently infected with transmissible gastroenteritis virus. Both peripheral blood leukocytes and intraepithelial leukocytes were capable of mediating antibody-dependent cell-mediated cytotoxicity and spontaneous cell-mediated cytotoxicity against PK-15 transmissible gastroenteritis cells. While the peripheral blood leukocytes mediated lower levels of specific 51Cr release in spontaneous cell-mediated cytotoxicity than in antibody-dependent cell-mediated cytotoxicity, the intraepithelial leukocytes were more effective in spontaneous cell-mediated cytotoxicity than antibody-dependent cell-mediated cytotoxicity.