导入NKG5—SV基因增强细胞杀伤活性的研究

目的研究免疫效应蛋白NKG5的拼接异构体基因NKG5-SV导入外周血自然杀伤细胞(NK细胞)对其杀伤活性的影响.方法Percoll细胞分离液分离外周血NK细胞,流式细胞仪检测分离后CD56阳性细胞浓度.以逆转录病毒为载体将NKG5-SV基因和对照基因LacZ导入NK细胞,Northernblot检测NKG5-SV的表达,K562细胞杀伤实验检测病人NK细胞转染NKG5基因前后和正常人NK细胞的NK活性.结果Percoll分离后的淋巴细胞CD56阳性细胞为77.44%,分离前为15.88%.病人NK细胞经NKG5-SV基因转染后NKG5-SVmRNA表达增高.病人NK细胞、NK-LacZ细胞、NK-NKG5-SV细胞和正常人NK细胞对K562的杀伤活性分别为5.96%±0.38%、6.03%±0.42%(P＞0.05)、27.67%±0.18%(P＜0.01)、30.33%±0.83%(P＜0.01).结论免疫效应蛋白基因导入NK细胞,可增强NK细胞的杀伤活性.     

龙牙楤木多糖抗肿瘤活性及对荷瘤小鼠免疫功能的影响

目的:探讨龙牙楤木多糖(AEPS)抗肿瘤活性及对荷瘤小鼠免疫功能的影响。方法:以S180肉瘤为肿瘤模型,检测龙牙楤木多糖对肿瘤生长的抑制活性;MTT法检测龙牙楤木多糖对S180肉瘤细胞、A549肺癌细胞、SMMC-7721肝癌细胞的体外抑制活性;以其对荷瘤小鼠免疫器官、血液淋巴细胞数量及淋巴细胞增殖影响、巨噬细胞活性和NK细胞杀伤活性来评价AEPS对荷瘤小鼠免疫功能的作用。结果:AEPS对S180肉瘤生长有显著的抑制作用,其中75 mg/(kg.d)剂量组抑瘤率最高达57.68%;AEPS对S180肉瘤细胞、A549肺癌细胞、SMMC-7721肝癌细胞生长的最高抑制率均达60%以上;AEPS显著提高荷瘤小鼠脾脏和胸腺质量以及血液淋巴细胞数量,促进淋巴细胞增殖反应,增加NK细胞杀伤活性和巨噬细胞活性。结论:AEPS有显著的抗肿瘤活性,并能直接作用于肿瘤细胞,抑制肿瘤生长,其抑瘤作用与机体免疫功能的增强有关。     

MiR-191过表达抑制NK细胞的肿瘤杀伤活性和细胞凋亡

目的研究miR-191过表达对自然杀伤细胞(natural killer cell,NK)的凋亡和肿瘤杀伤活性的影响。方法通过免疫磁珠阴性分选方法从小鼠脾脏中分离NK细胞,体外用IL-2刺激扩增5～7 d后,采用流式细胞术分析NK细胞的细胞毒活性和凋亡情况。制备小鼠骨髓、脾脏、淋巴结和胸腺的单细胞悬液,计算细胞总数后,采用流式细胞术分析NK细胞的比例和数量。结果与野生型(WT)小鼠相比,miR-191过表达转基因TgmiR-191小鼠NK细胞的凋亡率及其对小鼠淋巴细胞瘤细胞RMA-S的特异性杀伤活性均显著降低。miR-191过表达可显著增加脾脏和淋巴结的NK细胞数量。结论 miR-191过表达对NK细胞的肿瘤杀伤活性和细胞凋亡有明显的抑制作用。     

亚硒酸钠对免疫功能影响的实验研究

硒对免疫功能的影响虽已有一些报导,但结果不甚一致。由于天然杀伤细胞(NK)、巨噬细胞(Mφ)等在肿瘤免疫中具有重要作用,笔者对亚硒酸钠(Na_2 SeO_3)在体内、外对小鼠脾细胞NK活性及Mφ毒性作用的影响进行了探讨。     

鳖血提取物对小鼠免疫功能正向调节作用的研究

目的 :研究鳖血提取物对小鼠的免疫调节功能。方法 :采用环磷酰胺为免疫抑制剂 ,获得免疫功能低下的小鼠动物模型。比较鳖血提取物作用前后 ,小鼠T细胞亚群的数量、NK细胞的杀伤功能 ,淋巴细胞的增殖功能以及细胞因子水平的变化。结果 :鳖血提取物对免疫功能低下小鼠的CD4 T细胞在外周血中的比例、NK细胞的杀伤活性、淋巴细胞的增殖功能 ,均具有正向调节作用 (P <0 .0 5 ) ,并呈剂量依赖性 ;能提高淋巴细胞分泌IFN γ的能力。结论 :鳖血提取物具有正向调节小鼠免疫功能的能力     

烫伤对大鼠免疫功能的影响及药膳饮食对其的调理作用观察

目的观察烫伤对大鼠免疫功能的影响及药膳饮食对其的调理作用。方法健康Wistar大鼠70只，体质量（200±20）g，雌雄各半，随机分成正常对照组（A组，n=10）、烧伤常规喂养组（B组，n=30）和烧伤药膳喂养组（C组，n=30）。A组37℃造成假伤后全部处死取材，B、C组大鼠造成30％TB-SAⅢ度烧伤伤后B组常规饲养，C组伤后2h开始以复温至37℃的药膳饮食灌胃，2ml/次，2次／d＋常规饲养。B、C组于伤后3、7、14d各取10只，无菌条件下取材送检，观察T淋巴细胞亚群和NK细胞活性、血浆IgA、IgG、IgM、C3、C4、含量、肠道sIgA含量的变化。结果①B、C组伤后CD3^＋、CD4^＋、CD4^＋／CD8^＋、NK细胞活性、IgA、IgG、IgM、C3、C4水平及肠道sIgA含量均低于A组，CD8^＋高于A组（P〈0．05或P〈0．01）；②C组与B组比较，各免疫指标均恢复快（P〈0．05或P〈0．01）。结论严重烫伤大鼠细胞免疫和体液免疫功能发生了改变；药膳饮食可以改善烫伤大鼠T淋巴细胞亚群分布，提高NK细胞活性，促进血浆IgA、IgM、IgG、C3、C4的恢复和肠黏膜细胞sIgA的分泌，从而改善机体的免疫功能。     

紫色杆菌LPS对小鼠脾细胞免疫活性的抑制作用

细菌内毒素或脂多糖(LPS)是机体内强烈的免疫调节剂。天然低毒性紫色杆菌LPS 体内处理小鼠,能促进脾细胞的分化、增殖,但降低脾细胞对 Con A 和同种细菌 LPS 的反应性,抑制混合淋巴细胞反应(MLR)和自然杀伤(NK)细胞的活性。LPS 处理供体小鼠还抑制其脾细胞在 F_1 小鼠内诱导的移植物抗宿主反应。应用混合培养方法,在 Con A和 LPS 诱导的淋巴细胞转化反应中分别检测到非特异性抑制细胞活性,但在MLR和NK 活性测定中未发现抑制细胞的存在。上述结果说明 LPS体内抑制T、B 淋巴细胞功能和 NK细胞活性,而这种抑制作用除由抑制细胞介导外,还存在其它尚未明瞭的机理。     

淫羊藿苷(ICA)对化疗后免疫抑制小鼠的免疫促进作用

目的:通过观察淫羊藿苷(ICA)对化疗药环磷酰胺(Cy)所致免疫抑制小鼠免疫功能的影响,探讨ICA促进免疫功能的作用及机理.方法:除正常组小鼠外,所有小鼠经腹腔注射Cy(300 mg/kg);第二天开始给予实验组小鼠灌胃不同剂量的ICA[150、80、40 mg/(kg·d)],阳性对照组小鼠尾静脉注射参芪扶正注射液(1 ml/d),模型组给予等量生理盐水,连续干预10天.所有小鼠均于末次给药12小时后处死,计算胸腺指数(TI)和脾指数(SI),光镜下计数外周血和脾淋巴细胞数量.MTT法检测小鼠脾淋巴细胞增殖反应.ELISA法检测TNF-α的含量.乳酸脱氢酶实验(LDH)检测小鼠脾脏NK和CTL细胞的活性.流式细胞术(FACS)检测脾淋巴细胞中NKT和CD3+T细胞的比例.结果:模型组小鼠以上各项免疫指标均有所下降.ICA处理组小鼠脾脏指数和胸腺指数均升高(P<0.01),脾淋巴细胞数量显著增加(P<0.01),但未达到正常对照组水平;ICA明显提高小鼠脾淋巴细胞的增殖反应,促进了小鼠脾NK和CTL细胞对肿瘤细胞的杀伤活性(P<0.01),提高了小鼠脾细胞TNF-α的产生(P<0.01).ICA处理组小鼠脾细胞中CD3+T、NKT细胞比例明显增加(P<0.01).结论:ICA能促进小鼠免疫功能,具有逆转化疗后小鼠免疫抑制状态的作用.     

重组人IL-18对辐射损伤后小鼠免疫功能的调节作用

目的:探讨重组人IL-18(IL-18)对辐射损伤后小鼠免疫功能的影响.方法:对32只小鼠分4组进行辐射损伤,然后分别用淋巴细胞转化实验、NK细胞细胞毒实验、T淋巴细胞亚群测定和血清中IgG测定方法观察IL-18对其免疫功能的调节作用.结果:重组人IL-18能够提高辐射损伤后小鼠的T、B淋巴细胞转化能力,增强NK细胞对肿瘤细胞的杀伤能力,上调CD4 T细胞的数量,但对IgG产生能力没有调节作用.结论:重组人IL-18对辐射后小鼠免疫功能损伤有促进恢复的作用.     

肝癌患者双弹头导向药物治疗对NK细胞活性和T细胞亚群的影响

目的探讨中晚期肝癌(HCC)患者接受抗甲胎蛋白抗体介导的双弹头导向药物治疗对细胞免疫功能的影响。方法采用碱性磷酸酶 -抗碱性磷酸酶法和51Cr释放试验 ,分别测定了HCC患者T细胞亚群数和NK细胞的活性。结果导向组患者用导向药物治疗后 ,其NK细胞和CD3 T细胞、CD4 T细胞数 ,CD4 /CD8 T细胞比值均无显著降低 ,CD8 T细胞数无明显升高(P均>0.05) ;而化疗组患者做化疗后 ,其NK细胞的活性和CD3 T细胞、CD4 T细胞数 ,以及CD4 /CD8 T细胞的比值均显著降低 ,CD8 T细胞数显著升高(P均<0.05)。结论该双弹头导向药物对HCC患者的细胞免疫功能无影响 ,而化疗患者的细胞免疫功能则明显下降。     

Natural killer (NK) cell activity during HIV infection: a decrease in NK activity is observed at the clonal level and is not restored after in vitro long-term culture of NK cells.

NK cell activity is impaired in HIV-infected patients. The mechanisms behind the altered NK functions are not clear, and conflicting data concerning NK and antibody-dependent cellular cytotoxicity (ADCC) activity have been reported. In order to investigate whether this impairment is also observed at the clonal level and whether it is related to a defect at the target cell binding and/or the post-binding level, we evaluated highly purified NK cell lines and cloned NK cells obtained from 22 HIV-infected patients at different stages of disease and compared them with normal controls for their ability to: (i) kill K-562 and U-937 cell lines using a 51Cr release assay; (ii) bind and kill K-562 and U-937 cells at the single cell binding level; (iii) release NK cytotoxic factor (NKCF), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma); (iv) kill anti-IgM preincubated Daudi cell line (ADCC activity). This study with cloned NK cells or NK cell lines from HIV-infected individuals showed: (i) a decrease in their lytic capability against target cell lines; (ii) a low ability to form conjugates with K-562 and U-937 cell lines with respect to controls; (iii) a decreased ability to kill bound target cells; (iv) low levels of released NKCF, TNF-alpha and IFN-gamma after incubation with U-937 cells. Taken together, these findings suggest that the impaired NK cell function during HIV infection is also observed at the clonal level and is related to defects both at the target and post-binding levels. However, the precise mechanisms remain to be determined. The inability to restore normal NK activity after long-term culture in the presence of high levels of recombinant IL-2 is in agreement with the hypothesis of a 'general anergic process' during HIV infection.     

Effects of X-RAY Irradiation on Natural Killer (NK) Cell System. II. Increased Sensitivity to Natural Killer Cytotoxic Factor (NKCF)

Irradiation with low-doses of X-rays of tumor cells elevated their susceptibility to lysis by natural killer (NK) cells in an accompanying paper. Cytotoxicity assays conducted at the single cell level revealed that X-ray irradiation of K562 cells did not affect the number of effector-target conjugates but increased the frequency of dead conjugated target cells. During interaction with K562 cells large granular lymphocytes released a soluble cytotoxic factor (NKCF) that killed the target cells. X-ray irradiation did not affect the NKCF stimulatory ability of K562 cells, while it elevated their sensitivity to the lytic effect of NKCF. In contrast to X-rays, exposure to ultraviolet (UV) radiation of K562 cells did not elevate their NK sensitivity but rather reduced it. Treatment with mitomycin C produced no effect on NK sensitivity. These results indicate that X-ray irradiation elevates the target sensitivity to NKCF, which may be involved in the increased NK sensitivity, and that the X-ray effect may be different from that of UV radiation or DNA synthesis inhibition.     

Effects of X-ray irradiation on natural killer (NK) cell system. II. Increased sensitivity to natural killer cytotoxic factor (NKCF)

Irradiation with low-doses of X-rays of tumor cells elevated their susceptibility to lysis by natural killer (NK) cells in an accompanying paper. Cytotoxicity assays conducted at the single cell level revealed that X-ray irradiation of K562 cells did not affect the number of effector-target conjugates but increased the frequency of dead conjugated target cells. During interaction with K562 cells large granular lymphocytes released a soluble cytotoxic factor (NKCF) that killed the target cells. X-ray irradiation did not affect the NKCF stimulatory ability of K562 cells, while it elevated their sensitivity to the lytic effect of NKCF. In contrast to X-rays, exposure to ultraviolet (UV) radiation of K562 cells did not elevate their NK sensitivity but rather reduced it. Treatment with mitomycin C produced no effect on NK sensitivity. These results indicate that X-ray irradiation elevates the target sensitivity to NKCF, which may be involved in the increased NK sensitivity, and that the X-ray effect may be different from that of UV radiation or DNA synthesis inhibition.     

Effects of X-RAY Irradiation on Natural Killer (NK) Cell System. II. Increased Sensitivity to Natural Killer Cytotoxic Factor (NKCF)

Abstract

Irradiation with low-doses of X-rays of tumor cells elevated their susceptibility to lysis by natural killer (NK) cells in an accompanying paper. Cytotoxicity assays conducted at the single cell level revealed that X-ray irradiation of K562 cells did not affect the number of effector-target conjugates but increased the frequency of dead conjugated target cells. During interaction with K562 cells large granular lymphocytes released a soluble cytotoxic factor (NKCF) that killed the target cells. X-ray irradiation did not affect the NKCF stimulatory ability of K562 cells, while it elevated their sensitivity to the lytic effect of NKCF. In contrast to X-rays, exposure to ultraviolet (UV) radiation of K562 cells did not elevate their NK sensitivity but rather reduced it. Treatment with mitomycin C produced no effect on NK sensitivity. These results indicate that X-ray irradiation elevates the target sensitivity to NKCF, which may be involved in the increased NK sensitivity, and that the X-ray effect may be different from that of UV radiation or DNA synthesis inhibition.     

Impaired release of natural killer cytotoxic factor in patients with primary Sjögren''s syndrome.

The impaired natural killer (NK) cell activity against K562 target cells of patients with primary Sjögren''s syndrome (primary SS) was re-examined in a 2-year follow-up study of 10 patients and 10 normal controls. The ability of blood mononuclear cells (BMNC) to form effector/target cell conjugates and to release NK cytotoxic factor (NKCF) were studied. NK cell activity of the patients was unchanged low (P less than 0.01) compared with the controls. The number of effector/target cell conjugates did not differ between patients and controls, whereas NKCF-release from interferon-stimulated BMNC was significantly (P less than 0.01) reduced in the patients with primary SS and positively correlated to the reduced NK cell activity (r = 0.85, P = 0.0002). The permanently low NK cell activity of patients with primary SS appears therefore, at least in part, to be due to an impaired release of NKCF and not to a defective ability of effector cells to recognize and/or adhere to target cells.     

Role of natural killer cytotoxic factors in the mechanism of target-cell killing by natural killer cells

Studies on the mechanism of cell-mediated cytotoxicity (CMC) have suggested a stimulus-secretion model and implicated a role of soluble cytotoxic mediators. Our studies in the natural killer (NK) system provide several lines of evidence for the involvement of natural killer cytotoxic factors (NKCF) in NK CMC and led to the development of a model for the NK lytic mechanism. This model delineates several interactions between NK cells and targets that are deemed necessary to achieve target-cell lysis. The first stage is the interaction of the effector with the target cell, resulting in contact and adhesion. This is presumably mediated by NK recognition structures and target-cell structures. Following binding, the target cell stimulates the NK cell to release NKCF. This step is functionally distinct from the initial effector-target binding. The trigger mechanism for release of NKCF appears to be dependent on protein kinase C. The released NKCF binds to NKCF binding sites on the target cell followed by processing or internalization and, ultimately, resulting in cell death. This model has been shown to be useful in investigating the mechanism of defective NK activity in certain disease states. Biochemical analysis and comparative studies suggest that NKCF is a distinct molecule from other cytotoxins studied to date. The studies in the NK CMC system supporting a role of cytotoxic mediators also suggest a possible role for cytotoxic factors in other cytotoxic systems. Furthermore, the selective susceptibility to lysis of tumor or infected cells by NKCF suggests a possible role of their effectiveness inin vivo therapy.     

Suppressed natural killer cell activity in patients with euthyroid Graves' ophthalmopathy

The purpose of the present study was to determine whether patients with euthyroid Graves' exophthalmopathy have an impaired NK cell function compared to patients with Graves' hyperthyroidism and healthy controls. The NK cell activity measured against K562 target cells was significantly suppressed (p less than 0.01) in patients with euthyroid Graves' ophthalmopathy, whereas the NK cell activity of patients with Graves' hyperthyroidism was not. Although interferon-alpha, interleukin-2 and indomethacin significantly enhanced (p less than 0.01) the NK cell activity in all three groups, none of these agents fully restored the defective NK cell activity in euthyroid Graves' ophthalmopathy. The concentrations in the blood of large granular lymphocytes and CD16 positive cells did not differ between the three groups, furthermore an immunosuppressive serum factor was not detected. The number of effector/target cell conjugates did not differ between patients and controls, whereas the interferon-alpha induced production of a soluble natural killer cytotoxic factor (NKCF) with specificity for NK sensitive target cells was suppressed in patients with Graves' euthyroid ophthalmopathy. We conclude that one of the mechanisms underlying the defective NK cell activity in patients with euthyroid ophthalmopathy may be an impairment of the release of NKCF from the NK cells.     

Autoantibodies as Predictive and Diagnostic Markers of Idiopathic Inflammatory Myopathies

The purpose of the present study was to determine whether patients with euthyroid Graves’ exophthal-mopathy have an impaired NK cell function compared to patients with Graves’ hyperthyroidism and healthy controls.

The NK cell activity measured against K562 target cells was significantly suppressed (p < 0.01) in patients with euthyroid Graves’ ophthalmopathy, whereas the NK cell activity of patients with Graves’ hyperthyroidism was not. Although interferon-α, interleukin-2 and indomethacin significantly enhanced (p < 0.01) the NK cell activity in all three groups, none of these agents fully restored the defective NK cell activity in euthyroid Graves’ ophthalmopathy. The concentrations in the blood of large granular lymphocytes and CD 16 positive cells did not differ between the three groups, furthermore an immunosuppressive serum factor was not detected. The number of effector/target cell conjugates did not differ between patients and controls, whereas the interferon-α -induced production of a soluble natural killer cytotoxic factor (NKCF) with specificity for NK sensitive target cells was suppressed in patients with Graves’ euthyroid ophthalmopathy. We conclude that one of the mechanisms underlying the defective NK cell activity in patients with euthyroid ophthalmopathy may be an impairment of the release of NKCF from the NK cells.     

Lysis of natural killer-sensitive and -resistant tumor cells by natural killer cytotoxic factors (NKCF)-containing liposomes

The lethal hit stage in NK cell-mediated lysis requires a complex series of events involving the release of NKCF, subsequent binding of these factors to the target cell, and susceptibility of the target cell to lysis by NKCF. Binding of NKCF alone is not sufficient because a number of tumor cells are able to bind NKCF without being lysed, suggesting the need for an additional processing step active on susceptible target cells. In the present study, we show that the interaction with liposome-incorporated NKCF renders NK resistant target cells sensitive to NKCF-mediated lysis. These results suggest that NKCF may mediate their cytotoxic effects through internalization of these factors into the cytosol.     

Activation of human blood lymphocytes and monocytes by the streptococcal preparation OK432: enhanced generation of soluble cytotoxic factors

The streptococcal preparation OK432 augments natural cytotoxicity of human blood lymphocytes and monocytes. It also enhanced the production of natural killer soluble cytotoxic factors (NKCF) when the effector cells interact with K562 cells. There was a good correlation between the OK432-induced enhancement of NK cell-mediated cytotoxicity and the released NKCF activity. OK432-pretreated monocytes secreted higher amounts of monocyte cytotoxic factors (MCF) than the untreated monocytes. With the monocytes the enhanced generation of MCF was not always accompanied by the increase in direct cell-mediated lysis of K562. OK432 treatment alone did not induce NKCF release from lymphocytes, and the presence of K562 in the culture was necessary. In contrast, monocytes generated MCF when exposed to OK432. In the supernatants of cocultures of OK432-activated effectors and K562 the NKCF and MCF activity was elevated two- to ten-fold. The OK432-induced augmentation of natural cytotoxicity exerted by lymphocytes and monocytes may be mediated through an increase in the synthesis, activation and/or release of NKCF and MCF.