LAK细胞体外杀伤直肠腺癌细胞的透射电镜观察

应用透射电镜观察LAK细胞与HR8348细胞相互作用时超微结构变化。结果显示:(1)效靶细胞接触、结合后,两种细胞质膜接触部位均有不同程度的增厚,电子密度增加,界限不清,形成粘着斑或连结状结构,这种结构是效靶细胞稳定结合的形态学基础。(2)部分LAK细胞突起插入靶细胞深部,少数LAK细胞胞体钻入靶细胞,表明LAK细胞具有与NK、CTL细胞相同的“钻瘤”功能。(3)效靶细胞接触后,两种细胞细胞器及其它细胞颗粒均向效靶接触侧集中,效靶接触部位含有丰富的微丝、微管、微管集中处溶酶体颗粒较多;推测微丝、微管及溶酶体在靶细胞溶解中起一定作用。(4)效靶细胞结合后,靶细胞出现鼓泡和环形穿孔,靶细胞呈凋落状或溶解性坏死。凋落状坏死可能是LAK细胞释放的非穿孔素性物质通过靶细胞膜上的受体,启动靶细胞内源性核酸酶,导致DNA裂解所致。后者可能与穿孔素作用,Ca~H、Mg~H离子内流,细胞水肿及溶酶体导致细胞自溶有关。     

LAK细胞杀伤直肠腺癌细胞时酶细胞化学定量观察

采用酶细胞化学技术及MIAS—300型图象分析仪观察了LAK细胞杀伤HR8348细胞不同时间的效靶细胞内SDH和AcPase的变化。结果显示:①效靶细胞共育不同时间的HR8348细胞内AcPase均明显高于对照组,并随时间延长,AcPase含量增加。靶细胞内SDH含量在共育30、60min时明显增加,90min后逐渐下降。②LAK细胞内AcPaSe在60、90、120min时酶含量较高,与对照组相比有非常显著的统计学差异(P<0.01)。SDH在效靶共育60,90,120,180、240min时含量明显增高,与对照组相比差异非常显著(P<0.01)。效靶细胞内AcPase、SDH含量变化说明两种细胞功能非常活跃。效靶细胞接触早期靶细胞内SDH变化可能与靶细胞抵御损伤而表现出细胞功能活跃有关。靶细胞内AcPase含量增加,是靶细胞内溶酶体活化的表现,是靶细胞自溶的物质基础。     

LAK细胞杀伤直肠腺癌细胞时酶细胞化学定量观察

采用酶细胞化学技术对LAK细胞杀伤HR8348细胞不同时间的效靶细胞内SDH和ACP酶进行动态定量观察。使用MIAS-300型图像分析仪分别检测SDH阳性粒子数及ACP酶灰度值变化。结果显示:1、效靶共育不同时间的HR8348细胞内ACP酶均明显高于对照组,ACP酶随效靶共育时间延长,ACP酶含量增加。靶细胞内SDH含量在共育30分钟时明显增加,60分钟后逐渐下降。2、LAK细胞内ACP酶在60、90、120分钟时酶含量较高,与对照组相比差异非常显著(P<0.01)。SDH在效靶共育60、90、120、240分钟与对照组相比差异非常显著(P<0.01)。效靶细胞内ACP、SDH含量变化说明两种细胞功能非常活跃。效靶接触早期细胞内SDH变化可能与靶细胞抵御损伤而表现出细胞功能活跃有关。靶细胞内ACP酶含量增加,是靶细胞内溶酶体活化的表现,是靶细胞自溶的物质基础。     

LAK细胞对直肠腺癌细胞杀伤的观察

采用体外培养方法,在恒温(37℃)和5％CO_2条件下,连续观察LAK细胞对直肠腺癌细胞系(HR8348)的结合率。杀伤活性及杀伤动态。结果表明:HR8348细胞对LAK细胞杀伤较敏感,在相同的效靶比例条件下,4h时效靶细胞结合率为52.0±3.6,LAK细胞杀伤率为49.0±2.3％。效靶细胞结合率及杀伤率呈正相关(P<0.01),LAK细胞的杀伤取决于效靶细胞的稳定结合。LAK细胞杀伤HR8348的动态过程是:效靶细胞主动趋向运动→相互识别→接触与结合→LAK细胞发挥杀伤作用→靶细胞溶解。LAK细胞杀伤作用随效靶共育时间延长乃效靶比例的增高而增强。     

LAK细胞体外杀伤直肠腺癌细胞的扫描电镜观察

应用扫描电镜观察体外LAK细胞与直肠腺癌细胞相互作用时的形态学变化。结果表明:LAK细胞与HR8348细胞均具有主动互相趋向运动,效靶细胞借其细胞突起及微绒毛相互接触。LAK细胞及肿瘤细胞的细胞突起及微绒毛由早期的平面接触逐渐发展为犬牙交错状结合。效靶细胞紧密结合后,靶细胞鼓泡,细胞膜出现环形穿孔。互相接触,结合的微绒毛及细胞突起在靶细胞的溶解过程中起到一个物理微桥的作用。LAK细胞分泌的杀伤物质通过微桥介导靶细胞的溶解。效靶细胞互相结合的微绒毛间形成关闭小室,它可能与维持局部杀伤物质浓度有关。     

原发性卵巢癌患者CD_3AK细胞和LAK细胞杀瘤活性比较

为探讨CD3AK细胞和LAK细胞杀瘤作用的特异性及靶细胞选择性，对比观察了3例原发性卵巢癌患者CD3AK细胞和LAK细胞对卵巢癌细胞系（3AO）和人早幼粒细胞白血病细胞系（HL60）的杀伤活性。结果表明：LAK细胞在培养第1～3周，各效靶比（10：1，20：1，40：1）对HL60的杀伤活性略高于对3AO的杀伤活性，但差异无显著性（P＞0．05）；CD3AK细胞在培养第1周，各效靶比（10：1，20：1，40：1）对HL60的杀伤活性均显著高于对3AO的杀伤活性（P＜0．05），而培养第2、3、4周时，对HL60和3AO的杀伤活性无显著区别（P＞0．05）。提示：LAK细胞和CD3AK细胞均缺乏杀瘤特异性，而CD3AK细胞具有一定的靶细胞选择性。     

人淋巴因子激活的杀伤细胞对肝癌细胞株凋谢作用的观察

用重组白细胞介素2从健康人未稍血中诱导出 LAK 细胞,此 LAK 细胞与 H_(7402)细胞共同培养后,用细胞涂片,盖片培养及透射电镜观察,可见 LAK 细胞能主动接近 H_(7402)细胞,两者以胞质突互相交错结合,然后 H_(7402)细胞则以典型谢方式死亡。这表明在引起靶细胞的死亡方式方面,LAK细胞与 NK 细胞和 CTL 细胞相同。结果还提示,细胞凋谢是细胞内的自动过程。     

丁酸钠抑制淋巴因子激活杀伤细胞杀瘤活性的研究

目的：探讨低浓度丁酸钠(sodium butyrate)对人结肠腺癌细胞SW1116表面细胞间黏附分子-1(ICAM-1)和癌胚抗原(CEA)表达的影响及其对淋巴因子激活杀伤(LAK)细胞杀伤活性的改变。方法：噻唑蓝(MTT)比色法检测经不同浓度丁酸钠处理4天后SW1116在不同效／靶比下对LAK细胞杀伤敏感性的改变，流式细胞术和免疫荧光法定量和定性检测丁酸钠对ICAM-1和CEA表达的影响。结果：MTT显示，经0．5、1和2mmol／L丁酸钠处理后，细胞对LAK细胞杀伤敏感性从74．6％下降至15．9％(效／靶为20)，且在一定程度上有浓度依赖性，当效／靶为10时亦呈类似变化，与之对应的ICAM-1阳性细胞数从92．2％下降至7．6％，而CEA阳性细胞数从1．2％上升至16．6％，荧光显微镜下与流式细胞术的改变相一致。结论：丁酸钠减弱LAK细胞对人结肠癌细胞SW1116的杀伤，这种作用可能与其降低ICAM-1同时增加CEA的表达，从而改变效／靶细胞的结合有关。     

LAK细胞可溶性因子介导K_(562)细胞的损伤

采用~(51)Cr释放方法探讨LAK细胞可溶性因子介导的K_(562)细胞的损伤。15例中有12例LAK细胞培养上清介导的K_(562)细胞的损伤为10.96±5.15％。将LAK细胞洗去培养上清后与K_(562)细胞共育6小时,5例此种效靶细胞共育后的上清介导的Ks6z细胞的损伤为12.41±6.54％。结果表明,LAK细胞杀伤K_(562)细胞的作用至少一部分是由LAK细胞分泌的可溶性因子完成的。     

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。     

An immunofluorescence study on the changes of microtubulin in human rectal adenocarcinoma cells killed by LAK cells in vitro

The human rectal adenocarcinoma cell line(HR8348)was treated withlympholdne activated killer(LAK)cells in vitro and the changes of the microtubulin inboth the effector and target cells were investigated with the aid of immunofluorescencemicroscopy.It was revealed that after the attachment of LAK cells to the tumor cells,theeffector-target conjugates formed and distribution of the microtubulin in both the effectorand target cells changed.In the LAK cells,the microtubulin concentrated mainly in thecontact region,forming a crescent-like structure,while in the target cells,themicrotubulin condensed into patches and fused with the crescent-like structure of the LAKcells,Eventually,the target cells degenerated and died.It was suggested that the lysis ofthe target cells may be related to the redistribution of the microtubulin in both theeffector and target cells.     

NK/LAK细胞对人口腔癌耐细胞药株杀伤活性的观察

目的明确免疫活性细胞对肿瘤细胞的杀伤活性是否与肿瘤的耐药性相关。方法采用连续形态学观察及MTT比色法,研究淋巴因子激活的杀伤(LAK)细胞及自然杀伤(NK)细胞,对人口腔癌耐药细胞株KBV200耐药性逆转前后及亲本敏感株KB的杀伤活性。(1)在肿瘤细胞与LAK细胞共育后3 h内连续在倒置显微镜下观察LAK细胞对上述3者的杀瘤效应;(2)采用MTT比色法检测NK及LAK对3株细胞的杀伤率。结果与KB细胞组相比,在KBV200和KBV200 维拉帕米组,LAK细胞出现在靶细胞周围的时间早、数量多,伸出伪足的LAK细胞比率高,出现集落样细胞团块时间亦早。NK、LAK细胞对KBV200细胞株耐药性逆转前后的杀伤率均明显高于敏感株KB(P<0.05),而对耐药株耐药性逆转前后的杀伤率无统计学差异(P>0.05);LAK对各株的杀伤活力均明显强于NK细胞(P<0.05)。结论免疫活性细胞对KBV200细胞株有较强的杀伤作用,逆转耐药性不降低免疫活性细胞杀伤活力,提示细胞过继免疫治疗可能成为控制化疗耐药病人病情发展的一个有效手段。     

NK／LAK细胞对人口腔癌耐细胞药株杀伤活性的观察

OBJECTIVE: To understand the relation between cytotoxic activity of immunologic effector cells and multidrug resistance of the tumor cells. METHODS: Continuous observation of the morphological changes and MTT colorimetry were employed to evaluate the cytotoxic activity of lymphokine-activated killer (LAK) cells and natural killer (NK) cells against multidrug-resistant (MDR) human oral carcinoma cell line-KBV200 (before and after reversal of MDR) and parental drug-sensitive cell line KB. The morphologic changes of LAK cells and the 3 target cell lines were observed continuously under inverted microscope 3 h after co-culture of LAK cells with one of three target cell lines respectively. The lysis rates of three tumor cell lines in response to co-culture with LAK or NK cells were determined using MTT colorimetry. RESULTS: In comparison with the parental drug-sensitive cell line KB, both KBV200 and its reserved cell line by verapamil (KBVV) showed earlier adherence and greater number of cells lysed by LAK. In MTT colorimetry assay, the cytotoxicity of both LAK and NK cells against the 3 cell lines was associated with the effector-to-target (E/T) cell ratio; the lysis rates of KBV200 and reversed KBV200 cells by verapamil in response to LAK and NK cells were higher than that of KB cells (P<0.05), but KBV200 and KBVV did not significantly differ (P>0.05). At the same E/T ratio, LAK cells possessed stronger cytotoxicity than NK cells against all the tumor cell lines (P<0.05). CONCLUSIONS: Immunologic effector cells possess strong cytotoxic activity against multidrug-resistant cell line KBV200. Modulation of MDR does not decrease the cytotoxic activity of the immunologic effector cells. The results of this study suggest that adoptive cell immunotherapy with immunologic effector cells may be of value in controlling the progress of MDR tumors.     

SEM observation on LAK cells killing the human rectal adenocarcinoma cells in vitro

ＳＥＭｏｂｓｅｒｖａｔｉｏｎｏｎＬＡＫｃｅｌｌｓｋｉｌｌｉｎｇｔｈｅｈｕｍａｎｒｅｃｔａｌａｄｅｎｏｃａｒｃｉｎｏｍａｃｅｌｌｓｉｎｖｉｔｒｏＤｉｎｇＹａｎｑｉｎｇ（丁彦青）；ＬｉＣｈｕｎｄｅ（李春德）；ＺｈａｎｇＪｉｕｈｕａ（张进华）；ｚｈｕＭｅｉ...     

Determination of IL-2 and cytotoxicity of killer cells in MTT colorimetry

Summary The activity of interleukine 2 (IL-2) in culture supernatants of lymphokine-activated killer (LAK) cells and tumor infiltrating lymphocytes (TIL) as well as cytotoxicity of LAK cells on cultured leukemic cells were determined by MTT colorimetry. The results showed that higher activity of IL-2 in culture supernatant of LAK and TIL cells was found it could be used to support the culture of IL-2 dependent cell lines. The significant cytotoxicity of LAK cells on leukemic cell lines could be found in vitro, and it was consistent with the ratio of effector cells to target cells. The number of living leukemic cells is consistently related with the concentration of formazan metabolite of MTT. It suggested that the numbers of living cells and cytotoxicity of LAK cells could be estimated by determination of formazan metabolite OD value.     

TEM observation on LAK cells killing the human rectal adenocarcinoma cells in vitro

ＴＥＭｏｂｓｅｒｖａｔｉｏｎｏｎＬＡＫｃｅｌｌｓｋｉｌｌｉｎｇｔｈｅｈｕｍａｎｒｅｃｔａｌａｄｅｎｏｃａｒｃｉｎｏｍａｃｅｌｌｓｉｎｖｉｔｒｏ￥ＤｉｎｇＹａｎｑｉｎｇ（丁彦青），ＬｉＣｈｕｎｄｅ（李春德），ＺｈａｎｇＪｉｎｈｕａ（张进华），ＺｈｕＭｅ...     

Electron microscopic observation on the killing of human lung carcinoma cells by lymphokine-activated killer cells (LAK cells) in vitro

The interactions and reactions between co-cultured LAK cells and target cells were observed under scanning and transmission electron microscopy. Close contacts between these two cells were necessary to effect and sustain the killing process. In the contact area, there were prominent changes of the cell surface structures of both cells. The active motility of LAK cells accelerated the detachment and disintegration of the target cells. Lack of dense (killing) granules in the cytoplasm of LAK cells and the apoptotic death pattern of the affected target cells suggest that a non-perforin-dependent mechanism might be involved in killing. Morphologically, the killing process of LAK cells was basically similar to that of CTL and NK cells.