抗人DR5单克隆抗体对白血病U937细胞凋亡的作用

目的：研究鼠抗人DR5单克隆抗体（mDRA-6）对白血病细胞U937的凋亡诱导作用。方法：光镜下观察mDRA。6作用后U937细胞的形态变化；流式细胞术检测15937细胞表面DR5表达率；MTT法检测mDRA-6对U937细胞生长的影响；AnnexinV—FITC／PI双染法流式细胞仪检测细胞凋亡率；琼脂糖凝胶电泳检测U937细胞DNA的片段化降解；JC-1单染流式细胞仪检测细胞线粒体膜电位改变。结果：mDRA-6作用U937细胞后呈现典型的细胞凋亡特征；MTT法检测显示mDRA-6作用U937细胞24小时死亡率为61．09％，并呈时间、浓度依赖性；流式细胞仪检测显示10mg/L的mDRA-6作用4小时，U937细胞凋亡率为79．12％；琼脂糖凝胶电泳显示DNA呈现明显梯状带型；mDRA-6作用U937细胞后线粒体膜电位明显下降。结论：mDRA-6能够诱导白血病U937细胞凋亡，是具有诱导细胞凋亡活性的功能性抗体。     

水飞蓟素在紫外线照射诱导A375-S2细胞凋亡中发挥抑制作用

目的：从天然药物中筛选出抑制紫外线照射诱导人黑色素瘤细胞A375-S2凋亡的有效单体。 方法： MTT法测定细胞生长抑制率；形态学观察，DNA凝胶电泳及LDH法；用半胱天冬酶活力检测试剂盒测定半胱天冬酶活力；用免疫印记法检测Bcl-2家族成员(Bcl-2, Bcl-xL和Bax)的表达。 结果： 紫外线照射(2.4 J/cm2, 5 min) 能显著诱导A375-S2细胞发生凋亡，其作用呈明显时间依赖性。形态学观察可见凋亡小体的形成，琼脂糖凝胶电泳可见凋亡典型的DNA梯带；水飞蓟素具有抑制紫外线照射 (2.4 J/cm2, 5 min) 诱导A375-S2细胞凋亡的作用，水飞蓟素作用于紫外线照射 (2.4 J/cm2, 5 min)的A375-S2细胞，培养12 h，使紫外线照射诱导的半胱天冬酶-9、半胱天冬酶-3的活力降低；免疫印记法检测发现水飞蓟素作用的A375-S2细胞 (紫外线照射) 中Bcl-2 蛋白和Bcl-xL蛋白的表达增加。 结论： 水飞蓟素明显抑制紫外线照射诱导的A375-S2细胞的凋亡，其抑制凋亡作用与半胱天冬酶途径和线粒体途径相关。     

人U937细胞吞噬IgA免疫复合物的研究

目的 对IgA Fc受体(FcaR Ⅰ)介导的U937细胞吞噬IgA免疫复合物进行研究。方法 以异硫氰酸荧光素(FITC)标记的8．9NIP／BSA为抗原，与抗NIP的IgA或IgA抗体结合，分别形成IgA免疫复合物(IgA IC)和IgG免疫复合物(IgG IC)，再与经佛波醇乙酯(PMA)刺激分化为单核细胞样的13937细胞孵育，流式细胞仪分析U937细胞吞噬IgA Ic和IgG IC的情况。结果 U937细胞表面：FcaR Ⅰ表达量高于3种IgG Fe受体(FcγRⅠ、FcγRⅡ、FcγRⅢ)。PMA刺激后细胞表面FcαRⅠ上调明显，吞噬IgAIC能力增加。FcαRⅠ介导的U937细胞对IgA IC的吞噬作用高于FcγRⅠ和FcγRⅡ介导的对IgG IC的吞噬作用，且这种吞噬作用是特异性的。在补体受体CRl和CR3作用下，U937细胞对IgA IC的吞噬作用有所增强。结论 FcαRⅠ介导单核细胞非常强的吞噬IgA IC的作用。     

玉竹提取物B抗肿瘤机制的初步研究

目的：研究玉竹提取物B(the extract B of Polygonsham odoratum,EB-PAOA)对S—180荷瘤鼠细胞因子产生水平的影响及诱导人结肠癌CL-l87细胞调亡的作用，以初步探讨EB-PAOA的抗肿瘤作用机制。方法：采用MTT法，检测EB-PAOA对S—180荷瘤鼠产生IL-2、IFN—γ、IL-1和TNF—α等细胞因子水平的影响；体外培养人结肠癌CL-l87细胞株，用MTT法测定EB-PAOA对CL-187细胞的抑制率；用电镜观察并确认有无凋亡细胞；通过流式细胞仪检测凋亡率。结果：EB-PAOA处理后的荷瘤鼠产生IL-2、IL-1和TNF—α的能力均有所增强；EB-PAOA能抑制CL-l87细胞的增殖；电镜下可见到大量凋亡细胞；流式细胞仪DNA直方图上出现了凋亡峰，凋亡率呈时间依赖性。结论：EB-PAOA抗肿瘤的作用机制可能是通过促进荷瘤鼠脾细胞分泌IL-2以及腹腔巨噬细胞分泌IL-1和TNF—α增强细胞免疫功能并具有直接诱导肿瘤细胞调亡作用而实现的。     

冬凌草甲素对急性白血病HL-60细胞的增殖抑制作用及其作用机制

目的探讨冬凌草甲素对急性白血病HL-60细胞的增殖抑制作用及其作用机制。方法以不同浓度的冬凌草甲素作用于体外培养的HL-60细胞，MTr法检测细胞生长抑制率，应用流式细胞仪检测细胞凋亡情况，瑞氏染色法观察细胞凋亡时的形态学变化。应用PCR．ELISA及RT—PCR法检测细胞凋亡前后端粒酶活性及端粒酶hTERT mRNA表达水平的变化。结果冬凌草甲素可显著的抑制HL-60细胞的生长，诱导细胞发生凋亡，并呈现出明显的量-与时-效关系。冬凌草甲素作用48—60h后在瑞氏染色图片上可见核浓缩及核碎裂等典型的细胞凋亡特征，同时端粒酶hTERT mRNA的表达水平及端粒酶活性均明显降低。结论冬凌草甲素能抑制HL-60细胞的生长及诱导细胞发生凋亡：降低端粒酶hTERT mRNA的表达水平及端粒酶活性可能是其重要作用机制之一。     

两型TNF-α对单核细胞系U937细胞功能的影响

目的：比较两型TNF—α对单核细胞系U937细胞功能的影响，以探讨跨膜型TNF—α在炎症中的作用。方法：通过吞噬、RT-PCR、Western blot及FACS等方法，比较两型TNF—α对U937细胞生物学功能(包括吞噬、细胞因子mRNA、胞质IκB—α及ICAM—1表达等)的影响。结果：分泌型TNF—α可明显促进U937细胞吞噬功能，使TNF—α、IL—1β及IL—8 mRNA积累增加，促进胞质IκB—α降解及提高黏附分子ICAM—1的表达；而跨膜型TNF—α对上述U937细胞的生物学功能则无明显影响：当二者联用时，跨膜型TNF—α与分泌型TNF—α亦无协同作用。结论：分泌型TNF—α对U937细胞具有明显地激活作用；而跨膜型TNF—α则无影响，提示两型TNF—α在炎症过程中的作用不同。     

槲皮素对U937细胞系抑制增殖和诱导凋亡作用的研究

目的 探讨黄酮类化合物槲皮素（Que）对人类单核细胞白血病U937细胞系的抑制增殖和诱导凋亡的作用。方法 应用MTT法检测不同浓度槲皮素对U937细胞的增殖抑制作用；AO／PI荧光染色后倒置荧光显微镜下观察细胞形态学变化；琼脂糖凝胶电泳测定细胞DNA的片段化；应用流式细胞仪检测细胞凋亡率及细胞周期分布。结果槲皮素能明显抑制U937细胞增殖，并存在剂量-效应关系和时间-效应关系；诱导U937细胞出现凋亡所具有的形态学和生化特征；随着槲皮素浓度升高，凋亡细胞和坏死细胞比例增加；将细胞特异性地阻滞在S期，出现凋亡峰。结论 槲皮素能抑制U937细胞增殖，诱导细胞凋亡，并具有细胞周期特异性。     

野生型p53基因导入对U937细胞分化、凋亡和CD36受体表达的影响

目的： 研究野生型p53基因重组腺病毒载体（AdCMV-p53）导入对U937细胞分化、凋亡和清道夫受体CD36表达的影响。 方法： AdCMV-p53导入U937细胞后，用细胞计数、细胞周期分析、台盼蓝染色排除法计数细胞悬液中的活细胞数目和NBT还原反应观察其对U937细胞生长、分化的影响；RT-PCR、免疫荧光和流式细胞分析检测AdCMV-p53导入对CD36表达的影响。 结果： AdCMV-p53可以高效导入U937细胞，野生型p53基因导入促进U937细胞向巨噬细胞分化，台盼蓝染色发现实验组阳性细胞数（64.6±9.2）%较对照组（14.2±5.5）%明显增多，吞噬能力增强；NBT还原反应实验组（49.7±12.6）%较对照组（6.3±1.8）%升高。RT-PCR和流式细胞分析检测，野生型p53基因导入使得CD36 mRNA转录增强，CD36蛋白表达增加。 结论： 野生型p53基因能影响细胞分化和凋亡，并上调清道夫受体CD36的表达，对于动脉粥样硬化的预防和基因治疗具有潜在意义。     

TNF-α对小鼠腹腔渗出细胞抗真菌活性的影响

目的：研究TNF—α对小鼠腹腔渗出细胞的激活和杀菌活性的效应。方法：以IL-12、IL-18、IFN—γ、TNF-α及抗体诱导小鼠腹腔渗出细胞和巨噬细胞，应用ELISA法测定细胞因子变化，Griess反应法测定培养上清液中NO的含量，检测巨噬细胞内新型隐球菌菌落数。结果：IL-12和IL-18联合应用协同诱导腹腔渗出细胞产生TNF—α，且能被IFN—γ抗体所抑制。IFN—γ、TNF-α协同诱导巨噬细胞产生NO和增强其杀伤新型隐球菌活性。结论：TNF—α在细胞因子相互调节诱导增强巨噬细胞杀真菌活性中起重要作用。     

中药牛膝提取物抗肿瘤活性的初步研究

目的　探讨中药牛膝提取物的抗肿瘤活性及其作用机制。方法　MTT法检测细胞抑制率;流式细胞仪分析细胞周期及凋亡情况;分别用生物化学法和ELISA法检测巨噬细胞因子TNF α和IL- 6的表达;用RT -PCR法检测巨噬细胞因子IL 6mRNA表达。结果　牛膝提取物在体外对两种肿瘤细胞株的增殖具有明显抑制作用,量效、时效关系显著(P <0 .0 1) ,细胞周期停滞于G0 G1 期,诱导细胞凋亡可能是其发生作用的机制之一;牛膝提取物虽然在体外不能促进小鼠脾细胞的生长增殖,但对巨噬细胞的吞噬功能却有较强的刺激作用(P <0 .0 1) ,并可促进细胞因子TNF -α和IL- 6的产生以及IL- 6mRNA的表达。结论　该提取物具有抗肿瘤作用,不仅对免疫功能没有抑制作用,而且可以通过延缓肿瘤细胞周期、诱导凋亡、增强巨噬细胞对肿瘤细胞的杀伤作用及分泌细胞因子如TNF- α、IL -6等参与其抗肿瘤机制。     

Oridonin-induced apoptosis of Jurkat cells and its mechanism

Jurkat cells in culture medium in vitro were exposed to different concentrations of oridonin. The proliferation rate of the cells was measured by MTT assay, cell apoptotic rate was detected by flow cytometry, morphology of cell apoptosis was observed by Hoechst 33258 fluorescence staining, DNA fragmentation was assayed by agarose gel electrophoresis, caspase-3 and poly(ADP-ribose) polymerase (PARP) expressions were detected by Western blotting using polyclonal anti-caspase-3 antibody and mouse anti-human PARP monoclonal antibodies, and caspase-3 activity was assayed with a colorimetric assay kit before and after apoptosis had occurred. Oridonin (over 32 mol/l) inhibited the growth of Jurkat cells and caused significant apoptosis. The suppression was both time-dependent and dose-dependent. Marked morphological changes of cell apoptosis, including condensation of chromatin and nuclear fragmentation, were clearly observed by Hoechst 33258 fluorescence staining, as well as by agarose gel electrophoresis. Western blotting showed cleavage of the caspase-3 zymogen protein (32 kDa), with the appearance of its 17 kDa subunit, and a cleaved 89-kDa fragment of 116 kDa PARP was also found, together with a concurrent increase in caspase-3 apoptotic activity. Oridonin can induce apoptosis in Jurkat cells via activation of caspase-3; the results indicate that oridonin might be an important potential anti-leukaemia reagent.     

兔抗人DR5抗血清诱导Jurkat细胞凋亡

制备兔抗人sDR5抗血清,检测它对Jurkat细胞的生长抑制和凋亡诱导作用。采用本室制备的sDR5免疫新西兰白兔,制备兔抗人sDR5抗血清,用ELISA法测定抗sDR5抗血清效价及抗血清的特异性。MTT试验分析它对Jurkat细胞生长抑制影响,倒置光显微镜和荧光显微镜观察抗sDR5抗血清对Jurkat细胞形态的影响,用AnnexinV/PI双染试剂盒检测Jurkat细胞凋亡率,琼脂糖凝胶电泳检测Jurkat细胞中DNA的片断化。结果:获得了高效价特异性兔抗人sDR5抗血清。兔抗人sDR5抗血清对Jurkat细胞具有显著的细胞生长抑制作用,并呈剂量依赖性。兔抗人sDR5抗血清处理后,Jurkat细胞可出现典型的细胞凋亡的形态特征:细胞膜皱缩,出泡,染色质浓缩,形成凋亡小体等。流式细胞术结果显示:兔抗人sDR5血清1/80、1/160作用Jurkat细胞2 h,细胞凋亡率分别为54.98%和34.13%。兔抗人sDR5抗血清可导致Jurkat细胞中的DNA片段化。本室制备的兔抗人sDR5抗血清能抑制Jurkat细胞生长和诱导Jurkat细胞凋亡。     

抗人DR5单克隆抗体——mDRA-6与顺铂协同杀伤白血病细胞HL-60作用研究

目的：观察抗死亡受体5（Death receptor 5,DR5）单克隆抗体--mDRA-6与顺铂（DDP）对HL-60细胞的协同杀伤作用.方法：DR5蛋白免疫BALB/c小鼠,融合筛选抗DR5杂交瘤细胞,制备抗DR5单抗--mDRA-6;流式细胞术测定顺铂对HL-60细胞表面DR5表达及细胞凋亡率;荧光显微镜下观察mDRA-6与顺铂协同作用下HL-60细胞形态变化;MTT法测定不同浓度的顺铂与mDRA-6对HL-60细胞存活的影响;琼脂糖凝胶电泳检测mDRA-6与顺铂联合对HL-60细胞DNA片段化的影响.结果：顺铂可诱导HL-60细胞表面DR5表达增加,mDRA-6与顺铂联用致HL-60细胞出现染色质浓缩、断裂,细胞出芽,凋亡小体形成等细胞凋亡形态学变化;250 ng/ml的mDRA-6作用于HL-60细胞10小时,细胞凋亡率为16.61%;0.16 μg/ml的DDP作用于HL-60细胞10小时,细胞凋亡率为2.35%;二者联合作用后,HL-60细胞凋亡率增至57.10%;mDRA-6与DDP联合作用HL-60细胞,DNA琼脂糖凝胶电泳显示明显“梯形”条带.结论：抗DR5单抗--mDRA-6与DDP对HL-60细胞具有强大的协同杀伤作用.     

同型半胱氨酸对培养的人脐静脉内皮细胞凋亡的影响

目的:研究同型半胱氨酸(Hcy)对培养的人脐静脉内皮细胞(hUVEC)凋亡的影响。方法:原代培养hUVEC,通过Hoechst 33258荧光染色观察细胞核形态改变,琼脂糖凝胶电泳检测 DNA片段,流式细胞术检测Annexin V/PI联合标记的细胞凋亡率, Western blot检测P53、Bax的表达及比色法测定caspase 3活性。 结果: 同型半胱氨酸诱导培养的hUVEC出现明显的凋亡形态学改变。琼脂糖凝胶电泳检测可见明显的"DNA ladder"图谱。Hcy诱导凋亡细胞数明显增加。同时促进Bax、P53的表达,使caspase 3活性显著增强。结论: 同型半胱氨酸诱导培养的hUVEC凋亡。     

人白细胞介素1β通过caspase途径诱导人黑色素瘤A375-S2细胞凋亡

目的:对interleukin-1β(IL-1β)诱导人黑色素瘤A375-S2细胞凋亡的信号转导途径进行研究。方法:使用倒置显微镜观察细胞形态学变化。通过MTT法测定IL-1β对A375-S2细胞的抑制作用以及细胞内半胱氨酸蛋白酶(caspases)与这种作用的关系。利用乳酸脱氢酶(LDH)测定法对IL-1β作用后细胞的损伤情况进行分析。琼脂糖凝胶电泳法检测IL-1β对细胞DNA降解的影响。结果: IL-1β对A375-S2细胞的抑制作用呈剂量和时间依赖性,在10^-9mol/L作用72 h时达到90%以上。caspase-1、-3、-8、-9和caspase-10的抑制剂能够部分抑制IL-1β早期诱导的细胞凋亡。 LDH活力测定显示,在IL-1β诱导的细胞死亡过程中,凋亡占主导地位,并呈现剂量和时间依赖性 。细胞经过10^-11mol/L IL-1β处理72 h后,出现凋亡典型的DNA梯状条带,与上述结果一致。 结论: IL-1β能够诱导人黑色素瘤A375-S2细胞凋亡,这种作用可能依赖于激活一类介导凋亡的caspase家族蛋白酶。     

Outbreak of fatal childhood viral infection in Sarawak, Malaysia in 1997: inocula of patients' clinical specimens induce apoptosis in vitro

Identification of the aetiologic agent(s) associated with an outbreak of fatal childhood viral infection in Sarawak, Malaysia, in mid 1997 remains elusive. It is reported here that African green monkey kidney (Vero) and human monocytic (U937) cells treated with inocula derived from clinical specimens of some of these fatal cases showed the presence of cellular genomic DNA degradation when the extracted DNA was separated by pulsed field gel electrophoresis (PFGE), oligonucleosomal DNA ladders characteristic of apoptotic cells when the infected cells' DNA was separated by agarose gel electrophoresis, and apoptotic cellular DNA fragmentation when cells were stained using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL). These results suggest that inocula derived from the patients' clinical specimens contain factors which stimulate apoptotic cellular responses in vitro.     

Apoptosis as a Mechanism of Lectin-Dependent Monocyte-Mediated Cytotoxicity

In this study we investigated the mechanisms of cytotoxicity mediated by pokeweed mitogen (PWM)-activated human peripheral blood monocytes. By using DNA electrophoresis and propidium iodide (PI)-DNA staining flow cytometry, we demonstrated that apoptotic cell death of target U937 cells and Raji cells was induced in lectin (PWM)-dependent monocyte-mediated cytotoxicity (LDMC). The LDMC-mediated DNA fragmentation in U937 cells and Raji cells was induced in lectin (PWM)-dependent monocyte mediated cytotoxicity(LDMC). The LDMC-mediated DNA fragmentation in U937 cells was completely inhibited by anti-TNFα monoclonal antibody (mAb), but not by the addition of monosaccharide (N-acetylglucosamine, GlcNAc, a sugar specifically recognized by PWM and a lectin-like receptor on monocytes). In contrast, GlcNAc inhibited the DNA fragmentation in Raji cells induced by LDMC which the anti-TNFα mAb had no effect. PWM was found to stimulate the production of nitric oxide (NO) from monocytes. The NO-production was enhanced in the presence of target Raji cells, while the enhancement was abolished by the treatment with GlcNAc. By flow cytometry, we found that PWM bound to tumour cells as well as monocytes, and inhibited the expression of HLA-DR antigen on tumour cells. These results suggest that the presence of lectin molecules on the surface of monocytes and tumour cells may bring the two cells together, thus facilitating the induction of apoptosis in target cells by triggering the production of cytolytic factors (TNF and NO) and the modification of target cell surface antigen (HLA-DR).     

Transglutaminase 2 null macrophages respond to lipopolysaccharide stimulation by elevated proinflammatory cytokine production due to an enhanced αvβ3 integrin-induced Src tyrosine kinase signaling

Transglutaminase 2 (TG2) is a protein crosslinking enzyme with several additional biochemical functions. Loss of TG2 in vivo results in impaired phagocytosis of apoptotic cells and altered proinflammatory cytokine production by macrophages engulfing apoptotic cells leading to autoimmunity. It has been proposed that TG2 acts as an integrin β(3) coreceptor in the engulfment process, while altered proinflammatory cytokine production is related to the lack of latent TGFβ activation by TG2 null macrophages. Here we report that TG2 null macrophages respond to lipopolysaccharide treatment by elevated IL-6 and TNFα production. Though TGFβ has been proposed to act as a feed back regulator of proinflammatory cytokine production in LPS-stimulated macrophages, this phenomenon is not related to the lack of active TGFβ production. Instead, in the absence of TG2 integrin β(3) maintains an elevated basal Src family kinase activity in macrophages, which leads to enhanced phosphorylation and degradation of the IκBα. Low basal levels of IκBα explain the enhanced sensitivity of TG2 null macrophages to signals that regulate NF-κB. Our data suggest that TG2 null macrophages bear a proinflammatory phenotype, which might contribute to the enhanced susceptibility of these mice to develop autoimmunity and atherosclerosis.     

Interleukin-6 regulates the cytotoxic effect of tumour necrosis factor on U937 cells.

In this study, we demonstrate that low but not high concentrations of interleukin-6 (IL-6) potentiate the cytotoxic effect of tumour necrosis factor-alpha (TNF-alpha) on U937 cells, in a dose-dependent manner. Killing of U937 cells by 100 U/ml of TNF-alpha, was maximally potentiated by 50 U/ml of IL-6. No potentiation of cell killing was observed when the concentration of IL-6 was increased to 4000 U/ml. At a concentration of 50 U/ml, IL-6 up-regulated TNF receptor expression but no change in TNF receptor number was observed when the concentration of IL-6 was increased to 4000 U/ml. Low concentrations of IL-6 can also induce sub-cytotoxic doses of TNF-alpha (0.1 and 0.33 U/ml) to kill U937 cells. Up-regulation of TNF receptors by IL-6 is dependent on de novo protein synthesis since receptor induction is abolished in the presence of cycloheximide. Taken together the data suggest that the potentiation of cell killing observed by a combination of these lymphokines is mediated in part by IL-6-induced changes in TNF receptor expression.