Sp1反义寡核苷酸抑制Jurkat T细胞hTERT表达而抑制端粒酶活性(英文)

目的:研究转录因子Sp1反义寡核苷酸(ODN)对Jurkat T细胞端粒酶活性和端粒酶催化亚基hTERT表达的影响。方法:设计Sp1反义ODN并用脂质体转染细胞,用端粒酶PCR-ELISA方法检测端粒酶活性;用逆转录PCR方法检测Sp1和hTERT mRNA水平,用Western blot检测蛋白水平。结果:sp1反义ODN(1μmol/L)明显抑制Jurkat T细胞Sp1 mRNA和蛋白表达,抑制率分别为44.8％(P<0.05)和57％(P<0.01),并抑制hTERT mRNA表达,抑制率为43.7％(P<0.01);当浓度从0.25到2.0μmol/L,Sp1反义ODN对端粒酶活性抑制率从27.1％到64.6％,呈明显的剂量依赖性关系。结论:Sp1反义寡核苷酸通过抑制hTERT mRNA表达而抑制端粒酶活性。     

Transcriptional changes facilitate mitotic catastrophe in tumour cells that contain functional p53

Exposure of Jurkat T lymphocytes containing functional p53 to nanomolar concentrations of bisanthracycline WP631 resulted in arrest at the G₂/M checkpoint and transient senescence-like phenotype in the presence of DNA synthesis. The cells entered crisis, became polyploid, showed aberrant mitotic figures, and died through mitotic catastrophe. Cell death was accompanied by changes in the expression profile of various oncogenes and tumour suppressor genes including the down-regulation of p53. The changed expression was confirmed for some of these genes using semi-quantitative RT-PCR, and the decline in p53 protein levels was established. Our results suggest that WP631 induced changes in cell cycle control pathways leading to death of Jurkat T cells through mitotic catastrophe, which occurred in the absence of caspase-2 and caspase-3 activities, rather than apoptosis.     

Effect of CD26/dipeptidyl peptidase IV on Jurkat sensitivity to G2/M arrest induced by topoisomerase II inhibitors

CD26/dipeptidyl peptidase IV (DPPIV) is a surface antigen with multiple functions, including a role in T-cell activation and the development of certain human cancers. We previously demonstrated that CD26/DPPIV enhanced sensitivity of Jurkat cells to doxorubicin. We now show that expression of CD26/DPPIV enhanced sensitivity of CD26 Jurkat transfectants to G(2)-M arrest mediated by the antineoplastic agent etoposide. The increased sensitivity to etoposide-induced G(2)-M arrest was associated with disruption of cell cycle-related events, including hyperphosphorylation of p34(cdc2) kinase, change in cdc25C expression and phosphorylation, and alteration in cyclin B1 expression. CD26/DPPIV-associated enhancement of doxorubicin and etoposide-induced G(2)-M arrest was also observed in serum-free media, suggesting an effect of CD26 on cell-derived processes rather than serum-derived factors. Importantly, our work elucidated a potential mechanism for the enhanced susceptibility of CD26-expressing Jurkat cells to the topoisomerase II inhibitors by demonstrating that CD26/DPPIV surface expression was associated with increased topoisomerase II alpha levels and enhanced enzyme activity. Besides being the first to show a functional association between the multifaceted molecule CD26 and the key cellular protein topoisomerase II alpha, our studies provide additional evidence of a potential role for CD26 in the treatment of selected malignancies.     

胆固醇缺乏对Jurkat细胞增殖及凋亡的影响

目的 探讨胆固缺乏时对Ｊｕｒｋａｔ细胞增殖功能及凋亡的影响。方法 用Ｌｏｖａｓｔａｔｉｎ抑制Ｊｕｒｋａｔ细胞内源性胆固醇的合成,以低浓度脂蛋白受体（ＬＤＬ－Ｒ）介导的低密谋脂蛋白（ＬＤＬ）内吞提供细胞外源性胆固醇,细胞处理１～３天后,用流式细胞仪分析各细胞周期相绫分布,并定量分析胆固醇缺乏对处理３天后细胞凋亡的影响。结果 Ｊｕｒｋａｔ经去脂血清培养基加Ｌｏｖａｓｔａｔｉｎ处理３天后,细胞受阻于Ｃｕ     

Activation of Caspases in p53-induced Transactivation-independent Apoptosis

Though p53 -induced apoptosis plays an important role in tumor suppression, the mechanism(s) by which p53 induces apoptosis is stillunclear. To elucidate the p53 -induced apoptotic pathway, we examined the role of p53 transactivation activity and caspase in J138V5C cells carrying a human temperature-sensitive ( ts ) p53 mutant (138Ala→Val). The results showed that p53 -induced apoptosis was not blocked by cycloheximide, which effectively prevented the expression of p53 target genes, indicating that transactivation was not essential for p53 -induced apoptosis in this system. Western blotanalysis showed that PARP, CPP32 and ICH-1 precursors were cleaved during apoptosis. The CPP32-preferential tetrapeptide inhibitor Ac-DEVD-CHO blocked the cleavage of ICH-1 and PARP precursors, suggesting that CPP32 or some other DEVD-sensitive caspase(s) is the upstream activator of ICH-1. We also examined the role of the Fas pathway by using Fas and Fas ligand-neutralizing antibodies. Both antibodies failed to block p53 -induced apoptosis, suggesting that the Fas pathway was not essential for p53 -induced apoptosis in this system. Taken together, our results indicate that p53 -induced, transactivation-independent apoptosis in Jurkat cells involves sequential activation of CPP32 or some other DEVD-sensitive caspase(s) and ICH-1, via a Fas-independent pathway.     

消银解毒方颗粒对Jurkat T细胞内JAK1/STAT3信号通路作用机制的研究

目的探讨消银解毒方颗粒对人急性T淋巴细胞白血病细胞株(Jurkat T)细胞内酪氨酸激酶1(JAK1)/信号转导和转录激活因子3(STAT3)信号通路的调控作用。方法以植物血凝素(PHA)和白细胞介素-6(IL-6)共同诱导Jurkat T淋巴细胞建立血热证银屑病T细胞异常活化病理细胞模型,将模型细胞接种于24孔板,制备消银解毒方颗粒、凉血方颗粒(消银解毒方颗粒拆方)、解毒方颗粒(消银解毒方颗粒拆方)、阿维A(阳性对照)、蒸馏水(阴性对照)的药理血清,择取最佳浓度药理血清及JAK/STAT信号通路阻断剂Filgotinb(GLPG0634)作用于模型细胞48 h,同时设空白细胞组。收集各组细胞,采用蛋白质印迹(Western blot)、实时定量聚合酶链式反应(real-time PCR)法检测JAK1、STAT3、糖蛋白(GP)130的蛋白表达与基因转录水平。结果模型组的JAK1、STAT3、GP130的蛋白和mRNA表达较空白组明显增高(P0.01)。与模型组相比,各实验组磷酸化酪氨酸溶酶(p JAK)1/JAK1、磷酸化信号转号和转录激活因子(p STAT)3/STAT3、GP130蛋白表达量均明显降低(P0.01);与模型组相比,各实验组JAK1、STAT3、GP130的mRNA表达量均降低(P0.05或P0.01)。结论消银解毒方颗粒能够通过抑制JAK1/STAT3信号通路的开放进而抑制T细胞异常活化,以发挥其治疗银屑病的作用。     

去甲斑蟊素诱导T淋巴细胞白血病Jurkat细胞凋亡作用及其机制

目的研究去甲斑蝥素(norcantharidin,NCTD)在体外对急性T淋巴细胞白血病细胞株Jurkat细胞凋亡的作用及其机制。方法取对数生长期的Jurkat细胞分别加入不同浓度的NCTD和(或)10 mg/L的长春新碱(VCR),倒置显微镜下观察不同时间Jurkat细胞形态、密度及结构的变化;采用MTT比色法测定NCTD及VCR两种药物作用于细胞72 h后对细胞增殖抑制作用的影响;实时荧光定量RT-PCR法对p53及survivin基因表达量进行检测。结果 NCTD及VCR作用组细胞形态出现不规则,胀大、变形现象明显,分布稀疏,大量细胞死亡,两者在相同时间点上对细胞的影响无明显差别。MTT法检测结果表明:NCTD增殖抑制作用呈现时间-浓度依赖关系,最佳浓度及时间在20 mg/L,作用72 h时。NCTD与VCR两者对Jurkat细胞均有抑制作用(P0.05),两者的抑制率相比无显著性差异(P0.05),且两者联合可增加细胞增殖抑制率(P0.05)。应用实时荧光定量RT-PCR技术定量表达p53及survivin基因,结果显示NCTD组p53基因表达较对照组细胞株明显上升(P0.001),相反survivin基因较对照组明显下降(P0.01)。结论(1)NCTD抑制Jurkat细胞株的增殖抑制作用具有时间-浓度依赖关系;(2)NCTD与VCR对细胞增殖均有抑制作用,两者的影响无显著性差异,且两者联合可增强对细胞的增殖抑制作用;(3)NCTD促进Jurkat细胞株细胞凋亡、抑制增殖的机制可能与上调p53基因表达量及下调survivin基因表达量有关。     

大蒜素对 Jurkat 细胞 CXCR4表达的影响

目的：探讨大蒜素对体外培养的 Jurkat 细胞中 CXCR4启动子活性的影响。方法：克隆人 CXCR4启动子序列,构建报告载体 PgL4.17- CXCR4,转染 Jurkat 细胞,G418筛选分组后,大蒜处理分高、中、低浓度3组（40μg/ ml、20μg/ ml、10μg/ ml）,以未做任何处理的转染细胞作为对照组,以注射用水处理的转染细胞作为注射水对照组,各组处理24h 后,在相应的条件下进行荧光素酶活性测定。结果：成功构建了含人 CXCR4启动子序列的 PgL4.17- CXCR4报告载体,6组荧光素酶活性比较,差异有统计学意义（P <0.001）,其中大蒜素处理组荧光素酶活性均低于对照组（P <0.001）。结论：大蒜素能降低 CXCR4启动子的表达,有效抑制体外培养的 Jurkat 细胞中的 CXCR4启动子活性。     

hTERT基因反义核酸对Jurkat细胞端粒酶活性影响

目的:检测hTERT基因反义核酸对Jurkat细胞端粒酶活性的影响及其机制。方法:TRAP法检测端粒酶活性,流式细胞仪检测hTERT蛋白表达,逆转录-多聚酶链式反应检测hTERTmRNA表达。结果:检测端粒酶活性,Jurkat细胞吸光度A值为0.492±0.051,hTERT反义核酸作用48hA值降为0.351±0.051,hTERT反义核酸作用72hA值降为0.238±0.024;检测hTERT蛋白表达,Jurkat细胞hTERT蛋白阳性率89.513%±3.389%,hTERT反义核酸作用48hhTERT蛋白阳性率低至77.237%±2.872%,hTERT反义核酸作用72hhTERT蛋白阳性率低至47.767%±1.326%。而hTERT正义核酸无上述作用。hTERT反义核酸作用Jurkat细胞48h、72h对hTERTmRNA表达无影响。结论:hTERT反义核酸降低Jurkat细胞端粒酶活性,机制可能是降低hTERT蛋白表达量,对hTERTmRNA无影响。