四氢异葎草酮诱导SGC-7901细胞凋亡作用的研究

观察四氢异棒草酮（Tetrahydro-isohumulone,TH）通过线粒体途径诱导SGC-7901细胞凋亡的机制．MTT法检测TH对SGC-7901细胞增殖的影响;荧光显微镜观察TH对SGC-7901细胞凋亡形态的影响;流式细胞仪分析SGC-7901细胞凋亡率;采用试剂盒检测Caspase-9的活性;Western blot法检测Caspase-3蛋白表达情况．2、8、32μg／mL的TH处理SGC-7901细胞72h后,SGC-7901细胞生长受到抑制,抑制率在一定范围内呈剂量、时间相关性;随着TH质量浓度的提高SGC．7901细胞的凋亡率也增加;TH各剂量均能够显著升高SGC．7901细胞内Caspase-9活性,并呈剂量依赖性;同时,TH能够上调SGC-7901细胞内Caspase-3的表达量．并且呈剂量依赖性．TH可通过诱导SGC-7901细胞凋亡发挥抗肿瘤作用,启动线粒体凋亡通路可能是TH诱导SGC-7901细胞凋亡的重要机制之一．     

超声激活血卟啉诱导艾氏腹水瘤细胞凋亡的细胞学研究

采用频率为1.34 MHz、声强为1w/cm2的超声结合血卟啉对EAT细胞凋亡模式进行了研究,探讨了声动力学疗法抗肿瘤的分子机制.超声激活血卟啉作用于EAT细胞后,不同时间点取材,通过扫描电镜观察EAT细胞的形态学变化;通过HO荧光染色观察凋亡细胞的形态学特征;利用免疫细胞化学方法检测caspase-8蛋白的活性变化.结果表明,超声激活血卟啉可诱导EAT细胞凋亡的发生,并且随时间的延迟凋亡现象愈加明显;免疫细胞化学方法表明超声处理后caspaSe-8蛋白表达活性显著增强,且于1h活化程度达到最高.研究表明,超声激活血卟啉可诱导EAT细胞凋亡,其作用的分子机制可能涉及Caspase-8依赖的膜受体凋亡信号调节通路.     

葛根黄酮提取物诱导HL-60细胞凋亡调控研究

用RT-PCR技术和蛋白质免疫印迹技术,检测caspase-8和caspase-3基因转录和蛋白质翻译水平的变化,以探讨葛根黄酮提取物诱导HL-60细胞凋亡的调控途径。结果表明,50μg/mL葛根总黄酮提取物可以上调caspase-8和caspase-3基因转录和蛋白质翻译水平,且其能力分别强于葛根素和大豆甙元单体;说明葛根黄酮提取物诱导HL-60细胞凋亡可能通过经F as死亡受体和/或线粒体途径共同介导执行,葛根黄酮提取物抑制诱导凋亡能力强于葛根素和大豆甙元单体。     

d-柠檬烯诱导白血病细胞凋亡的机制研究

探讨传统中药中挥发油成分d-柠檬烯在HL-60细胞株中的抗肿瘤作用机制。体外细胞培养、凋亡检测、流式细胞术和蛋白免疫印迹等分子生物学技术进行机制研究。d-柠檬烯可以抑制HL-60细胞增值的同时可以诱导HL-60细胞的凋亡。d-柠檬烯可引起细胞内活性氧(ROS)的蓄积、线粒体膜电位(MMP)的下降和Caspase-8活化。抗氧化剂N-乙酰半胱氨酸(NAC)可以阻断d-柠檬烯诱导的细胞内活性氧(ROS)的蓄积,但不能阻断线粒体膜电位(MMP)的下降和Caspase-8活化。d-柠檬烯诱导的HL-60细胞凋亡机制是通过活性氧非依赖的Caspase-8活化来实现的。     

昆布多糖对结肠癌LoVo凋亡中Caspase-8、3、7作用

探讨昆布多糖对人结肠癌LoVo细胞增殖的影响以及凋亡相关的Caspase-8、Caspase-3、Caspase-7活性的变化.分别以不同质量浓度的昆布多糖处理LoVo细胞,采用Hoechst 33258染色在荧光倒置显微镜下观察细胞形态变化;Caspase-8、Caspase-3、Caspase-7活性检测试剂盒检测Caspase-8、Caspase-3、Caspase-7活性.昆布多糖作用LoVo细胞72 h后,随着昆布多糖质量浓度的增高(400,800,1 600μg/mL),凋亡的细胞数也随之增加.昆布多糖作用LoVo细胞24 h后,随着昆布多糖质量浓度的增高(400,800,1 600μg/mL),Caspase-8、Caspase-3、Caspase-7的活性逐渐升高并存在剂量依赖性(P0.05).昆布多糖具有明显的诱导细胞凋亡的作用,且伴随Caspase-8、Caspase-3、Caspase-7活性升高通过Caspase信号传导通路诱导LoVo细胞凋亡.     

HL-60多药抗性细胞抗HT诱导凋亡机制的研究

用1mg·L-1三尖杉酯碱(Harringtoning,HT)处理人早幼粒急性白血病HL-60细胞2h,诱导细胞出现明显的凋亡(Apoptosis,Apo)形态学和生化特征,包括染色质凝集、Caspase-3(天冬氨酸特异的半胱氨酸蛋白水解酶-3)活化、DNA断裂等.来自HL-60的多药抗性细胞HR20和HT9抗HT诱导的凋亡,一般认为是P-糖蛋白(P-GP-170)对药物的外排作用引起细胞对凋亡的抗性.但用2mg·L-1 CsA(Cysclosporine A)逆转抗性细胞对药物的外排作用,1mg·L-1 HT作用48h仍不能诱导HR20和HT9细胞凋亡,说明HR20和HT9多药抗性细胞抗HT诱导凋亡的机制不仅仅在于细胞膜上的P-糖蛋白对药物的外排作用,可能还有凋亡相关因子或其他因素参与作用.Caspase-3是细胞凋亡通路中的一个中心环节,HR20和HT9抗HT诱导的Caspase-3活化,这可能是HR20和HT9不能凋亡的一个重要原因.     

中华补血草多酚诱导白血病细胞HL-60凋亡的研究

采用MTT法研究了中华补血草根多酚提取物对HL-60细胞的抑增殖效果;AO/EB染色、DNA片段化检测和Annexin V-FITC流式细胞术等方法检测了多酚提取物对HL-60细胞诱导凋亡结果.Western blotting检测HL-60细胞株中Bcl-2、Bax蛋白的表达量.结果表明,中华补血草多酚能明显抑制HL-60细胞增殖,诱导HL-60细胞凋亡并具有质量浓度依赖性;Western blotting检测经药物处理的细胞内Bax蛋白显著升高,而Bcl-2蛋白表达水平明显降低,差异具有统计学意义(P<0.05),其凋亡机制可能与下调Bcl-2蛋白、上调Bax蛋白表达量相关.     

骨髓基质细胞HS-5对HL-60细胞凋亡的影响

为研究骨髓基质细胞系HS-5对急性髓系白血病细胞系HL-60凋亡的影响,本实验分两组:HS-5与HL-60(HS-5:HL-60=1×105/m L:1×106/m L)接触共培养组(实验组)和HL-60(1×106/m L)单独培养组(对照组)。采用流式细胞术(FCM)检测HL-60细胞在24、48、72 h的早期凋亡率;瑞氏染色观察HL-60细胞的形态变化;RT-PCR法检测HL-60细胞的抗凋亡基因bcl-2和bcl-xl的表达情况。结果显示:FCM测得在24、72 h实验组与对照组HL-60细胞的凋亡率差异无统计学意义(P0.05),而在48 h时实验组较对照组HL-60细胞的凋亡率显著降低(P0.05),此时瑞氏染色见对照组比实验组细胞变的更不规则,同时有抗凋亡基因bcl-2和bcl-xl的表达上调(P0.05)。由此可知,HS-5与HL-60细胞接触培养能保护HL-60细胞免于快速凋亡,部分原因是通过上调HL-60细胞的抗凋亡基因实现的。     

3-MA 对阿霉素诱导 K562、K562 / ADM 细胞凋亡及其相关基因 mRNA 表达的影响

目的：通过自噬抑制剂3-甲基腺嘌呤（3-MA）对阿霉素（ADM）诱导白血病K562细胞及K562／ADM细胞的细胞效应和自噬基因Beclinl、凋亡抑制基因SurvivinmR．NA表达的变化观察,探讨自噬在细胞凋亡中的作用和机制．方法：体外培养K562和K562／ADM细胞,采用MTT法分别检测ADM及3-MA预处理对K562、K562／ADM细胞增殖的影响,流式细胞仪检测细胞凋亡率,实时RT—PCR法检测细胞自噬及凋亡相关基因（Beclinl、Survivin）mRNA表达的变化．结果：ADM可抑制K562与K562／ADM细胞增殖,且抑制作用呈现浓度与时间依赖性．ADM诱导组K562与K562／ADM细胞在24h、48h、72h细胞凋亡率均较空白对照组明显提高（P〈0．05）．在ADM诱导前,经3-MA预处理可使ADM诱导的K562与K562／ADM细胞抑制率和细胞凋亡率均较单用ADM显著提高（尸〈0．05）,Bedin1、SurvivinmRNA相对表达量均较单用ADM明显下降（P〈0．05）,呈正相关（r=0．827,P〈0．01）．结论：ADM可抑制K562、K562／ADM细胞的生长,并诱导细胞凋亡．3-MA通过抑制细胞的自噬可增强ADM诱导白血病细胞K562、K562／ADM的凋亡,其机制可能与下调BeclinlmRNA表达,而使Survivin表达受抑制有关．     

二烯丙基二硫诱导人白血病细胞系HL-60细胞凋亡的研究

目的研究二烯丙基二硫(diallyl disulfide,DADS)诱导人急性髓性白血病细胞系HL-60细胞凋亡和对细胞周期的影响。方法采用MTT、流式细胞仪等方法检测DADS对HL-60的增殖抑制,诱导凋亡以及细胞周期分布的影响。结果20μΜDADS作用HL-60细胞48h,MTT显示DADS对HL-60细胞有明显的抑制增殖作用;流式细胞仪分析结果显示DADS对HL-60细胞有明显的G2/M期阻滞作用;用丫啶橙染色法观察到不同阶段的凋亡细胞形态学改变,且DADS呈时间依赖性诱导HL-60细胞凋亡。结论DADS引起HL-60细胞G2/M期阻滞并诱导凋亡可能是其抗肿瘤的机制之一。     

Pleiotropic defects in lymphocyte activation caused by caspase-8 mutations lead to human immunodeficiency

Apoptosis is a form of programmed cell death that is controlled by aspartate-specific cysteine proteases called caspases. In the immune system, apoptosis counters the proliferation of lymphocytes to achieve a homeostatic balance, which allows potent responses to pathogens but avoids autoimmunity. The CD95 (Fas, Apo-1) receptor triggers lymphocyte apoptosis by recruiting Fas-associated death domain (FADD), caspase-8 and caspase-10 proteins into a death-inducing signalling complex. Heterozygous mutations in CD95, CD95 ligand or caspase-10 underlie most cases of autoimmune lymphoproliferative syndrome (ALPS), a human disorder that is characterized by defective lymphocyte apoptosis, lymphadenopathy, splenomegaly and autoimmunity. Mutations in caspase-8 have not been described in ALPS, and homozygous caspase-8 deficiency causes embryonic lethality in mice. Here we describe a human kindred with an inherited genetic deficiency of caspase-8. Homozygous individuals manifest defective lymphocyte apoptosis and homeostasis but, unlike individuals affected with ALPS, also have defects in their activation of T lymphocytes, B lymphocytes and natural killer cells, which leads to immunodeficiency. Thus, caspase-8 deficiency in humans is compatible with normal development and shows that caspase-8 has a postnatal role in immune activation of naive lymphocytes.     

RIP3 mediates the embryonic lethality of caspase-8-deficient mice

Apoptosis and necroptosis are complementary pathways controlled by common signalling adaptors, kinases and proteases; among these, caspase-8 (Casp8) is critical for death receptor-induced apoptosis. This caspase has also been implicated in non-apoptotic pathways that regulate Fas-associated via death domain (FADD)-dependent signalling and other less defined biological processes as diverse as innate immune signalling and myeloid or lymphoid differentiation patterns. Casp8 suppresses RIP3-RIP1 (also known as RIPK3-RIPK1) kinase complex-dependent necroptosis that follows death receptor activation as well as a RIP3-dependent, RIP1-independent necrotic pathway that has emerged as a host defence mechanism against murine cytomegalovirus. Disruption of Casp8 expression leads to embryonic lethality in mice between embryonic days 10.5 and 11.5 (ref. 7). Thus, Casp8 may naturally hold alternative RIP3-dependent death pathways in check in addition to promoting apoptosis. We find that RIP3 is responsible for the mid-gestational death of Casp8-deficient embryos. Remarkably, Casp8(-/-)Rip3(-/-) double mutant mice are viable and mature into fertile adults with a full immune complement of myeloid and lymphoid cell types. These mice seem immunocompetent but develop lymphadenopathy by four months of age marked by accumulation of abnormal T cells in the periphery, a phenotype reminiscent of mice with Fas-deficiency (lpr/lpr; also known as Fas). Thus, Casp8 contributes to homeostatic control in the adult immune system; however, RIP3 and Casp8 are together completely dispensable for mammalian development.     

细胞凋亡的基因调控研究进展

细胞凋亡的调控是细胞生理死亡和肿瘤等疾病发生的重要机制。分别对线虫和哺乳类动物细胞凋亡的基因调控研究进展进行了综述 ,阐述了bcl 2基因家族、c myc基因、p5 3基因、白细胞介素Ⅰβ转化酶基因家族、Fas/FasL基因对细胞凋亡的调控作用。     

Functional complementation between FADD and RIP1 in embryos and lymphocytes

FADD is a common adaptor shared by several death receptors for signalling apoptosis through recruitment and activation of caspase 8 (refs 1-3). Death receptors are essential for immune homeostasis, but dispensable during embryogenesis. Surprisingly, Fadd(-/-) mice die in utero and conditional deletion of FADD leads to impaired lymphocyte proliferation. How FADD regulates embryogenesis and lymphocyte responses has been a long-standing enigma. FADD could directly bind to RIP1 (also known as RIPK1), a serine/threonine kinase that mediates both necrosis and NF-κB activation. Here we show that Fadd(-/-) embryos contain raised levels of RIP1 and exhibit massive necrosis. To investigate a potential in vivo functional interaction between RIP1 and FADD, null alleles of RIP1 were crossed into Fadd(-/-) mice. Notably, RIP1 deficiency allowed normal embryogenesis of Fadd(-/-) mice. Conversely, the developmental defect of Rip1(-/-) lymphocytes was partially corrected by FADD deletion. Furthermore, RIP1 deficiency fully restored normal proliferation in Fadd(-/-) T cells but not in Fadd(-/-) B cells. Fadd(-/-)Rip1(-/-) double-knockout T cells are resistant to death induced by Fas or TNF-α and show reduced NF-κB activity. Therefore, our data demonstrate an unexpected cell-type-specific interplay between FADD and RIP1, which is critical for the regulation of apoptosis and necrosis during embryogenesis and lymphocyte function.     

小剂量阿糖胞苷体外诱导HL-60细胞凋亡的作用

阿糖胞苷是白血病的重要药物,其小剂量化疗仍用于临床治疗白血病,并取得了一定的疗效,但其作用机制尚未完全阐明,可能与药物诱导细胞分化或凋亡有关.文中用TUNEL法和流式细胞仪分析小剂量Ara-C(10-8M)对HL-60细胞周期和细胞凋亡的影响,用免疫组化法和原位杂交法分别检测P53蛋白和p53mRNA、bcl-2mRNA表达水平的改变,以探讨bcl-2和p53基因表达与小剂量阿糖胞苷诱导HL-60细胞凋亡的关系.结果表明:小剂量Ara-C能诱导HL-60细胞凋亡,其分子机制可能与P53蛋白、p53mRNA表达增加和bcl-2mRNA表达降低有关.     

丙戊酸诱导多药耐药白血病细胞HL-60／ADR凋亡的研究

目的研究丙戊酸对多药耐药白血病细胞HL-60／ADR的抑制作用及其机制。方法采用MTT比色试验观察丙戊酸对多药耐药细胞HL-60／ADR的抑制作用。采用光镜技术观察细胞形态结构的改变,利用流式细胞术检测细胞凋亡率。结果（1）丙戊酸能明显抑制HL-60／ADR细胞的生长,药物浓度相当于生药32．65mg／mL时,即具有显著的抑制作用,抑制率呈剂量和时间依赖性,半数抑制浓度0c．50）相当于生药66．44mg／mL。（2）形态学改变呈典型的凋亡特征。（3）丙戊酸能诱导多药耐药细胞HL-60／ADR凋亡,凋亡率也呈时间和剂量的依赖性。结论丙戊酸对多药耐药细胞HL-60／ADR的生长具有极强的抑制作用,诱导其凋亡是其机制之一。     

The CED-4-homologous protein FLASH is involved in Fas-mediated activation of caspase-8 during apoptosis

Fas is a cell-surface receptor molecule that relays apoptotic (cell death) signals into cells. When Fas is activated by binding of its ligand, the proteolytic protein caspase-8 is recruited to a signalling complex known as DISC by binding to a Fas-associated adapter protein. A large new protein, FLASH, has now been identified by cloning of its complementary DNA. This protein contains a motif with oligomerizing activity whose sequence is similar to that of the Caenorhabditis elegans protein CED-4, and another domain (DRD domain) that interacts with a death-effector domain in caspase-8 or in the adapter protein. Stimulated Fas binds FLASH, so FLASH is probably a component of the DISC signalling complex. Transient expression of FLASH activates caspase-8, whereas overexpression of a truncated form of FLASH containing only one of its DRD or CED-4-like domains does not allow activation of caspase-8 and Fas-mediated apoptosis to occur. Overexpression of full-length FLASH blocks the anti-apoptotic effect of the adenovirus protein E1B19K. FLASH is therefore necessary for the activation of caspase-8 in Fas-mediated apoptosis.     

一个新的靶点的反义bcl—2寡核苷酸诱导HL—60细胞凋亡的研究

目的：探讨针对bcl-2mRNA蛋白编码区的反义寡核苷酸对HL-60细胞bcl-2蛋白表达和凋亡的作用。方法：应用台盼蓝拒染法和流式细胞仪检测HL-60细胞的活性和bcl-2蛋白表达和细胞凋亡的情况。结果：10μmol/L和20μmol/L的反义寡核苷酸能抑制HL-60细胞bcl-2蛋白表达和诱导细胞调亡,并且针对bcl-2mRNA蛋白编码区的反义寡核苷酸比针对bcl-2mRNA翻译起始区的反义寡核苷酸作用更强。结论：针对bcl-2mRNA蛋白编码区的反义寡核苷酸能抑制HL-60细胞bcl-2蛋白表达和诱导细胞调亡。     

CD95's deadly mission in the immune system

Apoptosis in the immune system is a fundamental process regulating lymphocyte maturation, receptor repertoire selection and homeostasis. Thus, death by apoptosis is as essential for the function of lymphocytes as growth and differentiation. This article focuses on death receptor-associated apoptosis and the role of CD95 (Apo-1/Fas)-mediated signalling in T-cell and B-cell development and during the course of an immune response. Gaining an insight into these processes improves our understanding of the pathogenesis of diseases such as cancer, autoimmunity and AIDS, and opens new approaches to rational treatment strategies.     

nm23-H1基因与人早幼粒白血病细胞HL-60增殖的相关性分析

目的探讨nm23-H1基因的表达与白血病细胞HL-60增殖之间的关系。方法：以25ng／mL阿糖胞苷处理HL-60细胞，MTT法测定细胞生长抑制率，NBT还原比色法判断细胞分化状况，RT-PCR检测nm23-H1基因表达的变化；构建nm23-H1基因的真核表达质粒pEGFP-N1-nm23-H1，转染HL-60细胞，通过细胞生长曲线和血清依赖性实验检测nm23-H1基因的过表达对HL-60细胞生长的影响。结果：小剂量Ara-C对HL-60细胞的生长呈时间依赖性抑制，作用4d后细胞NBT还原能力增强且nm23-H1基因的表达下调；转染nm23-H1基因的HL-60细胞生长加快、血清依赖性下降。结论：Ara-C对HL-60细胞增殖的抑制作用与下调nm23-H1基因的表达有-定关系；nm23-H1基因在HL-60细胞中的过表达有促细胞增殖的作用，即增高了HL-60细胞的恶性程度。