环氧合酶2基因沉默诱导人肝癌细胞凋亡的作用观察

目的 观察作用于环氧合酶2(COX-2)mRNA的发卡状COX-2(COX-2 shRNA)真核表达质粒对人肝癌HepG2细胞增殖和凋亡的影响.方法 设计靶向COX-2基因的RNA干扰序列,构建COX-2 shRNA表达载体,用阳离子脂质体Lipofectamine 2000转染体外培养的HepG2细胞.半定量RT-PCR检测COX-2 mRNA表达水平的变化,筛选有效抑制COX-2基因表达的序列.采用CCK-8细胞计数法检测COX-2 shRNA对细胞增殖的影响,流式细胞仪测定细胞凋亡率和细胞周期,Western blotting检测细胞内COX-2和Bcl-2蛋白表达水平.结果 测序结果显示成功构建了2个表达COX-2shRNA的质粒载体,转染HepG2细胞后COX-2 mRNA表达下降至阴性对照组的41.66％和77.79％.选择沉默效率较佳者转染细胞并经G418筛选,获得稳定表达COX-2 shRNA的细胞,胞内COX-2 mRNA表达水平下降了75.30％,细胞增殖活性下降了 29.33％.在稳定表达COX-2 shRNA、错义序列以及未转染的细胞中,细胞凋亡率分别为17.83％、4.63％和4.47％,G0/G1期细胞比例分别为73.93％、61.2％和57.630％,而S期细胞比例分别为13.43％、26.03％和26.90％.Westernblotting检测显示,表达COX-2 shRNA的HepG2细胞内COX-2蛋白水平下降至表达错义序列组的31.25％(P＜0.01),Bcl-2蛋白水平下降至表达错义序列组的54.93％(P＜0.01),而转染错义质粒组与未转染组之间比较差异无统计学意义.结论 构建的真核表达载体pSilencer 2.1-U6-COX-2 shRNA能有效沉默人肝癌HepG2细胞内COX-2基因的表达,并具有抑制细胞增殖、促进细胞凋亡和细胞周期阻滞、降低胞内抗凋亡蛋白(H)Bcl-2水平的作用.     

钩藤酸E通过线粒体途径诱导HepG2细胞凋亡

目的研究钩藤酸E诱导人肝癌HepG2细胞凋亡的机制。方法采用MTT法分析确定钩藤酸E对肿瘤细胞及对小鼠骨髓体外增殖抑制作用,显微镜观察分析细胞凋亡的形态学变化,乳酸脱氢酶测定分析细胞死亡机制,免疫印迹实验检测线粒体相关蛋白的表达。结果钩藤酸E可明显地抑制HepG2细胞生长,且呈明显的量效关系和时效关系,而对小鼠骨髓细胞无抑制作用。Hoechst33258荧光染色可见凋亡小体产生,乳酸脱氢酶分析证明钩藤酸E引起细胞死亡为凋亡和坏死同时进行。免疫印迹结果显示上调的Bax和下调的Bcl-2和Bcl-xL表达证实了钩藤酸E诱导的HepG2细胞凋亡是通过线粒体途径发挥作用,并导致细胞色素C的释放。结论钩藤酸E可明显地诱导HepG2细胞凋亡,并经历了线粒体信号转导途径。     

三氧化二砷对人肝癌细胞凋亡及Bcl-2、caspase-3蛋白表达的影响

目的研究三氧化二砷（arsenic trioxide,As2O3）诱导HepG2细胞凋亡及其机制。方法采用MTT法、形态学观察及流式细胞术方法检测As2O3对HepG2细胞的生长抑制、凋亡作用的影响,用Western blot检测As2O3处理后HepG2细胞内凋亡相关蛋白Bcl-2和caspase-3的表达。结果As2O3对HepG2细胞有明显的抑制增殖作用,且呈浓度和时间依赖性,细胞呈典型的凋亡形态学改变,As2O3呈时间依赖性诱导HepG2细胞凋亡。As2O3处理不同时间后,Bcl-2蛋白表达水平下调,caspase-3蛋白表达水平升高。结论As2O3具有诱导人肝癌HepG2细胞凋亡的作用,其机制可能与其下调Bcl-2蛋白的表达,促进caspase-3活化有关。     

JTV1对K562细胞增殖和凋亡的影响及其机制

目的观察上调JTV1基因的表达对人白血病K562细胞系增殖与凋亡的影响并探讨其机制。方法将携带有JTV1基因的pcDNA3.1-JTV1载体转染人K562细胞作为阳性转染组,转染pcDNA3.1空载体作为空载体转染组,未转染的K562细胞为未转染组。通过集落形成实验检测各组K562细胞的增殖能力,采用流式细胞术与Annexin-PI双染色结合分析细胞周期和细胞凋亡率,同时采用RT-PCR法检测JTV1基因及凋亡相关基因Bcl-2、Bax、C-myc mRNA水平的变化,采用Western blotting检测基因JTV1及凋亡相关基因Bcl-2、Bax、C-myc蛋白水平的变化。结果集落形成实验结果显示上调JTV1基因表达后K562细胞的增殖能力明显降低。流式细胞术与Annexin-PI双染色检测结果显示,与空载体转染组及未转染组相比,阳性转染组K562细胞的G期细胞比例明显升高(P<0.05);阳性转染组中Bax基因的mRNA水平和蛋白水平较空载体转染组及未转染组均明显上升,而Bcl-2基因和C-myc基因的mRNA水平和蛋白水平则明显下调(P<0.05)。结论 JTV1基因过表达可能通过抑制基因Bcl-2和C-myc的表达增强基因Bax的表达,从而抑制K562细胞系的增殖并促进其凋亡。     

hTERT-siRNA表达载体的构建及对MCF-7细胞生长、端粒酶活性的抑制作用

目的 构建人端粒酶逆转录酶(hTERT)基因的RNA干扰(RNAi)表达载体,并研究该载体对乳腺癌MCF-7细胞端粒酶活性及细胞增殖的影响,为针对端粒酶的乳腺癌基因治疗提供新的途径.方法 设计针对人端粒酶逆转录酶催化亚基(hTERT)的干扰靶序列TGTTCAGCGTGCTCAACTA,构建重组siRNA表达质粒pGenesil-hTERT,同时构建不针对任何基因的阴性对照重组pGenesiL-HK.两种重组质粒经酶切、电泳分析和测序鉴定后,用脂质体转染法分别转染乳腺癌MCF-7细胞,应用端粒酶重复序列扩增聚合酶链反应(TRAP-PCR)及聚丙烯酰胺凝胶电泳检测端粒酶活性,流式细胞仪测定细胞凋亡率.结果 酶切电泳测序分析表明插入序列正确,重组质粒构建成功.转染pGenesil-hTERT的MCF-7细胞,凝胶电泳见端粒酶特征性条带明显减少,端粒酶活性受到明显抑制pGenesil-hTERT转染细胞后凋亡率较对照组明显升高(P＜0.01),且转染48h凋亡率最高,达54.7%±2.41%.结论 hTERT-siRNA可有效抑制乳腺癌MCF-7细胞端粒酶活性、促进细胞凋亡,此法有望应用于肿瘤基因治疗.     

靶向p38MAPK的siRNA通过干扰线粒体途径弱化光激发酞菁锌诱导Lovo细胞的凋亡

目的探讨RNAi沉默p38MAPK基因表达对光激发TαPc Zn诱导的人结肠癌Lovo细胞线粒体凋亡途径干扰的机制。方法运用siRNA靶向干扰p38MAPK基因,采用RT-PCR和Western blot法分别检测细胞内p38MAPK的干扰效率,同时用Western blot法检测AIF、Bcl-2、Cyto-c及Caspase-3的表达,JC-I染色检测沉默p38MAPK基因对ΔΨm的影响。结果与对照组比较,光作用下siRNA-p38MAPK转染组中p38MAPK的mRNA和蛋白水平显著下调(P0.01)。在光激发下,TαPc Zn明显诱导Lovo凋亡,ΔΨm降低,从线粒体释放到细胞质的AIF、Cyto-c增加,Bcl-2表达减少,同时caspase-3发生断裂激活。而在siRNA沉默p38MAPK后,光激发TαPc Zn诱导细胞凋亡的作用减弱,ΔΨm有所增加,细胞质中AIF、Cyto-c释放减少,caspase-3激活减弱,Bcl-2表达增加。结论光激发TαPc Zn作用下,沉默p38MAPK基因可明显干扰线粒体途径,进而减弱光激发TαPc Zn所诱导的细胞凋亡。     

靶向HBV C基因的siRNA在HepG2.2.15细胞中对HBV的抑制作用

目的研究靶向HBV C基因的短发夹状RNA(shRNA)表达质粒对HepG2.2.15细胞中HBV的抑制作用。方法设计构建两个靶向HBV C基因的shRNA表达质粒S1和S2,以及用于对照的非同源shRNA表达质粒S3。转染HepG2.2.15细胞后,通过RT-PCR和ELISA方法分别检测细胞中HBV C基因表达和细胞上清中HBV DNA、HBsAg和HBeAg表达。结果在转染后24 h时,HBV C基因mRNA表达量下降58%～83%,HBV DNA下降了2.44～3.36个log值,HBsAg和HBeAg的表达水平分别降低了51%～79%和50%～77%。S1、S2联合运用可以提高对HBV的抑制作用,而S3无抑制效果。结论靶向HBV C基因的shRNA表达质粒S1、S2及其联合运用,均能有效地抑制HepG2.2.15细胞中HBV的复制和表达。     

丁酸钠对肝癌Hep G_2细胞株Bcl-2表达的影响

目的：研究丁酸钠（NaB）引起HepG2细胞凋亡的作用及其分子机制。方法：用不同浓度丁酸钠处理HepG2细胞后，用逆转录-聚合酶链反应（RT—PCR）检测Bcl-2mRNA的表达丰度；Hochest3342核染色后观察HepG2细胞核凋亡形态的变化；原位切口末端标记法（TUNEL）检测细胞凋亡。结果：丁酸钠在mRNA水平明显抑制Bcl-2基因的转录；荧光显微镜下可见丁酸钠处理后细胞核出现高度凝聚、边缘化，并可见凋亡小体出现；TUNEL法证明出现细胞凋亡。结论：丁酸钠通过下调Bcl-2的转录和表达，诱导肝癌HepG2细胞的凋亡。     

siRNA靶向抑制PKR基因及其对宫颈癌Hela细胞凋亡的影响

目的研究siRNA（small interfering RNA,siRNA）对宫颈癌Hela细胞中双链RNA（dsRNA）激活的蛋白激酶（protein kinase regulated by dsRNA,PKR）基因的抑制作用,观察其对dsRNA诱导的细胞凋亡的影响。方法采用RNAi技术,对宫颈癌Hela细胞PKR基因进行特异性抑制;用阳离子脂质体与化学合成的Pre-designed anti-PKR siRNA构建转染复合体,反转染Hela细胞72h后提取总蛋白,用Western blot检测PKR蛋白表达水平的变化,用流式细胞仪检测细胞的凋亡率,激酶活性检测法测定半胱氨蛋白水解酶-3（caspase-3）活性变化。结果 siRNA能降低PKR基因的蛋白表达;未转染组的Hela细胞在加入dsRNA后,细胞凋亡明显增加,caspase-3激酶活性增强（P〈0.05）。与对照组相比,转染组的Hela细胞在加入dsRNA后,细胞凋亡显著减少,caspase-3激酶活性降低（P〈0.05）。结论 siRNA可特异有效地干扰宫颈癌Hela细胞内PKR基因的表达,从而影响肿瘤细胞凋亡。dsRNA可通过激活PKR,增强caspase-3激酶活性,而促进Hela细胞的凋亡。     

Spred2基因对人肝癌细胞凋亡韵影响

目的观察Spred2对人肝癌细胞（HepG2）凋亡的影响。方法用阳离子脂质体瞬时转染pcDNA3．0,pcDNA3．0-Spred2到HepG2细胞,建立过表达Spred2的细胞模型。膜联蛋白（annexlFl）V．FITC／PI（7．AAD）双标通过流式细胞仪检测细胞凋亡;蛋白质印迹检测SprcdZ的表达水平。结果特柒Spred2基因能明显诱导HepG2细胞凋亡。另外,转染Spred2基因能显著地增强5一FU诱导HepG2细胞凋亡。结论Spred2对人肝癌细胞凋亡有调控作用。     

Mitochondrial and intracellular free-calcium regulation of radiation-induced apoptosis in human leukemic cells.

PURPOSE: To investigate the mechanisms and pathways of X-ray apoptosis in Molt-4 cells, focusing on mitochondrial and cytosolic Ca2+ ([Ca2+]i) regulation. MATERIALS AND METHODS: X-irradiated Molt-4 cells and cell extract (CE) were used to analyse: (1) induced apoptosis (Giemsa stain), (2) p53, Bcl-2 and Bax expressions (immunoblot), (3) mitochondrial potential deltapsi(m) and (4) [Ca2+]i (flow cytometry), (5) caspase-3 activity, and (6) roles of [Ca2+]- and caspase-3-mediated pathways by inhibiting either or both pathways for induced apoptosis. RESULTS: Molt-4 cells were sensitive to apoptosis since 5 Gy induced 57 and 94% apoptosis at 6 and 24 h. After 5Gy, p53 was accumulated that upregulated Bax but which repressed Bcl-2 with time, resulting in a 7-fold increase in Bax/Bxl-2 at 6 h. Predominant Bax reduced deltapsi(m), and low-deltapsi(m) cells increased 45 min earlier than apoptosis after 5 Gy. Caspase-3 was activated in apoptotic CE. The caspase-3 inhibitor Ac-DEVD-CHO inhibited apoptosis and DNA-ladder formation by approximately 50%, suggesting a approximately 50% role of caspase-3-activated DNase (CAD). [Ca2+]i was increased after 5 Gy. [Ca2+]i-chelating BAPTA-AM (5 microM) and/or DNase gamma-inhibiting Zn2+ (0.5 mM) inhibited approximately 50% of induced apoptosis and DNA-laddering, indicating a 50% participation of Ca2+/Mg2+-dependent DNase gamma. CONCLUSIONS: The p53-Bax-mitochondria-caspase-3-CAD pathway and the [Ca+2]i-mediated DNase gamma pathway were involved in the regulation of X-ray apoptosis in sensitive Molt-4 cells.     

去甲斑蝥素和紫杉醇联合诱导白血病细胞K562凋亡

目的探讨去甲斑蝥素(norcantharidin,NCTD)和紫杉醇(paclitaxel,PTX)联合诱导白血病细胞K562凋亡的效应及机制。方法采用流式细胞术分析细胞凋亡和线粒体膜电位;采用免疫印迹技术检测细胞内凋亡相关信号分子Bim、Bax和Bcl-2表达。结果 7.5μmol/L NCTD单独作用可诱导(2.6±0.7)%白血病细胞K562发生凋亡;0、2.5、5和10nmol/L的PTX诱导细胞凋亡的百分率分别为(0.8±0.3)%(、4.7±1.9)%(、7.2±2.6)%和(9.4±2.9)%;而NCTD(7.5μmol/L)与PTX联合作用(2.55、和10 nmol/L)后,细胞凋亡率分别增高至(8.5±2.2)%(、19.3±3.1)%和(23.4±4.1)%。药物联合后Bim和Bax表达增强,而Bcl-2表达减少。结论 NCTD通过线粒体途径凋亡信号的转导,增强PTX诱导K562细胞凋亡的效应。     

hTERT启动子调控caspase 3基因的特异性表达及其抑癌作用

目的：探讨端粒酶催化亚基(KFERT)启动子调控caspase3(半胱天冬酶-3)基因在端粒酶阳性肿瘤细胞中表达的特异性及在体内外的抑癌作用。方法：构建hTERT启动子调控的caspase3基因的真核表达载体，通过RTPCR、瞬时转染、MTT法、流式细胞术和动物实验方法，检测hTERT启动子调控caspase3基因表达对肿瘤细胞生长的影响。结果：hTERT启动子调控caspase3基因在端粒酶阳性肿瘤细胞HepG2中有明显表达，而在正常人胚肺成纤维细胞(human embryonic lung fibroblast，HEL)中未见表达；caspase3表达能对端粒酶阳性肿瘤细胞HepG2、HeLa、Glc和A549细胞的增殖产生明显抑制作用，抑制率为10．5％-37．9％；同时也可以诱导HepG2、HeLa、Glc和A549细胞发生凋亡，凋亡率为15．39％-35．19％；而对端粒酶阴性的正常细胞HEL没有明显的影响。动物实验结果显示，hTERT启动子调控的caspase3基因转染对HepC2细胞的体内增殖具有抑制作用。结论：hTERT启动子调控的caspase3基因表达载体是一种有应用潜力的肿瘤基因治疗载体。     

重离子辐射对人外周血T淋巴细胞生物学性能的影响

目的研究重离子对人外周血T淋巴细胞增殖、凋亡等生物学性能的影响并探讨其机制,为肿瘤放射治疗的辐射防护提供实验依据和基础。方法 Ficoll分离法分离人外周血T淋巴细胞,采用12C重离子束坪区照射,照射样品能量为70 MeV、LET=29 keV/μm,照射剂量为1.0和2.0 Gy,剂量率为0.5 Gy/min。分别于照射后12、24h,RT-PCR检测凋亡相关基因Bcl-2,Bax,Caspase3,Caspase8和Caspase9的表达;于照射后24、48 h,CCK8法检测细胞增殖能力;于照射后24、48 h,采用AnnexinV-PE/7-AAD、AnnexinV-FITC/PI法检测凋亡发生,并采用RT-PCR检测凋亡相关蛋白Bcl-2、Bax和Caspase3的表达。结果重离子照射可明显抑制人外周血T淋巴细胞的增殖,随着剂量增大,抑制作用更加明显。同时,重离子照射可促进T淋巴细胞的凋亡,特别是对于晚期凋亡的诱导作用（P〈0.01）。RT-PCR检测结果显示,重离子辐射可抑制抗凋亡蛋白Bcl-2的表达,促进促凋亡蛋白Bax和Caspase3的表达（P〈0.01）。结论重离子辐射可显著影响抑制T淋巴细胞的增殖并促进其凋亡。     

Gamma irradiation of human leukaemic cells HL-60 and MOLT-4 induces decrease in Mcl-1 and Bid, release of cytochrome c, and activation of caspase-8 and caspase-9

PURPOSE: Apoptosis is significantly controlled by proteins of Bcl-2 (B-cell lymphoma 2) family promoting cell death or maintaining cell survival. We selected two representatives of Bcl-2 family (anti-apoptotic Mcl-1 - myeloid cell line-1 and pro-apoptotic Bid - Bcl-2 homology domain 3 interacting death agonist), cytochrome c (cyt-c), and two initial caspases (-8 and -9) to evaluate their function in ionizing radiation (IR)-induced apoptosis in human leukaemic cell lines diverging in p53 (TP53 tumor suppressor gene) status. MATERIALS AND METHODS: A total of 30 microg of proteins of whole-cell lysates or 10 microg of mitochondrial protein fractions were electrophoretically separated and analyzed by Western-blotting. RESULTS: Here we show that in both HL-60 (p53 null) and MOLT-4 (p53 wild type) leukaemic cells the amount of Mcl-1 initially increased after irradiation by sublethal but not by lethal dose and later (when apoptosis occurred) it decreased in a dose-dependent manner. Caspase-8 was cleaved and afterwards the amount of Bid decreased as it was truncated. We also found cyt-c release from the inner mitochondrial membrane space into cytoplasm to be dose-dependent and it was followed by induction of apoptosis. In the p53-null cells caspase-8 was activated prior caspase-9, whereas the cells harboring p53 exhibited a simultaneous activation of both initial caspases. CONCLUSION: IR induced a decrease in Mcl-1, activation of Bid, caspase-8, and -9, and release of cyt-c. Presented data indicate that both extrinsic and intrinsic apoptosis signalling pathways were activated in HL-60 and MOLT-4 cells upon exposure to IR regardless to the p53 status.     

Radioresistant Sf9 insect cells undergo an atypical form of Bax-dependent apoptosis at very high doses of γ-radiation

Abstract

Purpose: To investigate the underlying mechanisms of cell-death at extremely high doses of radiation in radioresistant Spodoptera frugiperda-9 (Sf9) insect cells.

Materials and methods: Morphology, cell proliferation and DNA-fragmentation analysis was performed at 500–2000 Gy. Changes in intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), cardiolipin oxidation and Annexin-V externalization were studied using flow-cytometry. Cytochrome-c release was measured using immunofluorescence microscopy. Inhibitors of apoptosis, i.e., Bongkrekic acid (BKA), Caspase-9 inhibitor (C9i), 5-(4-fluorosulfonylbenzoyl) adenosine hydrochloride (FSBA) and Cyclosporin-A (CsA) were used to dissect apoptotic mechanism at many classical steps. Caspase-3 activity was measured using a caspase-activity assay kit.

Results: A dose-dependent induction of typical apoptosis was observed at extremely high doses, marked by extensive apoptotic body formation. However, certain atypical responses such as cellular hypertrophy and the lack of phosphatidylserine-externalization were observed during the initial hours after radiation. Loss of mitochondrial membrane potential observed at 48 h following a 2000 Gy dose was accompanied by an increase in ROS that caused significant cardiolipin oxidation leading to cytochrome-c release, caspase activation and internucleosomal DNA fragmentation. Inhibitors of B-cell lymphoma-2 (Bcl-2)-associated X protein (Bax)-mediated cytochrome-c release, apoptosome formation and caspase-9 effectively prevented radiation-induced apoptosis, strongly suggesting the role of Bax-dependent cell death mechanism.

Conclusions: Our study demonstrates that the Sf9 insect cells display good homology with human cells in the mitochondria-dependent events during radiation-induced apoptosis, although doses eliciting similar responses were 50–200 times higher than human cells. Factors upstream to mitochondrial damage remain pertinent for a thorough understanding of this extreme radioresistance displayed by lepidopteran cells.     

RNA干扰p21基因对X射线诱导细胞周期解耦联的影响

目的 研究RNA干扰p21基因对X射线诱导细胞周期解耦联的影响。方法构建RNA 干扰质粒pSilencer3.1-H1 neo-p21,并采用脂质体转染将质粒转入EL-4细胞。 采用单克隆抗体免疫荧光标记及流式细胞术 (FCM) 检测蛋白表达的变化。采用碘化丙啶(PI)荧光标记及FCM检测多倍体细胞数的变化。结果量效实验证实, 1.0~4.0 Gy X射线照射后24 h,EL-4细胞中P21蛋白表达与对照组相比较显著增高。 时效实验证实, 4.0 Gy X射线照射后8~72 h, EL-4细胞中P21蛋白表达与对照组相比较显著增高。 实验证实, 0.5~6.0 Gy X射线照射后,EL-4细胞的八倍体细胞数未见明显变化,X射线照射不诱导EL-4细胞周期解耦联。RNA干扰实验证实, p21基因沉默后, P21蛋白表达于 4.0 Gy照射后24 及48 h, 干扰质粒组与空质粒对照组相比较显著降低;与此同时,干扰质粒组的八倍体细胞数与空质粒组相比较显著增高。结论RNA干扰p21基因使P21蛋白表达抑制后,X射线照射可诱导EL-4细胞周期解耦联。提示,P21蛋白可能在电离辐射诱导细胞周期解耦联中起重要作用。     

过表达的p27kip1对肝癌细胞的抑制作用

目的探讨过表达的p27kip1对肝癌细胞(HHCC)的抑制作用.方法利用脂质体介导法将真核表达载体pcDNA3.1/Myc-His(+)C-p27kip1基因导入HHCC中,用Western blot检测p27kip1是否导入到HHCC中;用平板克隆形成实验、软琼脂克隆形成实验检测过表达的p27kip1对HHCC的抑制作用;应用流式细胞仪、电子显微镜探讨过表达的p27kip1抑制HHCC生长的可能机制.结果Western blot检测表明,转染p27kip1的HHCC 100%表达p27kip1蛋白.过表达的p27kip1对HHCC有明显的抑制作用,抑制率为49.09%～56.00%;过表达的p27kip1可以封闭G1期的进程和诱导HHCC发生凋亡,凋亡百分比为12.3%.结论过表达的p27kip1可以通过诱导凋亡抑制肝癌细胞生长.