流感病毒诱导肿瘤细胞凋亡及其机制的研究

目的：观察流感病毒对肿瘤细胞是否具有凋亡诱导作用,并进一步研究FasL在此过程中的作用．方法：流感病毒以20m．o．i．感染Hela、Raji、SMMC-7721和SPC-A-1等肿瘤细胞,于诱导后不同时间观察细胞超微结构改变,DNA琼脂糖凝胶电泳检测梯状条带,PI染色流式细胞仪检测细胞DNA含量,Annexin-V FITC／PI流式细胞仪进行凋亡定量分析,并用生物素-亲和素ELISA测定流感病毒诱导后细胞培养上清中sFasL浓度的变化。结果：经流感病毒诱导后,四种肿瘤细胞均显示凋亡的形态学改变,细胞DNA电泳出现梯状条带,流式细胞仪测定表明细胞凋亡率增高,且显示一定的时间效应;在上述过程中,细胞培养上清中sFasL浓度有明显增高。结论：流感病毒可诱导上述肿瘤细胞发生凋亡,其发生机制可能与FasL表达增加有关。     

甲型流感病毒H3N2诱导狗肾传代细胞凋亡的研究

目的探讨甲型流感病毒对体外培养的狗肾传代细胞系(MDCK)的凋亡诱导作用.方法用不同血凝单位的甲型流感病毒感染MDCK细胞,经HE染色、琼脂糖凝胶电泳以及流式细胞术等方法检测凋亡情况.结果甲型流感病毒感染MDCK后,在细胞形态上呈现凋亡特有变化;电泳出现典型梯状条带;Annexin-V染色流式细胞仪检测出在一定范围内,细胞凋亡率与病毒的浓度呈正相关.结论甲型流感病毒能够诱导体外培养的MD-CK细胞凋亡,且在一定范围内,细胞凋亡率与病毒的浓度呈正相关.     

减毒麻疹病毒诱导子宫内膜腺癌细胞凋亡及其机理

目的：探讨体外感染减毒麻疹病毒（MV）对子宫内膜腺癌细胞（JEC）的凋亡诱导作用及相关机理。方法：MTT比色法动态监测MV感染对JEC细胞增殖活力的影响;琼脂糖凝胶电泳以及流式细胞术检测细胞凋亡;免疫细胞化学法检测MV感染后JEC细胞的Fas、FasL及TGF-β表达,分析凋亡机制。结果：MV感染6 d内,促进细胞的生长;感染6 d后,抑制细胞的生长,最终造成细胞死亡。流式细胞术结果显示：各感染浓度组JEC细胞的凋亡率比正常对照组增高（P〈0.05或P〈0.01）,随感染时间的延长,细胞的凋亡率呈上升的趋势,感染12 d时,凋亡率与感染浓度呈正相关（r=0.77）,且琼脂糖凝胶电泳出现特征性DNA＂Ladder＂凋亡条带;此时,感染细胞内Fas及TGF-β的表达增强（P〈0.01）,而FasL始终未表达。结论：MV感染能够抑制JEC细胞增值,并诱导其凋亡,JEC细胞凋亡机制可能与Fas、TGF-β的表达有关。     

DETECTION OF B LYMPHOMA CELLS UNDERGOING APOPTOSIS BY ANNEXIN—V ASSAY

Objecte.To quantitatively analyze apoptotic and secondary necrotic cells under apoptosis conditions.Methods.The cells of Burkitt lymphoma (BL) cell line Raji were incubated with 1.0μmol/L dexamethaone(DEX) for 2,4 and 8h respectively,then stained with Annexin V-FITC (fluorescein isothiocyanate conjugated)which was used to detect the exposed phosphatidylserine(PS) on the epimembrane resulting from a loss of phospholipid asymmetry in the early stage of apoptosis ,and also stained with propidium iodide(PI)which allows analysis of secondary necrotic cells related with cell membrane and DNA damage that probably represent late stage of apoptosis,then apoptotic cells were quantified by flow cytometry(FCM).Furthermore,Annexin^ /PI^- and Annexin^ /PI^ cells were sorted by fluoresence-activated cell sorter(FACS),and identified by electron microscopy(EM)and DNA gel electrophoresis.Reuslts.The percentage of apoptotic cells was fund to increase with the incubation time(r=0.97).This method was senitive with low detection limit(0.02%) ,and was reproducible with low coefficient variance (CV)(4.2%).Meanwhile,the Annexin^ /PI^- and Annexin^ /PI^- cells were identified as apoptotic and necrotic cells under EM,and DNA extracted from the Annexin^ /PI^- cell was characteriazed by “ladder pattern“.Conclusions.Annexin-V assay is a specific,sensitive,accurate,reproductive and quantitative method for analyzing apoptotic cells.     

单核细胞增多型李氏忒菌诱导小鼠胸腺细胞凋亡

以流式细胞仪 (FCM)分析 ,DNA琼脂糖凝胶电泳分析及细胞形态学改变为指标 ,研究单核细胞增多型李氏忒菌 (LM)感染对小鼠胸腺细胞凋亡的诱导作用。结果发现 ,LM能诱导小鼠胸腺细胞产生典型的细胞凋亡形态学改变 ;FCM分析显示特征性的凋亡峰 ;琼脂糖凝胶电泳分析显示胸腺细胞出现典型的DNA“梯状带” ;胸腺细胞凋亡于LM(5× 10 5CFU)感染后 8h出现 ,48h达高峰 ;胸腺萎缩于LM感染后 16h出现 ,48h达最低水平 ;胸腺细胞凋亡百分率随LM感染剂量增加而增高。提示 ,LM以时间和剂量依赖方式诱导小鼠胸腺细胞凋亡。     

单核细胞增多型李氏忒菌诱导小鼠胸腺细胞凋亡

The murine thymocyte apoptosis induced by Listeria monocytogenes(LM) was detected with morphology, FCM, and DNA electrophoresis. The results were that LM elicited typical morphological changes of thymocyte apoptosis; the typical apoptosis peak was displayed with FCM, and typical "ladder pattern" with agarose gel electrophoresis. The apoptotic cells were found at 8 h after the mice had infected LM and reached climax at 48 h. The thymus weight significantly reduced at 16 h, and reached the lowest at 48 h after the mice had infected LM. The percentage of apoptotic cells was raised with the increasing of LM. These results suggest that LM induces thymocyte apoptosis in dose- and time-dependent manner.     

沙眼衣原体感染细胞抗凋亡及Bim的表达

目的:研究沙眼衣原体(D血清型)感染的Hela229细胞Bim蛋白质的表达及抵抗凋亡的情况。方法:Western-blot检测沙眼衣原体感染和未感染的Hela229细胞Bim蛋白质的表达水平。凋亡诱导剂etoposide作用Hela229细胞后,琼脂糖凝胶电泳检测DNA Ladder带;流式细胞仪检测凋亡率。结果:Hela229细胞在未感染沙眼衣原体时可检测到Bim的表达;在感染24小时、48小时后均未检测到Bim的表达。经etoposide作用后,未感染的Hela229细胞检测到DNALadder带;流式细胞仪检测的凋亡率为90.64%。感染24小时的Hela229细胞,未检测到DNA Ladder带;凋亡率为11.50%,与未感染的Hela229细胞诱导后的凋亡率比较有统计学意义(P<0.05)。结论:沙眼衣原体感染的Hela229细胞Bim蛋白质的表达下降;并能抵抗etoposide诱导的细胞凋亡。     

Annexin V for flow cytometric detection of phosphatidylserine expression on lymphoma cells undergoing apoptosis]

OBJECTIVE: To quantitatively analyze apoptotic and secondary necrotic cells under apoptosis conditions. METHODS: The cells of Burkitt lymphoma cell line Raji were incubated with 1.0 mumol/L dexamethasone (DEX) for 2, 4 and 8 h, then stained with Annexin V-FITC (fluorescein isothiocyanate conjugated) which was used to detect the exposure of phosphatidylserine (PS) on the out membrane resulting from a loss of phospholipid asymmetry in the early stage of apoptosis, and also stained with propidium iodide (PI) which allows the analysis of secondary necrotic cells related with cell membrane and DNA damage, then apoptotic cells was quantified by flow cytometry (FMC). Furthermore, Annexin+/PI- and Annexin+/PI+ cells were sorted by fluoresence-activated cell sorter (FACS), and identified by electron microscopy (EM) and DNA gel electrophoresis. RESULTS: The results revealed that the percentage of apoptotic cells was increased and correlated well with incubation time (r = 0.97). The sensitivity of this method was shown by its detection limit 0.02%; the method was reproducible, and the coefficient variance (CV) was 4.2%. Meanwhile, the Annexin+/PI- and Annexin+/PI+ cells were identified as apoptotic and necrotic cells under EM, and the DNA extracted from the Annexin+/PI- cells was characterized by "ladder pattern". CONCLUSION: Annexin V assay for analyzing apoptotic cells is specific, sensitive, accurate, reproducible and quantitative for apoptosis investigation.     

Effect of adenovirus-mediated p27 gene expression on the proliferation and apoptosis of HL-60 and Raji cell lines

Background p27 is an essential mediator of cell cycle control,which plays a key negative role in the proliferation and tumorigenesis of certain cell types. Here, we designed this study to explore the possible effects of p27 on the proliferation and apoptosis of HL-60 and Raji cell lines. Methods HL-60 and Raji cells were transfected with p27 via an adenovirus-mediated approach. The efficiency of Adp27 infection and the expression of p27 mRNA and protein were evaluated by X-gal staining, RT-PCR, and flow cytometry. The proliferation and apoptosis of HL-60 and Raji cells were estimated by means of trypan blue staining, M‘l-r assay, Annexin V/PI, and DNA ladderelectrophoresis. Results The infection efficiencies in HL-60 and Raji cells were 40.3% and 32.0%, respectively. RT-PCR and flow cytometry showed that there was significant expression of p27 mRNA and protein in HL-60 and Raji cells infected with Adp27; on the other hand, uninfected HL-60 cells showed faint traces of p27 mRNA and protein and Raji cells showed nearly no signs of p27 mRNA and protein. As demonstrated by a cell growth curve and by an MTT assay, strong time-dependent proliferation inhibition was apparent in HL-60 and Raji cells infected by Adp27. After 72 hours of infection, the Annexin V^ /PI^- apoptotic cell rates in HL-60 and Raji cell lines were 46.9% and 35.7%, respectively, significantly higher than in the control groups (4.7% and 5.6%, respectively). Typical DNA ladder bands were detectable in HL-60 and Raji cells after 48 hours of Adp27 infection. Conclusions Adenoviral vector-mediated p27 gene transfection of HL-60 and Raji cells leads to the inhibition of cellular proliferation and the promotion of cell apoptosis. This technique may provide an approach to gene therapy for leukemia or lymphoma.     

Antineoplastic mechanism of Octreotide actionin human hepatoma

Objectives To investigate whether apoptosis can be induced by Octreotide in human hepatoma cells in vitro and elucidate the antineoplastic mechanism of Octreotide in hepat oma. Methods A cultured human hepatoma cell line, BEL-7402, was exposed to Octreotide and ap optosis was evaluated by cytochemical staining (Hochesst 33 258), transmiss ion electron microscopy, agarose gel electrophoresis and flow cytometry (FCM).Results After exposure to 0.2 μg/ml Octreotide, apoptosis with nuclear chromatin cond ensation as well as fragmentation, cell shrinkage and the formation of apoptotic bodies was observed using cytochemical staining and transmission electron micros copy. A DNA ladder in agarose gel electrophoresis was also displayed. FCM show ed that the apoptotic cell number rose with an increase in the concentration of Octreotide (0-2 μg/ml). There was a positive correlation between Octreotide concentration and apoptotic rate in BEL-7402 cells (r=0.809, P&lt;0.05) .Conclusion Apoptosis in human hepatoma cells can be induced by Octreotide, which may be rel ated to the mechanism of antineoplastic action ofOctreotide in hepatoma.     

抗人DR5单克隆抗体(mDRA-6)对Jurkat细胞作用研究

目的:观察抗肿瘤坏死因子相关凋亡诱导配体(TRAIL)的死亡受体5(DR5)单克隆抗体—mDRA-6对Jurkat细胞的凋亡作用。方法:制备抗人DR5单抗-mDRA-6;检测Jurkat细胞表面DR5表达率;荧光显微镜下观察mDRA-6作用下Jurkat细胞形态变化;MTT法计算mDRA-6对Jurkat细胞存活的影响;FITC-AnnexinⅤ及PI双染流式细胞仪检测mDRA-6对Jurkat细胞凋亡率影响;琼脂糖凝胶电泳检测mDRA-6对Jurkat细胞DNA片段化的作用。结果:Jurkat细胞表面DR5表达率为94.8%;mDRA-6使Jurkat细胞染色质边集、断裂,细胞出芽,凋亡小体形成;MTT法显示mDRA-6具有明显的Jurkat细胞杀伤作用,1.563 mg/L的mDRA-6可使Jurkat细胞死亡60.50%;AnnexinⅤ及PI双染显示0.3mg/L的mDRA-6作用10 h,Jurkat细胞凋亡率达43.22%;10 mg/L mDRA-6作用HL-60细胞3h,DNA琼脂糖凝胶电泳显示明显的“梯形”条带。结论:抗DR5单抗-mDRA-6能够诱导Jurkat细胞凋亡。     

化癥胶囊方剂诱导前列腺癌细胞系PC-3凋亡的实验研究

目的：明确化癥胶囊方剂对前列腺癌细胞PC-3的诱导凋亡作用。方法：PC-3细胞与适宜浓度化癥胶囊方剂共同孵育于1640培养液中,采用一系列细胞凋亡定性、定量检测方法研究化癥胶囊方剂诱导的PC-3细胞凋亡,如行DNA凝胶电泳、流式细胞仪DNA含量分析以检测凋亡。结果：化癥胶囊方剂可诱导PC-3细胞发生凋亡特征性的生化改变,如DNA提取及琼脂糖凝胶电泳分析显示,化癥胶囊方剂可诱导PC-3细胞染色体DNA片段化,形成凋亡特征性的“DNA梯状(DNA ladder)”电泳图谱;流式细胞仪检测可见有低G1期细胞出现。在试验范围内凋亡比率与药物浓度呈一定相关。结论：化癥胶囊方剂可诱导PC-3细胞凋亡,其作用与药物浓度呈一定相关性。     

四种检测细胞凋亡方法的比较

目的：比较４种细胞凋恨检测方法的优缺点。方法：以ｂｕｆａｌｉｎ诱导ＨＬ６０细胞凋亡为例,选择４例方法即电镜（ＥＭ）、ＤＮＡ电泳、原位缺口标记法（ＴＵＮＥＬ）和流式细胞术（ＦＣＭ）检测细胞凋亡,并对凋亡率进行了比较。结果：ＥＭ和ＤＮＡ电泳技术是定性说明凋亡的可靠方法,而ＴＵＮＥＬ和ＦＣＭ擅长于定量检测凋亡细胞,ＥＭ、ＴＵＮＥＬ和ＦＣＭ检测的凋亡率具有显相关性。结论：研究细胞凋亡时宜选用既特异、灵敏     

Growth inhibition and apoptosis induction in human hepatoma cells by tanshinone II A.

In order to study the effect of tanshinone II A on growth and apoptosis in human hepatoma cell line BEL-7402 in vitro, the human hepatoma cell line BEL-7402 was treated with tanshinone II A at various concentrations for 72 h. Growth suppression was evaluated by MTT assay; apoptosis-related alterations in morphology and biochemistry were ascertained under cytochemical staining (Hoechst 33258), transmission electron microscopy (TEM), and DNA agarose gel electrophoresis. Apoptotic rate was quantified by flow cytometry (FCM). The results showed that Tanshinone II A could inhibit the growth of hepatoma cells in a dose-dependent manner, with IC50 value being 6.28 micrograms/ml. After treatment with 1-10 micrograms/ml tanshinone II A for 72 h, BEL-7402 cells apoptosis with nuclear chromatin condensation and fragmentation as well as cell shrinkage and the formation of apoptotic bodies were observed. DNA ladder could be demonstrated on DNA electrophoresis. FCM analysis showed hypodiploid peaks on histogram, and the apoptotic rates at 5 micrograms/ml concentration for 12 h, 24 h, 36 h, 48 h and 72 h were (2.32 +/- 0.16)%, (3.01 +/- 0.35)%, (3.87 +/- 0.43)%, (6.73 +/- 0.58)% and (20.85 +/- 1.74)% respectively, which were all significantly higher than those in the control group (1.07 +/- 0.13)%. It is concluded that Tanshinone II A could induce human hepatoma cell line BEL-7402 apoptosis, which may be related to the mechanism of growth inhibition.     

巯基乙酸-碲化镉量子点对人肝细胞毒性和DNA损伤的诱导作用

目的：研究巯基乙酸（TGA）包覆的碲化镉(CdTe)量子点（QDs）对人正常肝HL-7702细胞的毒性和DNA损伤的作用,为QDs毒理学研究和安全性评价提供实验依据。方法：实验设立对照组和不同浓度CdTe QDs作用组（浓度分别为6.25、12.50、25.00和50.00 mg/L）。CdTe QDs作用于HL-7702细胞后,分别采用MTT法检测细胞增殖变化,单细胞凝胶电泳法（SCGE）检测细胞DNA损伤情况,PI单染流式细胞术（FCM）检测细胞周期变化,AO/EB双荧光染色法和Annexin Ⅴ- FITC/PI双染FCM检测细胞凋亡情况。结果：MTT法,CdTe QDs可抑制HL-7702细胞增殖,并存在剂量-效应和时间-效应关系;SCGE和PI单染FCM法,CdTe QDs作用于HL-7702细胞24 h后,随着剂量的增加,DNA损伤率逐渐增高,G0/G1期细胞百分率显著下降,S期和G2/M期细胞百分率明显上升（P<0.05）;AO/EB检测,CdTe QDs作用后细胞出现凋亡形态学改变;Annexin Ⅴ-FITC/PI双荧光标记FCM法,CdTe QDs染毒可促进细胞凋亡,各剂量组细胞凋亡率与对照组比较差异有统计学意义（P<0.05）。结论：CdTe QDs可影响细胞存活,引起DNA损伤、细胞周期阻滞,并诱导细胞凋亡。     

Growth Inhibition and Apoptosis Induction in Human Hepatoma Cells by Tanshinone Ⅱ_A

Summary:In order to study the effect of tanshinone Ⅱ_A on growth and apoptosis in human hepatomacell line BEL-7402 in vitro,the human hepatoma cell line BEL-7402 was treated with tanshinone Ⅱ_Aat various concentrations for 72 h.Growth suppression was evaluated by MTT assay;apoptosis-relat-ed alterations in morphology and biochemistry were ascertained under cytochemical staining(Hoechst33258),transmission electron microscopy(TEM),and DNA agarose gel electrophoresis.Apoptoticrate was quantified by flow cytometry(FCM).The results showed thst Tanshinone Ⅱ_A could inhibitthe growth of hepatoma cells in a dose-dependent manner,with IC_(50) value being 6.28μg/ml.Aftertreatment with 1—10 μg/ml tanshinone Ⅱ_A for 72 h,BEL-7402 cells apoptosis with nuclear chro-matin condensation and fragmentation as well as cell shrinkage and the formation of apoptotic bodieswere observed.DNA ladder could be demonstrated on DNA electrophoresis.FCM analysis showedhypodiploid peaks on histogram,and the apoptotic rates at 5     

8-溴-7-甲氧基白杨素诱导白血病Jurkat细胞凋亡

目的:研究8-溴-7-甲氧基白杨素(8-bromo-5-hydroxy-7-methoxychrysin,BrMC)诱导人急性T淋巴细胞白血病Jurkat细胞凋亡作用。方法:体外培养Jurkat细胞。FITC标记annexinⅤ/PI染色流式细胞术(FCM)分析细胞凋亡率。ELISA法测定细胞组蛋白/DNA碎片。二氯荧光素二乙酯(2'7'-dichlorofluoresein diacetate,DCFH-DA)荧光探针FCM检测细胞活性氧。结果:BrMC以剂量依赖方式诱导annexinⅤ阳性细胞和组蛋白/DNA碎片增加(P<0.05)。BrMC促进细胞活性氧生成,10 mmol/L N-乙酰半胱氨酸(NAC)预孵育能有效阻断Jurkat细胞活性氧生成,并减弱其诱导细胞凋亡效应。结论:BrMC具有诱导Jurkat细胞凋亡作用,其作用与促进细胞活性氧生成有关。     

鱼藤素对乳腺癌细胞MDA-MB-231线粒体通透性转换孔的作用

目的研究鱼藤素对乳腺癌细胞MDA-MB-231线粒体通透性转换孔的作用。方法MTT法检测鱼藤素对MDA-MB-231细胞的增殖抑制作用;AnnexinⅤ-FITC/PI双染流式细胞术检测细胞凋亡率;罗丹明123单染法观察线粒体膜电位变化;蛋白免疫印迹法检测细胞质中Cytc的表达;分光光度法检测caspase-3蛋白活性;Fluo-3/AM荧光指示剂检测细胞内钙离子浓度变化;RT-PCR和蛋白免疫印迹法检测Bcl-2和Bax mRNA及其蛋白表达。结果鱼藤素对MDA-MB-231细胞增殖具有明显的抑制作用,呈时效和量效依赖关系(P0.05);鱼藤素处理细胞后,细胞线粒体膜电位降低,细胞质内Cytc蛋白表达水平升高,caspase-3活性明显提高,与未处理组相比差异具有统计学意义(P0.01)。流式细胞术检测显示细胞凋亡率和细胞内钙离子浓度随鱼藤素浓度的增加而增多;RT-PCR和蛋白印迹检测结果发现鱼藤素能使Bcl-2 mRNA和蛋白表达减少,而Bax表达增加。结论鱼藤素对乳腺癌MDA-MB-231细胞的增殖抑制、诱导凋亡作用可能与细胞内钙超载,Bcl-2和Bax表达改变影响线粒体通透性转换孔开放,诱导线粒体膜电位降低及Cytc释放有关。     

针对病毒性心肌炎的细胞凋亡研究

目的 探讨柯萨奇病毒致细胞死亡时是否存在凋亡，以及凋亡百分率。方法 柯萨奇病毒感染心肌细胞和HeLa细胞以后，应用TUNEL原位标记、DNALadder、Hoechst3325B染色和Annexin—V／PI染色观察细胞凋亡发生情况；并通过流式细胞仪分析细胞凋亡发生率。结果 Hoechst33258染色发现病毒感染后的细胞核出现强亮度的蓝色荧光，可观察到凋亡细胞的典型变化；Armexin—V／PI染色可见HeLa细胞膜显强绿色荧光。细胞核显强红色荧光；感染组细胞DNA出现阶梯状条带影。通过流式细胞仪检测被PI染色的细胞DNA．发现病毒感染组出现凋亡峰，凋亡率为49．5％。结论 柯萨奇病毒致病毒性心肌炎时细胞不仅存在凋亡，而且是细胞主要死亡形式。     

Arsenic trioxide-induced apoptosis of human malignant lymphoma cell lines and its mechanisms.

OBJECTIVE: To investigate the responses of human Burkitt lymphoma cells to arsenic trioxide (As2O3) and the possible mechanisms. METHODS: Epstein-Barr virus (EBV)-positive human B-lymphoma Raji cell line and EBV-negative human B-lymphoma BJAB cell line were used as in vitro models to assess the cell apoptosis by morphology and DNA agarose gel electrophoresis. Protein expression was analyzed using Western blotting. RESULTS: After 24-hour treatment with the 2, 5 and 10 micromol/L As2O3, the concentrations of As2O3 achievable in vivo, cell apoptosis was induced in human Burkitt lymphoma BJAB cells at the rates of 47.6%+/-4.8% (Mean+/-SD, n=3), 66.4%+/-5.1%, 87.0%+/-7.3% and at 35.5%+/-3.8%, 51.5%+/-6.2%, 62.2%+/-7.9% respectively in Raji cells, corresponding to the concentration of As2O3. EBV-infected Raji cell line was less sensitive to As2O3 than EBV-negative BJAB cell line (P<0.05). As2O3-induced apoptosis was accompanied by down-regulation of Bcl-xL protein expression and activation of apoptosis protein caspase-3, as identified by Western blotting. CONCLUSION: As2O3 exerts apoptosis-inducing effects on human Burkitt lymphoma cells through down-regulation of Bcl-xL protein expression and activation of apoptosis protein caspase-3, and may serve as a candidate therapeutic agent against malignant lymphoma for both systemic and local therapies.