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1.
大鼠肝卵圆细胞增殖模型的建立与优化 总被引:11,自引:2,他引:11
目的 建立大鼠肝卵圆细胞增殖模型,并观察2-乙酰氨基芴(2-acetaminofluorene,AAF)剂量与模型动物肝卵圆细胞增殖程度间的关系。方法 选择体重150g左右的雄性Wistar大鼠,通过胃管灌喂AAF,每日1次,连续4d,第5日行三分之二肝切除(手术当日不灌喂AAF),第6日继续灌喂,连续1周。AAF剂量分别按2.5、5、10、20mg/kg给予,对照组给予生理盐水灌喂。各组手术后复隔2-3d分别取3只大鼠肝组织作常规组织学观察及免疫组织化学染色。结果 对照组大鼠肝组织内未见到肝卵圆细胞,2.5mg/kg剂量组及5mg/kg剂量组仅见少量肝卵圆细胞增生,而10、15、20mg/kg三个剂量组肝组织内均见明显的肝卵圆细胞增殖反应;肝卵圆细胞胞浆细胞角蛋白19(CK19)、OV6及波形蛋白染色均呈阳性,胞核增殖细胞抗源染色呈阳性。结论 AAF剂量为10-20mg/kg时可获得满意的大鼠肝卵圆细胞增殖模型。 相似文献
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目的 探讨miR-21调控肝卵圆细胞活化和增殖过程的可能机制.方法 采用2-乙酰氨基芴(2-AAF)/部分肝切除法诱导SD大鼠肝卵圆细胞活化模型,分别于0、6、12、24、72、168 h处死动物,同时以单纯肝切除的相应时间点作为对照,分别提取各标本RNA逆转录后行SYBR Green实时荧光定量PCR,检测各组miR-21的表达,行两样本t检验,并用生物信息学方法分析其调控的miRNA转录分子和靶基因.实时荧光定量PCR检测其靶基因表达,Westem blot法检测其靶基因蛋白表达,进行两样本均值的t检验,以P< 0.05为差异有统计学意义. 结果 成功建立大鼠肝卵圆细胞活化模型,检测miR-21的扩增信号,miR-21在实验组表达12h开始升高,24 h达到峰值,随后开始降低,168 h再次升高;而对照组6h开始升高,24h后开始下降后基本恢复原水平.实验组与对照组表达比较,实验组在6 h miR-21的表达低于对照组,t=3.029,P=0.039,差异有统计学意义;而在24 h和168 h的表达高于对照组,t值分别为-3.433、-5.105,P值均<0.05,差异有统计学意义.根据三个数据库的综合评分,结合相关文献,我们选取Smad7为研究的靶基因.检测两组Smad7mRNA表达,对照组中在6h略有下降,后开始升高,在24 h达到峰值后开始下降恢复原水平;在实验组中,6h升高后至24 h达到峰值,随后开始降低,168 h又略有升高.对照组Smad7蛋白表达从6h开始降低,到24 h达到最低后开始升高;实验组6h升高,随后下降,168 h达到最低.Smad7 mRNA表达量变化趋势与miR-21表达变化趋势基本一致,Smad7蛋白表达和miR-21表达变化趋势呈负相关. 结论 成功建立SYBR Green实时荧光定量PCR检测大鼠miR-21的方法,证实miR-21在肝卵圆细胞活化与增殖中起重要作用,Smad7作为miR-21的靶基因可能参与这一过程. 相似文献
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Marina Sánchez‐Hidalgo Melanie Lee Catalina A. de la Lastra Juan M. Guerrero Graham Packham 《Journal of pineal research》2012,53(4):366-373
Abstract: Melatonin exerts strong anti‐tumour activity via several mechanisms, including anti‐proliferative and pro‐apoptotic effects in addition to its potent antioxidant activity. Several studies have investigated the effects of melatonin on haematological malignancies. However, the previous studies investigating lymphoid malignancies have been largely restricted to a single type of malignancy, Burkitt’s lymphoma (BL). Thus, we examined the actions of melatonin on the growth and apoptosis in a small panel of cell lines representing different human lymphoid malignancies including Ramos (Epstein–Barr virus–negative BL), SU‐DHL‐4 (diffuse large B cell lymphoma), DoHH2 (follicular B non‐Hodgkin lymphoma) and JURKAT (acute T cell leukaemia). We showed that melatonin promotes cell cycle arrest and apoptosis in all these cells, although there was marked variations in responses among different cell lines (sensitivity; Ramos/DoHH2 > SU‐DHL‐4 > JURKAT). Melatonin‐induced apoptosis was relatively rapid, with increased caspase 3 and PARP cleavage detected within 0.5–1 h following melatonin addition. Moreover, there was evidence for rapid processing of both caspase 9, as well as a breakdown of the mitochondrial inner transmembrane potential. On the contrary, caspase activation was detected only in SU‐DHL‐4 and Ramos cells following melatonin treatment suggesting that the extrinsic pathway does not make a consistent contribution to melatonin‐induced apoptosis in malignant lymphocytes. Although all cell lines expressed the high‐affinity melatonin receptors, MT1 and MT2, melatonin‐induced caspase activation appeared to be independent these receptors. Our findings confirm that melatonin could be a potential chemotherapeutic/preventive agent for malignant lymphocytes. However, it is necessary to take into account that different lymphoid malignancies may differ in their response to melatonin. 相似文献
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目的 研究肝星状细胞(HSC)在体外培养的卵圆细胞向成熟肝细胞分化过程中所起的作用.方法 (1)将卵圆细胞分别与原代培养的HSC混合共培养(混合共培养组),采用Millicell小室不接触共培养(不接触共培养组),卵圆细胞单独培养作为对照组.在共培养后第7、14、21天观测,采用Western blot和荧光定量PCR检测肝细胞核因子4 α(HNF-4 α)、白蛋白和甲胎蛋白(AFP)、细胞角蛋白19(CK-19)的表达量;(2)电子透射显微镜观察卵圆细胞超微结构的变化;(3)过碘酸希夫染色检测各时间点卵圆细胞内糖原颗粒的含量;(4)酶联免疫吸附法检测各时间点卵圆细胞分泌白蛋白的含量.统计学处理采用重复测量资料的多因素方差分析和LSD-t检验.结果 (1)卵圆细胞第7、14、21天表达HNF-4 α和白蛋白的mRNA的量(相对与共培养之前的表达量的倍数):混合共培养组HNF-4 α分别为(1.9±0.2)倍、(10.7±1.2)倍、(12.0±1.3)倍;白蛋白mRNA的量分别为(5.7±1.6)倍、(110.7±13.7)倍、(173.6±22.3)倍;不接触共培养组HNF-4 α分别为(1.4±0.1)倍、(3.2±0.6)倍、(8.9±1.4)倍;白蛋白mRNA的量分别为(2.9±1.4)倍、(22.3±8.5)倍、(96.3±16.3)倍.共培养组(混合共培养与不接触共培养组)的表达量均高于对照组,LSD-t值分别为32.98,10.08;13.38,7.96; P值均<0.01,差异有统计学意义.第7、14、21天AFP、CK-19的表达量:混合共培养组AFP分别为(1.1±0.2)倍、(0.2±0.0)倍、(0.0±0.0)倍;CK-19分别为(0.2±0.1)倍、(0.0±0.0)倍、(0.0±0.0)倍;不接触共培养组AFP分别为(1.0±0.2)倍、(0.2±0.1)倍、(0.1±0.0)倍;CK-19分别为(0.6±0.1)倍、(0.1±0.0)倍,(0.0±0.0)倍.共培养组(混合共培养与不接触共培养组)的表达量均低于对照组.LSD-t值分别为37.99,34.50;13.59,22.46;P值均<0.01,差异有统计学意义.(2)卵圆细胞共培养后白蛋白分泌量明显增加:混合共培养组:14 d为(15.30±0.09)ng/ml、21 d(20.98±0.12)ng/ml,不接触共培养组14 d为(11.41±0.13)ng/ml,21 d为(15.12±0.17)ng/ml,混合共培养组高于不接触共培养组,LSD-t=251.94,P<0.01,差异有统计学意义.(3)随着共培养时间的延长,卵圆细胞内质网、线粒体和高尔基体等细胞器逐渐丰富,且细胞间形成毛细胆管结构.(4)过碘酸希夫染色显示共培养后卵圆细胞内出现大量红色糖原颗粒.结论 HSC可以诱导卵圆细胞向成熟肝细胞分化,HSC除了通过细胞因子等可溶性因子途径发挥作用外,与卵圆细胞的直接接触也有着重要作用. 相似文献
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The purpose of this study was to elucidate the mechanism of spontaneous and rapid cell death of cultured gastric pit cells. Gastric pit cells have a rapid cell turnover rate in vivo. We here show that guinea pig gastric pit cells in culture undergo spontaneous and rapid apoptotic DNA fragmentation, which may represent the rapid cell turnover cycle of gastric pit cells in vivo. This spontaneous apoptotic DNA fragmentation required the presence of fetal calf serum in the culture media. Furthermore, the spontaneous apoptotic DNA fragmentation was prevented by protein synthesis and caspase inhibitors. 相似文献
6.
Fas/Fas—L在重型肝炎患者肝组织中的表达及其在肝细胞凋亡中的意义 总被引:2,自引:0,他引:2
目的 研究Fas/Fas-L系统在重型肝炎患者肝细胞中的表达情况及在肝细胞凋亡中的意义。方法 采用免疫组织化学方法对20例住院的重型肝炎病人之活检肝组织进行了Fas抗原及Fas配体表达的检测,并运用末端转移酶标亡技术(TUNEL)同时检测细胞原位凋亡的情况。结果 所检测20例重型肝炎肝组织中,Fas抗原在肝细胞中呈强阳性表达;浸润的淋巴细胞上大量表达Fas-L,肝细胞中也有散在分布;敏例标本均见凋亡细胞,分布在炎症坏死区及周边和小叶内。结论 Fas/Fas-L系统过量表达,引起大量肝细胞死亡,参与重型肝炎的发病,Fas途径引起的细胞凋亡可能是重型肝炎发病过程中的重要机制之一。所检标本中,凋亡和坏死同时存在,这两种不同的死亡方式有重型肝炎的发病上存在着关联性。 相似文献
7.
A peptide inhibitor of cytochrome c/inositol 1,4,5-trisphosphate receptor binding blocks intrinsic and extrinsic cell death pathways
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Boehning D van Rossum DB Patterson RL Snyder SH 《Proceedings of the National Academy of Sciences of the United States of America》2005,102(5):1466-1471
Apoptotic stimuli augment intracellular calcium concentration through inositol 1,4,5-trisphosphate receptors (IP3R) on endoplasmic reticulum calcium stores. We previously discovered an apoptotic cascade wherein cytochrome c binds to IP3R early in apoptosis, resulting in dysregulated calcium release. Here we show that cytochrome c binding to IP3R depends on a cluster of glutamic acid residues within the C terminus of the channel. A cell permeant peptide derived from this sequence displaces cytochrome c from IP3R and abrogates cell death induced by staurosporine treatment of HeLa cells and Fas ligand stimulation of Jurkat cells. Small-molecule inhibitors of cytochrome c/IP3R interactions may prove useful in treating disorders associated with inappropriate intrinsic and extrinsic apoptotic signaling. 相似文献
8.
肝干细胞是一类具有自我更新与增殖分化能力的细胞,能产生表现型与基因型和自己完全相同的子细胞.起源于前肠内胚层,在胚胎发育过程中以肝细胞的形式存在,在成年哺乳动物肝中以小卵圆细胞存在,表现为核大而胞质小并具有特殊的细胞标记.正常情况下这类细胞处于静止期,增生分裂非常慢.当切除或药物损伤后,这类细胞开始活化增生,迅速从静止期进入增殖期.近年来的研究已经证实,肝卵圆细胞在肝细胞严重受损和分裂增生受抑制时呈现出向肝细胞和胆管上皮细胞双向分化的潜能,是一种肝脏的干细胞,目前已经成为热点.肝卵圆细胞不仅在急慢性肝功能不良、晚期肝硬化等肝脏病变,在胰腺病变引起的糖尿病等疾病研究中也开始引起兴趣.但如何发现和获得肝卵圆细胞始终是解决此类问题的关键.本文就肝卵圆细胞的分子标志物的研究进展作一综述. 相似文献
9.
10.
Yun‐Feng Shan Wei‐Ping Zhou Xiao‐Yong Fu He‐Xing Yan Wen Yang Shu‐Qin Liu Hui‐Fang Cao Bin Kang Meng‐Chao Wu Hong‐Yang Wang 《Liver international》2006,26(2):240-247
Abstract: Background: Human gankyrin gene product (p28GANK) is a novel oncogenic protein ubiquitously overexpressed in hepatocellular carcinoma and also plays a role in cell cycle progression in normal hepatocytes and liver regeneration. However, little is known about the physiological role of p28GANK in the liver oval cell activation and proliferation. We investigated the possible involvement of p28GANK in oval cell‐mediated liver regeneration and cell cycle progression. Methods: We examined the different p28GANK expression in 2‐acetylaminofuorene/partial heptectomy (2‐AAF/PH) rats, as a model of oval cell activation, and PH rats by Western blot and immunohistochemistry. Oval cells isolated from 2‐AAF/PH rat model were cultured in our study. p28GANK expression was examined in the oval cells after mitogenic stimulation. Results: In 2‐AAF/PH rats, p28GANK was expressed in the activated oval cells and located in the nucleus. p28GANK protein expression was increased in 2‐AFF/PH rats after hepatectomy lasting for 96 h when retinoblastoma maintained hyperphosphorylation status at Ser‐795. The isolated oval cells express AFP, OV6, CK19, CD34, CD45, c‐kit and albumin. After epidermal growth factor stimulation, p28GANK protein was up‐regulated in oval cells from 24 to 72 h, which coincided with increased expression of CyclinD1, CDK4 and decreased of Rb protein. Conclusions: p28GANK expression was increased in oval cell‐mediated liver regeneration and oval cells after mitogenic stimulation. Thus, p28GANK may play a role in oval cell‐mediated liver regeneration and liver oval cell cycle progression. 相似文献
11.
大鼠肝卵圆细胞的分离培养及脾内移植研究 总被引:11,自引:1,他引:11
目的 观察肝卵圆细胞在同种异体大鼠脾内移植的演变结果,为肝干细胞移植治疗临床肝功能衰竭提供实验依据。方法 采用改进的梯度离心法分离肝卵圆细胞,体外培养鉴定后移植入2/3肝切除的同种异体大鼠脾脏内。结果 每只模型大鼠肝脏中可分离获得约1.69×10~5/ml肝卵圆细胞。体外培养的肝卵圆细胞呈现上皮细胞的生长特点,对OV6、细胞角蛋白19及甲胎蛋白染色呈阳性反应,对白细胞共同抗原染色呈阴性反应。肝卵圆细胞植入异体大鼠脾脏内可形成岛屿状肝组织结构,形成“肝化脾”。结论 大鼠肝卵圆细胞具有肝干细胞的生物学特征,在一定条件下可分化为肝细胞及胆管上皮细胞。 相似文献
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肝卵圆细胞是一类存在于成年肝脏、具有自我更新和多向分化潜能的肝干细胞。当肝脏发生严重损伤且肝细胞再生障碍时,卵圆细胞被激活并大量增殖,参与肝脏损伤的修复与重建。肝卵圆细胞增殖分化是多因素作用的结果。现就肝卵圆细胞的定位、来源及调控机制的研究进展作一概述。 相似文献
13.
肝脏具有很强的再生能力,当发生轻、中度肝损害时,肝细胞可经增殖和分化等复杂过程完成肝再生;而在肝细胞大量损害或肝细胞增殖能力被抑制时,卵圆细胞可增殖并分化为肝细胞或胆管细胞.然而,目前研究结果已初步证实肝细胞癌的发生也源起于卵圆细胞,其分子机制仍有待深入研究[1-3].
Polo样激酶1(Plk1)属于保守的丝氨酸/苏氨酸激酶家族,在调控细胞周期进程和细胞分裂方面起着十分重要的作用[4-6].研究结果表明,Plk1在肝癌中的表达增强[7-8].我们在前期研究中也发现Plk1基因在肝细胞癌中差异表达[9].但是Plk1在肝卵圆细胞中的表达及其病理意义鲜见报道.本研究以二乙酰胺基芴(2-AAF)为致癌物诱导大鼠肝卵圆细胞增殖,与单纯肝再生组织进行对比分析,探讨Plk1在肝卵圆细胞中的表达及其与癌前状态的关系. 相似文献
14.
丁酸钠诱导体外培养的大鼠肝卵圆细胞分化为成熟肝细胞 总被引:13,自引:0,他引:13
目的探讨分化刺激剂丁酸钠对体外培养的大鼠肝卵圆细胞分化的影响。方法从喂养含0.1%乙硫氨酸的胆碱缺乏性饮食4~6周的人鼠肝脏中分离出盱卵圆细胞,用免疫细胞化学和逆转录聚合酶链反应(RT-PCR)等方法对其进行鉴定。用0.75mmol/L酸钠处理大鼠肝卵圆细咆后.姬姆萨染色观察细胞表型改变,western blot检测细胞白蛋白的表达水平。结果免疫细胞化学结果显示分离出的细胞既表达成熟肝细咆的标志物白蛋白,也表达胆管细胞的标志物细胞角蛋白19,RT-PCR结果显示这些细胞还表达干细胞的标志物c-kit,但不表达造血干细胞的标志物CD34,表明这些细胞是大鼠肝前体细胞——肝卵圆细胞。0.75mmol/L丁酸钠能诱导大鼠肝卵圆细胞出现明显的表型改变,细胞变大,变圆,核浆比减小,且双核细胞数增多,约占总细胞数的50%左右,同时western blot的结果显示0.75mmol/L丁酸钠能够提高大鼠肝卵圆细胞白蛋白的表达水平。。结论分化刺激剂丁酸钠能诱导体外培养的大鼠肝卵圆细胞向成熟肝细胞分化。 相似文献
15.
急性肝损伤大鼠肝卵圆细胞活化增殖的研究 总被引:1,自引:0,他引:1
目的 用D-氨基半乳糖 (D-galactosamine ,D-GalN)联合胆总管结扎 (CommonBiliaryDuctLigation ,CBDL)建立大鼠急性肝损伤模型 ,观察肝损伤后再生过程中肝卵圆细胞 (HepaticOvelCell,HOC)的活化和增殖。 方法 雄性SD大鼠 ,胆总管结扎离断术后予腹腔注射D-GalN 2 .0 g/kg ,对照组不做任何处理。处理后第 1、2、3、4、5及 6天 6个时间点测血清肝功能 ,取肝组织分别行苏木精 伊红染色、电镜、免疫组织化学及RT-PCR检测 ,并对符合HOC形态学特征且胞浆OV-6阳性染色的细胞进行计数。结果 肝功能损害以第 2天明显 (P <0 .0 1) ;对照组肝组织内未见HOC ,处理组各时间点均见有不同程度的HOC增殖反应 ,以第 3天HOC计数最多 (P <0 .0 5 ) ;HOC胞质AFP、OV 6染色阳性 ,胞核BrdU染色阳性 ;RT-PCR示模型组CGT mRNA表达明显增多。结论 用D GalN2 .0 g/kg联合CBDL可获得较满意的大鼠HOC增殖模型 ,在急性肝损伤时HOC被活化和不同程度的增殖 ,参与了肝再生过程 相似文献
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Caspase介导的Fas凋亡途径 总被引:9,自引:0,他引:9
凋亡是导致细胞死亡的调节性生理过程.Fas在各种疾病和细胞凋亡的发生中起到首要作用.Caspase为半胱氨酸蛋白酶家族,是凋亡的中心调节者,一旦Caspase-8,10被激活,引起Caspase的断裂及激活下游的Caspase-3,6,7,作用于Bcl-2家族某些成员如Bad、一些骨架蛋白和RB蛋白等并使其断裂,从而导致细胞凋亡.本文重点讨论了Caspase-3,-8的激活机制及其在Fas诱导细胞凋亡中的作用. 相似文献
17.
Chen-Xuan Wu Qi Zou Zheng-Yan Zhu Ying-Tang Gao Yi-Jun Wang 《World journal of gastroenterology : WJG》2009,15(12):1506-1511
AIM: To evaluate the effect of intrahepatic transplantation of hepatic oval cells (HOC) on fulminant hepatic failure (FHF) in rats.
METHODS: HOC obtained from rats were labeled with green fluocescent protein (GFP) or 5, 6- carboxyfluorescein diacetate succinmidyl ester (CFDASE). Cell fluorescence was observed under fluorescent microscope at 6, 24, 48 and 72 h after labeling. CFDA- SE labeled HOC (5 × 10^6 cells each rat) were injected into livers of rats with FHF induced by D-galactosamine. Serum albumin (ALB), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (TBil) levels were measured at different time points. Liver function of rats was examined on days 3, 7, 14 and 21 after HOC transplantation.
RESULTS: The positive rate of GFP and CFDA-SE labeled HOC was 10% and 90%, respectively, with no significant change in cell viabilities. The survival rate was higher in HOC transplantation group than in control group, especially 48 (9/15 vs 6/15) and 72 h (9/15 vs 4/15) after HOC transplantation. The serum ALT, AST and TBil levels were decreased while the serum AIb level was increased after HOC transplantation. Fluorescence became faded and diffused in liver tissues, suggesting that proliferation and differentiation occur in transplanted HOC.
CONCLUSION: CFDA-SE is superior to GFP in labeling HOC, although fluorescence intensity is decreased progressively with cell division. HOC transplantation can improve the liver function and increase the survival rate of recipients. 相似文献
METHODS: HOC obtained from rats were labeled with green fluocescent protein (GFP) or 5, 6- carboxyfluorescein diacetate succinmidyl ester (CFDASE). Cell fluorescence was observed under fluorescent microscope at 6, 24, 48 and 72 h after labeling. CFDA- SE labeled HOC (5 × 10^6 cells each rat) were injected into livers of rats with FHF induced by D-galactosamine. Serum albumin (ALB), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (TBil) levels were measured at different time points. Liver function of rats was examined on days 3, 7, 14 and 21 after HOC transplantation.
RESULTS: The positive rate of GFP and CFDA-SE labeled HOC was 10% and 90%, respectively, with no significant change in cell viabilities. The survival rate was higher in HOC transplantation group than in control group, especially 48 (9/15 vs 6/15) and 72 h (9/15 vs 4/15) after HOC transplantation. The serum ALT, AST and TBil levels were decreased while the serum AIb level was increased after HOC transplantation. Fluorescence became faded and diffused in liver tissues, suggesting that proliferation and differentiation occur in transplanted HOC.
CONCLUSION: CFDA-SE is superior to GFP in labeling HOC, although fluorescence intensity is decreased progressively with cell division. HOC transplantation can improve the liver function and increase the survival rate of recipients. 相似文献
18.
肝细胞凋亡、坏死、衰老与卵圆细胞的激活和扩增密切相关,此外,卵圆细胞介导肝脏再生是肝损伤后再生的重要组成部分。目前已有多个实验证实了多种细胞因子、激素及神经递质参与此过程。回顾了近年研究结果,简要介绍TWEAK/Fn14、Hedgehog及甲状腺激素等多个信号通路与卵圆细胞介导肝脏再生的关系及研究进展,阐明各个信号通路在卵圆细胞介导肝脏再生中起的作用,了解其调节肝脏再生的机制,或许能对未来临床用药指明靶点。 相似文献
19.
This study was undertaken to define the role that apoptosis may play in inducing cellular injury and death in gastric mucosa exposed to aspirin. Apoptosis was characterized by DNA gel electrophoresis, terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, and DNA-histone-associated complex formation. A human gastric cell line (AGS) was exposed to physiologic concentrations (3 to 50 mM) of aspirin. Both time- and concentration-dependent effects on apoptosis were noted, which were effectively prevented by the caspase inhibitor z-VAD-fmk. Accordingly, the role of caspases in aspirin-induced apoptosis was also evaluated. Early activation of caspase-8 and caspase-9 was demonstrated, indicating a role for both receptor and mitochondrial pathways, respectively, in the apoptotic process. Corresponding activation of effector caspases-3, -6, and -7 was also evident, as was cleavage of PARP. We conclude that physiologically relevant concentrations of aspirin induces apoptosis in human gastric cells through a caspase-mediated mechanism. 相似文献