【作者】 高小玲；

【导师】 王丕龙；

【作者基本信息】 重庆医科大学 ， 消化内科， 2010， 博士

【摘要】 第一部分DHA对肝癌HepG2、前列腺癌PC-3、胃癌SGC7901及结肠癌Caco2细胞增殖抑制的研究目的:比较双氢青蒿素(DHA)对四种肿瘤细胞的体外生长抑制作用,以筛选出最敏感的细胞株进行作用机制的研究。方法:噻唑蓝(MTT)比色法评价DHA对人肝癌HepG2,前列腺癌PC-3,胃癌SGC7901及结肠癌Caco2细胞的增殖抑制作用;流式细胞术检测DHA对上述四种细胞凋亡率的影响。结果:四种细胞的各对照组与DMSO组之间细胞增殖比较无统计学差异(P>0.05);DHA对每种细胞的抑制率与DHA浓度和作用时间正相关(P<0.05),相同时间,HepG2各浓度组的抑制率与其他三种细胞相应浓度组比较有明显差异(P<0.01,除外25μmol/L DHA 24h组P<0.05);作用24h后,每种细胞株各DHA浓度组的细胞凋亡率均明显高于对照组(P<0.01)。四种细胞株中,DHA对HepG2细胞的抑制作用最强,其中DHA(200μmol/L)作用48h时达最高增殖抑制率(86.46±19.65)% , DHA(100μmol/L)作用24时达最高凋亡率(31.74±2.80)%。结论:DHA明显抑制四种细胞株的增殖并诱导其凋亡,其中HepG2最为敏感。第二部分DHA诱导人肝癌HepG2细胞凋亡启动因素的研究目的:探讨DHA诱导人肝癌HepG2细胞凋亡的启动因素。方法:用0、50、100和200μmol/L DHA作用HepG2细胞6h、24h后,采用荧光探针2’,7’-二氯二氢荧光黄双乙酸钠(DCFH-DA)及Fluo-3AM装载检测细胞内活性氧(ROS)含量及Ca2+浓度;采用流式细胞仪检测线粒体膜电位(ΔΨm)变化及透射电镜观察DHA作用HepG2后细胞超微结构的变化。结果:与对照组相比,DHA作用细胞后,细胞内荧光度值显著增加(P<0.01),且具有明显的浓度与时间依赖性,而具有浓度依赖的胞浆游离Ca2+浓度增加及线粒体膜电位降低均出现在DHA作用24h组;DHA100μmol/L作用24h透射电镜观察,可见大量凋亡细胞及内质网显著扩张。抗氧化剂NAC(5mmol/L)可明显抑制DHA的上述作用。结论:DHA明显增加细胞中ROS水平及Ca2+浓度,降低线粒体膜电位,ROS的产生可能位于上游环节。第三部分DHA诱导人肝癌HepG2细胞内质网应激凋亡信号转导机制的研究目的:研究DHA诱导人肝癌HepG2细胞凋亡的内在作用机制。方法:用DHA、DHA+NAC及DHA+SP600125作用HepG2细胞, MTT比色法检测细胞增殖抑制的效果;流式细胞术检测细胞凋亡的情况;扫描电镜观察DHA作用后细胞超微结构的变化;逆转录-聚合酶链反应(RT-PCR)检测CHOP, XBP1及ATF4 mRNA的表达,蛋白印迹技术(Western Blot)及免疫细胞化学检测CHOP, p-JNK, Bax及Bcl-2蛋白的表达。结果:DHA对HepG2细胞的增殖抑制率与DHA浓度和作用时间相关(P<0.05),NAC在24h及48h均可抑制DHA的作用,除JNK的特异性抑制剂SP600125在48h高浓度组(50,100,200μmol/L)对DHA的作用没有明显影响外,在其他组中可部分抑制DHA的作用;DHA组24h细胞凋亡率明显高于对照组,且与DHA浓度正相关(P<0.05),NAC及SP600125均可抑制DHA的作用;电镜观察显示,DHA作用后细胞表面微绒毛减少和消失,凋亡细胞可见大小不等球状的突起及凋亡小体,坏死细胞的细胞膜破裂,胞浆外溢;RT-PCR检测发现DHA可上调CHOP, XBP1u、XBP1s及ATF4 mRNA的表达,Western Blot及免疫细胞化学检测发现DHA上调CHOP及Bax蛋白的表达,下调Bcl-2表达,NAC可抑制DHA对上述相关基因与蛋白的调节;Western-Blot在DHA作用6h时检测出JNK的磷酸化,NAC及SP600125均可抑制p-JNK的表达。结论:DHA激活UPR的三条信号通路(PERK、ATF6、IRE1),通过激活CHOP, JNK而诱导内质网应激凋亡途径,ROS在这一过程中具有重要的作用。第四部分DHA对裸鼠人肝癌HepG2细胞移植瘤生长抑制的研究目的:研究DHA对裸鼠人肝癌HepG2移植瘤生长的抑制作用。方法:采用皮下接种肝癌HepG2细胞悬液的方法构建裸鼠的移植瘤模型,观察DHA在体内对HepG2细胞的生长抑制作用;逆转录-聚合酶链反应(RT-PCR)及免疫组化检测CHOP、Bax、Bcl-2及p-JNK的表达情况;透射电镜观察肝癌组织超微结构的改变。结果:与对照组相比,瘤体积及瘤重量明显降低(P<0.05),抑瘤率达50.91%,而对照组与DHA组裸鼠体重无明显差异(P>0.05);RT-PCR及免疫组化显示DHA可上调肿瘤组织中CHOP、Bax的表达水平,下调Bcl-2的表达,差异有统计学意义(P<0.05),p-JNK在两组中均无表达;透射电镜观察,DHA治疗后,移植瘤组织可见细胞凋亡形态学改变,内质网腔明显扩张、线粒体肿胀。结论:DHA可抑制裸鼠人肝癌HepG2细胞移植瘤的生长,内质网应激凋亡途径可能参与了DHA的这一作用。更多还原

【Abstract】 PART ONE STUDIES ON THE PROLIFERATION INHIBITORY EFFECTS OF DHA ON HUMAN HepG2, PC-3, SGC7901 AND Caco2 CELL LINESOBJECTIVE:To compare the inhibitory effects of dihydroartemisinin （DHA） on four human carcinoma cell lines in vitro, and select the most sensitive cell lines for further investigation.METHODS: The proliferation inhibition of DHA on human HepG2, PC-3, SGC7901 and Caco2 cell lines was evaluated using MTT chromatometry; Flow cytometry analysis was used to detect the apoptosis of the four cell lines.RESULTS: There was no significant difference in proliferation of control and DMSO group of four cell lines （P>0.05）. Proliferation inhibitory rate of each cell lines was positively correlated to DHA concentration and treatment time（P<0.05）, and inhibitory rates of various DHA concentration groups of HepG2 had significant difference compared with that of other three cell lines at the same treatment time（P<0.01, except for 25μmol/L DHA 24h treatment time P<0.05）. The apoptotic rates of each DHA concentration groups after 24h treatment were markedly higher than that of the control groups in four cell lines （P<0.01）. DHA had the stongest effect on HepG2 among the four cell lines, with the highest inhibitory rate of （86.46±19.65） % at DHA 200μmol/L for 48 h, and the highest apoptotic rate of （31.74±2.80） % at DHA 100μmol/L for 24 h.CONCLUSION: DHA can efficiently inhibit proliferation and induce apoptosis of HepG2, PC-3, SGC7901 and Caco2 cell lines; HepG2 is the most sensitive to DHA among the four cell lines.PART TWO STUDIES ON THE INITIATORS OF APOPTOTIC INDUCTION OF DHA ON HUMAN HepG2 CELL LINESOBJECTIVE:To investigate the initiators of apoptotic induction of DHA on HepG2 cell lines.METHODS: HepG2 cells were treated with 0, 50,100 and 200μmol/L DHA for 6, 24 h. The fluorescent probe 2,7-dichlorodihydrofluorescein diacetate （DCFH-DA） and Fluo-3AM were employed to detecte the levels of reactive oxygen species（ROS） and intracellular calcium concentration, respectively; Flow cytometer was used to measure the mitochondrial membrane potential （ΔΨm）, and the ultrastructural changes of HepG2 cells treated with DHA were observed using transmission electron microscope （TEM）.RESULTS:Compared with control group, intracellular fluorescent intensity markedly increased （P<0.01） after DHA treatment in a time- and concentration-dependent manne; However, the increase of [Ca2+]i and the reduction ofΔΨm, which were concentration-dependent, occurred only in 24h treatment group（P<0.01）. After 24h treatment of DHA 100μmol/L, a lot of apoptotic cells as well as significant distention of endoplasmic reticulum lumina were obserbed using TEM. The above effects of DHA could be markedly attenuated by antioxidant N-acetylcysteine （NAC） 5mmol/L.CONCLUSION: DHA significantly increases ROS generation and [Ca²⁺]_i, reduces the level ofΔΨm. ROS generation is probably upstream event.PART THREE STUDIES ON ENDOPLASMIC RETICULUM STRESS-INDUCED APOPTOTIC SIGNAL TRANSDUCTION MEDIATED BY DHA IN HUMAN HepG2 CELL LINESOBJECTIVE : To study the molecular mechanism of apoptosis induced by DHA in HepG2 cell linesMETHODS: Human HepG2 cells were treated with DHA, DHA+NAC and DHA+SP600125. The inhibitory effect on cell proliferation was assayed by MTT; Apoptotic induction was measured by Flow cytometry; Ultrastructural changes of HepG2 cells treated with DHA were observed with scanning electron microscope （SEM）; The expression of CHOP, XBP1 and ATF4 mRNA were detected using reverse transcription-polymerase chain reaction （RT-PCR）; Levels of CHOP, p-JNK, Bax and Bcl-2 protein expression were determined using Western Blot and immunocytochemistry.RESULTS: Proliferation inhibitory rate of HepG2 cells treated with DHA correlated with DHA treatment time and concentration （P<0.05）. NAC blocked the effect of DHA treatment for both 24h and 48h, which could also be partially attenuated by JNK specific inhibitor SP600125 except for DHA at high concentration （ 50, 100, 200μmol/L） 48h groups; Compared to the control group, the apoptotic rates of HepG2 cells in DHA group were significantly higher and had a positive correlation with DHA concentration（P<0.05）. Both NAC and SP600125 could attenuate the DHA effect. SEM showed decreasing and even deminish of microvilli on the cellular surface after DHA treatment, varying sized of ball-shaped membrane blebbing on the cellular surface of apoptotic cells, apoptosis body, broken plasma membranes and cytoplasma outflow in necrotic cells; RT-PCR showed up-regulation of mRNA expression of CHOP, XBP1u, XBP1s and ATF4 by DHA while Western-Blot and immunocytochemistry showed up-regulation of protein expression of CHOP and Bax, and down-regulation of Bcl-2, NAC inhibited the effects of DHA on these genes and protein expression; Phosphorylation of JNK was detected at 6h DHA treatment time by Western-Blot, both NAC and SP600125 blocked the phosphorylation of JNK induced by DHA.CONCLUSION: DHA could activate the three signal transduction pathways of UPR （PERK、ATF6、IRE1）, and induce the endoplasmic reticulum stress-mediated apoptotic pathway via CHOP and JNK activation. ROS plays an important role in this mechanism.PART FOUR STUDIES ON INHIBITION OF HepG2 XENOGRAFT TUMOR IN NUDE MICEOBJECTIVE:To investigate the inhibitory effect of DHA on growth of HepG2 xenograft tumor in nude mice.METHODS: The nude mice model of human hepatocarcinoma was established by subcutaneous injection with HepG2 cell suspension, and growth inhibition of the xenograft tumor induced by DHA was observed in vivo; Reverse transcription-polymerase chain reaction （RT-PCR） and immunohistochemistry were employed to evaluate CHOP, Bax, Bcl-2 and p-JNK expression; Ultrastructural changes of xenograft tumor tissues were observed by transmission electron microscope （TEM）.RESULTS: Compared to control group, the volume and weight of tumor of DHA group were significantly decreased （P<0.05）, and the inhibitory rate was 50.91%. No significant difference in body weight between control and DHA group was recorded; RT-PCR and immunohistochemistry showed the expression of CHOP and Bax were up-regulated, while that of Bcl-2 was down-regulated, and the differences were statistically significant （P<0.05）, and no expression of p-JNK in both groups; TEM indicated apoptosis morphological changes including significant distension of endoplasmic reticulum lumina and swelling of mitochondria in xenograft tumor histocytes after DHA treatment.CONCLUSION: DHA can inhibit the growth of HepG2 xenograft tumor in nude mice, and endoplasmic reticulum stress-induced apoptosis pathway may be involved in this action of DHA.更多还原