【作者】 伍婧；

【导师】 罗荣城；

【作者基本信息】 南方医科大学 ， 肿瘤学， 2008， 硕士

【摘要】 原发性肝癌（primary hepatic carcinoma,PHC）是常见的恶性肿瘤之一。我国是世界上肝癌的高发地区,肝癌死亡率居全国各种肿瘤的第2位,并且近10年来死亡率一直呈上升趋势。尽管近年来肝癌研究已取得了很大的进步,疗效也得到明显改观,但就肝癌整体疗效而言仍很不理想。目前,肝细胞癌的治疗方法主要包括手术治疗、肝动脉栓塞化疗（TACE）、冷冻治疗、射频消融、体外放疗、放射性粒子植入、生物治疗等。由于肝癌起病隐袭,多数患者就诊时肿瘤已进展至晚期,能行手术切除的肝癌尚不足25%。肝动脉栓塞化疗被证明有效,但并非所有病人都适合。全身化疗对晚期肝癌作用有限,单药化疗或联合化疗,其有效率均不足20%。故寻找一种合理有效的治疗晚期PHC方案具有重要意义。索拉非尼（Sorafenib,nexavar）是一种双芳基尿素类口服多激酶抑制剂,其抗肿瘤机制包括两个方面。一方面可靶向作用于肿瘤细胞及肿瘤血管上的丝氨酸/苏氨酸激酶及受体酪氨酸激酶,通过抑制受体酪氨酸激酶KIT和FLT-3,以及Raf/MEK/ERK途径中丝氨酸/苏氨酸激酶,抑制肿瘤细胞增殖;另一方面,通过上游抑制受体酪氨酸激酶VEGFR和PDGFR,及下游抑制Raf/MEK/ERK途径中丝氨酸/苏氨酸激酶,抑制肿瘤血管生成。Scott M.Wilhelm等发现Sorafenib能通过抑制Raf-1及其相关的激酶、野生型或V599E突变型B-RAF的活性来阻断Raf/MEK/ERK信号传导通路,还能显著抑制一些酪氨酸激酶受体的活性,包括干细胞因子受体（KIT）、Fms样酪氨酸激酶（FLT-3）、血管内皮生长因子受体-2（VEGFR-2）、血管内皮生长因子受体-3（VEGFR-3）、血小板衍生生长因子受体-β（PDGFR-β）等。Li Liu等证实Sorafenib能下调肝癌细胞株PLC/PRF/5与HepG2的MEK、ERK蛋白磷酸化水平和cyclinD1水平,并且能下调eIE4E与抗凋亡蛋白Mcl-1水平,通过抑制MEK/ERK信号传导途径与MEK/ERK非依赖信号传导途径来抑制肝癌细胞增殖和诱导肝癌细胞凋亡。索拉非尼治疗肝癌的Ⅲ期双盲随机对照临床（SHARP）研究显示:与安慰剂相比,索拉非尼显著延长晚期HCC患者的总体生存达44%。索拉非尼组与安慰剂组中位OS分别为10.7个月及7.9个月,两组间差别有统计学意义。与安慰剂组相比,索拉非尼组的中位TTP更长（5.5个月对2.8个月）,疾病控制率（DCR）更高（43%对32%）。已有临床试验表明,Sorafenib与化疗药如氮烯咪胺、紫杉醇、卡铂以及其他靶向药物如吉非替尼、贝伐单抗联用,能提高化疗药及其他靶向药物的抗肿瘤疗效。索拉非尼被确立成为第一个批准治疗晚期HCC的靶向治疗药物。三氧化二砷（Arsenic trioxide,As₂O₃）为中药砒霜、复方青黛的主要成分,我国学者将其用于白血病的治疗,取得了显著的成就。已有多项体内、体外研究发现As₂O₃对肝癌细胞有抑制增殖和诱导凋亡作用。Sorafenib和As₂O₃在治疗肝细胞癌中抑制增殖、促进凋亡的机制不同,两药联合可多靶点,多途径治疗肝细胞癌。本研究主要从体外实验研究Sorafenib和As₂O₃联合对肝细胞癌的抑制作用,分析两药联合的性质,为进一步的临床应用提供实验依据。本研究共分两个部分:第一部分索拉非尼联合三氧化二砷对肝癌细胞株增殖抑制作用的研究观察Sorafenib、As₂O₃单药和联用对HepG₂肝癌细胞株的增殖的抑制,比较两药联合作用与单药作用的差异以及分析两药的交互效应。一、研究方法及内容1.细胞培养:HepG2细胞在37℃、5%的CO₂孵箱中培养,培养基为含10%胎牛血清、1%的双抗（青霉素和链霉素）的RPMI 1640。细胞为上皮样细胞,每2～3d传代1次,并取对数生长期的细胞用于实验。2.取对数生长期HepG2细胞种板,培养24 h后加入药物。实验分组:Somfenib6、3、1.5μmol/L和As₂O₃ 4、2μmol/L分别组成单药组和Sorafenib+As₂O₃联合用药组,另设不加药的对照组,加药后分别培养24、48、72 h后,终止培养,加入MTT液,用酶标仪（波长570 nml）测定各孔的OD值。根据下列公式计算对HepG2细胞的抑制率:抑制率=（1-实验药物组OD值/空白对照组OD值）×100%3.统计方法:所有数据采用SPSS 13.0统计软件进行统计处理,实验数据用均数±标准差（（?）±s）表示。两样本均数的比较采用Independent-Samples T Test,多组间均数比较采用One-way ANOVA,post hoc test方差齐性采用LSD法,方差不齐的采用Dunnett T3法。交互效应分析采用factorial analysis。P＜0.05有统计学意义。二、主要结果1.MTT法检测显示,不同浓度的Sorafenib、As₂O₃单药对HepG2细胞均有抑制增殖的作用（P＜0.05）。Sorafenib与As₂O₃联合作用于HepG2细胞分别较两单药抑制作用更强（P＜0.05）。2.Sorafenib和As₂O₃联用在24h、48h、72h均有协同作用（P＜0.05）。三、主要结论两药单用及联用对HepG2细胞均有抑制增殖的作用,两药联用较之单药的抑制作用更强,两药联用的性质为协同。第二部分索拉非尼联合三氧化二砷对肝癌细胞株的凋亡诱导作用及机制研究观察Sorafenib、As₂O₃单药和联用对HepG₂肝癌细胞的凋亡的诱导作用,探讨两药单用与联用诱导凋亡的可能机制。一、研究方法及内容1.实验分组:Sorafenib 4μmol/L,As₂O₃ 3μmol/L,联合组Sorafenib 4μmol/L+As₂O₃ 3μmol/L,另设不加药的对照组。细胞培养:HepG2细胞在37℃、5%的CO₂孵箱中培养,培养基为含10%胎牛血清、1%的双抗（青霉素和链霉素）的RPMI 1640。细胞为上皮样细胞,每2～3 d传代1次,并取对数生长期的细胞用于实验。2.Annexin V-FITC/PI双染检测细胞凋亡。取对数生长期的人肝癌细胞株HepG2,用0.25%胰蛋白酶消化,加入含10%胎牛血清的RPMI-1640培养液制备成浓度为2×10⁵/ml的细胞悬液,接种2.5ml/孔于6孔培养板中,使其细胞数为5×10⁵个/孔。培养24h后取出培养板,对照组加入RPMI-1640培养液0.5ml;索拉非尼组添加稀释的Sorafenib药液0.5ml至终浓度3μmol/L;三氧化二砷组加入稀释的As₂O₃药液0.5ml至终浓度4μmol/L;联合用药组加入稀释的As₂O₃药液0.25ml至终浓度4μmol/L,稀释的索拉非尼药液0.25ml至终浓度为3μmol/L。置于37℃、5%CO₂培养箱继续培养48h。取出培养板,胰蛋白酶消化,收集细胞离心2000rpm×5min,弃去上清液。PBS洗涤。加入500μL的Binding Buffer悬浮细胞,加入5μL AnnexinV-FITC混匀后,加入5μL Propidium Iodide,混匀。室温避光反应5～15min。1小时内进行流式细胞仪检测。3.罗丹明123染色检测细胞线粒体膜电位变化。细胞培养及药物处理同前述。加药后48h收集细胞。加入罗丹明123染液10μg/mL。37℃,5%CO₂细胞培养箱孵育10 min,离心以培养基洗细胞两次。重悬细胞于培养基中,37℃,5%CO₂培养60 min。流式细胞仪检测。计算相对荧光强度,相对荧光强度=加药组荧光强度/对照组荧光强度。4.比色法检测caspase-3的活化程度。细胞培养及药物处理基本同前述。加药后48h收集细胞。用PBS洗涤细胞2次。在收集的沉淀细胞中加/250μL冰冷LysisBuffer,吹打均匀。置冰上裂解30min,其间涡旋振荡3～4次,每次10 s。4℃,离心（10,000 rpm）1 min。小心吸取上清转移至新的管中,并放置冰上待用。取1μL上清,常规方法BCA法测定其中的蛋白浓度。吸取50μL含200μg蛋白的细胞裂解上清。加入50μL的2×Reaction Buffer,使用前每50μL 2×Reaction Buffer加入0.5μL DTT。加入5μL Caspase-3 Substrate并于37℃避光孵育4h。用酶标仪在λ=405nm测定其吸光值。通过计算OD_处理组/OD_对照组的倍数来确定处理组Caspase-3活化程度。5.统计方法:所有数据采用SPSS 13.0统计软件进行统计处理,实验数据用均数±标准差（（?）±s）表示,均数比较采用one-way ANOVA。post hoc test方差齐性采用LSD法,方差不齐的采用Dunnett T3法。P＜0.05有统计学意义。二、主要结果1.As₂O₃ 4μmol/L与Sorafenib 3μmol/L及联合组作用于HepG2细胞24h后检测细胞的早期凋亡率,As₂O₃ 4μmol/L单药、Somfenib 3μmol/L单药、As₂O₃ 4μmol/L+Sorafenib 3μmol/L联合组及对照组的差异有统计学意义（P＜0.05）。进一步两两比较显示,联合组较Sorafenib单药、As₂O₃单药、对照组的早期凋亡率的差异均有统计学意义（P＜0.05）。较之As₂O₃、Sorafenib单药组,联合组对HepG2细胞凋亡诱导作用更强。2.检测线粒体膜电位罗丹明123染色后的荧光强度,计算相对荧光强度。As₂O₃4μmol/L单药、Sorafenib 3μmol/L单药、As₂O₃ 4μmol/L+Sorafenib 3μmol/L联合组及对照组的差异有统计学意义（P＜0.05）。进一步两两比较显示,联合组较Sorafenib单药、As₂O₃单药、对照组的相对荧光强度变化均有统计学意义（P＜0.05）。即较之As₂O₃、Sorafenib单药组,药物联合作用后HepG2细胞线粒体膜电位下降更大。3.比色法检测caspase-3的活化程度。As₂O₃ 4μmol/L单药、Sorafenib 3μmol/L单药、As₂O₃ 4μmol/L+Sorafenib 3μmol/L联合组及对照组的OD_处理组/OD_对照组的差异有统计学意义（P＜0.05）。进一步两两比较显示,联合组较Sorafenib单药、As₂O₃单药的Caspase-3活化程度均有统计学意义（P＜0.05）。较之As₂O₃、Sorafenib单药组,药物联合作用于HepG2细胞后Caspase-3活化程度更大,诱导细胞凋亡的作用更强。三、主要结论与Sorafenib、As₂O₃单独用药比较,联合用药诱导凋亡的作用更强。联合用药凋亡诱导作用增强可能与两药作用于信号传导通路的不同靶位,导致caspase-3蛋白活化增加、线粒体凋亡途径激活增强有关。更多还原

【Abstract】 Primary hepatic carcinoma（PHC） is considered as one of the most common cancer in the world.And it has been ranked as the second cause of cancer mortality in China.The potentially curative therapies of late stage hepatocellular carcinoma （HCC） are low effective,so finding active and reasonable treatments is significant.Sorafenib is a multikinase inhibitor that has shown to block tumor cell proliferation and angiogenesis by inhibiting serine/threonine kinases,as well as the receptor tyrosine kinases vascular endothelial growth factor receptor 2（VEGFR2）, vascular endothelial growth factor receptor 3（VEGFR3）,platelet-derived growth factor receptor（PDGFR）,FLT3 and C-KIT.In 2007 ASCO manual meeting,a phaseⅢrandomized placebo-controlled trial（SHARP trial） was reported.It confirmed that Sorafenib improved 44%of HCC patients’ survival time than the placebo,MST was 10.7 months and TTP was 5.5 months in the Sorafenib team.At the end of 2007, FDA approved sorafenib for patients with inoperable liver cancer.Arsenic trioxide （As₂O₃） has shown remarkable curative effect in the treatment of acute promyelocytic Leukemia（APL）.Studies have showed that As₂O₃ could inhibition of HCC in vitro and in vivo.As Sorafenib and As₂O₃ has different mechanism of inducing apoptosis and inhibiting proliferation on tumor cell,it is possible that the two drugs can treat HCC through different ways and targets.This study was designed to investigate that the nature of sorafenib combined with As₂O₃ on hepatocellular carcinoma cells.The first part:Inhibitory proliferation effect of Sorafenib combined with Arsenic trioxide on hepatocellular carcinoma cellsMethods:1.HepG2 human HCC tumor cells were cultured with RPMI 1640 containing 10%fetal calf serum.Cells in exponentially growing period were chosen for experiment.HepG2 cells were divided into 4 groups:control group,Sorafenib-treated group,As₂O₃-treated group and combination treatment group.MTT assay analyzed cells treated with Sorafenib（1.5、3、6μmol/L） alone,As₂O₃（2、4μmol/L） alone,Sorafenib combined As₂O₃ and cells of control group for 24, 48 and 72 hours respectively.The inhibitory rates of cells were calculated according OD value.2.Statistical analysis:All datas were analysised by SPSS 13.0 statistical software. Datas were expressed as（?）±s.The inhibitory rates were analyzed by One-way ANOVA followed by post hoc test.P＜0.05 was considered to be significant.Results:1.The MTT result indicated:Sorafenib,As₂O₃ alone or together could inhibit the proliferation of HepG2 cells（P＜0.05）.Sorafenib combined with As₂O₃ enhanced inhibition of proliferation in HepG2 cells（P＜0.05）.2.There was a synergistic effect in Sorafenib combined with As₂O₃ at 24h,48h,72h on HepG2 cells（P＜0.05）.Conclusion:Sorafenib,As₂O₃ alone or together could inhibit the proliferation of HepG2 cells, the combination of Sorafenib and As₂O₃ showed stronger inhibition.The nature of the interaction of combination was synergistic.The second part:Induction apoptosis effect of Sorafenib combined with Arsenic trioxide on hepatocellular carcinoma cellsMethods:1.HepG2 human HCC tumor cells were cultured as the above mentioned.Cells of exponentially growing period were chosen for experiment.HepG2 cells were divided into 4 groups:control group,Sorafenib-treated group（Sorafenib 3μmol/L）, As₂O₃-treated group（As₂O₃ 4μmol/L） and combination treatment group（Sorafenib 3μmol/L+As₂O₃ 4μmol/L）.2.Group as the above mentioned,the apoptosis rate of HepG2 cells labeled by Annexin V-FITC/PI was observed by FCM.3.Group as the above mentioned,cells collected and that changes of mitochondrial membrane potential（Δψm） labeled by Rhodamine123 was examined by FCM.4.Group as the above mentioned,the activity of caspase-3 were examined by colorimetric.5.Statistical analysis:All datas were analysised by SPSS 13.0 statistical software. Datas were expressed as（?）±s.The data were analyzed by One-way ANOVA followed by post hoc test.P＜0.05 was considered to be significant.Results:1.Compared with untreated control,Sorafenib,As₂O₃ alone and together induced more HepG2 cells apoptosis（P＜0.05）.Induction of cell apoptosis was enhanced by combinaton of Sorafenib and As₂O₃ compared with each agent alone.2.The mitochondrial membrane potential（Δψm） was decreased by As₂O₃,sorafenib alone or combination.The effect of the agents combined was stronger than that alone （P＜0.05）.3.The activity of the caspase-3 increased in the three treated groups.At the same time,the combination group increased more significantly than the Sorafenib group, As₂O₃ group alone respectively.Conclusion:Sorafenib combined with As₂O₃ showed synergistic effect on induction apoptosis of HepG2 cells.The mechanisms were closely related to the promotion of apoptosis by influence several signaling pathways such as the mitochondria-dependent and caspase- dependent ways.更多还原