【作者】 侯文静；

【导师】 刘彦；

【作者基本信息】 第二军医大学 ， 妇产科学， 2008， 博士

【摘要】 近年来紫杉醇联合顺铂的化疗方案(PT方案)已广泛应用于临床治疗卵巢癌,尤其是卵巢癌晚期无法手术的姑息治疗、一线化疗失败或多次化疗失败的转移性卵巢癌,为了防止化疗时紫杉醇药物发生严重的过敏及恶心呕吐等反应,在接受化疗前的所有患者应事先进行地塞米松预防用药。但近来许多研究表明,地塞米松能选择性抑制紫杉醇诱导一些肿瘤细胞的凋亡,下调机体的免疫反应,导致肿瘤组织转移,影响紫杉醇在恶性肿瘤化疗中的疗效,那么地塞米松对紫杉醇诱导卵巢癌细胞凋亡的作用如何,是否拮抗紫杉醇诱导卵巢癌细胞凋亡从而降低紫杉醇的疗效?因此,本项目以人卵巢癌细胞株SKOV-3为材料,通过体外实验探讨地塞米松预处理对紫杉醇诱导卵巢癌细胞凋亡产生的影响、发生的可能机制,并建立SKOV-3裸鼠移植瘤,通过体内实验进一步探讨地塞米松对紫杉醇抗肿瘤疗效的影响,对今后在临床工作中如何正确认识和使用地塞米松以及开发紫杉醇的给药途径必将产生深远的影响。第一部分地塞米松对紫杉醇诱导人卵巢癌细胞株SKOV-3细胞凋亡的保护作用目的探讨地塞米松(Dex)对紫杉醇(PTX)抑制人卵巢癌细胞株SKOV-3细胞增殖及诱导SKOV-3细胞凋亡的影响。方法(一)MTT法测定细胞增殖1、用含不同浓度紫杉醇(10 nM、20 nM、50 nM、100nM)的培养液培养SKOV-3细胞24小时,测定紫杉醇对SKOV-3细胞增殖的影响。2、用不同浓度地塞米松(0、0.001 uM、0.01 uM、0.1 uM、1uM)对SKOV-3细胞进行预处理24h后,每组中分别再用含不同浓度的紫杉醇(0、10 nM、20 nM、50 nM、100nM)培养液培养细胞24h,测定不同浓度的地塞米松对不同浓度的紫杉醇进行预处理后对SKOV-3细胞增殖的影响。(二)TUNEL联合DAB染色从形态学观察地塞米松对紫杉醇诱导人卵巢癌细胞株SKOV-3细胞凋亡的影响。(三)PI单染法测定地塞米松对紫杉醇诱导人卵巢癌细胞株SKOV-3细胞凋亡及细胞周期的影响。结果(一)PTX能够抑制人卵巢癌细胞株SKOV-3细胞增殖,且与PTX的浓度有关,浓度越高,抑制率越大。(二)0.001～1uM浓度范围内地塞米松对紫杉醇抑制人卵巢癌细胞株SKOV-3细胞增殖有不同程度的保护作用。(三)TUNEL法联合DAB染色进行形态学观察,对照组中极少见到凋亡细胞,PTX组出现大量DAB染的胞核呈棕黄色的凋亡细胞,表现为核固缩,染色质边聚,凝集成块,并可见散在棕色的凋亡小体。但预先用地塞米松进行处理后再用PTX处理组出现凋亡细胞较单用紫杉醇明显减少。凋亡指数(AI)在对照组和Dex组分别为3.5±0.25%和4.0±0.26%两组间无差异,在Dex+PTX组与PTX组为11.7±1.72%和30.8±1.15%,两组与对照组比较差异显著(P＜0.01),且Dex+PTX组与PTX组比较,Dex+PTX组凋亡指数降低,有明显差异(P＜0.01)。(四)PI单染检测细胞凋亡率表明地塞米松进行预处理后再用紫杉醇组细胞凋亡率为5.55±0.53%较单用紫杉醇组细胞凋亡率13.90±1.62%明显降低(P＜0.01),而且在地塞米松拮抗紫杉醇诱导SKOV-3细胞凋亡中,用地塞米松预处理后再用紫杉醇组与单用紫杉醇组比较,细胞周期中G2/M期无明显差异(P＞0.05)。结论(一)地塞米松对紫杉醇抑制人卵巢癌细胞株SKOV-3细胞增殖有保护作用,且与地塞米松的给药浓度有关。(二)地塞米松能拮抗紫杉醇诱导人卵巢癌细胞株SKOV-3细胞凋亡,但与细胞周期中G2/M期停滞无关。第二部分地塞米松拮抗紫杉醇诱导人卵巢癌细胞株SKOV-3细胞凋亡的可能机制目的从IAP家族中抗凋亡基因Survivin和Bcl-2家族中抗凋亡基因Bcl-2、Bcl-X_L两条通路,探讨地塞米松拮抗紫杉醇诱导人卵巢癌细胞株SKOV-3细胞凋亡的可能机制,并为下一步的动物实验奠定基础。方法将细胞用不同药物处理后,用半定量RT—PCR检测凋亡相关基因Bcl-2、Bcl-X_L and Survivin mRNA的表达。Western blot检测Bcl-X_L和末端半胱氨酸蛋白酶Cleaved-caspase-3蛋白的表达。结果(一)用地塞米松进行预处理后再用紫杉醇组(Dex+PTX组)与单用紫杉醇组(PTX组)比较,Dex+PTX组Bcl-X_L mRNA、Survivin mRNA的表达明显高于PTX组(P＜0.01)。(二)Bcl-2蛋白家族成员Bcl-2 mRNA的表达:Dex组和Dex+PTX组Bcl-2蛋白家族成员Bcl-2 mRNA的表达较对照组无差异(P＞0.05),Dex+PTX组与PTX组之间比较也无差异(P＞0.05)。(三)Bcl-X_L蛋白水平表达:Dex组较对照组升高(p＜0.01),而且用地塞米松进行预处理后再用紫杉醇组(Dex+PTX组)与单用紫杉醇组(PTX组)比较,Dex+PTX组Bcl-X_L蛋白表达明显高于PTX组(P＜0.01)。(四)Cleaved caspase-3蛋白水平表达:Dex组和Dex+PTX组较对照组明显降低(p＜0.01),而且用地塞米松进行预处理后再用紫杉醇组(Dex+PTX组)与单用紫杉醇组(PTX组)比较,Dex+PTX组Cleaved caspase-3蛋白表达明显低于PTX组(P＜0.01)。结论地塞米松对紫杉醇的抗凋亡作用可能通过上调IAP家族中Survivin和Bcl-2家族中Bcl-X_L抗凋亡基因的表达,下调caspase3活性,从而抑制人卵巢细胞株SKOV-3细胞凋亡。第三部分地塞米松在人卵巢癌细胞株SKOV-3裸鼠移植瘤中对紫杉醇抗肿瘤疗效的影响目的通过建立人卵巢癌细胞株SKOV-3裸鼠移植瘤模型进行体内实验,评价地塞米松进行预处理后,对紫杉醇抗肿瘤作用的影响,并进一步分析其作用机理。方法(一)建立人卵巢癌细胞株SKOV-3裸鼠移植瘤的模型。(二)分组及给药:(1)对照组:生理盐水(含0.001%无水乙醇),腹腔内注射;(2)地塞米松组:1mg/kg,腹腔内注射;(3)紫杉醇组:20mg/kg,尾静脉注射;(4)地塞米松+紫杉醇组:在其它三组给药前12小时,预先给予地塞米松1mg/kg,腹腔内注射,然后给予紫杉醇20mg╱kg,尾静脉注射。三天一个疗程,共6个疗程。(三)观察移植瘤的生长,绘制裸鼠移植瘤的生长曲线,实验结束称瘤体的重量,计算肿瘤生长抑制率。(四)免疫组化分析:Bcl-X_L和Cleaved caspase-3蛋白的表达,细胞增殖标记物Ki-67的表达。(五)光镜和透射电镜下观察组织形态和细胞超微结构改变。结果(一)在紫杉醇对SKOV-3裸鼠移植治疗中,用地塞米松进行预处理后再用紫杉醇组,移植瘤的体积较单用紫杉醇组增大(P＜0.01)。(二)治疗结束后称瘤重,Dex+PTX治疗组瘤重为0.79±0.09g,PTX组瘤重为0.50±0.08g,Dex+PTX组较PTX组瘤重升高(P＜0.01)。Dex+PTX组和PTX组抑制率分别为45.14%和65.28%,PTX组较Dex+PTX组抑制率大约增加18—25%。(三)HE染色后,PTX治疗组绝大部分细胞变大,细胞质较其它组明显减少,细胞表现为空泡和凋亡特征。在Dex+PTX组中也有一些细胞表现为空泡和凋亡特征,但明显少于PTX组。电镜下Dex+PTX组和PTX组可见核固缩,染色质边聚,凝集成块等细胞凋亡征象,但Dex+PTX组中细胞凋亡征象少于PTX组。(四)免疫组化结果表明,Dex+PTX组与PTX组相比,在Dex+PTX组中Ki-67细胞增殖标记物、Bcl-X_L蛋白表达升高,Cleaved caspase-3蛋白表达降低,有显著性差异,P＜0.0125。结论(一)体内实验证实地塞米松能降低紫杉醇对人卵巢癌SKOV-3裸鼠移植瘤治疗效果。(二)体内实验证实地塞米松能拮抗紫杉醇诱导的人卵巢癌SKOV-3裸鼠移植瘤细胞凋亡,可能通过上调Bcl-2家族中Bcl-X_L抗凋亡基因,下调Cleaved-caspase3的活性。更多还原

【Abstract】 In recent years,the chemotherapy of paclitaxel combined cisplatin has been widely used in the treatment of ovarian cancer,particularly for advanced that could not be operated,drug-refractory and metastatic ovarian cancer.Dexamethasone was routinely used as a premedication in the clinical applicantion of paclitaxel to prevent hypersensitivity and nausea.However,recently studies discovered that dexamethasone could selectively inhibit paclitaxel-induced apoptosis in a number of human solid tumor cells.It can downregulate the immune response and contribute to tumour metastasis.Pretreatment with dexamethasone interfered with the therapeutic efficacy of Paclitaxel.How is the influence of dexamethasone on paclitaxel-induced apoptosis in ovarian cancer cells? If dexamethasone can inhibit paclitaxel-induced apoptosis in ovarian cancer cells and reduce the therapeutic efficacy of paclitaxel.Therefore,we performed in vitro experiments with human ovarian cancer SKOV-3 cell line to evaluate the effects and mechanisms of dexamethasone on human ovarian cancer cell apoptosis induced by paclitaxel and to explore the influence of dexamethasone on antitumor therapeutic efficacy of paclitaxel in vivo through development of animal models bearing ovarian xenograft tumor.This finding may have a potential implication on how to use correctly dexamethasone in the clinical practice and to develop new drug route of paclitaxel.PartⅠProtective effect of dexamethasone on apoptosis of human ovarian cancer cell line SKOV-3 induced by paclitaxelObjective To observe the effects of dexamethasone on apoptosis of human ovarian cancer cell line SKOV-3 induced by paclitaxel.Methods Human ovarian cancer cell line SKOV-3 was pretreated with dexamethasone at different concentrations for 24 hours,then was treated with Paclitaxel at different concentrations for 24 hours.MTT assay was used to examine the cell proliferation and the growth inhibition of SKOV-3 cell after treatments with dexamethasone and paclitaxel.The cell apoptotic rate of human ovarian cancer cell line SKOV-3 induced by paclitaxel in the presence or absence of dexamethasone was detected by TUNEL assay.The change of cell cycle and percentage of apoptosis of human ovarian cancer cell line SKOV-3 induced by paclitaxel in the presence or absence of dexamethasone was measured by PI-staining,and then analyzed by FCM.Results Paclitaxel can inhibit the cell proliferation of human ovarian cancer cell line SKOV-3 and was relative with the concentration.It was revealed that 0.001 uM～luM dexamethasone can protected differently the inhibition of human ovarian cancer cell line SKOV-3 proliferation induced by Paclitaxel.TUNEL and DAB staining assay showed that there was rarely apoptotic cells in control group,but there were many apoptotic cells with DAB-stained buffy nuclers,karyopycnosis or chromatin aggregation in human ovarian cancer cell line SKOV-3 induced by paclitaxel in the presence or absence of dexamethasone.The apoptotic rate was 3.5±0.25%and 4.0±0.26%in control group and Dex group with no difference between each other.The apoptotic rate was 11.7±1.72% and 30.8±1.15%in paclitaxel in the presence or absence of dexamethasone group,with significant difference between each other(p＜0.01).The apoptosis percentage of human ovarian cancer cell line SKOV-3 induced by paclitaxel and pretreated with dexamethasone measured by PI-staining was 5.55±0.53%,which was less than the apoptosis percentage 13.90±1.62%treated only with Paclitaxel(p＜0.01).The percentage of cell arrested by PTX in the G2/M phase was no significant difference in paclitaxel in the presence or absence of dexamethasone group(p＞0.05).Conclusion Dexamethasone can protected differently the inhibition of human ovarian cancer cell line SKOV-3 proliferation induced by paclitaxel and was relative with the concentration of dexamethasone.Dexamethasone can inhibit apoptosis of human ovarian cancer cell line SKOV-3 induced by paclitaxel without affecting the ability of paclitaxel to induce mitotic division arrest in the G2/M phase.PartⅡPossible mechanisms of dexamethasone inhibit apoptosis of human ovarian cancer cell line SKOV-3 induced by PaclitaxelObjective To investigate the possible mechanisms of dexamethasone inhibiting apoptosis of human ovarian cancer cell line SKOV-3 induced by paclitaxel.The experiment will settle ground work for the further study in vivo.Methods The expression levels of Survivin,Bcl-2,Bcl-X_L mRNA were determined by semi- quantiative RT-PCR after cells were treated by different methods.Western blot analysis was used to detect the expression levels of Bcl-X_L anti-apoptosis protain and activity of Caspase-3.Results The expression of Bcl-X_L,Survivin mRNA treated with dexamethasoneprior to paclitaxel were higher than treated only with paclitaxel(p＜0.01).The expression of Bcl-2 mRNA treated with dexamethasone prior to paclitaxelwas nearly same compared with treated only with paclitaxel(p＞0.05).The expression of Bcl-X_L protain in dexamethasone group is higher than control group(p＜0.01).The expression of Bcl-X_L protain treated with dexamethasone prior to paclitaxel was higher than treated only with paclitaxel(p＜0.01).The expression of Cleaved caspase-3 protain treated treated with dexamethasone prior to paclitaxel was lower than treated only with Paclitaxel(p＜0.01).Conclusion Pretreatment with dexamethasone can inhibit the paclitaxel -induced apoptosis of human ovarian cancer cell line SKOV-3.The possibbe mechanism is down- regulation of caspase-3 activity by Bcl-X_L,Survivin anti- apoptosis pathways.PartⅢDexamethasone intereferes with therapeutic efficacy of paclitaxel against human ovarian cancer cell line SKOV-3 xenograft tumorsObjective We performed in vivo experiments through development of human ovarian cancer cell line SKOV-3 xenograft tumor models.We evaluate the potential effect of dexamethasone on the antitumor activity of paclitaxel in ovarian cancer cell line SKOV-3 xenograft tumors and analyze the mechanism furtherly.Methods We firstly developed the model of human ovarian cancer cell line SKOV-3 xenograft tumors,then mice were randomly divided into 4 groups(10 mice per group) and treated with various regimens on day 0(1) Control;(2) Dexamethasone alone at 1 mg/kg,ip;(3) Combination of dexamethasone and paclitaxel,in which dexamethasone was administered 12 h before paclitaxel treatment;(4) Paclitaxel alone at 20 mg/kg,iv.The same treatment regimens were repeated every 3 days for a total of 6 cycles.We observed the growth of human ovarian cancer cell line SKOV-3 xenograft tumors and drew the curve of xenograft tumors.After the mice were killed,the tumors were removed and their weights were measured.The inhibition rate of xenograft tumors growth was calculate.Immunohistochemistry assay the expression of Bcl-X_L,Cleaved caspase-3 protain and proliferation marker Ki-67 of human ovarian cancer cell line SKOV-3 xenograft tumors after 6 cycles of treatments.Under the microscope and transmission electric micro- scope,we observed the tissue morphous and ultramicrostructure.Results Nude mice bearing SKOV-3 xenograft tumors were treated with paclitaxel with pretreatment of dexamethasone,the xenograft tumors volumes were bigger than those in mice treated with paclitaxel alone(p＜0.01).The weight of xenograft tumors at the end of experiments for treated with paclitaxel with or without dexamethasone were 0.79±0.09g and 0.50±0.08g,respectively.The weight of xenograft tumors treated with paclitaxel with dexamethasone was more than treated with paclitaxel without dexamethasone.The inhibitory rate of paclitaxel alone was 65.28%.However,when the mice was pretreatment with dexamethasone,the IR of paclitaxel on SKOV-3 xenograft tumors dropped to 45.14%,which was 18-25%less than those in mice treated with paclitaxel alone.SKOV-3 xenograft tumors stained with HE,most xenograft tumor cells after the treatment of paclitaxel alone were found to become much larger,and the cellularity was reduced significantly when compared with that in any other groups.In addition,the SKOV-3 xenograft tumor cells exhibited typical apoptotic characteristics.In the group treated with combination of dexamethasone and paclitaxel,there were also many xenograft tumor cells exhibiting vacuolization and apoptotis features,but less than that in the group treated with paclitaxel alone.It was showed by electronic microscope that karyopycnosis or chromatin aggregation was more in the group treated with paclitaxel alone than in the group administered with dexamethasone prior to paclitaxel.The immunochemical results suggested that the expression of proliferation marker Ki-67 and Bcl-X_L protain in the group administered with dexamethasone prior to paclitaxel was significantly higher when compared with that in the group treated with paclitaxel alone(p＜0.0125).The expression of Cleaved caspase-3 protain in the group administered with dexamethasone prior to paclitaxel was significantly lower when compared with that in the group treated with paclitaxel alone(p＜0.0125).Conclusion Pretreatment with dexamethasone significantly interfered with the therapeutic activity of paclitaxe in human ovarian cancer cell line SKOV-3 xenograft tumors in vivo.Dexamethasone can inhibit paclitaxel-induced apoptosis in human ovarian cancer cell line SKOV-3 xenograft tumors in vivo.The possibbe mechanism is down-regulation of caspase-3 activity by up-regulation of Bcl-X_L anti- apoptosis pathways.更多还原