【作者】 刘川；

【导师】 吴小候；

【作者基本信息】 重庆医科大学 ， 外科学， 2008， 博士

【摘要】 近年来,人们发现凋亡过程受到抑制在肿瘤的发生、发展中起到非常关键的作用。其中IAPs在细胞的凋亡中扮演非常重要的角色,某些IAPs成员异常高表达常引起组织细胞凋亡受阻,与肿瘤的发生密切相关。目前已发现的人类IAP家族有8个,包括NAIP、ILP-2、c-IAPl、c-IAP2、XIAP、Bruce、survivin及Livin。其中Livin是最近发现的IAPs家族新成员,主要在一些肿瘤组织细胞中高表达,在正常组织不表达或极少表达,可能成为肿瘤治疗的靶点。我们的前期研究在膀胱癌患者尿液中也发现有Livin mRNA的表达,而健康志愿者无表达。目的:在我们的前期工作基础上,我们拟利用反义核酸技术,在体、内外阻断膀胱癌细胞Livin mRNA及蛋白质的表达,观察癌细胞的生长以及凋亡是否受到影响,以明确Livin在膀胱癌细胞中的作用,并初步探讨其机制。为以Livin为靶基因的膀胱癌的治疗提供理论依据。方法:一.设计、合成全硫代修饰Livin反义寡核苷酸序列及其功能鉴定。1.参考文献设计、筛选、合成全硫代修饰Livin反义寡核苷酸。同时设计一条错义序列寡核苷酸作为对照。2.制作Livin反义寡核苷酸- Lipofectamine?2000复合物,将其转染入5637膀胱癌细胞中。3.荧光显微镜观察转染效率。4.用RT-PCR、Western blot、细胞免疫荧光检测转染后膀胱癌细胞Livin mRNA及蛋白表达的变化。二.细胞分组根据向培养细胞中加入的成分不同,分为: (1)反义组:加入反义寡核苷酸; (2)错义组:加入错义的寡核苷酸; (3)脂质体组:只加入等量稀释的脂质体; (4)PBS(磷酸缓冲液)组:只加入等量的PBS。三.Livin反义寡核苷酸对人膀胱癌细胞株5637生物学活性的影响1.以不同浓度的Livin反义寡核苷酸转染培养的膀胱癌细胞,用MTT法测定细胞增殖活性。从中筛选出适当的Livin反义寡核苷酸浓度,进行下一步实验。2.Livin反义寡核苷酸对膀胱癌细胞的生物学效应①激光共聚焦显微镜、透射电子显微镜观察膀胱癌细胞转染Livin反义寡核苷酸后的形态学变化。②AO/EB双染色、Annexin V-FITC细胞凋亡检测试剂盒流式细胞仪检测细胞凋亡情况。四.Livin反义寡核苷酸诱导荷瘤鼠膀胱癌组织凋亡的研究1.裸鼠膀胱癌移植瘤模型的建立采用皮下注射法建立裸鼠膀胱癌移植瘤模型。2.给药方法及分组采用瘤体内5点注射法,根据注射核酸的不同,将裸鼠随机分为对照组(瘤体内注射错义寡核苷酸)和反义组(瘤体内注射反义寡核苷酸)。3.Livin反义寡核苷酸对荷瘤鼠肿瘤组织Livin表达的影响用RT-PCR、Western blot检测检测转染后膀胱癌细胞Livin mRNA及蛋白表达的变化。4.Livin反义寡核苷酸对荷瘤鼠肿瘤组织的生物学效应①用游标卡尺测量、计算肿瘤体积,绘制癌细胞体内生长曲线。接种后第30天剥瘤称重。②H.E染色光镜观察肿瘤组织转染Livin反义寡核苷酸后的形态学变化。③TUNEL法检测荷瘤鼠肿瘤细胞凋亡情况。④免疫组化检测荷瘤鼠肿瘤caspas-3的表达情况。结果:一.Livin反义寡核苷酸能有效转染入膀胱癌细胞内并抑制Livin mRNA及蛋白的表达。将FITC标记的Livin ASODN转染膀胱癌细胞后,在荧光显微镜下观察,见约55%的细胞染上绿色荧光,说明Livin ASODN能有效地转染入5637细胞中。RT-PCR扩增后显示5637细胞表达livinα和livinβ,但反义组的电泳带亮度明显低于错义组、脂质体组及PBS组,而后三组间亮度相似。反义组livin两个片段的mRNA相对表达量(0.46±0.05)较错义组(0.88±0.05)、脂质体组(0.92±0.04)、PBS组(0.91±0.06)明显减弱,P<0.01,而各对照组间比较无显著性差异,P>0.05。采用Western blot检测各组细胞中livin蛋白的表达,结果反义组livinα和livinβ杂交带亮度明显低于错义组、脂质体组和PBS组。细胞免疫荧光染色后用激光共聚焦显微镜观察,发现反义组癌细胞标记livin的绿色荧光明显减弱,且见有的细胞体积明显缩小,完全无绿色荧光。其余各对照组无明显变化。说明Livin反义寡核苷酸能有效抑制膀胱癌细胞Livin mRNA及蛋白的表达。二.Livin反义寡核苷酸对人膀胱癌细胞增殖活性的影响MTT法发现Livin反义寡核苷酸能抑制人膀胱癌细胞的增殖活性,且其抑制作用呈明显的浓度依赖性。我们据此选择160nmol/L的寡核苷酸浓度进行下一步实验。三.Livin反义寡核苷酸对膀胱癌细胞的生物学效应①细胞形态的变化:激光共聚焦显微镜、透射电子显微镜下见反义组可见较多细胞体积肿大、细胞器肿胀、变性坏死。有的细胞体积缩小,发生凋亡,甚至形成凋亡小体。②细胞凋亡率的变化:AO/EB双染色、Annexin V-FITC标记凋亡细胞,流式细胞仪检测细胞凋亡率,结果发现反义组的细胞凋亡率(46.39±9.23)%显著高于PBS组(4.54±1.84)%、脂质体组(5.70±1.61)%及错义组(5.10±1.56)%,P﹤0.01。而后三组间凋亡率差异无显著意义,P﹥0.05。三.Livin反义寡核苷酸对荷瘤鼠肿瘤组织的抑制作用1.Livin反义寡核苷酸对荷瘤鼠肿瘤组织Livin表达的影响RT-PCR扩增后显示,反义组荷瘤鼠肿瘤组织表达livinα和livinβ的电泳带亮度明显低于对照组。Western blot检测荷瘤鼠肿瘤组织livin蛋白的表达,结果反义组livinα和livinβ杂交带亮度明显低于对照组。免疫组化也显示反义组livin表达减弱(其面积积分光密度为:0.1661±0.1028,对照组为0.2490±0.1483,P﹤0.05)说明Livin反义寡核苷酸在体内也能有效抑制膀胱癌细胞Livin mRNA及蛋白的表达。2.Livin反义寡核苷酸对对荷瘤鼠肿瘤组织的生物学效应①肿瘤生长的变化:通过测量、计算肿瘤体积,绘制癌细胞体内生长曲线。发现从肿瘤接种后的第18天开始至接种后第30天,反义组肿瘤体积显著小于对照组,P<0.05。接种后第30天剥瘤称重,反义组瘤块重量为1.31±0.88g,明显轻于对照组(2.41±0.41g),P<0.05。说明Livin反义寡核苷酸能在体内抑制了肿瘤的生长。②H.E染色光镜观察肿瘤组织转染Livin反义寡核苷酸后的形态学变化:可见反义组肿瘤组织有局灶变性、坏死现象及炎症细胞浸润。③肿瘤细胞凋亡情况:TUNEL法检测荷瘤鼠肿瘤组织凋亡指数,结果发现反义组为19.60±5.91,明显高于对照组(3.48±2.35),P﹤0.05。④免疫组化检测荷瘤鼠肿瘤caspas-3的表达情况:反义组肿瘤组织caspas-3表达显著强于对照组(面积积分光密度分别为:反义组0.4501±0.1618,对照组0.3601±0.1065,P﹤0.05)。说明Livin反义寡核苷酸抑制了Livin的表达后,解除了Livin对caspas-3的抑制作用,从而使caspas-3的表达增强。结论:一.人膀胱癌细胞株5637表达Livin两个异构体。二.Livin在人膀胱癌细胞的增殖及凋亡过程中扮演重要角色。三.Livin抑制膀胱癌细胞凋亡的可能机制之一是其抑制了Caspase-3的活性。四.我们合成的Livin反义寡核苷酸在体内、外均能有效抑制膀胱癌细胞Livin mRNA及蛋白的表达、抑制癌细胞的生长、诱导癌细胞凋亡。五.Livin反义寡核苷酸可能成为膀胱癌基因治疗的潜在方法之一。更多还原

【Abstract】 Background:The inhibitor of apoptosis proteins (IAPs) are a family of highly conserved cell apoptosis inhibitors that have been found in yeast, invertebrates and vertebrates. The basal level of apoptosis is tightly controlled by endogenous IAPs in mammalian cells. The dysregulation of IAPs expression results in Tumorigenesis. Up to now, eight IAP-family members have been identified in human’s cells: NAIP, c-IAP1 (MIHB, HIAP-2), c-IAP2 (HIAP-1, MIHC, API2) .XIAP (hILP, MIHA, ILP-1), Survivin, BRUCE (apollon), ILP-2 and Livin (ML-IAP, KIAP). Livin is one of the novel human IAPs family members, which encodes negative regulatory proteins that prevent cell apoptosis. It was expressed during the development of embryonal. Interestingly, it was not detected in human adult tissues but expressed in the transformed cell lines and a variety of human tumors, including melanoma, bladder cancer, renal cancer, lung cancer, neuroblastomas and leukemia. It could be involved in carcinogenesis and it was important for cell survival in carcinomas. Clinical experiments also demonstrated that livin may be involved in the progression of superficial bladder cancer and used as a marker of early recurrence. In 30 patients with bladder cancer, Livin expression was measured by reverse transcription-PCR. In this study, patients with increased Livin had a shorter time to relapse compared with patients without increased Livin expression (3.5 versus 25.8 months).Our former study was to detect of Livin expression in urine of bladder cancer patients by using RT-PCR. We found that 42 of 52(86.5%)patients with bladder cancer showed positive Livinαsignals in urine,while none showed positive Livinβsignals in urine;none showed positive Livinαand Livinβsignals in urine from non-cancerous patients or healthy volunteers.It shows that Livinαis applicable to detect cancer cells in urine of patients with bladder cancer and may be helpful in screening bladder cancer.Objective:To investigate the effect of antisense o1igonuc1eotides targeting livin on the inhibition of livin mRNA and protein expression and apoptosis in human bladder cancer cells in vitro and in vivo. To discuss the mechanism of inhibition effect to bladder cancer cells apoptosis by Livin gene. To investigate the potency of Livin gene as a new target for bladder cancer’s treatment.Methods:1. Specific phosphorothioate antisense oligodeoxynucleotide targeting Livin was synthesized, and phosphorothioate missense oligodeoxy- nucleotide was synthesized as control nucleotide. And were transfected into bladder cancer cells.2. The inhibiton effect of Livin antisense oligodeoxynucleotide (ASODN) on the biological effect of bladder cancer cell line (5637).(1)Transfected Livin ASODN into bladder cancer cells in different concentrations. The inhibition of the proliferating of 5637 cells was assayed by MTT method.(2) Investigate the effect of antisense o1igonuc1eotides targeting livin on the inhibition of livin mRNA and protein expression by RT-PCR, Western blot and immunofluorescence in 5637 cells.(3)Biological effect of Livin ASODN on 5637 cells①Morphology of 5637 cells transfected with Livin ASODN was observed by electromicroscope and confocal laser scanning microscope (CLSM).And the expression and location of livin were observed by CLSM.②Apoptosis rate of 5637 cells transfected with Livin ASODN was assayed by AO/EB stain and flow cytometry.3. Investigate the inhibitory effects of Livin ASODN on development and growth ability of Human bladder cancer cells xenograft in nude mice and it’s mechanism.(1) A human bladder cancer animal model was set up by hypodermic injection of 5637 cells into nude mice.(2) The tumor-bearing mice were randomized into 2 groups: ASODN group , and MSODN group. The o1igonuc1eotides were separately injected into the lumor bodies of mice.(3) Investigate the effect of livin ASODN on the inhibition of livin mRNA and protein expression by RT-PCR, Western blot and immuno- histochemistry of human bladder cancer cells xenograft in nude mice.(4) Biological effect of Livin ASODN on xenograft in nude mice.①The tumor weight and volume were measured.②Morphology of xenograft in nude mice was observed by microscope.③Apoptosis index of xenograft in nude mice was assayed by TUNEL stain.④The expression of caspase-3 of xenograft in nude mice was detected by immunohistochemistry.Results:1. Livin ASODN was transfected into bladder cancer cells, the transfection rate was about 55%.2. The effect of Livin ASODN on the inhibition of livin mRNA and protein expression.The expression of livinαand livinβin 5637 cells was detected by RT-PCR and Western blot.After the transfection of Livin ASODN, the expression of livin mRNA and protein was decreased (P<0.01), which was detected by RT-PCR and Western blot.In RT-PCR procedure and gel electrophoresis, the luminosity ratio of livin strip and GAPDH strip were represented the intensity of the expression of livin. The luminosity ratio of Livin ASODN group was 0.46±0.05, and the control group was 0.88±0.05 (MSODN group), 0.92±0.04 (Lipo group), 0.91±0.06 (PBS group) respectively.In Western blot procedure, the luminosity ratio of livin strip was smaller than the control group too.The fluorescence signal of livin observed by CLSM was significantly decreased.3. Biological effect of Livin ASODN on 5637 cells①Livin ASODN could inhibit the proliferating activity of bladder cancer cells, and the inhibiting effection depended on the concentration of Livin ASODN.②Under electromicroscope and confocal laser scanning microscope, the morphology changes of 5637 cells transfected with Livin ASODN was: intumesce, degeneration, cellular organ swelling, some of cells necrosis and to form apoptotic body.③The apoptosis rate of 5637 cells transfected with Livin ASODN was increased assayed by AO/EB stain and flow cytometry. The apoptosis rate was (46.39±9.23)%( ASODN group), (5.10±1.56)% (MSODN group), (5.70±1.61)% (Lipo group), (4.54±1.84)% (PBS group), P﹤0.01.4. Experimentation of xenograft in nude mice(1) The effect of livin ASODN on the inhibition of livin mRNA and protein expression.The expression of livin mRNA and protein detected by RT-PCR, Western blot and immunohistochemistry of human bladder cancer cells xenograft in nude mice was decreased.In RT-PCR and Western blot procedure, the luminosity of livin strip was smaller than the control group.The expression of livin detected by immunohistochemistry of human bladder cancer cells xenograft in nude mice was decreased compare to control group(AIOD: 0.1661±0.1028 VS 0.2490±0.1483), P﹤0.05.(2) Biological effect of Livin ASODN on xenograft in nude mice①Tumor sizes were significantly smaller in Livin ASODN group than MSODN group from 18 to 30 days after inoculation, P < 0.05. Tumors weight were significantly lighter in Livin ASODN group(1.31±0.88g) than those in MSODN group(2.41±0.41g) at 30 days after inoculation,P<0.05②Necrosis foci and inflammatory cell infiltration were observed in xenograft in nude mice of Livin ASODN group by microscopy.③Apoptotic index detected by TUNEL stain in xenograft in nude mice of Livin ASODN group(19.60±5.91) was significantly higher than MSODN group(3.48±2.35), P﹤0.05.④The expression of Caspase-3 detected by immunohistochemistry in xenograft in nude mice of Livin ASODN group (AIOD: 0.4501±0.1618)was increased compare to MSODN group(AIOD: 0.3601±0.1065), P﹤0.05.Conclusions:1. Human cancer cell line 5637 could express Livinαand Livinβwhich was detected by RT-PCR and Western blot.2. Livin plays an important role in the inhibition of apoptosis and proliferation with bladder cancer cells.3. One of the possible mechanical of the inhibition of apoptosis by Livin was the inhibition of the Caspase-3.4. Livin ASODN could remarkably down-regulate the expression of livin gene in 5637cells, induce apoptosis and inhibit tumor growth.5. Livin ASODN might be a potential target and strategies for the treatment of bladder cancer.更多还原