【作者】 李凡；

【导师】 任国胜；

【作者基本信息】 重庆医科大学 ， 普外科， 2009， 博士

【摘要】 第一部分凋亡抑制蛋白Livin在人乳腺癌中的表达及意义目的:探讨在人乳腺癌细胞和乳腺上皮细胞中Livin的表达情况,研究Livin在乳腺癌组织、良性肿瘤组织及癌旁乳腺组织的表达及意义,以及人乳腺癌组织中Livin的表达与临床病理特征的关系。方法:采用免疫细胞化学、Western blot和Real-time-quantitive- RT-PCR等方法检测Livin在乳腺癌细胞株MCF-7、ZR-75-30、MDA-MB-231、MDA-MB-435s与乳腺上皮细胞株HBL-100中的表达情况。应用免疫组织化学方法测定Livin在人乳腺癌组织与乳腺良性肿瘤、癌旁乳腺组织的表达。结果:①Livin在4株乳腺癌细胞株中的表达明显高于乳腺上皮细胞株(P<0.05)。②在4株乳腺癌细胞株中Livin的表达无明显差异(P>0. 05)。③Livin在乳腺癌组织,良性肿瘤及癌旁乳腺组织的表达水平上结果有明显差异,乳腺癌组织的Livin表达明显高于良性肿瘤及癌旁乳腺组织(P<0.05)。④Livin在乳腺癌组织T≤2cm组中的表达明显低于T>2cm组(P<0.05)。⑤Livin在淋巴结转移阳性的乳腺癌中表达明显高于淋巴结转移阴性的乳腺癌(P<0.05)。⑥Livin在乳腺癌中组织学III级组的表达高于I和II级组的表达(P<0.05)。⑦Livin在乳腺癌c-erBb-2(+)组的表达与c-erBb-2(-)组的表达无显著差别(P>0.05),在ER(-)组的表达与ER(+)组中的表达无显著差别(P>0.05)。结论:在乳腺癌细胞株中Livin的表达显著增高;Livin在乳腺癌组织中表达明显增强。Livin的表达在T>2cm、有淋巴结转移、组织学III级的乳腺癌组织中的表达增高,与c-erBb-2和ER的表达未见显著相关性。第二部分Livin蛋白siRNA真核表达载体的构建和鉴定目的:在人乳腺癌细胞中构建及鉴定Livin蛋白siRNA真核表达载体。探讨Livin siRNA对人乳腺癌细胞株MCF-7和MDA-MB-231中Livin mRNA及蛋白表达的下调作用。方法:构建三个靶向Livin的siRNA真核表达载体si-Livin1、si-Livin2、si-Livin3和si-HK(阴性对照质粒);大量提取重组质粒,分别转染人乳腺癌细胞MCF-7和MDA-MB-231后,通过Real-time- quantitative-RT-PCR和Western blot技术分别检测干扰前后乳腺癌细胞Livin蛋白和mRNA水平的表达。结果:成功构建了针对Livin基因的3个特异性siRNA表达载体。它们不同程度地抑制了乳腺癌细胞Livin基因的表达,以si- Livin2抑制效率最高。si-Livin2作用于人乳腺癌细胞株MCF-7和MDA-MB-231后能明显抑制其Livin基因的mRNA及蛋白表达水平(P<0.05)。结论:靶向Livin的真核表达载体si-Livin1、si-Livin2、si-Livin3均能不同程度的抑制人乳腺癌细胞Livin mRNA和蛋白表达,以si-Livin2干扰效果最好。第三部分靶向Livin的siRNA真核表达载体对乳腺癌细胞株生物学功能的影响及可能机制目的:研究Livin蛋白siRNA真核表达载体作用于人乳腺癌细胞株后对其生物学功能的影响,探讨可能的作用机制。方法:采用LipofectamineTM 2000转染si-Livin2质粒到人乳腺癌细胞MCF-7和MDA-MB-231中,通过实时定量PCR、Western blot技术检测干扰前后乳腺癌细胞Caspase-3、7、9和Smac/DIABLO在mRNA、蛋白水平的表达;采用MTT法检测对细胞增殖的抑制作用,流式细胞法测细胞周期,TUNEL法检测细胞凋亡以及细胞对阿霉素化疗的增敏作用,观察细胞超微结构变化。结果:Si-Livin2转染48 h后,与未转染组相比,MCF-7和MDA- MB-231细胞增殖活力受到抑制;细胞周期重新分布,G0/G1期细胞比例明显增多(P<0.05),S期细胞比例明显减少(P<0.05);细胞凋亡率显著升高(P<0.05),细胞对阿霉素的化疗敏感性增加;电镜下可见典型的凋亡细胞特征。细胞内Caspase-3、7、9和Smac/ DIABLO的mRNA、蛋白表达有不同程度的上升(P<0.05)。结论:Livin siRNA通过上调Caspase-3、7、9和Smac/DIABLO表达,有效地抑制乳腺癌细胞增殖,诱导凋亡,对阿霉素的治疗可起到增敏辅助作用。第四部分Livin siRNA对人乳腺癌裸鼠移植瘤的抑制作用目的:研究Livin siRNA真核表达载体对人乳腺癌细胞MDA-MB -231裸鼠移植瘤的生长抑制作用,探讨可能的作用机制。方法:将人乳腺癌细胞株MDA-MB-231注射入裸鼠皮下,建立裸鼠皮下移植瘤模型。分别向移植瘤中注射生理盐水,si-HK和si-Livin2质粒,检测观察肿瘤的生长情况,H.E染色观察肿瘤组织的病理改变,透射电镜观察肿瘤组织超微结构,TUNEL法检测细胞凋亡情况,免疫组织化学检测肿瘤Livin蛋白的表达变化,Western blot检测肿瘤Livin,Caspase-3和Caspase-9的表达变化。结果:si-Livin2导入裸鼠皮下移植瘤后35天,其肿瘤体积明显低于空白组和对照组肿瘤,病理学检查及TUNEL检测发现,实验组肿瘤生长受到抑制,细胞凋亡指数显著高于空白组和对照组(P<0.05);与空白组和对照组比较,实验组肿瘤Livin表达受到抑制,Caspase-3和Caspase-9蛋白的相对表达含量均有所升高。结论:Livin siRNA能显著降低人乳腺癌裸鼠移植瘤Livin的表达,抑制MDA-MB -231裸鼠移植瘤的生长。更多还原

【Abstract】 PART ONEEXPRESSION AND SIGNIFICANCE OF THE APOPTOTIC INHIBITOR LIVIN IN HUMAN BREAST CANCERObjective:To study the expression and significance of the apoptotic inhibitor Livin in human breast cancer.Methods:Immunocytochemistry, Real-time-quantitive-RT-PCR and Western blot were applied to detect the expression of Livin in human breast cancer cells(MCF-7,ZR-75-30, MDA-MB-231, MDA-MB-435s)and breast epithelial cell(HBL-100). Immunohistochemistry was applied detect the expression of Livin in human breast cancer, benign tumor and tumor-adjacent tissues.Results:①Livin expression of the breast cancer cells were obviously higher than those of breast epithelial cell(P<0.05).②There was no significant difference of Livin expression level between the four breast cancer cells.③Livin expression level of human breast cancer was obviously higher than that of tumor-adjacent tissues and benign tumor.④Livin expression level in T≤2cm group of human breast cancer was obviously lower than that in T>2cm group (P<0.05).⑤Livin expression level in axillary lymph node negative group was obviously lower than that in axillary lymph node positive group (P<0.05).⑥Livin expression level in grade III group of human breast cancer was obviously higher than that in grade I and II group (P<0.05).⑦There were no significant differences of Livin levels between in CerBb-2(+)group and C-erBb-2(-)group (P>0.05), ER(-)group and ER(+) group (P>0.05) .Conclusion:The expression level of Livin is increased obviously in breast cancer cell lines. Livin expression level increased in T>2cm, axillary lymph node positive, grade III tumors, but it had no relation with the CerBb-2 and ER status.PART TWOCONSTRUCTION AND IDENTIFICATION OF LIVIN EUKARYOTIC EXPRESSION VECTOR IN BREAST CANCER CELLSObjective: To construct and idendify the siRNA eukaryotic expression vectors targeting to Livin gene and to study the suppressing effect of Livin-siRNA on the Livin gene expression in MCF-7 , MDA-MB-231 cells.Methods: Three siRNA eukaryotic expression vectors targeting Livin(si-Livin1,si-Livin2,si-Livin3) and negative control vector (si-HK)were constructed. The recombined plasmid was extracted in middle quantity and transfected into MCF-7 and MDA-MB-231 cells by LipofectamineTM 2000. Real-time-quantitive-RT-PCR and Western blot were used to detect Livin mRNA and protein respectively.Results: Three specific siRNA eukaryotic expression vectors targeting Livin and negative control vector were successfully constructed. The Livin siRNA could inhibit the expression of Livin mRNA and protein to varying degree, and si-Livin2 resulted in the highest inhibiting rate.The Livin mRNA and protein expression were decreased significantly in MCF-7 and MDA-MB-231 breast cancer cells by si-Livin2(P<0.05).Conclusion: Three specific siRNA eukaryotic expression vectors targeting Livin (si-Livin1,si-Livin2,si-Livin3)could inhibit the expression of Livin mRNA and protein to varying degree .The effect of si-Livin2 is better than that of si-Livin1 and si-Livin3.PART THREE THE EFFECT OF LIVIN-TARGETED siRNA ON BIOLOGICAL BEHAVIOR OF HUMAN BREAST CANCER CELLS AND POSSIBLE MECHANISMObjective:To investigate the effect of Livin-targeted siRNA on the biological behaviour of breast cancer cell lines MCF-7 and MDA-MB-231 and to study its possible mechanism.Methods: si-Livin2 was transfected into MCF-7 and MDA- MB- 231 cells by LipofectamineTM 2000.The changes of Caspase-3,7,9 and Smac/ DIABLO expressions were detected by Real-Time-quantitative-PCR and Western blot.The effect of suppressing proliferation of MCF-7 and MDA- MB-231 cells was detected by MTT assay. Cell cycles were determined by flow cytometry. Cells ultramicrostructure changes were observed by electron microscope.The effect of inducing MCF-7 and MDA- MB-231 cells apoptosis and inhanced chemotherapy sensitivity of breast cancer cells treated to adriamycin was detected by TUNEL assay.Results: Forty-eight hours after si-Livin2 transfection, compared with the blank and si-HK group, the proliferation of MCF-7 and MDA-MB-231 cells was strongly inhibited(P<0.05). The cells percentage of the G0/Gl phase was increased and the S phase was decreased significantly ( P < 0.05 ) .Rate of apoptosis increased significantly(P<0.05). Typical morphological characteristic of apoptotic cells was observed by electron microscope. Chemotherapy sensitivity of breast cancer cells to adriamycin was enhanced. The mRNA and protein expressions of Caspase-3,7,9 and Smac/DIABLO were all increased in both si-Livin2 transfected groups(P<0.05), but there were no change of the other control groups.Conclusion:Through upregulating the expressions of Caspase-3,7,9 and Smac/DIABLO,Livin-targeted siRNA can inhibit proliferation and induce apoptosis in MCF-7 and MDA-MB-231 cells,and enhance cells chemotherapy sensitivity to adriamycin significantly.PART FOURINHIBITION EFFECT OF LIVIN SIRNA ON THE GROWTH OF HUMAN BREAST CANCER CELL XENOGRAFT IN NUDE MOUSEObjective:To study the inhibitory effect of Livin siRNA eukaryotic expression vector on human breast cancer MDA-MB-231 cell xenograft in nude mice and its mechanism.Methods: MDA-MB-231 cells were transplanted into subcutaneous tissue of nude mice to establish tumor xenograft. Si-Livin2 vectors were injected into tumor xenograft. The tumor growth was monitored. Tumor morphology was observed with HE staining.Ultramicrostructure changes of tumor tissue were observed by electron microscope. Cell apoptosis was detected by TUNEL assay. Immunohistochemistry was applied to detect the expression of Livin in tumor xenograft. The expressions of Livin, Caspase-3,9 protein in tumor tissues were detected by Western blot.Results:The tumor volume was significantly smaller in si-Livin2 group than in blank and control group(P<0.05).Necrosis and cell apoptosis were found in treated group. Typical morphological characteristic of apoptotic cells was observed in treated group by electron microscope. The apoptosis rate of tumor cells was significantly higher in treated group than in blank and control group. The expression of Livin protein was significantly lower in treated group than in blank and control group (P<0.05). Caspase-3 and Caspase-9 expressions were upregulated in treated group compared with other control groups(P<0.05).Conclusion: Livin siRNA can silence the Livin expression in human breast cancer cell xenograft and inhibite the growth of MDA-MB-231 cell xenograft in nude mice.更多还原