【作者】 田甜；

【导师】 任国胜；

【作者基本信息】 重庆医科大学 ， 外科学， 2009， 博士

【摘要】 第一部分REGγ基因真核表达质粒的构建及其稳定转染细胞株的建立目的:构建REGgamma（REGγ）基因的真核表达重组体pcDNA3.1-REGγ,并建立稳定转染REGγ基因的乳腺（癌）细胞株,为进一步深入研究REGγ基因的功能奠定基础。方法:提取MCF-7细胞的总RNA为模板,通过RT-PCR获取全长REGγcDNA,经限制性内切酶EcoRI、EcoRV酶切后与pcDNA3.1连接构建重组体。以Lipofectamine2000将重组体导入HBL-100、MCF-7、MDA-MB-231细胞,由G418筛选出稳定转染细胞株。并经免疫细胞化学、细胞免疫荧光、Western Blot、RT-PCR等方法检测各株细胞的REGγ表达。结果:经内切酶酶切及DNA序列分析证实重组体构建成功。经免疫细胞化学、细胞免疫荧光、Western Blot、RT-PCR等检测证实稳定转染细胞株中其REGγ的表达与未转染的同种细胞相比均明显增强,有显著性差异（P＜0.01）;REGγ在乳腺癌细胞株的表达明显高于乳腺上皮细胞株（P＜0.05）;在两株乳腺癌细胞株中,MDA-MB-231表达的REGγ高于MCF-7（P＜0.05）。结论:真核表达重组体pcDNA3.1-REGγ构建成功,并经抗生素筛选获得了稳定高表达REGγ的细胞株,为进一步研究REgγ的功能奠定了基础。同时发现在乳腺癌细胞株中REGγ的表达明显高于乳腺上皮细胞株;在乳腺癌细胞株中,恶性潜能高的细胞株REGγ的表达高于恶性潜能低的细胞株。第二部分REGγ基因在人乳腺癌细胞中的功能研究目的:探讨REGγ基因对乳腺癌细胞增殖、凋亡及其对癌基因的影响。方法:通过软琼脂集落形成试验、采用噻唑蓝（MTT）比色法、流式细胞仪（FCM）检测REGγ对细胞增殖及细胞周期的影响;免疫细胞化学检测增殖细胞核抗原（proliferative cell nuclear antigen,PCNA）、bcl-2、fas、c-myc的表达;分光光度法检测Caspase-3活性变化;AnnexinV-FITC的FCM检测来了解细胞凋亡的变化;以透射电子显微镜（TEM）观察转染REGγ引起的细胞超微结构改变;Western Blot检测REGγ对其靶蛋白p21和SRC-3及癌基因CyclinD1的影响。结果:MTT法及FCM检测提示,增加REGγ表达可使细胞生长加速、细胞倍增时间缩短、S+G₂+M的增殖期细胞数明显增加。软琼脂集落形成实验表明,转染REGγ基因的细胞集落形成率高、克隆形成时间短、克隆存活时间长。PCNA的免疫细胞化学检测结果显示,转染REGγ基因的细胞PCNA表达明显增强（p＜0.01）。Caspase-3分光光度法检测显示,实验组细胞的OD值普遍低于对照组（p＜0.05）;Annexin V-FITC的FCM检测显示,实验组细胞的凋亡率明显低于对照组（p＜0.01）;免疫细胞化学检测发现,转染REGγ基因的细胞表达bcl-2增强（p＜0.05）、表达fas减弱（p＜0.05）、表达c-myc增强（p＜0.05）;TEM观察转染REGγ基因的细胞表现为核仁肥大,线粒体、高尔基体丰富或扩张,未见凋亡小体形成;Western Blot检测发现转染REGγ基因的细胞,其p21和SRC-3表达减弱（p＜0.05）、但癌基因CyclinD1表达增强（p＜0.01）。结论:REGγ基因能促进乳腺癌细胞的生长及增殖,抑制乳腺癌细胞凋亡。其机制可能与REGγ基因参与了细胞增殖和细胞凋亡基因的调控有关。第三部分转染REGγ基因对乳腺癌细胞裸鼠移植瘤生长的影响目的:研究REGγ基因转染乳腺癌MDA-MB-231细胞后在裸鼠体内的成瘤作用及其机制。方法:以稳定高表达REGγ基因的细胞株为实验组、转染空载体及未施加处理因素的细胞为对照组。将此3组细胞接种于裸鼠皮下,观察移植瘤的生长状况并计算抑瘤率;RT-PCR检测REGγ基因在瘤组织中的表达;FCM检测瘤组织的肿瘤浸润淋巴细胞（TIL）中CD16的表达及肿瘤细胞周期和细胞凋亡;免疫组化检测移植瘤中p21的表达。结果:与对照组比较,实验组移植瘤生长速度较快、体积较大、瘤重增加（P＜0.05）;RT-PCR检测显示REGγ基因在瘤组织中的表达增加（P＜0.01）;FCM检测提示CD16阳性率明显降低,G₀/G₁和G₂/M期细胞减少,S期细胞明显增多,肿瘤细胞的凋亡率明显降低（P＜0.05）;P21的表达明显降低（P＜0.05）。结论:REGγ基因在体内具有促进乳腺癌发生、发展的作用,其机制可能与其具有加速细胞周期、抑制细胞凋亡,抑制NK细胞活化以及对p21的特异性降解有关。更多还原

【Abstract】 PART ONEEUKARYOTIC EXPRESSION PLASMID CONSTRUCTION OF REGgamma GENE AND CONSTRUCTION OF THE STABLE TRANSFECTIVE CELL LINEObjective.To construct the stable transfective cell line with the eukaryotic expression vector for human REGγ,cDNA to laid the foundation for further study of the function of REGγ.Methods:REGγcDNA was obtained by RT-PCR of total RNA extracted from the human breast cancer cell MCF-7,and it was digested by the restriction endonuclease EcoRI and EcoRV before connection with pcDNA3.1.The recombinant was transfected into HBL-100,MCF-7 and MDA-MB-231 cell by using lipofectamine2000.The stable transfected cell line was selected in medium containing antibiotic（G418）.The expression of REGγ,detcected by immunocytochemistry,Western Blot,immunofluoresc ence and RT-PCR.Results:The recombinant was digested by restriction endonuclease and confirmed correct by sequencing,overexpression of REGγin the stable transfected cell line by contrast with untransfective cell line（P＜0.01）;The expression of REGγ,in breast cancer cell lines were obviously higher than those of breast epithelial cell（P＜0.05）;REGγlevels of MDA-MB-231 were obviously higher than that of MCF-7（P＜0.05）.Conclusions.The construction of the eukaryotic expression vector for REGγwas successful,and the stable transfective cell line for overexpression REGγwas obtained by G418 selection,simultaneously, REGγexpressed in breast cancer cell lines were higher than those of breast epithelial cell;MDA-MB-231 was higher than that of MCF-7 within breast cancer cell lines.This work may pave the way for further study of the functions of REGγ.PART TWOFUNCTION STUDY OF REGγIN HUMAN BREAST CANCER CELL LINESObjective:To study the function of REGγgene in cell proliferation, apoptosis,and the effect for oncogene in human breast cancer cells.Methods:The effect of REGγgene expression on cell proliferation and cell cycle was examined by soft agar conoly formation test,MTT assay and flow cytometry（FCM）;Immunocytochemistry was performed to detect the differences in proliferative cell nuclear antigen（PCNA）,bcl-2,fas and c-myc in breast cancer cells;Apoptosis was measured via FCM,and caspase 3 expression was determined using spectrophotometry; Ultrastructural changes in breast cancer cells were observed with transmission electron microscopy（TEM）.The effect of REGγgene on target protein P21,SRC-3 and CyclinD1 was detected by Western Blot.Results:MTT assay and FCM indicated that Cell growth was accelerated,the doubling time was shortened and the proportion of cells in proliferative phase in tansfected with REGγgroup was obviously higher than that of untransfected with REGγ.Soft agar conoly formation test indicated that the cell colony forming efficiency was increased,the time of colony forming was shortened and survival time of colony was long in tansfected with REGγ,in contrast to untransfected with REG_γ.The level of PCNA measured by immunocytochemistry indicated that the expression of PCNA was remarkably higher in cells transfected with the REGγ,gene （P＜0.01）.The optical density（OD） was obviously lower in cells transfected with REGγby using spectrophotometry of caspase 3（P＜0.05）, and the rate of apoptosis was lower in cells transfected with REGγ（P＜0.01）.The expression of bcl-2 and c-myc were enhanced in cells transfected with recombinant,on the contrary,the expression of fas was decreased（p＜0.05）.Karyosome hypertrophy,mitochondrion abundance, Golgi apparatus abundance and ectasis were seen with TEM.Apoptotic bodies were not observed.Western blot revealed that the expression of p21 and SRC-3 were highly decreased in cells stably transfected with the recombinant compared with the control group（P＜0.05）,but the expression of CyclinD1 was reinforced（p＜0.01）.Conclusions:The REGγgene promotes cell proliferation and inhibits apoptosis in breast cancer cells.The mechanism may be relevant to REGγparticipation in regulation of cell proliferation and apoptosis Cell cycle,ApoptosisPART THREEEFFECT OF TRANSFECTION OF REG./GENE ON THE GROWTH OF XENOGRAFTED HUMAN BREAST CANCER IN NUDE MICEObjective:To study the tumorigenesis of human breast cancer MDA-MB-231 cells in nude mice after transfection with REGγgene（a kind of proteasome activating factor） and the related mechanism.Methods:REGγgene was transfected into MDA-MB-231 cells in pcDNA3.1 vector via lipofectin mediation.The stable clones were selected by G418 600mg/L.The cells without transfection and transfected with empty vector were regarded as control groups.Three groups of cells were inoculated subcutaneously into nude mice.The growth of transplanted tumors was observed and the tumor inhibition ratio was calculated.The expression of REGγgene in tumor tissues was detected by RT-PCR.The expression of CD16 in tumor-infiltrating lymphocytes（TIL） and the cell cycle and apoptosis were determined by flow cytometry.The expression of p21 in transplanted tumors was examined by immunohistochemistry.Results.As compared with control group,xenografted tumor in REGγgene transfection group showed faster growth speed,larger tumor volume, and increased tumor weight（p＜0.05）.The expression of REGγgene in tumors was markedly increased（p＜0.01）.The expression of CD16 in TIL was greatly decreased（p＜0.05） and the cell proportion in G₀/G₁ phase and G₂/M phase decreased significantly but the cell percentage in S phase was obviously increased.The apoptotic ratio and the expression of p21 were obviously reduced（p＜0.05）.Conclusions:REGγgene promotes the tumorigenesis and progress of breast cancer cells in nude mice.The mechanism may be explained by its effects of accelerating cell cycle,inhibiting apoptosis,suppressing the activation of NK cells,and specifically degrading p21.更多还原