【作者】 甘露；

【导师】 任国胜；

【作者基本信息】 重庆医科大学 ， 普外科， 2008， 博士

【摘要】 第一部分REGγ在人乳腺癌细胞及乳腺上皮细胞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REGγ的表达情况。方法:采用免疫细胞化学和western blotting等方法检测REGγ在乳腺癌细胞株MCF-7、MDA-MB-231与乳腺上皮细胞株HBL-100中的表达情况。结果:①REGγ在乳腺癌细胞株与乳腺上皮细胞株的表达水平上有明显差异,2株乳腺癌细胞的REGγ表达明显高于乳腺上皮细胞株（P＜0.05）。②在2株乳腺癌细胞株中,MDA-MB-231的REGγ高于MCF-7（P＜0.05）。结论:在乳腺癌细胞株中REGγ的表达明显高于乳腺上皮细胞株;乳腺癌细胞株中,恶性潜能高的细胞株REGγ的表达强于恶性潜能低的细胞株。第二部分REGγ在乳腺癌组织、良性肿瘤组织及癌旁乳腺组织中的表达及意义目的研究REGγ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REGγ的表达与临床病理特征的关系。方法应用免疫组织化学方法测定REGγ在乳腺癌组织、良性肿瘤组织及癌旁乳腺组织的表达情况。结果①REGγ在乳腺癌组织,良性肿瘤（纤维腺瘤）及癌旁乳腺组织的表达水平上结果差别有明显差异,乳腺癌组织的REGγ表达明显高于良性肿瘤及癌旁乳腺组织（P＜0.05）。②REGγ在乳腺癌组织的表达在T≤2cm组中明显低于T＞2cm组（P＜0.05）。③REGγ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）。④乳腺癌c-erBb-2（+）组的表达高于c-erBb-2（-）组（P＜0.05）。⑤ER（-）组的REGγ表达明显高于ER（+）组的（P＜0.05）。⑥在乳腺癌中,组织学Ⅲ级组的REGγ表达与Ⅰ和Ⅱ级组的差别无统计学意义（P＞0.05）。结论REGγ在乳腺癌组织中表达明显增强。REGγ的表达在T＞2cm、有淋巴结转移、c-erBb-2（+）及ER（-）乳腺癌组织中表达增高,与肿瘤分级无关。第三部分REGγ蛋白shRNA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REGγ蛋白shRNA表达载体。方法按照Elbashir等及Reynolds的设计原则设计并合成针对REGγ蛋白编码基因REGγ的shRNA,并克隆入转录载体pGenesil-1,构建重组质粒pshRNAREGγ-1,pshRNAREGγ-2和pshRNAREGγ-HK（阴性对照质粒）;大量提取重组质粒,采用Lipofectamine^TM 2000转染人乳腺癌细胞MDA-MB-23 1和MCF-7,并用G418筛选出稳定转染细胞,应用real-time-quantitative RT-PCR和Western Blot法分别在mRNA和蛋白水平检测REGγ基因的干扰效果。结果成功构建三个重组质粒,分别为pshRNAREGγ-1,pshRNAREGγ-2和阴性对照质粒pshRNAREGγ-HK。Real-timequantitative RT-PCR检测显示REGγ基因在mRNA转录水平被显著抑制,pshRNAREGγ-1,pshRNAREGγ-2在两株细胞中分别抑制到未转染细胞细胞的36%,48%和38%,46%。Western Blot检测结果与realtime-RT-PCR相似,pshRNAREGγ-1,pshRNAREGγ-2在两株细胞中分别将REGγ蛋白抑制到未转染细胞细胞的38.23%,45.12%和36.43%,48.46%。pshRNAREGγ-2干扰效果较好。结论重组质粒pshRNAREGγ-1,pshRNAREGγ-2均能有效抑制人乳腺癌细胞REGγ蛋白的表达,以pshRNAREGγ-2干扰效果最好。第四部分REGγ蛋白shRNA作用乳腺癌细胞株后其相关蛋白的变化及意义目的研究REGγ蛋白shRNA作用于不同人乳腺癌细胞后其相关基因在mRNA和蛋白水平的变化以及对细胞生长、细胞周期及凋亡等的影响。方法采用Lipofectamine^TM 2000转染pshRNAREGγ-2质粒到人乳腺癌细胞MDA-MB-231和MCF-7中,并用G418筛选出稳定转染细胞。采用Western blotting、real-time quantitative PCR、免疫细胞化学检测转染前后不同乳腺癌细胞后相关蛋白SRC-3、PCNA和p21在蛋白水平和mRNA水平的变化;电镜和Tunel法检测转染前后细胞凋亡的改变;流式细胞仪检测细胞周期的变化;MTT法检测细胞生长曲线的变化。结果pshRNAREGγ-2转染乳腺癌细胞后,real-time-quantitativeRT-PCR结果显示两株细胞的REGγ的mRNA水平均下降,但SRC-3、PCNA和p21的mRNA水平无明显变化;Western blot和免疫细胞化学则显示REGγ蛋白水平明显下降,PCNA表达下降,SRC-3有不同程度增加（P＜0.05）,在MDA-MB-231细胞中p21蛋白水平有明显增加（P＜0.05）,但在MCF-7细胞中无明显改变（P＞0.05）。电镜和TUNEL结果显示MDA-MB-231凋亡小体明显增多,但MCF-7的凋亡小体无明显变化。流式细胞仪检测细胞周期显示,在MDA-MB-231细胞中,S期细胞比例明显减少,G1期细胞比例明显增加（P＜0.05）;而在MCF-7细胞中则是S期细胞比例明显增加,而G1期细胞比例明显减少（P＜0.05）。MTT显示MDA-MB-231细胞生长减慢而MCF-7细胞的生长速度增加。结论REGγ在不同的乳腺癌细胞株中的作用不同,在MDA-MB-231细胞中,REGγ主要通过降解p21促进细胞生长,而在MCF-7细胞中,REGγ通过降解SRC-3抑制细胞生长。更多还原

【Abstract】 PART ONE EXPRESSION AND SIGNIFICANCE OF REGγIN HUMAN BREAST CANCER CELLS AND BREAST EPITHELIAL CELLObjective:To study the expression and significance of REGγin human breast cancer cells and breast epithelial cell.Methods:Western blotting and immunocytochemistry were applied to detect the expression of REGγin human breast cancer cells（MCF-7、MDA-MB-231） and breast epithelial cell（HBL-100）.Results:①REGγlevels of the breast cancer cells were obviously higher than those of breast epithelial cell（P＜0.05）.②REGγlevels of MDA-MB-231 were obviously higher than that of MCF-7.Conclusion:The expression level of REGγis increased obviously in breast cancer cell lines,and is more important in the cell line with higher potency of malignancy. PART TWO EXPRESSION AND SIGNIFICANCE OF REGγIN HUMAN BREAST CANCER,BENIGN TUMOR AND TUMOR-ADJACENT TISSUESObjective:To study the expression and significance of REGγin human breast cancer,benign tumor and tumor-adjacent tissues.Methods:Immunohistochemistry was applied to detect the expression of REGγin human breast cancer,benign tumor and tumor-adjacent tissues.Results:①REGγlevel of human breast cancer were obviously higher than that of tumor-adjacent tissues（P＜0.05）.②REGγlevel in T≤2cm were obviously lower than those in T＞2cm group the pathological gradeⅢgradeⅠandⅡ（P＜0.05）.③REGγlevel in axillary lymph node negative were obviously lower than those in axillary lymph node positive.④REGγlevel of CerBb-2（+） group were obviously higher than those of c-erBb-2（-） group（P＜0.05）.⑤REGγlevel of ER（-） group were obviously higher than those of ER（+） group and（P＜0.05）.⑥There were no significant differences of REGγlevels between with gradeⅢgroup and gradeⅠandⅡgroup（P＞0.05）.Conclusion:REGγlevel increased in T＞2cm,axillary lymph node positive,c-erBb-2（+） and ER（-） tumors,but the expression level had no relation with pathlogical grade PART THREE CONSTRUCTION AND IDENDIFICATION OF RECOMBINED REGγshRNA PLASMID IN BREAST CARCINOMA CELLSObjective:To construct and idendify the recombined small hairpin RNAs（shRNA） plasmid targeted to REGγgene,which can knock down the REGγgene of breast carcinoma cells.Methods:Small hairpin RNAs（shRNA） targeted to REGγcoding gene were designed and synthesized according to the principle mentioned by Elbashir and Reynolds.It was cloned into pGenesil-1 which worked as a transcription vector to construct recombined plasmid named pshRNAREGγ-1,pshRNAREGγ-2 and pshRNAREGγ-HK（negative control plasmid）.The recombined plasmid was extracted in middle quantity and transferred into breast carcinoma cells by Lipofectamine^TM 2000.Realtime-quantitive-RT-PCT and western blotting were used to detect REGγmRNA and protein respectively.Results:Three recombined plasmids:pshRNAREGγ-1, pshRNAREGγ-2 and pshRNAREGγ-HK were constructed successfully. Stable transfected cell lines were gained by G418 screen after transfection by Lipofetamine^TM 2000.REGγmRNA was suppressed to 36%,48%和38%,46%and in contrast to untransfected cell lines detected by realtime-quantitive-RT-PCR,and REGγprotein was knocked down to 38.23%,45.12%and 36.43%,48.46%measured by western blotting in cells transfected by pshRNA REGγ-1 and pshRNAREGγ-2 plasmid in MDA-MB-231 cell and MCF-7 cell respectively,pshRNAREGγ-2 is better than pshRNA REGγ-1.Conclusions:REGγprotein in breast carcinoma cells can be knocked down effectively by recombined plasmid pshRNAREGγ-1 and pshRNA REGγ-2.The effect of pshRNAREGγ-2 is better than that of pshRNA REGγ-1. PART FOUR STUDY OF THE SIGNIFICANCE AND ALTERATION OF RELATIVE PROTEINS IN pshRNAREGγPLASMID TRANSFECTED BREAST CANCER CELL LINESObjective:To investigate the influence on the cell growth,cell cycle, apoptosis and the change of levels for relative proteins by treated with shRNAREGγ-2 in different breast cancer cell lines.Methods:The variations of mRNA and protein level of REGγ、SRC-3、PCNA and p21 in different pshRNAREGγ-2 plasmid transfected breast cancer cell lines were tested by real-time-quantitative-RT-PCR and western blotting as well as immunocytochemistry respectively;The cell proliferation rate and apoptosis were measured by MTT、flow cytometer and transmission electron microscope、dTdT mediated dUTP nick end labeling（TUNEL） method.Results:The REGγmRNA and protein levels were significantly decreased in both transfected groups,but there were no change of the other three proteins’ mRNA levels:SRC-3、PCNA and p21,and the variations of these proteins levels were:PCNA level was significantly decreased,and SRC-3 level was significantly increased in both cell lines （P＜0.05）;p21 level was increased in MDA-MB-231cell（P＜0.05） but was no significant change in MCF-7cell.There were more apoptotic bodies were detected in MDA-MB-231 cell（P＜0.05） than those in MCF-7cell;the G1 phase cells percentage was increased with the decrease of S phase cells in MDA-MB-231 cell（P＜0.05） and the transfected cell growth was stepped down,but on the contrary,the S phase cells was increased in MCF-7cell（P＜0.05） with an accelarated cell growth.Conclusions:REGγhas different roles in MDA-MB-231cell and MCF-7cell.REGγstimulates cell proliferation by degradation of p21 and increases the PCNA in MDA-MB-231cell,and inhibits the proliferation of MCF-7 cell by degradation of SRC-3.更多还原