【作者】 章涛；

【导师】 周岐新；

【作者基本信息】 重庆医科大学 ， 药理学， 2007， 博士

【摘要】 第一章hPPARα基因载体构建及其高效转染表达体系的建立目的:克隆人过氧化物酶体增殖物激活受体α基因(hPPARα),构建真核细胞表达载体phPPARα-IRES2-EGFP,转染293细胞并检测转染细胞hPPARα表达水平,以建立其高效转染表达体系,为进一步研究PPARα功能和建立基于PPARα为靶点的药物筛选分子平台奠定基础。方法:采用逆转录法(RT)从HepG2细胞总RNA中获得hPPARαcDNA全长序列,再用分别带有BamHI和SalI酶切位点的hPPARα基因扩增上、下游引物,经PCR扩增获得hPPARα基因。用BamH I和Sal I双酶切消化载体pIRES2-EGFP和PCR产物,胶回收后进行hPPARα与pIRES2-EGFP连接,构建含hPPARα基因的重组质粒phPPARα-IRES2-EGFP。重组质粒经CaCl2法转化DH5α感受态细胞,卡那霉素筛选阳性克隆,并用BamH I和Sal I双酶切及测序鉴定重组质粒phPPARα-IRES2-EGFP中插入的hPPARα基因的完整性和忠实性。重组质粒phPPARα-IRES2- EGFP转染293细胞,荧光显微镜观察GFP报告基因表达强度和转染效率,并对转染细胞的hPPARα基因表达进行荧光定量PCR、免疫细胞化学和Western blot检测。结果:从HepG2细胞总RNA中经逆转录-聚合酶链式反应(RT-PCR)扩增得到1520 bp预期片段大小的hPPARαcDNA全长序列。重组质粒phPPARα-IRES2-EGFP经酶切及测序鉴定,其插入的hPPARα基因序列与GeneBank数据库提交的相应序列(NM001001928/gi:61744437)完全吻合。phPPARα-IRES2-EGFP转染的293细胞,其GFP报告基因表达强,转染效率高达83±9%;重组质粒转染的293细胞hPPARαmRNA表达水平比空载体pIRES2-EGFP转染组高3个数量级,提示导入的重组质粒能够在mRNA水平高效表达hPPARα;免疫细胞化学和western blot检测表明,转染phPPARα-IRES2-EGFP的293细胞高表达hPPARα蛋白。结论:成功克隆了hPPARα基因全长cDNA序列,构建了含hPPARα基因的真核表达载体phPPARα-IRES2-EGFP;并在转染的293细胞中获得hPPARα基因的高效表达;建立了phPPARα-IRES2-EGFP重组质粒体外高效转染表达体系。这为进一步研究PPARα受体功能和建立基于PPARα受体靶点的药物筛选分子平台奠定了基础。第二章hPPARδ基因载体构建及其高效转染表达体系的建立目的:克隆人过氧化物酶体增殖物激活受体δ基因(hPPARδ),构建真核细胞表达载体phPPARδ-IRES2-EGFP,转染293细胞并检测转染细胞中hPPARδ的表达水平,以建立phPPARδ-IRES2-EGFP载体高效转染表达体系,为进一步研究PPARδ受体功能和建立基于PPARδ为靶点的药物筛选分子平台奠定基础。方法:以hPPARδ基因全长cDNA为模版,设计分别带有BamH I和Sal I酶切位点的hPPARδ基因扩增上、下游引物,采用RT-PCR法自HepG2细胞总RNA中获得含hPPARδ全长基因编码序列。用BamH I和Sal I双酶切消化载体pIRES2-EGFP和RT-PCR产物,胶回收后对2者进行连接,构建含hPPARδ基因的载体phPPARδ-IRES2-EGFP;重组质粒phPPARδ-IRES2-EGFP转化DH5α感受态细胞,卡那霉素筛选阳性克隆,分别对挑选的阳性克隆用BamH I和Sal I双酶切及测序,以鉴定重组质粒phPPARδ-IRES2-EGFP中插入的hPPARδ序列的完整性和可靠性。重组质粒phPPARδ-IRES2-EGFP转染293细胞,荧光显微镜观察报告基因GFP表达强度和转染效率,并对转染细胞的hPPARδ基因表达进行荧光定量PCR、免疫细胞化学和Western blot检测。结果:采用RT-PCR从HepG2细胞总RNA中扩增得到1436 bp预期片段大小的hPPARδcDNA全长序列,重组质粒phPPARδ-IRES2- EGFP经酶切及测序鉴定,其插入的hPPARδ序列与GeneBank数据库中提交的相应序列( AY919140/gi:60115374 )完全吻合。phPPARδ-IRES2-EGFP转染的293细胞,其GFP报告基因表达强,转染效率高达85±10%;转染细胞hPPARδmRNA表达水平比空载体转染对照组高3个数量级,提示导入的重组质粒能够在mRNA水平高效表达hPPARδ;免疫细胞化学和Western blot检测均表明,转染phPPARδ-IRES2-EGFP的293细胞高表达hPPARδ蛋白。结论:成功的克隆了hPPARδ基因,构建了含hPPARδ真核表达载体phPPARδ-IRES2-EGFP;并在转染的293细胞中获得了hPPARδ高效表达,建立了phPPARδ-IRES2-EGFP重组质粒体外高效转染表达体系。这为进一步研究PPARδ受体功能和基于PPARδ受体靶点的药物筛选分子平台的建立奠定了基础。第三章hPPARγ1基因载体构建及其高效转染表达体系的建立目的:克隆人过氧化物酶体增殖物激活受体γ1基因(hPPARγ1),构建真核细胞表达载体phPPARγ1-IRES2-EGFP,转染293细胞并检测转染293细胞中hPPARγ1表达水平,以建立其高效转染表达体系,为进一步研究PPARγ1受体功能和基于PPARγ1受体靶点的药物筛选分子平台的建立奠定基础。方法:设计分别带有Xho I和Sma I酶切位点的hPPARγ1基因扩增上、下游引物,采用RT-PCR法自HepG2细胞总RNA中获得含hPPARγ1基因全长编码序列。用Xho I和Sma I双酶切载体pIRES2-EGFP和RT-PCR产物,胶回收后对2者进行连接,构建含hPPARγ1基因的重组质粒phPPARγ1-IRES2-EGFP;重组质粒转化DH5α感受态细胞,卡那霉素抗性筛选阳性克隆。用Xho I和Sma I双酶切筛选的阳性克隆质粒,并测序鉴定重组质粒phPPARγ1-IRES2-EGFP中插入的hPPARγ1的完整性和可靠性。重组质粒phPPARγ1-IRES2-EGFP转染293细胞,荧光显微镜观察GFP报告基因表达强度和转染效率,并对转染细胞的hPPARγ1基因表达进行荧光定量PCR、免疫细胞化学和western blot检测。结果:从HepG2细胞总RNA中经RT-PCR获得1520 bp预期片段大小的hPPARγ1全长序列。重组质粒phPPARγ1-IRES2-EGFP经酶切及测序鉴定,其插入的hPPARγ1基因序列与GeneBank数据库中提交的相应序列( NM005037/gi:62865855, NM138711/gi:62865854, NM138712/gi:62865852)完全吻合。重组质粒转染的293细胞,其GFP报告基因表达强,转染效率高达83±11%;转染的293细胞hPPARγ1 mRNA表达水平比空载体转染对照组高3个数量级,提示导入的重组质粒能够在mRNA水平高效表达hPPARγ1;免疫细胞化学和western blot检测均表明,转染phPPARγ1-IRES2-EGFP的293细胞高表达hPPARγ1蛋白。结论:成功克隆了hPPARγ1基因,构建了含hPPARγ1真核表达载体phPPARγ1-IRES2-EGFP;并在转染的293细胞中获得hPPARγ1基因的高效表达,建立了phPPARγ1-IRES2-EGFP体外高效转染表达体系。这为进一步研究PPARγ1受体功能和建立基于PPARγ1为靶点的药物筛选分子平台奠定了基础。第四章小檗碱抑制人乳腺癌MDA-MB-231细胞生长与PPARγ受体的关系目的:研究小檗碱和罗格列酮对人乳腺癌细胞株MDA-MB-231体外生长的影响及其与PPARγ受体的关系,以评价其在人乳腺癌治疗上的应用潜力。方法:采用噻唑蓝(MTT)法,分析不同浓度(1×10-8、1×10-7、1×10-6、1×10-5、1×10-4 mol/L)和不同作用时间下小檗碱和罗格列酮对MDA-MB-231细胞的体外生长抑制效应;结合PPARγ拮抗剂GW9662,研究小檗碱和罗格列酮对MDA-MB-231细胞增殖的影响与PPARγ受体激动的关系;流式细胞仪检测罗格列酮干预后MDA-MB-231细胞周期变化和细胞凋亡情况;并用TUNEL法,对小檗碱和罗格列酮作用后的MDA-MB-231细胞凋亡情况进行原位检测。结果:小檗碱和罗格列酮可抑制MDA-MB-231细胞的生长,并呈明显的量-效和时-效关系,小檗碱和罗格列酮干预MDA-MB-231细胞24 h的半数抑制浓度(IC50)分别为0.21和4.3μmol/L;PPARγ受体拮抗剂GW9662可部分逆转罗格列酮对MDA-MB-231细胞增殖的抑制作用(P<0.05),但不能逆转小檗碱的相应作用;随着罗格列酮使用浓度的增高,MDA-MB-231细胞G0/G1期细胞比例逐步上升,而S期比例逐步下降;高浓度罗格列酮(100μmol/L)还可诱导MDA-MB-231细胞凋亡,TUNEL法检测的凋亡发生率18.4±3.1%与Annexin V-FITC法检测的结果一致(16.6±2.7%)(P>0.05)。而小檗碱诱导MDA-MB-231细胞凋亡作用则更为明显,TUNEL法检测1×10-6、1×10-5和1×10-4 mol/L小檗碱干预MDA-MB-231细胞的凋亡发生率分别为17.7±3.2%、59.3±4.5%和89.6±5.7%。结论:小檗碱和罗格列酮均能明显抑制MDA-MB-231细胞生长,但与罗格列酮不同,前者的作用不通过PPARγ受体介导。罗格列酮可使MDA-MB-231细胞阻滞于G1期,高浓度时还可诱导其发生凋亡,而小檗碱诱导MDA-MB-231细胞凋亡的效应则更为显著。小檗碱和罗格列酮均有望成为治疗乳腺癌的有效药物。更多还原

【Abstract】 CHAPTER ONE The construction of phPPARα-IRES2-EGFP recombinant plasmid and establishment of its high efficient tranfection and expression systemOBJECTIVE: To clone human peroxisome proliferator-activated receptorαgene (hPPARα) and construct a eukaryotic expression vector of phPPARα-IRES2-EGFP carrying hPPARαgene. Transfect the 293 cellls with the vector and detect the expression of hPPARαgene in transfected 293 cells in order to establish an ideal molecular platform for further studying the functions of hPPARαand screening ligands.METHODS: Primer pairs which contained cutting sites of BamHI and SalI endonucleases were designed for the amplification of hPPARαgene from total RNA of HepG2 cells by RT-PCR. The pIRES2-EGFP plasmids and the PCR product of hPPARαgene were both excised by BamHI and SalI endonucleases. The products of enzyme digestion were recovered from agarose gel and ligated with each other. The recombinant plasmids were transformed into DH5αcompetent cells prepared by CaCl2 method, and cultured in LB agar medium which contained kanamycin for selecting positive clones. The integrity and fidelity of hPPARαgene contained in the recombinant plasmids from positive clones were identified by double digestion of BamH I and Sal I endonucleases and sequencing. Correct recombinant plasmids were then transfected into 293 cells. The expression intensity and transfection efficiency of EGFP reporter gene were observed with fluorescence microscope. Real-time quantitative PCR, immunocytochemistry, and western blot assays were used to analyze the expression of hPPARαat both mRNA and protein levels in transfected 293 cellls.RESULTS: hPPARαgene sequence contained in recombinant plasmid phPPARα-IRES2-EGFP was verified by enzymes digestion and sequence analysis. The sequence of inserted hPPARαgene was same as the corresponding sequence found in GeneBank database (NM001001928/gi:61744437). In the 293 cells high expression of GFP reporter gene from the recombinant plasmids can be seen under fluorescence microscope and the transfection efficiency of the plasmids was 83%±9%. Real-time quantitative PCR result showed that hPPARαmRNA expression level in transfected 293 cells was 3 orders higher than that of pIRES2-EGFP-transfected control. The results from western blot assay and immunocytochemistry staining showed that there exsited a high expression of hPPARαprotein in transfected cells.CONCLUSION: The recombinant plasmid phPPARα-IRES2-EGFP has been constructed successfully with highly efficient expression in transfected 293 cells, which provides a useful tool for analyzing hPPARαgene’s function as well as establishing a molecular platform with which the candidates of unknown hPPARαligands or activators can be found.CHAPTER TWO The construction of phPPARδ-IRES2-EGFP recombinant plasmid and establishment of its high efficient tranfection and expression systemOBJECTIVE: To clone human peroxisome proliferator-activated receptorδgene (hPPARδ) and construct a eukaryotic expression vector of phPPARδ-IRES2-EGFP carrying hPPARδgene, which was used to transfect 293 cellls. Detect the hPPARδgene expression in transfected 293 cells for the purpose of establishing a screening ideal platform for the natural ligands of hPPARδand studying its functions.METHODS: Primer pairs which contained cutting sites of BamHI and SalI endonucleases were designed and used to amplify the hPPARδgene from total RNA of HepG2 cells by RT-PCR. Both the PCR product of hPPARδgene and pIRES2-EGFP plasmids were excised by BamHI and SalI endonucleases. The products of enzymes digestion were recovered from agarose gel and then ligated with each other. The recombinant plasmids were transformed into DH5αcompetent cells prepared by CaCl2 method, and cultured in LB agar medium which contained kanamycin to select positive clones. Doubly digesting of BamHI and SalI endonucleases and sequencing were used to identify the integrity and fidelity of hPPARδgene contained in the recombinant plasmid from the positive clones. The correct recombinant plasmid was transfected into 293 cells for the purpose of expressing cloned hPPARδgene. Real-time quantitative PCR, immunocytochemistry, and western blot assays were used to analyze the expression of hPPARδat mRNA and protein levels in the transfected 293 cellls.RESULTS: hPPARδgene sequence contained in recombinant plasmid phPPARδ-IRES2-EGFP was verified by enzyme digestion as well as sequence analysis. The sequence of inserted hPPARδgene was in accordance with the corresponding sequence found in GeneBank database (AY919140/gi:60115374). High expression of GFP reporter gene can be seen in transfected 293 cells under fluorescence microscope and the transfection efficiency of phPPARδ-IRES2-EGFP was 85%±10%. hPPARδgene contained in phPPARδ-IRES2-EGFP plasmid was found to display a high efficient expression of mRNA and protein which could be detected by real-time quantitative PCR, immunocytochemistry, and western blot, respectively.CONCLUSION: The recombinant plasmid phPPARδ-IRES2-EGFP has been constructed successfully with highly efficient expression in transfected 293 cells, which provides a useful tool for analyzing hPPARδgene’s functions as well as establishing a molecular platform for discovering unknown hPPARδligands or activators.CHAPTER THREE The construction of phPPARγ1-IRES2-EGFP recombinant plasmid and establishment of its high efficient tranfection and expression systemOBJECTIVE: To clone human peroxisome proliferator-activated receptorγ1 gene (hPPARγ1) and construct a vector of phPPARγ1-IRES2 -EGFP carrying hPPARγ1 gene. Transfect 293 cellls with this construted vector and detect the hPPARγ1 gene expression in transfected 293 cells in order to establish an ideal molecular platform for screening natural ligands and studying hPPARγ1 gene’s functions. METHODS: Primer pairs which contained cutting sites of Xho I and Sma I endonucleases were designed and used to amplify the hPPARγ1 gene from total RNA of HepG2 cells by RT-PCR. Both the PCR product of hPPARγ1 and pIRES2-EGFP plasmid were excised by Xho I and Sma I endonucleases. The products of enzyme digestion were recovered from agarose gel and then ligated with each other. The recombinant plasmids were transformed into DH5αcompetent cells prepared by CaCl2 method, and cultured in LB agar medium containing kanamycin for selecting positive clones. Doubly digesting of Xho I and Sma I and sequencing were used to identify the integrity and fidelity of hPPARγ1 gene contained in the recombinant plasmid from the positive clones. The correct recombinant plasmid was transfected into 293 cells for the purpose of expressing cloned hPPARγ1 gene. Real-time quantitative PCR, immunocytochemistry, and western blot assays were used to analyze the expression levels of hPPARγ1 mRNA and protain in the transfected 293 cellls.RESULTS: hPPARγ1 gene sequence contained in recombinant plasmid phPPARγ1-IRES2-EGFP was verified by enzyme digestion as well as sequence analysis. The sequence of inserted hPPARγ1 gene was in accordance with the corresponding sequence found in GeneBank database (NM005037/gi:62865855,NM138711/gi:62865854,NM138712/gi:62865852). The high expression of GFP reporter gene can be seen in transfected 293 cells under fluorescence microscope and the transfection efficiency of phPPARγ1-IRES2-EGFP was 83%±11%. hPPARγ1 gene contained in phPPARγ1-IRES2-EGFP plasmid was found to display a highly efficient expression at both of mRNA and protein levels.CONCLUSION: The recombinant plasmid phPPARγ1-IRES2-EGFP has been constructed successfully with highly efficient expression in transfected 293 cells, which provides a useful tool for analyzing hPPARγ1 gene’s functions as well as establishing a molecular platform by which the candidates of unknown hPPARγ1 ligands or activators can be found.CHAPTER FOUR Anti-proliferation effects of berberine on human breast cancer cell line MDA-MB-231 and its relationship to PPARγOBJECTIVE: To investigate the antitumor effects of berberine and rosiglitazone on human breast cancer cell line MDA-MB-231 and evaluate their potential application value for breast cancer therapy.METHODS: Cytostatic effects of berberine and rosiglitazone on MDA-MB-231 cells were measured by MTT assay. MDA-MB-231 cells were treated with berberine and rosiglitazone alone or in combination with PPARγantagonist GW9662 to investigate the effects of berberine and rosiglitazone on the cells proliferation and their relationship to PPARγ. Cell-cycle kinetics was assessed by flow cytometer. Apoptotic cells were determined both by Annexin V staining and TUNEL assay.RESULTS: MTT analysis demonstrated that both berberine and rosiglitazone inhibited growth of MDA-MB-231 cells in a concentration and time-dependent manner with IC50 values of 0.21and 4.3μmol/L at 24 h after the drugs added into the culture, respectively. PPARγselective antagonist GW9662 could, at least in part, reverse the inhibitory effect of rosiglitazone on proliferation of MDA-MB-231 cells, but could not reverse the inhibitory effect made by berberine. Cell cycle analysis showed that the percentage of G0/G1 phase cells increased, S phase cells decresed, and cells were arrested in G1 phase with increasing concentrations of rosiglitazone. Detectable signs of apoptotic cell death caused by rosiglitazone occurred only at the concentration of 100μmol/L and the similar apoptosis rates from TUNEL assay and Annexin V assay were observed (18.4%±3.1% and 16.6%±2.7%) (P>0.05). Apparent apoptosis of MDA-MB-231 cells induced by berberine were observed by TUNEL assay and the apoptotic rates induced by 1×10-6, 1×10-5 and 1×10-4 mol/L berberine were 17.7%±3.2%, 59.3%±4.5%, and 89.6%±5.7%, respectively.CONCLUSION: Contrary to rosiglitazone which inhibited the growth of MDA-MB-231 cells via PPARγactivation, the berberine’s effect of growth inhibiting of MDA-MB-231 cells was independent of PPARγ. The apoptotic rates of MDA-MB-231 cells induced by berberine were higher than rosiglitazone made.These results suggested that berberine and rosiglitazone might be an effective and promising agents for the treatment of breast cancer.更多还原