Construction Of Human PPAR Series Vectors & Establishment Of Their High Efficient Tranfection And Expression System

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.