Effect Of Hypoxia Inducible Factor-1α On Regulatory Mechanism Of Hepatocyte Growth Factor Expression

Objective: Part 1: (1) To study the survival, proliferation and apoptosis effects of CoCl2 on rat cardiomyocytes and Hela cells. (2) To investigate the expression level of HGF/c-Met mRNA in rat cardiomyocytes and Hela cells under hypoxia, mimicked by CoCl2 treatment or pcDNA3-HIF-1α tranfection. (3) Bioinformatic analysis of human HGF promoter to find the probably regulation factor of HGF under hypoxia. Part 2: (1) To investigate the regulatory mechanism of HIF-1α on TGF- β2 expression according to the bioinformatic analysis.. (2) Observe the effect of recombinant TGF- β2 on the expression of HGF proteinMethods: Part 1: (1) The survival, proliferation and apoptosis effects of CoCl2 on primary cultured neonatal rat cardiomyocytes and human cervical tumor Hela cells were detected by MTT and flow cytometer assay. (2) Construct the pcDNA3-HIF-1α eucaryotic expression vector. (3) Detect apoptotic protein Bcl-2 and Bax by Western blot in Hela cells, when treated with CoCl2 and tranfected with pcDNA3-HIF-1α. (4) RT-PCR was performed to detect the HGF mRNA level in rat cardiomyocytes, when treated by CoCl2 for different time. (5) Western blot was performed to detect the HIF-1α protein level in rat cardiac myocytes, when treated by CoCl2 for 12h. (6) Real time PCR was performed to detect the HGF/c-Met mRNA level in Hela cells, when treated with CoCl2 and transfected with pcDNA3-HIF-1α. (7) Effect of CoCl2 on HGF protein expression in Hela cells were measured by ELISA. (8) Bioinformatic analyse of human HGF promoter. Part 2: (1) Construct pGL3-EPO-HRE firefly luciferase reporter vector and wide type pGL3-TGF-β2 promoter and its relevant five mutants firefly luciferase reporter vectors. (2) Western blot and dual-luciferase repoter gene assay were performed to detect the expression and DNA...