The Inhibitive Effects And Mechanisms Of PPARγ1 On The Extracellular Matrix Of Mesangial Cells

PART I:The establishment of plasmid over-expressing mPPARγ1 and functional identification in mesangial cellsObjective: Peroxisome proliferator-activated receptorγ (PPARγ) belongs to the muclear receptor superfamily, which are ligand-activated transcription factors. In general PPARγ is used as PPARγ1. PPARγ1 has two kinds of ligands. Fatty acids (FAs), FA-dirived compounds and 15d-PGJ2 are natural ligands for PPARγ1. Thiazolidinediones (TZDs) are high-affinity synthetic ligands for PPARγ1. Owing to the critical role of PPARγ1 play in lipid transportion and metabolism, Recent studies suggest that PPARγ1 is involved in a series of disorders such as diabetic nephropathy, obesity, atherogenesis, hypertension and some tumor. And its effect does not limit to the metabolism. It appears to block directly the progression of inflammation, sclerosis and proliferation without influencing metabolism. It is generally believed that TZDs exert those functions through activation of PPARγ1. But someone brought forward the concept that those effects come from TZDs' pharmacological property not from PPARγ1. However it remained unclear whether the effects originated from PPARγ1 or the ligand. To further investigate the mechanisms of PPARγ1 on the renal diseases we construct a plasmid expressing PPARγ1 and identify its functions on transfected mesangial cells. Methods: Wild type full length of mouse PPARγ1 (mPPARγ1/WT) cDNA was ligated into an expressing vector pIRES-EGFP. This constructed pIRES-EGFP-mPPARγ1/WT was then transfected into cultured glomerular mesangial cells facilitated by lipofectin. We screened the condition of the highest effiency of transfection by flowcytometry and MTT. The expression level of PPARγ1 was determined by RT-PCR and Western blot, as well as by specific PPRE binding activity. A vector expressing dominant negative type of mPPARγ1, pIRES-EGFP- mPPARγ1/DN, which lacks the biological activity of PPARγ1, was also constructed and transfected by using the same technology to serve as a control throughout the study.Results: Restriction enzymatic analyses and DNA sequencing confirmed the accuracy of constructed and selected plasmids. By the flowcytometry and MTT we got the optimum proportion of plasmid and lipofectin was 2ug/3ul per ml. The mRNA and protein level of PPARγ1 in mesangial cells reached the peaks 48 hours after transfection. The activity of PPARγ1 in the two types of plasmids was different. PPARγ1/WT got the highest actibvity at the timepoint of 48 hours. PPARγ1/DN kept low consistent activity approximately throughout of the experiment.Conclusions: The constructed expressing vector pIRES2-EGFP-mPPARγ1/WT provides a useful tool for further study on the effects and mechanisms of PPARγ1.