The Establishment And Amplification Of PPARγ2 Agonists Screening Models In The Evaluation Of Functional Regulation Of Blood Sugar Of Black Tea Extracts

With economic conditions improving, lifestyle changing, the prevalence of diabetes has risen greatly, especially for typeⅡdiabetes. Researches showed that insulin resistance play an important role on the occurrence and development of typeⅡdiabetes. Peroxisome proliferators-activated receptor gamma2 was the members of typeⅡnuclear hormone receptor superfamily, which was ligand-activated transcription factor. Presently, there are several known ligands and agonists. Thiazolidinediones was the peroxisome proliferators-activated receptor gamma2 agonist, recently approved for the treatment of typeⅡdiabetes mellitus. In order to adjust synthesis, transportation and utilization of glucose and metabolism of fat, the transcription of many genes which correlated with insulin effect were regulated. Consequently, it bated insulin resistance and enhanced insulin effect. This research established peroxisome proliferators-activated receptor gamma2 agonists screening models for screening hypoglycemic activity of black tea, in the programs of discovering new anti-diabetic drugs. The main research results can be listed as follows:1. Construction, amplification and extraction of PcDNA3.1(+)-PPARy2 expression vectors and PG13-PPRE×3-luc reporter vectors, which were sequenced that no misincorporated mutations and in the correct reading frame.2. In this experiment, two methods (boiling method and water-bath method) were used to extract short DNA fragments which was co-precipitated with absolute ethanol and 3 mol/L sodium acetate together. Results showed:the recovery rate of short DNA fragments in water-bath method is higher than boiling method. Water-bath method combined with ethanol precipitation for extracted short DNA fragments had many advantages, such as economical and convenient which should be advocated to use widespread.3. This research established peroxisome proliferators-activated receptor gamma2 agonists screening models previously. PcDNA3.1(+)-PPARy2 expression vectors, PG13-PPRE×3-luc reporter vectors and PRL-TK internal reference vectors were transiently co-transfected into HEK 293T cells. The effect of rosiglitazone on the signal transduction of PPARy2 was determined by examing the Luc activity. The effect of rosiglitazone on the proliferation function of cells was determined by CCK8 method. The cells were treated by rosiglitazone at 0.1,1,10,100,1000μmol/L for different time, and then tested absorbance at 450 nm. The results showed no effect of the proliferation of HEK 293T cells when the cells treated by rosiglitazone at 0.1,1,10,100,1000μmol/L for 24 hours, but made a difference at 100,1000μmol/L concentration for starting from 48 hours. The optimal transfection formula was determined by comparing the expression level of Luc in HEK 293T cells transfected by different ratio of expression vectors, reporter vectors and internal reference vectors. The results showed the optimum ratio of 3 vectors is 0.06/0.12/0.02 in sequence. The Luc expression level inducted by rosiglitazone was dose and time dependent manner. The highest expression level of Luc in HEK 293T cells, inducted by rosiglitazone was at 0.1μmol/L for 24 hours. Consequently, optimal induction concentration of rosiglitazone was 0.1μmol/L, time was 24 hours in the meanwhile.4. The model was used to screen PPARy2 excited activity from eight kinds of black tea extract, in which Tianjian Dark Tea,Golden Tips, Brick Tea, Fu Brick Tea and Qianliang Tea exhibited obvious positive signal, while Jin Hua Tiancheng Tea, Longrun Pu'er Tea and Liu Pao Tea exhibited a little..