The Research Of Phospholipid Transfer Protein Gene Expression In3T3-L1Preadipocytes During The Induced Differentiation Process

ObjectiveThe study aimed to i) compare the activity of serum phospholipid transfer protein(PLTP) between normal people and obese patients in order to preliminarily understand therelationship between PLTP with obesity, ii) to determine the changes of PLTP geneexpression in mouse embryonic fibroblasts (3T3-L1preadipocytes) during the process ofinduced differentiation in vitro, and iii) to explore the relationship between PLTP geneexpression with preadipocyte differentiation and lipid accumulation. Our data will providea new approach for the treatment of obesity and AS.Methods1. Detect the serum PLTP activity of normal people and obese patients:50-60yearsnormal (18.5≤Body Mass Index (BMI)<25) and obese (30≤BMI) of11cases each wereselected for the experiment. We pumped people’s blood, selected the serum aftercentrifugating the blood, and serum PLTP activity was determined by Human PLTPactivity assay kit.2. Culture and induce3T3-L1preadipocytes: the3T3-L1preadipocytes were culturedin vitro using pre-adipocyte expansion medium, and were seeded in six-well plates whenthe cells were70-80%confluent. Until the culture reached100%, seeded the cells withpre-adipocyte expansion medium for2-3days. Then induced the3T3-L1preadipocytes by1-methyl-3-isobutyl xan(IBMX), Dexamethasone and Insulin for2days. In thesubsequent induction process, the cells were seeded with adipocyte maintenance medium.3. Stain the induced cells with oil red O: selected the cells which were induced for0, 2,4,8,11,14days respectively, and then stained them using oil red O. The changes ofcells’ morphology and lipid aggregation were observed by inverted microscope.4. Detect the changes of PLTP mRNA in3T3-L1preadipocytes during the process ofinduced differentiation: extracted the total RNA of the cells which were induced for0,2,4,9,12days respectively. The changes of PLTP mRNA were detected by real-timequantitative PCR.5. Detect the changes of PLTP protein in3T3-L1preadipocytes during the process ofinduced differentiation: extracted the total protein of the cells which were induced for0,2,4,9,12days respectively. The changes of PLTP protein were detected by western blot.Results1. The obese patients’ PLTP activity was higher than the normal people’s PLTPactivity, about twice as high as the normal people’s. This result layed the theoreticalfoundation for the following experiments that PLTP in3T3-L1preadipocytes werechanging during the process of induced differentiation.2. After staining3T3-L1preadipocytes by oil red O, the color of the petri dish wasdeepening with the increased number of induced days in the visible spectrum. The cells’volums were becoming bigger and more round with the increased number of induced days.Under an inverted microscope, the mature adipocytes containing red lipid droplets whichwere cultured for11days or more were accounted for90%or more in the field of vision.3. The changes of PLTP mRNA: the levels of PLTP mRNA in3T3-L1preadipocyteswere increasing with the increased number of induced days. On the second and forth day,the levels of PLTP mRNA were not changed significantly (P>0.05). Compaired with thecells induced for0day, the levels of PLTP mRNA were increased significantly (3.3times,P<0.01,3.7times, P<0.01and5times, P<0.01, in the cells induced for6,9and12daysrespectively）.4. The changes of PLTP protein: at different stages of the process of induceddifferentiation, the levels of PLTP protein and PLTP mRNA were consistent in generalaccording the results of western blot. On the second and forth day, the levels of PLTPprotein were not changed significantly (P>0.05). Compaired with the cells induced for0day, the levels of PLTP protein were increased significantly (3.2times, P<0.05,4.1times,P<0.01and3.4times, P<0.05, in the cells induced for6,9and12days respectively）.Conclusion1. The obese patients’ PLTP activity was higher than the normal people’s PLTPactivity, which indicated PLTP and obesity were positively correlated. 2.3T3-L1preadipocytes were gradually differentiating into mature fat cells with theincreased number of induced days. On the11th day, the induction rate reached the highestlevel, and the mature fat cells no longer increased with the increased number of induceddays. The mRNA levels of PLTP and intracellar lipid increased simultaneously.3. The protein levels of PLTP in3T3-L1preadipocytes were increasing with theincreased number of induced days, which indicated PLTP may be involved even regulatedthe maturation process.