Research On The GDM Insulin Resistance And Expression Of Omentin-1,Chemerin And Vaspin In Vitro

BackgroundGestational diabetes mellitus(GDM)is a kind of diabetes mellitus which is discovered or occurred during the period of pregnancy for the first time and threaten pregnant women and their offspring seriously.So far the cause of GDM is unclear.Numerous studies agree that GDM is a kind of metabolic disease caused by a variety of factors.Insulin resistance and disorder of glycolipid metabolic caused by the insulin resistance is now recognized as one of the pathogenesis of GDM physiological and pathological basis.While the abnormality of insulin signaling pathway is the main cause of insulin resistance.In recent years,the relationship between the fat factors from fat tissue(adipokines)and GDM has got more and more public attentions and become a new research hotspot.In our previous studies,we had found that Omentin-1 appeared significantly reduced but Chemerin and Vaspin appeared significantly increased in GDM patients' serum,omental adipose and placenta tissue.The results showed that Omentin-1,Chemerin,Vaspin may have correlations with GDM insulin resistance.But the preliminary study is confined to the serum and histological studies in the human body,it just reveals the facial phenomenon that existed between the three adipokines and GDM.Meanwhile,it brings a new problem,that is whether there is a causal relatioship among Omentin-1,Chemerin,Vaspin and GDM insulin resistance?if there is,the changes of adipokines lead to the occurrence of GDM insulin resistance?or the occurrence of GDM insulin resistance leads to changes of these adipokines?In order to further explore if there is a necessary connection between Omentin-1,Chemerin,Vaspin and GDM insulin resistance,this study will based on the previous research and built model of original generation cultivation,proliferation and differentiation of GDM epiploon preadipocyte,using fat cells of GDM culture in vitro as the carrier to detect the expression of mRNA and protein of insulin receptor substrate-1/2(IRS-1/2)and phosphatidy inositol 3 kinase(PI3K)of fat cells in the downstream of the insulin receptor signaling molecules in an over expression state of different degrees of Omentin-1,Chemerin and Vaspin,as well as the changes of IRS-1/2 phosphorylation levels and glucose uptake rates of cell.By detecting signal expression changes after the insulin receptor,to explore inner link between Omentin-1,Chemerin,Vaspin and GDM insulin resistance from classic insulin signaling pathways(IRS-1/2,PI3K),so to provide clues for GDM's pathogenesis and treatment strategies.Part 1 Original generation cultivation,proliferation and differentiation model establishment of GDM epiploon preadipocyte cellsObjectiveTo establish the model of original generation culture and proliferation and differentiation of the GDM epiploon preadipocyte cells,so to lay the foundation for the follow-up study of GDM.MethodsUsing improved cell culture method,with greater omentum pure fat tissue of GDM cesarean section patients as raw material,made adipocyte cells primitive culture and batches,drew growth curve of batches preadipocyte cells.Induced the adipocyte cells into differentiation,staining oil red O on differentiated cells and testing the dynamic changes of gycerol phosphate dehydrogenase(GPDH)and fat contents,RT-PCR detecting adiponectin mRNA,so to identify whether the preadipocyte cells having been induced into mature fat cells.carrying on experiment of cells' cryopreserved and recoveried.Results1.There were some cells attached the wall after 6 h culturing,adherent rate of 13h was 70%,all cells almost attached the wall after 24h.2.The cultured preadipocyte were spindle cells,the proliferation of the cells was strong and the cells began to proliferate rapidly on the fourth day.It can be seen that the doubling time was about 48 h from the growth curve.3.On the seventh day,the preadipocyte of original generation turned from spindle into oval or round shape,the cells began to appear fat particles,and the extended cells still maintained spindle shaped and without fat droplets.4.On the 9 day,the original generation of preadipocyte has started to appear a lot of fat particles,while the extended cells still remained a spindle to the ninth days,the single layer of cells integrated basically and arranged closely,oil red O staining showed that still no colored.5.After preadipocyte passaged in the primary 4 days,the cells morphology and growth condition were strikingly similar to preadipocyte of original generation,almost being spindle cell and the uniform sizes.After 5-6 days,the cells entered the phase of rapid proliferation,arranged in parallel,and completed the integration of the single layer on the seventh days.6.Under the induced action of a certain concentration of insulin(10?g/ml)and dexamethasone(lumol/L),GPDH started rising and increasing rapidly.About after 9 days,the GPDH reached the peak and remained at a high level.After 3 days,some of the cells began to appear single scattered lipid droplets.With the extension of differentiation time,lipid droplets increased gradually and surrounded the nucleus in the cytoplasm.After about 11 days,lipid droplets reached the peak and were consistent with the quantitative results of oil red O staining.7.RT-PCR tested the mRNA of adiponectin.The electrophoresis results showed the expression of adiponectin mRNA in the differentiation of fat cells and no mRNA expression of adiponectin in the preadipocyte.Conclusion1.There have preadipocyte in omental adipose tissues of GDM,the components of preadipocyte with improved method by research groups were homogeneous,the proliferation is strong,the doubling time is about 48h.It can extend in succession and have a large number of amplification,the proliferation of cells rapidly within the 5 generation generally,and then the proliferation and differentiation rate begin to decrease.2.During the course of the culture and proliferation of the preadipocyte of original generation,it will naturally appear to differentiate into mature adipocytes,while preadipocyte after subculture will lost the ability to differentiate and may be associated with some inducible factors of the body that the original generation cells still carry.3.The preadipocyte can be induced to differentiate,a certain amount of insulin(10 g/ml)and dexamethasone(1umol/L)play an important role in the differentiation of preadipocyte and is the starting and promoting factor,the differentiation rate after induction is high and can up to 80%.4.The increase of the content of fat in the cells after induction is about 6 days later than GPDH,which shows that the appearance of enzymes is earlier than fat.5.The preadipocyte before subculturing can be cryopreserved and recoveried,after the appropriate induction can be differentiated into mature fat cells,the build of fat cell model make a good preparation for the follow-up of fat cells in vitro experimental study.Part 2 Effect of Omentin-1,Chemerin and Vaspin over expression in vitro on insulin signal transduction pathwayObjectiveUsing preadipocytes of GDM in vitro culture as the carrier to explore the expression of mRNA and protein of insulin receptor substrate-1/2(IRS-1/2)and phosphatidy inositol 3 kinase(PI3K)in the downstream of the insulin receptor signaling molecules of fat cells,as well as the changes of IRS-1/2 phosphorylation levels and glucose uptake rates of cell in an over expression state of Omentin-1,Chemerin and Vaspin,to explore inner link between Omentin-1,Chemerin,Vaspin and insulin resistance of GDM from the insulin signal transduction pathways of IRS-1/2 and PI3K(P85a).MethodsReviving,subculturing and differentiating preadipocyte of GDM,building Omentin-1,Chemerin and Vaspin over expression plasmid and using escherichia coli expression system to carry on the transformation,culture and plasmid extraction.Using 3 different over expression gradient(1.0?g,2.5?g and 5.0?g)to transfect fat cells of third generations with human source(each concentration tansfected 6 holes,3 series and a total of 18 data),with no transfection group as contrast;Using Q-PCR to detect mRNA expression of Omentin-1,Chemerin,Vaspin,IRS-1/2 and PI3K(P85a),using Western Blot to detect protein expression of Omentin-1,Chemerin,Vaspin,IRS-1/2,PI3K(P85a)and tyrosine phosphorylation levels of IRS-1/2,and[3H]-2-deoxidation-D-glucose uptake assay to detect the changes of glucose uptake of cells in different concentration trasfection groups.Using the mean value of group with O.Oug as standard when did statistic analysis,and the other 3 groups were standardized and quantified with the correspond ratio of x±S and the software of SPSS 20.0 was used for statistical disposition.Results1.With the elevating concentration gradient of transfection,all of the mRNA and protein expression of Omentin-1,Chemerin and Vaspin increased(all P<0.05),the constructed over expression carrier was efficient.2.With the increasing Omentin-1 expression,the mRNA and protein expression of IRS-1 and PI3K(P85)increased obviously in adipose cells(all P<0.05),the mRNA and protein expression of IRS-2 had no obvious change(all P>0.05),the IRS-1 phosphorylation level increased obviously(P=0.031),phosphorylation of IRS-2 had no obvious change(P=0.685),the glucose uptake rate elevated(P=0.024).3.With the increasing Chemerin expression,the mRNA and protein expression of IRS-1/2 and PI3K(P85a)in adipose cells had no obvious changes(all P>0.05),but the phosphorylation level of IRS-1 increased obviously(P=0.041),the phosphorylation level of IRS-2 had no obvious change(P=0.585),glucose uptake rate of fat cells elevated slightly,but had no Statistical differences(P=0.064).4.With the increasing Vaspin expression,the mRNA and protein expression of IRS-1/2 and PI3K(P85a)in adipose cells had no obvious changes(all P>0.05),the IRS-1 and IRS-2 tyrosine phosphorylation level had no obvious changes(all P>0.05),the glucose uptake rates had no obvious changes too(P=0.656).Conclusion1.There exists close relationship between Omentin-1 and GDM insulin resistance,the expression of Omentin-1 may activated signal pathways of IRS-1 and PI3K(P85)by some way and lead to increase of IRS-1 expression and activation of phosphorylation,and promote the excitation and expression of PI3K and improve glucose uptake rates.Omentin-1 plays a role of insulin sensitization and has no obvious relationship with IRS-2.2.There may have a certain relationship between Chemerin and GDM insulin resistance.With increasing Chemerin expression,although the mRNA and protein expression of IRS-1,IRS-2 and PI3K(P85a)have no obvious changes,the degree of IRS-1 phosphorylation increased and glucose uptake rate of cell also increased slightly.The specific mechanism is unclear and remains to be further researched.3.There is no necessary link between Vaspin and GDM insulin resistance.The high expression of Vaspin in GDM serum and omental adipose tissue is probably a compensation mechanism of obesity,glycolipid metabolic disorder and insulin resistance of GDM,it is a result of the interaction among fat accumulation,hyperglycemia,hyperlipidemia and Vaspin in the GDM body.