Effects Of Renin-angiotensin System Inhibitor On The Differentiation Of Human Preadipocytes And Expression Of Adiponectin

Objectives:The renin-angiotensin system(RAS) plays a major role in regulating blood pressure and cardiovascular function.The increased activity of RAS contributes to the development of many diseases,such as hypertension,cardiac failure,artherosclerosis,insulin resistance and so on.There exists fairly renin-angiotensin system in the region adipose tissue,the major effect factor of angiotoninâ…¡(Angâ…¡) participates the regulation of region body fat,contribution fat metabolism,by endocrine secretion,paracrine secretion and autocrine.In our experiment,our research is based on human preadipocyte,approaching:The cultural method of human preadipocyte.;The effects of RAS inhibitor on the differentiation of Human Preadipocytes and the expression of APN.Methods:1,Preadipocytes from 16 adult female of abdominal region subcutis and omentum,all the subjects were empty stomach,taked blood,detected blood sugar, serum Crea,cholesterol,triglyeride and insulin,et al.Adipose tissue was thoroughly flushed,cutted into small pieces,treated to asepsis,disconnected and removaled the fibrous tissue and blood vessel,tissue digestion,spallated red blood cell,cell filtration, trypan blue rejection experiment to observe living cell rate and count,derivated and differentiated the cell from 3-6 generation,use different RAS inhibitor to intervention till cell harvesting.Base of different cell treatment,divided to 4 groups,ACEI,ARB, TZD and normal control.MTT chromatic test to detect cell vigour,Stained by Oil Red O to detect the content of lipid in adipocyte,Glycose consumption test to detect the cell insulin sensibility,detected the activity preadipocyte differentiation marker--G3PDH,detected the secretory of APN,PCR(absolutely quantitative method) to detect APN mRNA expression.Results:1,Establish the human preadipocyte primary culture model.The primary culture of human preadipocyte adherenced in 24h,shaped mostly fusiform, small amounts rod-shape or triangle,proliferated about 5-7 days can be confluens. After go down to the future generation,the growth velocity speed up obviously,and can be confluens again after 2-4 days.After 1-2 passage,the cells form uniformity fusiform,Oil Red O stain are negative.Derivated and differentiated the cell from 3-6 generation,after 4-5 days around the nuclear small refraction particle can be seen, they are lipid droplet verificated by Oil Red O stained.Lipid droplet augmentation gradually and confluence,cell rounding at the same time,the nuclear is squeezed to one side,and form to be full of a great quantity lipid droplet round cells,they are the ripe adipocyte,Oil Red O stain are positive.This process to achieve peak about 14 days,after 21-28 days as over-mature cells are get off.2,Effects of RAS inhibitor on the differentiation of human preadipocytes and insulin sensitivity.Subcutis and omentum source preadipocyte,the cell vigour,lipid content,G3PDH activity and glycose consumption contrast have statistical significance,all of are higher than the control group.The omentum source ARB group each differentiated index all are higher than TZD group.3,Effects of RAS inhibitor on the APN secretary of human preadipocytes.The secretory volume of APN from the mature adipocyte after differentiation are higher than the preadipocyte undifferentiation.The treatment group are higher than nomal contrast group.The omentum source ACEI and ARB group cell are higher than TZD group,the subcutis source are the opposite.4,Effects of RAS inhibitor on the APN mRNA expression of human preadipocytes.In the subcutis source or omentum source preadipocyte groups,the APN mRNA in each intervention groups are higher than the control group.The omentum source adipocyte ACEI and ARB group APN mRNA level are higher than the TZD group,the subcutis source are the opposite.5,Effects of tissue source on the adipocyte differentiation index and mRNA expression.On the basic condition the subcutis source preadipocyte vigour are higher than omentum,RAS inhibitor can cancel this difference;On the basic condition the subcutis source cell lipid content are degrade,but this difference disappear between ACEI and TZD group.The location specificity of glycose consumption and G3PDH vigour only occur in the TZD group,subcutis source are all higher than omentum source.The location specificity of the secretory volume of APN occur in the TZD group(subcutis source are higher than omentum source) and ARB group(omentum source are higher than subcutis source);The souce of tissue have no ffect to the secretory volume of APN of the undifferentiation and non-treatment groups.The difference of location specificity of APN mRNA expression are shown as omentum source are higher than subcutis source in the control group and RAS inhibitor group,there's no difference in the TZD group.Conclusions:Through the enzyme digestion method,we got the fusiform cell of uniform-component,vigorous multiplication and cell differentiation.This cell had three typical characteristics like preadipocyte.â‘ it was from adipose tissue,fusiform shape,seldom or none of lipochondria in the intracytoplasm.â‘¡Rapid multiplication, got close to the doubling time with collagenoblast.â‘¢It changed to adipose cell after forming monolayer confluens,it included the appearance of enzyme lipochondria and insulin play an important promoting role.Induced-differentiation mature adipocyte is c formed typically as a ring,which is accredited as masculine through Oil Red O dying.In our reseach,preadipocyte get from omentum and abdominal subcutis,we find the APN level is very low before differentiation,Although the gene expression exist difference after differentiation,the secretion of APN at subcutis and omentum source human preadipocyte have no difference in the basic condition.RAS inhibitor enhanced preadipocyte differentiation,improving cell insulin sensitivity,boosting APN secretion and mRNA expression;and it played dominated effect in internal organs compared with TZD.Regression analysis show that,the expression and secretory volume of the omentum source cell APN mRNA are mostly affected by the treatment,however,the influential factor of subcutis source are rather complex, the possible reason can be concerned with the vigour of omentum source preadipocyte are lower in the basic condition.