The Study Of Adipose Tissue Extract To Adipogenic Differentiation Of Adipose Derived Stem

Clinically, patients with trauma, tumor surgery, birth defects as well asinfected patients, requires a lot of adipose tissue to repair soft tissue defects.However, there are many limitations of self-body adipose transplantationwhich is commonly used. The adipose tissue engineering is an emergingtechnology that reconstructs the soft tissues with stem cells, of which stemcells and the adipogenic factor play a key role. Due to its wide variety ofsources, easily obtained, multi-directional differentiation potential, adiposestem cells have been widely used in tissue engineering research. Adiposetissue is not only an energy storage organ, but also can secrete largeamounts of cytokines. We found that adipose stem cells can differentiate tothe adipose cells when using adipose tissue blocks to culture adipocytes byprimary culture. Adipose tissue secretions may contain adipogenic factorswhich can induce adipose stem cells adipogenic differentiation. Now theadipogenic factors in vitro researched maturely include: insulin,indomethacin and so on, and few studies on the physiological adipogenicfactors. This subject obtained adipose stem cells by tissue primary culture, and to explore the relationship between adipose tissue secretions andadipogenic. The research of physiological adipogenic factors will lay thefoundation for adipose tissue engineering for clinical.Methods：1. Culture rat adipose stem cells with adipose tissue block primaryculture method, and observe cells’ morphology and growth condition by theinverted microscope. When culturing the primary cell to the fourthgeneration, induce them respectively with the adipogenic, osteogenic, aswell as into neural inducer, and then do oil red O staining, Alizarin redstaining and immunofluorescence experiments.2. Collect the first three days’ culture medium as ATE with adiposetissue block primary method, take the fourth generation rat adipose stemcells which grew well, induce rat adipose stem cells into adipocytes withATE, and then do oil red O staining experiment.3. Culture adipose tissue block with culture medium containing FBSor not, collect ATE to induce the rat adipose stem cells into adipocytes,and then do oil red O staining experiments.4. Collect non-FBS medium to obtain ATE, divide into100μg/ml,250μg/ml,500μg/ml component, and induce the fourth generation ratadipose stem cells. Then observe the adipogenesis state, do oil red Ostaining experiments to detect the adipogenic rate, do the cell proliferationand migration experiments. 5. Gather the adipose tissue blocks secreted factor by ultrafiltrationtube and divide it into five different groups according to the molecularweight, and then induce the fourth generation of adipose stem cellsrespectively. Detect using Western blot and immunofluorescence stainingexperiments for each group.6. As adipose stem cells secreted proteins as control, analyze adiposetissue secretions by mass spectrometry; explore adipogenic factors in ATEwith information science. And then test and verify by western blotexperiment.Results：1. Cell obtained by adipose tissue block method grows well, candifferentiate to the adipose cells, osteoblasts and nerve cell.2. When ATE have induced three days, it was visible that there werelipid droplets in some of the fourth generation adipose stem cell cytoplasm,and on the seventh days, lipid droplets merged. There were no lipid dropletsin the negative control group.3. Whether getting ATE with FBS or not, there were also mature lipiddroplets in cytoplasm of ADSCs on the seventh day. And the adipogenicrate of two groups has no statistical difference.4.500μg/ml group has high adipogenic rate comparing with100μg/mland50μg/ml groups. Different concentrations of ATE are inhibition ofADSCs’ proliferation, but have no significant difference between groups. ATE has no effect on cell migration.5. On the seventh day，it was visible that the rat adipose stem cells inthe induction group whose molecular weight greater than100KDa began todifferentiate adipogenesis, and the rest of the group had no adipogenic. Theresults of Western blot and fluorescence immune experiment showed thatthe expression of adipogenic protein is positive in the induction groupwhose molecular weight greater than100KDa.6. Identify differentially expressed proteins whose molecular weightgreater than100KDa by mass spectrometry, they are Col I, CP, IgG2a, andFN. And western bolt experiment has once again proven the reliability ofthe results.Conclusions：1. The cells obtained by adipose tissue block method have thebiological characteristics of adipose stem cells; they have a good ability ofmulti-directional differentiation.2. ATE can induce adipose stem cells adipogenic differentiation, andATE contains adipogenic factors.3. FBS has no effect for adipogenic capacity of ATE.4. Consistent with stem cell adipogenic differentiation mechanism,the adipogenic capacity of500μg/ml group is stronger than lowerconcentration groups. ATE is able to inhibit cell proliferation, but thisinhibition is not unlimited. ATE has no effect on cell migration. 5. Factors capable of adipogenic differentiation in adipose tissueblock secretion is focused on the group whose molecular weight greaterthan100KDa. And this laid a foundation for further studies on newphysiological adipogenic factor.6. It is confirmed by our experiments that Col I, CP, IgG2a and FNare indeed present in adipose tissue secretions; its expression level ishigher than pure adipose stem cells secreted proteins. It might be anadipogenic factor.