Induction And Differentiation Of Preadipocytes In The Orbit Of Thyroid-associated Ophthalmopathy (TAO) And Observation Membrane Surface By Atomic Force Microscopy (AFM)

Objectives: Culture human orbital preadipocytes and investigate the induction and differentiation processes in vitro and identify it. Find out the best methods observing preadipocytes by Atomic force microscopy (AFM). Investigate the membrane surface ultrastructures of the preadipocytes in the orbit of normal human and TAO patients, the preadipocytes are observed and compared by AFM. Investigate the membrane morphologic change during the differentiation of preadipocytes in different period by AFM, and explore the pathogenesis.Methods:①Orbital fat tissue form TAO patients (n=15) and normal human (n=15) in primary culture.②Tissue culture methods and enzyme-digesting methods were used in primary culture of human orbital preadipocytes and compared. We observed the morphology, we observed the proliferation and the differentiation process of human orbital preadipocytes by MTT, we stained it with oil red O, we used immuocytochemistry to detect S-100.③Use different concentrate of glutaric dialdehyde fixed preadipocytes, and choose the best one as finally fixing solution. TAO and normal human orbital preadipocytes membrane were observed by AFM, the course of the differentiation of TAO and normal human orbital preadipocytes membrane were observed by AFM.Results:①Tissue culture methods and enzyme-digesting methods are reliable to obtain enough preadipocytes. Enzyme-digesting methods can get purer and more preadipocytes than tissue culture methods can, and the cell is fitter for AFM sample.②Use enzyme-digesting methods can get preadipocytes, which have some features: the cell is come from fat tissue, fusiform shape. The growth curve is"S"shape, the log phase growth is 3-11 day, and S-100 is weakly positive. We could see lipid droplets when preadipocytes were differentiated on the 2th day. Mostly preadipocytes were differentiated into mature adipocyte on the 16th day, they can be dyed in red by oil red O.③TAO and normal human orbital preadipocytes membrane are observed by AFM. when the scanning scope is 80μm×80μm, there is no evident difference. When the scanning scope is 1μm×1μm, the two cells appear many inequal of sizes granulo-material. The membrane of TAO preadipocytes are rougher, and the roughness is: 36.480±8.146nm. The membrane of normal human preadipocytes are smoother, and the roughness is: 32.189±7.819nm. The cell membrane of TAO is obviously rougher then normal cells (P<0.01).④The preadipocytes in the orbit of TAO and normal human can be differentiated into mature lipid-filled adiocytes, and the course of differentiation are observed by AFM. When the scanning scope is 80μm×80μm, with the time is lasting, the shape of cell is become polygon or round. When the scanning scope is 1μm×1μm, with the time is lasting, cell membrane is become rougher. And in the same day, the orbital preadipocytes of TAO is more roughness than normal human preadipocytes (P<0.05).Conclusions:①Enzyme-digesting methods can get more and purer cells than tissue culture methods.②Human orbital preadipocytes can be differentiated into mature lipid-filled adiocytes.③S-100 can be as an index to observe lipid droplets.④Using AFM, TAO and normal human orbital preadipocytes membrane are observed and 3D imaging of cells are observed, AFM is considered as an upstanding of observing the cell membrane morphous.⑤The cell membrane of TAO is obviously rougher than normal cells. In the course of cell differentiation of human orbital preadipocytes, the roughness of cell membrane is become more and more larger. And in the same day, the preadipocytes of TAO are more roughness than normal human preadipocytes.