The Establishment Of Preadipocytes And Myogenic Satellite Cells Co-culture System And Primary Study On The Growth Characteristics Of Cocultures In Procine

There is a close interaction between fat and muscle tissue by autocrine and paracrine,which plays a significant role in fatty deposition and muscle development, and have important regulated function on the establishment substance and energy metabolism. So establishment of adipocytes and myocytes co-culture system will help to study on the interaction between them.The present research established a eligible system for co-culture ofpreadipocytes and myogenic satellite cells, on the base, and methods of MTT spectrophotometry,Oil Red O staining, Desmin immunohislochemistry staining, HE staining and semiquantitative RT-PCR were used to study on the growth characteristics of cocultures in porcine adipose and muscle tissue by distracting and purifying preadipocytes and myogenic satellite cells from nascent pigs.This research includes following several aspects:1 The effect of different concentration rates on the proliferation of cocultures in Seeding were tested by the method of MTT colorimetry. The results show that growth rate of cocultures on 9th day in culture is more than other 12 rates as preadipocytes and myogenic satellite cells were seeded in the rate of 2.5×10⁷/L: 1×10⁸/L.2 The effect of adherenced time after seeding on the proliferation of cocultures were tested by the method of blood cell counting. The results show that the rate of adherenced cells is increased following time, but get to stabilization from 48 to 66 hours, and a part of cocultures in culture begins to proliferating after 66 hours.3 The effect of medium types and concentrations of blood serum contained in medium on survival rates of cocultures were tested by the rate of coherenced cells. The results show that using of serum is more useful for culture than a serum-free medium,DMEM/F12(1:1) medium is more benefit for co-culture than DMEM, RPMI and M199 medium, and the surivival rates of cocultures is increased as the concentration of serum contained in medium.4 To study on the growth morphology and characteristics of cocultures, Morphologic survey from 2nd～15th day and MTT spectrophotometry in 2nd～9th day were adopted. The results show that the morphology of cocultures was fusiform shape extending before confluence, but after confluence it changed into irregular with coinciding and interlace with each other. The growth rate is more than adipocytes and myocytes in culture, a rapid growth until 7th day,but keep on slow later growth to 9th day,which is different from adipocytes and myocytes.5 To study the effect of co-culture on the differentiation of preadipocytes and myogenic satellites cells, the methods of Oil Red O staining, HE staining were applied. The results show that the cells in coculture filled lipid appeared later and fewer than adipocytes as the control group. Polykaryocytes fuse into myotubes was visible by HE stanining but also later and less standing than myocytes as the control group.6 To study the effect of co-culture on the differentiation of preadipocytes and myogenic satellites cells, the methods of semiquantitatives RT-PCR also was applied. The results show that the mRNA levels of LPL and Myf₅ were tested by semiquantitative RT-PCR on both 5th and 10th day in co-culture, but it is difficulted to detected the mRNA of PPARr₂ and MyoG on 5th day, and until 10th day low levels were detected.