| Objectives: Embryonic stem cells (ES cells) derived from inner cell mass of the preimplanted blastocyst or from primordial germ cells(PGCs) early embryo, are highly undifferentiated totipotent or pluripotent stem cells that have unlimited growth and differentiation potential. With feeder layer or some other inhibited or inducing differentiated cytokines, ES cells can differentiate into certain tissues and cell types to be used in basical research and cure disease. Now fibroblast cells are widely used as feeder layer in many countries, other cells as feeder layer are rare, we still not found some research of Sertoli cells as feeder layer. Sertoli cells secret SCF which can promote the growth of ES cells and induce the cells into cells cycle .What is more, SCF is the only factor to regulate ES cells. It is also reported that SCF can inhibit cell apoptosis factors to promote growth of ES cells. Our research is to use use several methods such as cell culture,immunohistochemistry,MTT growth curve to establish PMEF and Sertoli cells feeder layer , separate and cultivate ES cells further. Compare the growth character of PMEF and Sertoli cells feeder layer, at the same time compare the influence of feeder layer on ES cells about the structure and biological characters. Materials and method: 1.Separation and culture of fibroblast cells and Sertoli cells Get pregnant kunming mouse(13.5-14.5dpc), separate the skin of the little embryo with 0.25%Trypsin for 15min 37℃, centrifugate,DMEM resuspend the cells, adjust the density to 1×106/ml, cultivate. Get male kunming mouse (after birth 15-16d), bring out the testis, mince, 0.25%Trypsin and 0.1%Collagenase, 37℃ 20min , centrifugate, DMEM resuspend the cells, adjust the density to 1×106/ml, cultivate . 2.Get cover glass with monolayer cells, HE staining,and Giemsa staining,immunohistochemistry, MTT growth line to verify the cells. Calculate the positive cells and deal with statistics software. 3.Make feeder layer Get fibroblast cells and Sertoli cells, deal with mitocin-C for 2.5-3.5hours, then make single cell solution with 0.25%trypsin, cultivate in the four-well plate covered with 0.1% Gelatin previously. 4. Separation and culture of ES cells Blastocysts of 3.5d were collected from uterin horns of kunming mouse, cultured on the fibroblast cell feeder layers. The ES cells were observed by phasecontrast microscope and identified by AKP staining. Pass the ES cells on fibroblast cells and Sertoli cells. Made MTT growth curve of ES cells on fibroblast feeder layer and Sertoli feeder layer.Result: 1.Fibroblast cells grow very fast and attach in 30minuts, after 24 hour , it will extend to a large extent. Sertoli cells grows slowly, nucleus is large and round. There are 1-2 nucleoli in the nucleus. The most obvious character is that much fat drop located in cell plasm. 2.HE staining shows that the tuber of fibroblast cell is long and normal, the nucleus of Sertoli cells is round, the nucleolus is very obvious. 3.The expression of Actin in Sertoli cells is much more than that in fibroblast cell . The difference between ratio of positive cells in two kind of cells is obvious.(p<0.01).The growth curve of fibroblast cells and Sertoli cells are both increasing. At first, fibroblast cells grow faster than Sertoli cells, after 5 days, it become slower, while Sertoli cells still grow gradually. The difference is not very notable.(P>0.05) 4.ES cells from ICM proliferate to some extent. ES cells attach the bottom of the cultivate bottle. ES cells push away the trophoblast cells. 5.The cells grows very fast and tightly packed in large nest and colonies in which it was difficult to recognize individual cells, each cellwas small, round or fusiform, with large nucleus and minimal cytoplasm. AKP was positive. ES cells on fibroblast cells have more clones than on Sertoli feeder layer. It is smaller than that on Sertoli cells . What is more, there are more little clones of ES cells or single ES cell around the big clone. The growth curv...
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