Preliminary Research On Polythylne Glycol (PEG)-Induced Fusion Of Yak Mammary Epithelial Cells And Mouse Myeloma Cells (SP2/0)

In this study, PEG was used to induce the cell-fusion between the yak mammary epithelial cells in culter and mouse myeloma SP2/0 cells. Optimization of fusion condition by altering the relative molecular weight of PEG, the density of the PEG6000, and the time of incubation were carried out. The results were demonstrated below:1. The yak mammary epithelial cells were obtained successfully by using collagenaseâ… digetion and outgrowth of migrating cells from fragment of tissue respectively. The two methods were compared in advantages and disadvantages. Observed the characteristics of the cells cultured in vitro.2. Detected the specific construction of the mammary epithelial cells by using indirect immunofluorescence technique. Cytokeratin was characteristically posed by the epithelial cells as cell constitutive protein. The result of immunology experiment by detecting the cytokeratin showed that the mammary epithelial cells were purified, and the cell obtained can be used to cell fuse.3. The propagating regularity of yak mammary epithelial cells was obtained, and it showed that the cell optical density decline apparently in primal 24h transfering , then the cell proliferate abundantly in the next days. The cell experience in stagnate period in the 7day, it demonstrated that yak mammary epithelial cells'growth curve assumed"S". Assure the cells'activity for cell fusion by collecting the cell in the exponential growth period.4. Plasmalemma of mouse myeloma SP2/0 cells were labeled red with DiI, and nucleus of yak mammary epithelial cells were labeled blue with DAPI.5. Observed the characteristic changes of the cell incubated with PEG by using inverted phase contrast microscope, the cell was shrunken, then the cell bulged and fuse when the PEG be diluted that the osmotic pressure was change.6. Different molecular weight (1000, 4000, 6000) of PEG were used to induce the fusion between mammary epithelial cells and mouse myeloma SP2/0 cells. Observe the changing of the cell fusion rate while concentration is 50% and the incubation time is 90s. The results showed that the fusion rate increased along with the rise of molecular weight of PEG. 7. Different concentration (40%, 45%, 50%) of PEG6000 were used to induce fusion between mammary epithelial cells and mouse myeloma SP2/0 cells. The results showed that the fusion rate increased along with the rise of concentration of PEG.8. PEG6000 in 50% was used to induce fusion between mammary epithelial cells and mouse myeloma SP2/0 cells. Different incubation time (30s, 90s, 3min, 5min) were used. The results showed that the maximum fusion rate, which is 17.4%,was acquired in concentration of 50% and incubated time of 90s.