Biological Characteristic Study Of Intestinal Epithelial And Neural Stem Cell From Beijing Fatty Chicken

To clarify the key biological characteristics of intestinal epithelial stem cell and neural stem cells from Beijing fatty chicken, the study researched on isolation, multiplication, identification, differentiation of two kinds of stem cells. The results are as follows:1. The IECs were isolated from Intestinal tissues of Beijing fatty chicken by the method of0.1g/L neutral protease I and300U/mL collagenase XI digestion. The suitable culture condition for IECs was DMEM/F-12+5%FBS+2mM L-glutamine+lOng/mL EGF+10ng/mL bFGF+sodium pyruvate. The IECs were oval or polygonal, adherent monolayer growth.2. The growth curve of different generations generations of chicken intestinal epithelial stem cell were typical of the "S" shape. In line with the general rules of cells grow in vitro. All of them experience the incubation phase, logarithmic phase, plateau phase. And with the increase of batches, division and proliferation ability of cells gradually become weak.3. The influences of cryoconservation and resuscitation on viability were evaluated.24tubes were cryopreserved, and the cell viability before frozen and after recovery was decrease with the increase of passage of cells. The main reason may be the mechnical and chemical damages of the cells suffered in the processes of freezing and recovery. But the cells viability was all above90%.4. We identified the IECs by the means of immunohistochemisty and RT-PCR. The result of immunofluorescence showed Musashil, DCAMKL1, LGR5, Bmil, Hesl positive, RT-PCR test results showed Musashil, Bmil, Hesl, CD29positive.5. NSCs were isolated using physical method, chicken NSCs can be cultured in vitro and have been passed to7generation. Spherical cells were grown in suspension. In the cell culture process, some cells occurred the phenomenon of natural differentiation, adherent growth and the specialized nerve cells emergenced.But most of the cells continued to grow like a ball. By replacing culture flasks, the cells of natural differentiation can be removed.6. The surface markers of NSCs, Nestin, Hesl, Musashil and Vimentin were detected by immunofluorescence and RT-PCR.7. The multi-lineage differentiation potential of NSCs was investigated and induced to differentiate into astrocytes, neurons and oligo-dendrocytes.We identified the NSCs by the means of immunohistochemisty and RT-PCR.NSCs have a high proliferation and multi-directional differentiation potential in vitro.