The Study Of Pluripotent Stem Cell Induced And Clutured In Vitro

Induced pluripotent stem cells (iPSC) was originally developed by Japanese scientist Yamanaka,using a retrovirus carrying four transcription factors:Oct4, Sox2, Klf4, c-Myc, were induced into mouse flbroblast cells, obtained cell types similar to embryonic stem cells on properties. Using the patient’s own cells obtained iPSC can reduce the immunological rejection, and it can avoid ES cells facing ethical problem very well. So the iPS cells have a broad application prospect in the establishment of animal model, drug screening and treatment. To investigate the feasibility by iPS cells induced from deaf human cells,the contents of this thesis include the following three parts:Firstly, induced mouse embryonic fibroblasts with genetype of P53knockout and wild-type by an inefficient system,and then analyzed the Dlkl-Dio3gene expression differences of two iPSC, for study the possible of P53signaling pathway and Dlkl-Dio3in association with somatic cell reprogrammi ng machanism.Secondly, induced the patient ear skin fibroblasts by non-integrated method, obtaining patient specific iPS cells, and lay the foundation for differentiation of human iPS cells for the next step.Thirdly, to be able to find a suitable reagent removal of mycoplasma conta-mination problems often encountered in cell culture.The main results obtained from the experiments are as follows:1.Using the inefficient inducing system, unfortunately, AP staining of iPS cells was weakly positive;By mesns of lentivirus can successfully induce mouse embryonic fibroblast, obtained iPS cells that AP staining was positive; RT-PCR detected the endogenous expression of pluripotency four factor Oct4, Sox2, Klf4and c-Myc,while exogenous factor four did not complete silence; Immunofluo rescence staining can detect the expression of nucleus, the cell membrane protein OCT4, NANOG, SSEA-1and SSEA-3.2. Using non-integrated Episomal plasmid was transfected into the patient ear skin fibroblasts, can obtain AP positive clone; Immunofluorescence staining detected the expression of pluripotency related nuclei, cell membrane protein OCT4, NANOG, SSEA-3, TRA-1-81.3. Adding the mycoplasma removing reagent from Selleckchem company into the culture medium of293T cells, mouse embryonic fibroblasts (MEF), human mesenchymal stem cells (MSC), human embryonic stem cells (hES) and other cells types, ultimately determine the effects of this reagent.The experiments by using lentivirus and non-integrated Episomal plasmid transfectd mouse and human somatic cells,established iPS cell lines; Although not a mouse iPS cell line in a relatively inefficient induction system was success, from another side confirmed that the BMP components in the serum may be a key obstacle in reprogramming of somatic cells.