| Human embryonic stem cells(hES cells) are pluripotent cells derived from inner cell mass of blastocyst at early stage of human embryo. On one hand,hES cells can maintain undifferentiated state under certain circumstance.On the other hand,it can be induced into large amounts of specific somatic cells including neural progenitor cells,cardiomyocytes, blood cells and insulin secreting cells in vitro for transplantation and to treat diseases such as neuronal degenerative disorders,cardiac infarcts, blood diseases and diabetes.Stem cell therapy contains the potential to revolutionize traditional medicine.From 1960s to the second half of 1990s,mouse embryonic stem cells(mES cells) and Primate embryonic stem cells(pES cells) was established successfully.On the base of researches on this,Thomoson and his colleagues reported the first derivation of human embryonic stem cell (hES cell) line in 1998.Since then,it had become one of the hottest and most attractive fields in biomedicine to exploit this human being biological resource with great prospect for widespread clinical applications.However,hES cells were derived form blastocyst at early stage of human embryo,there were a lot of debates on its ethics and brought restriction on its researches and developments.At the beginning of 2009, US Food and drug administration(FDA) approved first human trials of hES cell research on spinal cord injury.Along with the approval of first human trial,researches on hES cells will be one of the hottest fields in biomedicine again,as well as the criterion will be established to support the researches.Our institute initiated the work of establishment and culturing of hES cells in 2001,and successfully established new lines in 2002.But at the beginning of hES cell research,the environment of culturing hES cells was instable,cells differentiated largely,some times even died. Routine culturing of hES cells is rigorous and complex than other cells. The culturing system consisted of a mitomitcally inactivated feeder cell layer and hES cell medium supplemented with kinds of factors.Firstly, we should prepare the feeder cell layer.Secondly,change feeder cell medium into hES cell medium after feeder cells attached to the dish. Finally,passage hES cell clones by cutting them into small pieces and change hES cell medium every day till next passage day.Usually,the feeder cell layer is made of mouse embryonic fibroblasts(mEFs).It is easily to get and no ethic problem.But the preparation process is complex,and the number of cell division was limited,so we need derive new batches after ran out of old ones.This shudy based on variables during derivation of mEFs,preparation of mouse feeder cells and the environment ourside hES cells,and focus on the potential safety risk of hES culturing in vitro that may effect the clinical application in order to offer stable and safety hES cells.The thesis included 2 parts as follows:1.Standardize the preparation process of mEF as feeder cells, established database for the administration of routine use of feeder cell by using Access in Microsoft Office.2.Discuss the toxicity of mitomycin C(MMC) on the culturing of hES cells in vitro and try to explain the mechanisms underlying karyontype changes in hES cells and DNA damage response defect of hES cells.
|
PDF Full Text Request