Study On Nanog Gene's Transfection To Epidermal Stem Cells And Its Effect On Epidermal Stem Cells' Biological Characteristics

Gene knock-out and over expression are important tools to study genes'function,providing plenty of information for the maintenance of stem cell pluripotency. Over expression of Nanog in Embryonic stem cells (ESCs) can sustain the pluripotency of inner cell mass (ICM) and ESCs independently of the LIF/Stat3 path. And forced expression of Nanog in NIH3T3 cells can promote these cells'proliferation. Epidermal stem cells are a kind of adult stem cells that have high self-renewal ability and can maintain the homeostasis of epidermis by producing functional cells when needed. Recent years have seen huge application potential of epidermal stem cells in tissue engineering and cell therapy. Despite the increasing evidence of epidermal stem cells'pluripotency,this ability and their self-renewal ability is still much lower than ESCs. This experiment is designed to transfect the key pluripotency gene of ESCs Nanog into epidermal stem cells and to test its effect on the biological characteristics of epidermal stem cells. The experiment is as follows:1.By cutting a piece of ear skin from goats, comparing two kinds of methods to obtain primary cells (direct enzyme digestion and tissue culture),two kinds of culturing medium (with serum and serum free),purifying epidermal stem cells by different ratio of attachment to collegenⅣ, it is found that direct enzyme digestion is more efficient in getting primary cells and have much less fibroblast cells than direct tissue culture and is better for epidermal stem cells'isolation and passage, medium with serum performs better in promoting goat epidermal stem cells'attachment and survival, and after purification by collegenⅣ, goat epidermal stem cells can be passaged successively and remain high viability after thawing,thus is suitable for transfection of Nanog gene.2.Through morphology observation,immunocytochemistry staining of some epidermal stem cells'specific markers,growing curve test and clone forming efficiency test of the cells,we found the cells are small,ball-like,dense,having strong refraction ability and proliferation ability,grow in clones,most expressβ1-integrin,p63 andα6-integrin, few express CK-14 and none is CD71 positive. These are in accord with typical characteristics of epidermal stem cells. The cell viability become weaker as they are passaged according to the growth curve of passage 3,5,and 7. Though the clone forming efficiency is decreasing,its statistical...