角膜干细胞增殖与分化表达实验研究

目的 了解角膜缘干细胞体外生长的增殖分化特性,检测上皮细胞生长因子对干细胞增殖的促进作用。方法 采用ＤＭＥＭ和Ｆ１２培养基进行兔角膜上皮细胞培养,用克隆形成率（ＣＦＥ）、免疫组化染色和蛋白质免疫印迹等方法检测细胞的增殖力和分化表达状况。结果角膜缘干细胞（ＬＣ）在本培养条件下能够正常传代生长５代,ＣＦＥ为（５．０７±２．３５）％,而角膜中央上皮细胞（ＣＣ）仅第１次传代生长,ＣＦＥ为（１．１２±０．８

A study on proliferation and differentiation of limbal stem cells

OBJECTIVE: To study the proliferation and differentiation of limbal stem cells (LCs) in vitro and examine the effects of epithelial growth factor (EGF) on cell proliferation. METHODS: The cells were cultured in DMEM/F12 medium. The proliferation and differentiation of cultured cells were studied by colony-forming efficiency (CFE), immunohistochemistry staining and Western blot examination. RESULTS: LCs could be passaged 5 times in this culture condition. The CFE of stem cells was (5.07 +/- 2.35)%, while that of the central epithelial cells (CCs) was (1.12 +/- 0.86)%. The rates of freezing and recovery were (0.43 +/- 0.22)% in LCs and (0.16 +/- 0.07)% in CCs. Limbal stem cells could be stimulated to proliferate better by EGF at the concentration of 5 and 20 ng/ml. The staining of keratin 3 (K3) to stem cells in the primary culture was negative, but positive in the second and third passages. However, the expression of K3 by Western blot examination was positive in the primary and second passages but negative in the fourth one. CONCLUSION: It is better for limbal stem cells to be used for transplantation and drug screening in the primary culture.