Preliminary Study Of Induction Of Induced Pluripotent Stem Cells From Rat Lens Epithelial Cells

Chapterl Primary Culture of Rat Lens Epithelial CellsPurpose:To establish a simple and effective rat lens epithelial cells in vitro culture methods, and to further understand their growth and differentiation rule.Methods:Lens anterior lens capsule of SD rat at postnatal:3-5days were dissociated from the eye balls. Pancreatic enzyme digestion method was adopted for the primary culture method of lens epithelial cells. Their morphological characteristics and growth rule were observed under a microscope, immunofluorescence were performed to identified lens epithelial cells and analyzed their purity, and each generation of cell growth rate and its growth curve were analyzed by MTT.Results:The enzymatic digestion method could culture the rat lens epithelial cells in a rapid way, cells showed flat irregular polygons, mosaic-like arrangement; after second generation cells began to grow in a monolayer and distributed way; after fourth generation, cells began to show a fibrosis trend way, and cell morphology took a form of fibroblast-like spindle; sixth generation cells began aging; from the beginning of the fifth-generation cell cytoplasm aA-crystallin decreased expression levels; fourth and fifth generation grew the largest, followed by the third generation of cells; third generation cells reached the logarithmic phase after24h in culture.Conclusion:The enzymatic digestion can get large numbers of rat lens epithelial cells in a short time, and the third-generation cell is an ideal object for study. Chapter2Preliminary Study of Induction of Induced Pluripotent Stem Cells from Rat Lens Epithelial CellsPurpose:To induce the induced pluripotent stem cells from the third-generation rat lens epithelial cells by importing them to Oct3/4, c-Myc, Sox2, Klf4four transcription factors.Methods:The logarithmic growth phase of lens epithelial cells were induced by differentiate Sendai virus that carry four pluripotency transcription factors, and were transplanted on feeder layer cells after transfected7days. The generated ES-like cells colonies’s forming was observed under a microscope, and their forming efficiency was calculated, immunofluorescence staining method was performed to identify the embryonic stem cells markers:SSEA-1, OCT4, and qRT-PCR method were performed to identify the embryonic stem cells marker genes: NANOG,REX1,OCT4.Results:The induced lens epithelial cells can generate RLEC-ES-like cell colonies, and the transfection efficiency was0.034%+0.0092%. RLEC-ES-like colonies were taken for embryonic stem cell marker SSEA-1and OCT4immunofluorescence staining was positive; qRT-PCR method was performed to identify embryonic stem cell marker genes and NANOG, REX1, OCT4expression was positive.Conclusion:The rat lens epithelial cells can be induced by Sendai virus that carry Oct4,c-Myc, Sox2, Klf4with four transcription factors to generate RLEC-ES-like colonies, and their morphology, protein, RNA and other levels had been identified.