Study On Goat Keratinocyte Stem Cells And Transfection Of GFP Gene

Keratinocyte Stem cells(KSCs) are playing an important role in woundhealing.Nowadays, using Keratinocyte Stem cells to reconstruct tissue and transgene has becomed an important direction in biomedicine field. Green Fluorescent Protein( GFP) is one of the most extensively applied labelling proteins, we can observe whether GFP gene has expressed no needing adding exogenous substrate. To offer basic information to the clinical application of KSCs ,this thesis focuses,in four aspects,on isolation and culture,identification of bio-characteristics, plasticity and GFP gene transfection .1. Isolation and culture of Goat KSCTissue separate culture: After rinse and deal with antibiotic,the skin block was cut into 0.01cm2 ,then attached to petri dish which covered with 0.1% glutin.Two hours later,cells was cultured in HDMEM/F12 ( 3:1 ) containing 20%NBS, 5μg/mL Insulin, 0.5μg/mL Hydrocortisone and 100unit antibiotics,changing culture medium every threes days.When observed cells migrated,decrease NBS from 20 % to 10 %.DispaseII enzyme digestion: After rinsed and dealed with antibiotics, the skin block was cut into about 0.5 cm×1 cm,digested with 4 mg/mL DispaseII enzyme in 40C for 16 hours. Epidermis was separated from dermis,cut into pieces, digested with 0.25% trypsin for about 5-7 mins. Then neutralized, filtrated, centrifuged and cultured with HDMEM/F12(3:1).Trypsin enzyme digestion: After rinsed and dealed with antibiotics, the skin block was cut into about 0.5 cm×1 cm, digested with 0.25% trypsin in 40C for 12 hours. The skin block was cut into pieces, then filtrated, centrifuged and cultured with HDMEM/F12(3:1).Tissue separate culture method is simpleand the tissue separate can be used repeatedly. But the migrating time of primary cells,about 8 days,is too long.It's need over 15 days to passage. Passaging of primary cells usually demand discriminating digestion. Seasons have remarkable effect on migrating time, passaging time and passaging numbers. In spring and summer,time of cells migrated from tissue ( 4.9±1.4 d )and passaging time of primary cell s (10.3±2.5 d) are short than in autumn and winter(10±1.5 d,20.3±1.7d). The passaging number (7.4±3.5)is biger than the latter(4.5±1.5),too.DispaseII enzyme digestion has a short period.It's can be passaged at 8 days. The passaging number is 6.5.Seasons have no remarkable effect on migrating time, passaging time and passaging numbers.But its disposal is bother.It need at least 2 tissues with1cm×1cm.In addition, after filtrated,it usually contains...