Biological properties of neonatal mouse olfactory ensheathing cells purified by differential attachment,cytosine arabinoside,and mitogen stimulation  

作　　者：Nan Zhao Jun Liu Hong Gao Gang Ma Jiangxia Pang Zhongtang Feng Tinghua Wang 

机构地区：[1]Department of Neurosurgery, Kunming First People's Hospital, Kunming 650011, Yunnan Province, China [2]Institute of Neuroscience, Kunming Medical College, Kunming 650031, Yunnan Province, China

出　　处：《Neural Regeneration Research》2009年第8期629-634,共6页中国神经再生研究（英文版）

摘　　要：BACKGROUND： Differential attachment, chemicals, and immunoaffinity absorption are frequently used to purify olfactory ensheathing cells （OECs）. Although purity is high （〉 90%）, the complex process, high cost, decreased cell activity, and cell loss limit their application. OBJECTIVE： To purify OECs using differential attachment, cytosine arabinoside （Ara-C）, and mitogen stimulation, and to analyze the biological characteristics of OECs. DESIGN, TIME AND SETTING： Molecular biology experiment of cell morphology and immunocytochemistry. The study was performed at the Institute of Neuroscience, Kunming Medical College between January 2005 and January 2007. MATERIALS： N2 was purchased from Gibico, USA; basic fibroblast growth factor （bFGF） from Invitrogen, USA; PCR master mix kit from Fermentas, USA; nerve growth factor （NGF） and brain-derived neurotrophic factor （BDNF） from Santa Cruz Biotechnology, USA; OEC specific immunological marker NGF receptor （p75NGFR） from ABCAM, UK; immunological markers of oligodendrocyte and Schwann cells, cyclic nucleotide 3＇ phosphohydrolase （CNPase）, from NeoMarkers, USA; inverted fluorescence microscope from Leica, Germany. METHODS： OECs were isolated from olfactory bulbs of mice provided by Institute of Cancer Research （ICR mice） at postnatal 1 2 days, and purified by differential attachment, Ara-C inhibition （5 mg/L）, and 10 μg/L mitogen bFGF and 0.5% N2 stimulation. MAIN OUTCOME MEASURES： OEC growth was observed under inverted microscope; cell purity, as well as expression of NGF and BDNF, was determined by means of immunocytochemistry; expression of β-NGF, BDNF, NT-3, platelet-derived growth factor-B （PDGF-B）, bFGF, epidermal growth factor （EGF）, NGF receptor TrkA, BDNF receptor TrkB, and NT-4 mRNA were detected by RT-PCR. RESULTS： The majority of in vitro cultured OECs was bipolar or tripolar, and purity was estimated to be 〉 92.4%. Immunocytochemistry demonstrated expression of p75NGFR, NGF, BDNF and CNPase. ThBACKGROUND： Differential attachment, chemicals, and immunoaffinity absorption are frequently used to purify olfactory ensheathing cells （OECs）. Although purity is high （〉 90%）, the complex process, high cost, decreased cell activity, and cell loss limit their application. OBJECTIVE： To purify OECs using differential attachment, cytosine arabinoside （Ara-C）, and mitogen stimulation, and to analyze the biological characteristics of OECs. DESIGN, TIME AND SETTING： Molecular biology experiment of cell morphology and immunocytochemistry. The study was performed at the Institute of Neuroscience, Kunming Medical College between January 2005 and January 2007. MATERIALS： N2 was purchased from Gibico, USA; basic fibroblast growth factor （bFGF） from Invitrogen, USA; PCR master mix kit from Fermentas, USA; nerve growth factor （NGF） and brain-derived neurotrophic factor （BDNF） from Santa Cruz Biotechnology, USA; OEC specific immunological marker NGF receptor （p75NGFR） from ABCAM, UK; immunological markers of oligodendrocyte and Schwann cells, cyclic nucleotide 3＇ phosphohydrolase （CNPase）, from NeoMarkers, USA; inverted fluorescence microscope from Leica, Germany. METHODS： OECs were isolated from olfactory bulbs of mice provided by Institute of Cancer Research （ICR mice） at postnatal 1 2 days, and purified by differential attachment, Ara-C inhibition （5 mg/L）, and 10 μg/L mitogen bFGF and 0.5% N2 stimulation. MAIN OUTCOME MEASURES： OEC growth was observed under inverted microscope; cell purity, as well as expression of NGF and BDNF, was determined by means of immunocytochemistry; expression of β-NGF, BDNF, NT-3, platelet-derived growth factor-B （PDGF-B）, bFGF, epidermal growth factor （EGF）, NGF receptor TrkA, BDNF receptor TrkB, and NT-4 mRNA were detected by RT-PCR. RESULTS： The majority of in vitro cultured OECs was bipolar or tripolar, and purity was estimated to be 〉 92.4%. Immunocytochemistry demonstrated expression of p75NGFR, NGF, BDNF and CNPase. Th

关 键 词：olfactory ensheathing cells cell culture IMMUNOCYTOCHEMISTRY RT-PCR 

分 类 号：R741[医药卫生—神经病学与精神病学]