Temperature sensitive simian virus 40 large T antigen immortalization of murine odontoblast cell cultures: establishment of clonal odontoblast cell line.
Temperature sensitive simian virus 40 large T antigen immortalization of murine odontoblast cell cultures: establishment of clonal odontoblast cell line.
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DOI：
10.3109/03008209509016988
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发表时间：
1995-01-01
影响因子：
2.9
通讯作者：
Snead, M L
中科院分区：
文献类型：
--
作者：
MacDougall, M;Thiemann, F;Snead, M L
在牙齿形成过程中，上皮-间充质相互作用导致外胚间充质细胞分化为成牙本质细胞，形成牙本质细胞外基质(DECM)。本研究的目的是通过逆转录病毒转染法将成牙本质细胞永生化，建立稳定的小鼠成牙本质细胞系。为了实现这一目标，我们利用了一种先前描述的成牙本质细胞单层培养系统，该系统支持牙乳头间充质(DPM)向成牙本质细胞分化、DECM的表达和分泌以及牙本质的生物矿化。首先解剖E-18瑞士Webster小鼠的下颌磨牙，分离DPM，分离牙髓细胞。牙髓细胞(5×10(5)个/孔)作为单层细胞，在添加10%胎牛血清、100单位/毫升青霉素和链霉素、50微克/毫升抗坏血酸的α-MEM中生长。培养物在37摄氏度、95%空气和5%二氧化碳的潮湿环境中保持6天，每两天更换一次培养基。用从Cre包装细胞中回收的含有温度敏感的SV-40大T抗原基因和新霉素(G418)抗性基因的重组缺陷型逆转录病毒进行永生化。用含8微克/毫升聚丙稀烯的CRE条件培养液感染培养24 h，换成含300微克/毫升G418的选择性培养液，在33℃下培养1个月，每隔3~5天更换一次。在96孔培养板上用连续稀释法将新霉素抗性细胞克隆成单个细胞，并在33℃的选择培养基中生长。
During tooth formation instructive epithelial-mesenchymal interactions result in the cytodifferentiation of ectomesenchymal cells into odontoblasts which produce the dentin extracellular matrix (DECM). The purpose of our study was to establish a stable murine odontoblast cell line by immortalization of odontoblasts using retrovirus transfection. In order to accomplish this goal, we utilized a previously characterized odontoblast monolayer cell culture system supportive of odontoblast cytodifferentiation from dental papilla mesenchyme (DPM), expression and secretion of a DECM and dentin biomineralization. First mandibular molars from E-18 Swiss Webster mice were dissected, the DPM isolated, and pulp cells dissociated. Pulp cells (5 x 10(5)/well) were plated as monolayers and grown in alpha-MEM supplemented with 10% FCS, 100 units/ml penicillin and streptomycin, 50 micrograms/ml ascorbic acid. Cultures were maintained for 6 days at 37 degrees C in a humidified atmosphere of 95% air and 5% CO2, with media changes every two days. Immortalization was performed using a recombinant defective retrovirus containing the temperature sensitive SV-40 large T antigen cDNA and the neomycin (G418) resistance gene recovered from CRE packaging cells. Cultures were infected for 24 h with CRE conditioned medium containing 8 micrograms/ml of polybrene, the media was replaced with selective media containing 300 micrograms/ml of G418, and the cultures incubated at 33 degrees C for one month with media changes every 3-5 days. Neomycin resistant cells were cloned by serial dilution to single cells in 96-well culture plates and grown in selection medium at 33 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)