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曹代男,段好冉,魏玉峰,葛研,龚世平.2018.红耳龟胚胎成纤维细胞体外培养.动物学杂志,53(5):733-741.
红耳龟胚胎成纤维细胞体外培养
In Vitro Culture of Red-eared Slider Embryonic Fibroblasts
投稿时间:2017-11-12  修订日期:2018-08-13
DOI:10.13859/j.cjz.201805008
中文关键词:  红耳龟  胚胎成纤维细胞  原代培养  细胞培养
英文关键词:Red-eared Slider, Trachemys scripta elegans  Embryonic fibroblasts  Primary culture  Cell culture
基金项目:国家重点研发计划项目(No. 2016YFC1201100),广东省科技计划项目(No. 2017A020219004),广东省科学院科技发展专项(No. 2017GDASCX-0107)
作者单位E-mail
曹代男 广东省生物资源应用研究所 caodn@giabr.gd.cn 
段好冉 广东省生物资源应用研究所 1241694770@qq.com 
魏玉峰 广东省生物资源应用研究所 15692001678@163.com 
葛研 广东省生物资源应用研究所 293591399@qq.com 
龚世平 广东省生物资源应用研究所 gsp621@163.com 
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中文摘要:
      红耳龟(Trachemys scripta elegans)是世界上最危险的100种入侵物种之一,该种对环境适应能力极强。目前尚未见有关红耳龟细胞体外培养的研究报道,本研究旨在探索和建立快速稳定的红耳龟胚胎成纤维细胞分离培养体系,为进一步从细胞层面研究红耳龟对极端环境的耐受机制提供材料和方法支撑。本研究取发育至13或14期的红耳龟胚胎,采用酶消化法和差速贴壁法获得红耳龟胚胎成纤维细胞,在体外进行原代培养和传代培养;采用噻唑蓝(MTT)比色法分别绘制红耳龟胚胎成纤维细胞在不同温度、接种浓度和血清浓度下的生长曲线;采用反转录PCR对培养的红耳龟胚胎成纤维细胞进行分子水平的鉴定;采用脂质体转染法对红耳龟胚胎成纤维细胞进行基因转染。结果表明:1)红耳龟胚胎成纤维细胞呈典型的梭形、不规则星形或多边形,达到一定密度时呈多层生长,排列紊乱,随着传代次数的增加,细胞体积增大,梭形细胞有拉长趋势,细胞可至少传至15代;2)红耳龟胚胎成纤维细胞在30 ~ 34 ℃条件下的生长状态良好,其生长速率随着温度的升高而增高;其接种密度不宜低于1.25 × 104 个/cm2;其最佳血清浓度为10%;3)反转录PCR检测到成纤维细胞标记基因vim和acta2在红耳龟胚胎成纤维细胞中表达;4)脂质体转染法可以介导外源绿色荧光蛋白和红色荧光蛋白进入到红耳龟胚胎成纤维细胞内表达,转染效率约为30%。本研究成功地在体外分离培养了红耳龟胚胎成纤维细胞,该细胞系可应用于外源基因转染和表达。
英文摘要:
      The Red-eared Slider (Trachemys scripta elegans) is one of the world′s top 100 worst invasive alien species and has a strong ability to adapt to the environment. This study aims to establish a rapid and stable in vitro culture system of Red-eared Slider embryonic fibroblasts, and to provide material and method support for further study on the tolerance mechanism of Red-eared Slider to extreme environment at the cellular level. Red-eared Slider embryonic fibroblasts were isolated from stage 13﹣14 Red-eared Slider embryo by trypsin enzyme digesting and differential adherence, then were primarily cultured and subcultured in vitro. The growth curves of Red-eared Slider embryonic fibroblasts under different temperatures, different inoculation densities and different serum concentrations were drawn by MTT (Thiazolyl Blue Tetrazolium Bromide) method. The cultured Red-eared Slider embryonic fibroblasts were identified using reverse transcription-PCR (RT-PCR). Red-eared Slider embryonic fibroblasts were transfected using Lipofectamine 3000. The results showed that: 1) Red-eared Slider embryonic fibroblasts were typical spindle-shaped, irregular star or polygons. When reached to a certain density, they grew in multiple layers and arranged in a disorderly manner. With the increase of passage times, the cell volume increased and the spindle-like cells elongated. Red-eared Slider embryonic fibroblasts could be cultured for at least 15 generations (Fig. 1). 2) The growth state of Red-eared Slider embryonic fibroblasts was good at 30﹣34 ℃, and the growth rate increased with the increase of temperature. The inoculation density of Red-eared Slider embryonic fibroblasts should not be less than 1.25 × 104 cells/cm2 and the optimum serum concentration of the cell culture was 10% (Fig. 2). 3) The expression of vim and acta2, two fibroblast markers, were detected in the cultured Red-eared Slider embryonic fibroblasts by RT-PCR (Fig. 3). 4) Liposomes could successfully mediate the expression of exogenous green fluorescent protein and red fluorescent protein in Red-eared Slider embryonic fibroblasts, and the transfection efficiency was about 30% (Fig. 4). This study successfully isolated and cultured Red-eared Slider embryonic fibroblasts in vitro and the cells could be used for the transfection and expression of exogenous genes.
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