Vero细胞
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Vero细胞亦称绿猴肾细胞[1],是一种非整倍性的非洲绿猴(属名:Chlorocebus)肾细胞系,最初由日本千叶大学的安村美博于1962年3月27日,分离自正常成年非洲绿猴的肾脏上皮细胞[2]。“vero”即“verda reno”的缩写,其中“verda reno”在世界语中有“绿色的肾脏”的意思,而“vero”则在世界语中表示“真相”[3] 。
特征
Vero细胞是连续的非整倍性细胞系,这意味着它的染色体数目异常,而且已知连续的细胞系可以通过许多分裂周期,不会老化,Vero细胞的干扰素分泌出现缺陷。它们与正常的哺乳动物细胞不同,在被病毒感染时不会分泌干扰素α/β[4]。然而,它们仍然具有干扰素-α/β受体(IFNAR),因此当将重组干扰素添加到其培养基中时,它们仍然可以作出反应。
Sun等合成了五种不同尺寸的草酸钙(COM)晶体,包括50 nm、200 nm、1 μm、3 μm、10 μm,并且比较这五种尺寸的草酸钙晶体对Vero细胞的损伤差异[5][6]。实验结果表明,COM的尺寸和聚集程度是影响晶体细胞毒性的重要原因,而细胞对晶体的内吞方式与晶体尺寸存在密切的关系[5]。Vero细胞内吞50nm和100 nm的COM晶体主要以网格蛋白介导的途径,内吞1 μm的COM晶体则主要以巨胞饮(macropinocytosis)的形式进行内吞作用,而Vero细胞难以内吞尺寸更大的微米晶体[7]。
2014年,有日本的研究人员确定Vero细胞的整个基因组序列[8]。Vero细胞的12号染色体具有纯合的〜9-Mb缺失,导致基因组中I型干扰素基因簇和细胞周期蛋白依赖性激酶抑制剂CDKN2A和CDKN2B的丢失[8]。尽管非洲绿猴先前被归类为草原猴(Cercopithecus aethiops),但是它们已经被归类为绿猴属(Chlorocebus)[9]。有基因组分析表明,Vero细胞源自雌性绿猴(Chlorocebus sabaeus)[8]。
细胞培养
Vero细胞在传代培养时的生长状况良好,并且发现细胞膜界线清晰和胞浆透明度较好的现象。Vero细胞的形态较为完整,细胞增殖的速度较快。Vero细胞传代后的第三天开始形成单层,传代细胞在第七天形成致密单层,此种致密单层在连续培养第十二天后逐渐老化,细胞在第十六天开始从培养瓶壁上脱落[10]。
Vero细胞在转瓶后,可以在细胞培养二十四小时后出现贴壁,细胞在培养三天后可以达到相对静止期,细胞培养的第五天可以长成单层,细胞培养到第十二天时发现其生长致密,而细胞培养至第十四天时则开始出现老化。转瓶后的细胞生长速度比转瓶前缓慢,然而单层细胞持续的时间比转瓶前更持久。进行支原体检查时未发现有支原体的生长及污染。细胞型分析结果表明Vero 细胞的核型没有发现明显的异常之处,而染色体数目也没有明显变化[10]。
研究用途
Vero细胞可以用于多种研究用途。Vero细胞在建立后不久,就被发现对多种类型的病毒高度敏感,其中包括猿猴空泡病毒40[11]、麻疹病毒[12]、风疹病毒[13]、节足动物携带性病毒[14]及腺病毒[15]等。后来被发现也容易感染细菌毒素,包括白喉毒素[16]、不耐热肠毒素(heat-labile enterotoxins)[17]和志贺氏样毒素[18][19]等。
Vero细胞可以筛选大肠埃希氏菌的毒素。在Vero细胞被建立后,这些毒素亦可以称为“Vero毒素”。由于与痢疾志贺氏菌(Shigella dysenteriae)分离出的志贺氏毒素相似,因此后来被称为志贺氏样毒素(Shiga-like toxin)[8]。
Vero细胞又可以作为锥虫目等真核寄生虫的宿主细胞[8]。此外,Vero细胞被广泛应用于病毒感染分子机制研究、疫苗及重组蛋白的生产[20][21][22],世界卫生组织甚至认可其作为疫苗生产细胞系,建议将其作为流感疫苗生产的替代基质。目前已知Vero细胞可以协助生产狂犬病[23]及水貂犬瘟热等疫苗[24],而用Vero细胞培养的流感疫苗可以更好地介导人体产生对流感的免疫应答[25]。
猪流行性腹泻病毒
Hofmann等通过在培养基中添加胰蛋白酶,证实猪流行性腹泻病毒(PEDV)除了能够在天然宿主的初始靶细胞上增殖外,还可以在Vero细胞中增殖。同时又发现胰蛋白酶对PEDV纤突糖蛋白的切割作用,增强病毒对Vero细胞的感染力[26]。Ye等通过构建稳定表达PEDV ORF3蛋白的Vero细胞,发现ORF3蛋白能够促进PEDV的增殖[27]。然而有研究显示不同的结果,例如Chen等发现orf3基因转译的提前终止,有利于PEDV适应Vero细胞,并且可以提高其在Vero细胞上的复制能力[28];而Sun等对非胰蛋白酶依赖PEDV 85-7的Vero细胞的研究表明,PEDV的复制并没有被orf3基因的突变或转译的提前终止显著影响[29]。另外,Li等构建缺失orf3基因的重组PEDV,发现orf3基因缺失株和携有全长orf3基因的重组病毒,在Vero细胞上的滴度相同,故而推测orf3基因不影响其在Vero细胞上的增殖[30]。
参考文献
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