Vero細胞
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Vero細胞亦稱綠猴腎細胞[1],是一種非整倍性的非洲綠猴(屬名:Chlorocebus)腎細胞系,最初由日本千葉大學的安村美博於1962年3月27日,分離自正常成年非洲綠猴的腎臟上皮細胞[2]。「vero」即「verda reno」的縮寫,其中「verda reno」在世界語中有「綠色的腎臟」的意思,而「vero」則在世界語中表示「真相」[3] 。
特徵
Vero細胞是連續的非整倍性細胞系,這意味着它的染色體數目異常,而且已知連續的細胞系可以通過許多分裂週期,不會老化,Vero細胞的干擾素分泌出現缺陷。它們與正常的哺乳動物細胞不同,在被病毒感染時不會分泌干擾素α/β[4]。然而,它們仍然具有干擾素-α/β受體(IFNAR),因此當將重組干擾素添加到其培養基中時,它們仍然可以作出反應。
Sun等合成了五種不同尺寸的草酸鈣(COM)晶體,包括50 nm、200 nm、1 μm、3 μm、10 μm,並且比較這五種尺寸的草酸鈣晶體對Vero細胞的損傷差異[5][6]。實驗結果表明,COM的尺寸和聚集程度是影響晶體細胞毒性的重要原因,而細胞對晶體的內吞方式與晶體尺寸存在密切的關係[5]。Vero細胞內吞50nm和100 nm的COM晶體主要以網格蛋白介導的途徑,內吞1 μm的COM晶體則主要以巨胞飲(macropinocytosis)的形式進行內吞作用,而Vero細胞難以內吞尺寸更大的微米晶體[7]。
2014年,有日本的研究人員確定Vero細胞的整個基因組序列[8]。Vero細胞的12號染色體具有純合的〜9-Mb缺失,導致基因組中I型干擾素基因簇和細胞週期蛋白依賴性激酶抑製劑CDKN2A和CDKN2B的丟失[8]。儘管非洲綠猴先前被歸類為草原猴(Cercopithecus aethiops),但是它們已經被歸類為綠猴屬(Chlorocebus)[9]。有基因組分析表明,Vero細胞源自雌性綠猴(Chlorocebus sabaeus)[8]。
細胞培養
Vero細胞在傳代培養時的生長狀況良好,並且發現細胞膜界線清晰和胞漿透明度較好的現象。Vero細胞的形態較為完整,細胞增殖的速度較快。Vero細胞傳代後的第三天開始形成單層,傳代細胞在第七天形成緻密單層,此種緻密單層在連續培養第十二天後逐漸老化,細胞在第十六天開始從培養瓶壁上脫落[10]。
Vero細胞在轉瓶後,可以在細胞培養二十四小時後出現貼壁,細胞在培養三天後可以達到相對靜止期,細胞培養的第五天可以長成單層,細胞培養到第十二天時發現其生長緻密,而細胞培養至第十四天時則開始出現老化。轉瓶後的細胞生長速度比轉瓶前緩慢,然而單層細胞持續的時間比轉瓶前更持久。進行支原體檢查時未發現有支原體的生長及污染。細胞型分析結果表明Vero 細胞的核型沒有發現明顯的異常之處,而染色體數目也沒有明顯變化[10]。
研究用途
Vero細胞可以用於多種研究用途。Vero細胞在建立後不久,就被發現對多種類型的病毒高度敏感,其中包括猿猴空泡病毒40[11]、麻疹病毒[12]、風疹病毒[13]、節足動物攜帶性病毒[14]及腺病毒[15]等。後來被發現也容易感染細菌毒素,包括白喉毒素[16]、不耐熱腸毒素(heat-labile enterotoxins)[17]和志賀氏樣毒素[18][19]等。
Vero細胞可以篩選大腸埃希氏菌的毒素。在Vero細胞被建立後,這些毒素亦可以稱為「Vero毒素」。由於與痢疾志賀氏菌(Shigella dysenteriae)分離出的志賀氏毒素相似,因此後來被稱為志賀氏樣毒素(Shiga-like toxin)[8]。
Vero細胞又可以作為錐蟲目等真核寄生蟲的宿主細胞[8]。此外,Vero細胞被廣泛應用於病毒感染分子機制研究、疫苗及重組蛋白的生產[20][21][22],世界衛生組織甚至認可其作為疫苗生產細胞系,建議將其作為流感疫苗生產的替代基質。目前已知Vero細胞可以協助生產狂犬病[23]及水貂犬瘟熱等疫苗[24],而用Vero細胞培養的流感疫苗可以更好地介導人體產生對流感的免疫應答[25]。
豬流行性腹瀉病毒
Hofmann等通過在培養基中添加胰蛋白酶,證實豬流行性腹瀉病毒(PEDV)除了能夠在天然宿主的初始靶細胞上增殖外,還可以在Vero細胞中增殖。同時又發現胰蛋白酶對PEDV纖突糖蛋白的切割作用,增強病毒對Vero細胞的感染力[26]。Ye等通過構建穩定表達PEDV ORF3蛋白的Vero細胞,發現ORF3蛋白能夠促進PEDV的增殖[27]。然而有研究顯示不同的結果,例如Chen等發現orf3基因轉譯的提前終止,有利於PEDV適應Vero細胞,並且可以提高其在Vero細胞上的複製能力[28];而Sun等對非胰蛋白酶依賴PEDV 85-7的Vero細胞的研究表明,PEDV的複製並沒有被orf3基因的突變或轉譯的提前終止顯著影響[29]。另外,Li等構建缺失orf3基因的重組PEDV,發現orf3基因缺失株和攜有全長orf3基因的重組病毒,在Vero細胞上的滴度相同,故而推測orf3基因不影響其在Vero細胞上的增殖[30]。
參考文獻
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