国际妇产科学杂志

国际妇产科学杂志 ›› 2013, Vol. 40 ›› Issue (6): 565-567.

• 论著 • 上一篇    下一篇

采用慢病毒载体干扰ALDH-1表达的宫颈癌Hela细胞稳转株的构建及意义


刘龙阳, 张帝开 , 易娟娟, 陈 勍 , 张丙忠 , 陈志辽, 梁金晓, 林仲秋   

  1. 510655 广州，中山大学附属六院妇科（刘龙阳，张帝开）；中山大学孙逸仙纪念医院妇科肿瘤科（陈 勍， 张丙忠，陈志辽，梁金晓，林仲秋）；佛山市妇幼保健院皮肤性病科（易娟娟）
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2013-12-15 发布日期:2013-12-15
  • 通讯作者: 林仲秋

The Significance and Successful Construction of Stable Transfection of Hela Cells with Lentiviral Vector

LIU Long-yang，ZHANG Di-kai，YI Juan-juan，CHEN Qing，ZHANG Bing-zhong，CHEN Zhi-liao，LIANG Jin-xiao，LIN Zhong-qiu   

  1. Department of Gynecology， Sixth Affiliated Hospital， Sun Yat-sen University（LIU Long-yang， ZHANG Di-kai）； Department of Gynecological Oncology， Sun Yat-sen Memorial Hospital， Sun Yat-sen University（CHEN Qing， ZHANG Bing-zhong， CHEN Zhi-liao，LIANG Jin-xiao，LIN Zhong-qiu）；Department of Dermatology and STD，Foshan Maternal and Child Health Hospital（YI Juan-juan）
  • Received:1900-01-01 Revised:1900-01-01 Published:2013-12-15 Online:2013-12-15
  • Contact: LIN Zhong-qiu

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摘要： 目的：采用干扰乙醛脱氢酶1（ALDH-1）表达的慢病毒载体转染宫颈癌Hela细胞，构建干扰ALDH-1表达的稳转株，以利进一步探讨干扰ALDH-1表达的宫颈癌Hela细胞株的生物学特性。方法：体外培养宫颈癌Hela细胞，加入梯度浓度的嘌呤霉素溶液进行筛选，确定最佳筛选浓度；取24孔板中对数生长的宫颈癌Hela细胞，吸弃旧培养液，加入400 μL新培养基，分别加入4种干扰慢病毒液（ALDH-1、ALDH1-1719、ALDH1-740、ALDH1-921，其中有一种具最佳干扰效果）及1种阴性对照慢病毒液（LV3-NC），同时均加入Polybrene液。其中5种慢病毒液的加入量均为50 μL（滴度均为1×108 TU/mL），Polybrene的加入量为0.5 μL（浓度为5 mg/L）；24 h后换新鲜培养液，72 h后荧光显微镜下观察荧光表达情况。然后采用最佳筛选浓度的嘌呤霉素溶液进行筛选，以获得稳定转染的干扰ALDH-1表达的宫颈癌Hela细胞株。结果：确定嘌呤霉素的最佳筛选浓度为0.9 mg/L；5种慢病毒载体均成功转染入宫颈癌Hela细胞中，经嘌呤霉素筛选2周后成功获得稳定转染细胞株。结论：干扰ALDH-1表达的宫颈癌Hela细胞稳转株成功构建。

关键词: 氧化还原酶类, 乙醛, 慢病毒属, 遗传载体, 转染, HeLa细胞

Abstract: Objective：To construct the stable transfection Hela cell lines with lentiviral vector which may interfer the expression of ALDH-1，and to further explore the biological characteristics between the expression of ALDH-1 and Hela cell lines. Methods：Hela cells were cultured in vitro，added the different concentration of puromycin to screen，，and to determine the best screening concentratioｎ；Hela cells were tested from 24-well plates，threw away the old culture medium， and added 400 μL of new medium to the plates，and then added four kinds of lentivirus solution（ALDH-1， ALDH1-1719， ALDH1-740， ALDH1-921，and there was a kind of lentivirus which has the best interfering effect） and one kind of negative control lentivirus solution （LV3-NC） to the plates， at last added Polybrene liquid to them. Every lentivirus solution volume was added about 50 μL （each of viral titers were 1×108 TU/mL），and the Polybrene was added about 0.5 μL （concentration of 5 mg/L） respectively. Fresh culture medium was changed after 24 hours，and observe the fluorescence after 72 hours. At last，using the best screening concentration of puromycin to screen the transfection Hela cells，and to get the stable transfection cell lines. Results：The best screening concentration of puromycin was 0.9 mg/L，lentiviral vectors were successfully transfected into Hela cells，and the stable transfected cell lines were successfully obtained after two weeks. Conclusions：Hela cells which has stable interference of ALDH-1 were successfully constructed.

Key words: Oxidoreductases, Acetaldehyde, Lentivirus, Genetic vectors, Transfection, Hela cells