p100蛋白表达抑制的HepG2肝癌细胞稳定株的建立  

Establishment of Human p100 Protein Stable Knockdown HepG2 Cell Line

作　　者：田潇[1] 尹洁[1] 丁建民[4] 史雪彬[2,3] 何津岩[2,3] 张毅[2,3] 高星杰[1] 经翔[4] 杨洁[1,2,3] 

机构地区：[1]天津医科大学基础医学研究中心,天津市300070 [2]天津医科大学免疫教研室,天津市300070 [3]天津市细胞与分子免疫学重点实验室,天津市300070 [4]天津市第三中心医院超声科,天津市300170

出　　处：《医学分子生物学杂志》2012年第2期93-97,共5页Journal of Medical Molecular Biology

基　　金：资助项目：国家高技术研究发展计划资助项目（863计划）（No.2007A,A02Z115）,国家自然科学基金（No.30970582,31100967,31170830）,国家自然科学重大研究计划培育项目（No.90919032）,国家教育部高等学校博士学科专项科研基金（No.20091202110001）,中国博士后科学基金（No.2011M500529）,天津市应用基础及前沿技术研究计划（No.12JCYBJC16700）

摘　　要：目的建立p100表达抑制的HepG2肝癌细胞稳定株，并初步探讨p100在HepG2肝癌细胞中的功能。方法用脂质体将含有真核细胞筛选标记Neo和GFP的p100 shRNA表达质粒转染入HepG2细胞。经G418耐药筛选稳定整合抗药基因的细胞单克隆；荧光镜检GFP阳性细胞单克隆，挑取单克隆；Western检测HepG2细胞稳定株HepG2（p100I）中p100表达的抑制效果；平板细胞克隆形成实验检测细胞克隆形成能力；MTS法检测细胞存活；划痕实验检测细胞迁移能力。结果成功获得了p100表达抑制的HepG2肝癌细胞稳定株HepG2（p100I），其中p100的表达明显降低。并且实验表明，该p100表达抑制稳定株的克隆形成能力，抵抗化疗药物Cisplatin诱导的细胞死亡的能力和迁移能力明显低于对照组细胞。结论p100表达抑制的HepG2肝癌细胞稳定株的建立为研究p100蛋白在肝癌中的作用提供了体外细胞系模型，基于此稳定株的研究，发现p100能够影响HepG2肝癌细胞的多种细胞功能。Objective To establish a p100 stable knockdown HepG2 cell line, and thus inves- tigate the function of p100 protein in hepatoearcinoma HepG2 cells. Methods The p100 shRNA ex- pressing plasmid which carry eukaryotic selection marker Neo and GFP was transfected into HepG2 ceils using Lipofectamine. G418-resistant and GFP-positive single clones were picked up following term selection culture. Knockdown of p100 in the selected cell line was evaluated by western blot. Plate colony formation assay was used to detect the colony formation ability. MTS assay was used to analyze the cell proliferative response to cisplatin treatment. Cell migration was determined with wound healing assay. Results A p100 knockdown HepG2 stable cell line, HepG2 （p100I）, was successfully established, which provides an in vitro model to research the function of p100 protein in hepatocellular carcinoma cells. Our experimental results showed that knockdown of p100 protein in HepG2 cells reduced overall the colony formation, resistance to cisplatin-induced cell death, and cell migration. Conclusion p100 protein affects several tumor-related biological behaviors of HepG2 hepatocellular carcinoma cells, such as cell growth, cell survival and cell migration, thereby prob- ably contributing to liver cancer progression.

关 键 词：人类p100蛋白 HEPG2肝癌细胞 SHRNA表达载体 G418耐药筛选 稳定株 

分 类 号：Q71[生物学—分子生物学]