蛋白磷酸酶2A催化亚基α显性负性突变体表达载体对肝癌细胞生长的影响
단백린산매2A최화아기α현성부성돌변체표체재체대간암세포생장적영향
Gene therapy using a dominant negative form of the protein phosphatase 2A catalytic subunit α driven by a hepatoma tissue-specific promoter achieves effective growth inhibition of hepatoma cells
  • 万方数据
    中华肝脏病杂志 중화간장병잡지
    CHINESE JOURNAL OF HEPATOLOGY
    2013年 6期 459-463 ,共5页

    李道明%李伟%陶敏%陈凯%龚斐然%徐泽宽%陈政

    리도명%리위%도민%진개%공비연%서택관%진정

    癌,肝细胞%甲胎蛋白类%磷酸甘油酸酯激酶%磷蛋白磷酸酶 癌,肝細胞%甲胎蛋白類%燐痠甘油痠酯激酶%燐蛋白燐痠酶
    암,간세포%갑태단백류%린산감유산지격매%린단백린산매
    Carcinoma,hepatocellular%Alpha-fetoprotein%Phosphoglycerate kinase%Phosphoprotein phosphatase
    目的 构建肝癌组织特异性启动子驱动的蛋白磷酸酶2A催化亚基α显性负性突变体(DN-PP2Acα)表达载体,研究其对肝癌细胞生长的影响.方法 将甲胎蛋白(AFP)基因的增强子和磷酸甘油酸激酶(pgk)基因的启动子组合形成的肝癌组织特异性AFpg启动子.采用定点突变将野生型蛋白磷酸酶2A催化亚基α(PP2Acα)突变为DN-PP2Ac α.将AFpg启动子和DN-PP2Ac α编码序列构建成AFpg启动子调控的DN-PP2Ac α表达载体pGL3-Basic-AFpg-PP2Acα.荧光素酶报告基因实验检测AFpg启动子的转录活性.质粒pcDNA3.1(+)、pGL3-Basic、pcDNA3.1 (+)-DN-PP2Acα、pGL3-Basic-AFpg、pGL3-Basic-AFpg-PP2Ac α分别转染L02、SK-Hep-1、HepG2和Hep3B细胞,Western blot检测PP2Ac蛋白表达水平,四甲基偶氮唑盐比色法检测细胞生长情况.成组t检验分析不同处理组间的细胞活力差异.结果 L02、SK-Hep-1、HepG2和Hep3B细胞中,AFpg启动子相对荧光素酶活性分别为3.61±1.07、3.46±0.66、98.70±18.60、88.70±19.53,AFpg启动子在表达AFP的细胞中具有高转录活性.pGL3-BaSic-AFpg-PP2Acα可特异性地使DN-PP2Ac α表达于AFP阳性肝癌细胞HepG2和Hep3B,并抑制其生长,转染72 h后,HepG2细胞活力下降42.65％±3.99％,Hep3B细胞活力下降39.87％±3.91％,与0h时比较,差异均有统计学意义(t值分别为13.0563和12.9920,P值均＜0.01).pGL3-BaSic-AFpg-PP2Ac α对AFP阴性细胞的生长无明显影响.结论 AFpg启动子调控的DN-PP2Acα表达载体特异性地抑制AFP阳性肝癌细胞的生长,可用于肝癌组织特异性基因治疗.
    목적 구건간암조직특이성계동자구동적단백린산매2A최화아기α현성부성돌변체(DN-PP2Acα)표체재체,연구기대간암세포생장적영향.방법 장갑태단백(AFP)기인적증강자화린산감유산격매(pgk)기인적계동자조합형성적간암조직특이성AFpg계동자.채용정점돌변장야생형단백린산매2A최화아기α(PP2Acα)돌변위DN-PP2Ac α.장AFpg계동자화DN-PP2Ac α편마서렬구건성AFpg계동자조공적DN-PP2Ac α표체재체pGL3-Basic-AFpg-PP2Acα.형광소매보고기인실험검측AFpg계동자적전록활성.질립pcDNA3.1(+)、pGL3-Basic、pcDNA3.1 (+)-DN-PP2Acα、pGL3-Basic-AFpg、pGL3-Basic-AFpg-PP2Ac α분별전염L02、SK-Hep-1、HepG2화Hep3B세포,Western blot검측PP2Ac단백표체수평,사갑기우담서염비색법검측세포생장정황.성조t검험분석불동처리조간적세포활력차이.결과 L02、SK-Hep-1、HepG2화Hep3B세포중,AFpg계동자상대형광소매활성분별위3.61±1.07、3.46±0.66、98.70±18.60、88.70±19.53,AFpg계동자재표체AFP적세포중구유고전록활성.pGL3-BaSic-AFpg-PP2Acα가특이성지사DN-PP2Ac α표체우AFP양성간암세포HepG2화Hep3B,병억제기생장,전염72 h후,HepG2세포활력하강42.65％±3.99％,Hep3B세포활력하강39.87％±3.91％,여0h시비교,차이균유통계학의의(t치분별위13.0563화12.9920,P치균＜0.01).pGL3-BaSic-AFpg-PP2Ac α대AFP음성세포적생장무명현영향.결론 AFpg계동자조공적DN-PP2Acα표체재체특이성지억제AFP양성간암세포적생장,가용우간암조직특이성기인치료.
    Objective To generate a gene delivery plasmid carrying the dominant negative form of the protein phosphatase 2A catalytic subunit α (DN-PP2Acα) driven by a hepatocellular carcinoma (HCC) tissuespecific promoter and investigate its ability to inhibit growth of cultured hepatoma cells.Methods The gene delivery plasmid was constructed by PCR-amplifying DN-PP2Acα from wild-type PP2Acα using sitedirected mutagenesis and then ligating the sequence-verified amplicon downstream of an alpha-fetoprotein enhancer and phosphoglycerate kinase promoter (AFpg) in the luciferase reporter vector pGL3-Basic.Following transfection into two AFP+ hepatoma cell lines (HepG2 and HepG3) and two AFP-hepatoma cell lines (SK-HEP-1 and L02),the transcriptional activity of the AFpg-driven DN-PP2Acα plasmid was tested using luciferase reporter gene assay and western blotting.The effect on cell growth was tested using MTT assay.Between group differences were assessed by t-test.Results The AFpg-driven DN-PP2Acα plasmid showed high transcriptional activity and protein expression in both HepG2 and Hep3B cells.At 72 h after transfection,the proliferation capacities were repressed by 42.65％ ± 3.99％ (P =0.0002) and 39.87％±3.91％ (P =0.0002) in AFP+ HepG2 and Hep3B cells,respectively (vs.untransfected).In contrast,the plasmid was transcriptionally inactive in and had no effect on proliferation of AFP-cells.Conclusion The AFpg-driven DN-PP2Acα plasmid exhibits selective cytotoxicity against AFP+ hepatoma cells,and may represent a useful gene therapy strategy to treat HCC.
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