【作者】 王金金；

【导师】 马春红；

【作者基本信息】 山东大学 ， 医学免疫学， 2009， 硕士

【摘要】 研究目的:寻找新型转录因子ZHX2抑制AFP表达的功能片段,并探讨其对肝癌细胞生长的影响,为阐明ZHX2的结构与功能奠定基础,同时也为探索新的肝癌基因治疗方法提供帮助。研究方法:一、新型转录因子ZHX2抑制AFP表达功能片段的寻找1、新型转录抑制因子ZHX2不同截短片段的克隆表达①融合表达载体p1ZHX2(1-501AA)-HA,p2ZHX2(242-338AA)-HA,p3ZHX2(242-501AA)-HA的构建根据PubMed中人ZHX2 cDNA序列(gi:63079684),利用Primer Premier5.0软件,设计扩增不同长度ZHX2片段的特异性引物,为便于克隆和表达,上下游引物分别包含KpnI、EcoRI酶切位点,并在下游引物引入HA-tag序列。以pcDNA-ZHX2-HA的cDNA为模板,PCR扩增人ZHX2基因两个锌指结构域(zinc finger domains)和前两个同源结构域(homoboxes,HD1,HD2),即1-501AA片段、242-338AA片段、242-501AA片段。PCR产物分别经KpnI,EcoRI双酶切,回收后分别克隆入pcDNA3.0载体。连接产物转化大肠杆菌JM109,LA平板筛选挑取阳性重组子,并经酶切、DNA测序分析鉴定,分别命名为p1ZHX2(1-501AA)-HA,p2ZHX2(242-338AA)-HA,p3ZHX2(242-501AA)-HA。②Western blot检测ZHX2不同截短片段在细胞中的表达p1ZHX2(1-501AA)-HA,p2ZHX2(242-338AA)-HA,p3ZHX2(242-501AA)-HA脂质体法转染肝癌细胞HepG2、HepG2.2.15及非洲绿猴肾细胞COS-7,48h后收集细胞,提取总蛋白,以HA单抗作为一抗,HRP标记的抗鼠抗体为二抗,western blot分析ZHX2不同截短片段蛋白的表达情况。2、ZHX2不同截短片段蛋白对肝癌细胞系AFP的抑制作用①肝癌细胞系HepG2、HepG2.2.15中过量表达ZHX2不同截短片段对AFP的影响ZHX2不同截短片段的质粒p1ZHX2(1-501AA)-HA,p2ZHX2(242-338AA)-HA,p3ZHX2(242-501AA)-HA分别转染肝癌细胞系HepG2、HepG2.2.15细胞,48h后收集细胞及培养上清,ELISA检测培养上清中AFP的表达,同时western blot检测细胞中不同截短片段融合蛋白的表达。②ZHX2不同截短片段蛋白对AFP核心启动子的抑制作用ZHX2不同截短片段的重组质粒各0.3μg分别与0.6μg AFP核心启动子报告质粒(phAF269)DNA共转染HepG2和HepG2.2.15细胞(各孔以pCDNA3.0 DNA将质粒补足至1μg),同时每孔转染20ng的pRL-TK,做为转染率内参照。转染48h后,收集细胞,进行双荧光素酶分析。③双荧光素酶报告基因检测按照双荧光素酶报告基因检测试剂盒说明书将细胞裂解后与底物混合,用荧光计数仪检测荧光强度M1与荧光强度M2。以M1与M2的比值表示荧光素酶强度。每次实验设2个复孔,同一转染实验至少重复3次。④统计分析采用GraphPad Prism 4统计分析软件进行数据处理,实验数据以X~-±stdv表示,各组间比较采用t检验进行处理,当P＜0.05时,统计学上具备显著性差异。二、ZHX2及ZHX2截短片段蛋白对肝癌细胞的生长作用的研究1.CCK-8检测ZHX2及ZXH2截短片段对肝癌细胞生长的作用取生长状态良好的HepG2和HepG2.2.15细胞分别按1×10~4个/孔接种于96孔板中,次日ZHX2及其截短片段重组质粒p3ZHX2(242-501AA)-HA,p2ZHX2(242-338AA)-HA,分别转染HepG2和HepG2.2.15细胞,转染之后每隔24h,用CCK-8检测细胞生长情况,连续检测五天,绘制生长曲线观察ZHX2及其截短片段对细胞生长的影响。2.集落形成实验检测ZHX2及其功能片段对肝癌细胞集落形成能力取生长状态良好的HepG2细胞按3×10~5个/孔接种于24孔板中,次日ZHX2全长及功能片段表达质粒瞬时转染HepG2,转染之后24h,将细胞经胰酶消化后,以适当的细胞密度接种于六孔板中,静置培养2-3周,之后用龙胆紫染细胞,肉眼计数细胞克隆数,计算克隆形成率。研究结果一、新型转录因子ZHX2抑制AFP表达功能片段的寻找1、新型转录抑制因子ZHX2不同截短片段的克隆表达①成功构建三个融合表达p1ZHX2(1-501AA)-HA,p2ZHX2(242-338AA)-HA和p3ZHX2(242-501AA)-HA以含有全长ZHX2基因的pcDNA3.0-ZHX2-HA质粒为模板,PCR扩增ZHX2的三个不同截短片段,构建真核融合表达质粒,命名为:p1ZHX2(1-501AA)-HA,p2ZHX2(242-338AA)-HA和p3ZHX2(242-501AA)-HA。重组质粒DNA分别进行单、双酶切,结果表明,KpnI/EcoRI双酶切三种质粒,分别可获得1587bp,338bp,827bp的目的基因片段,与预期大小一致;进一步DNA测序证明三个重组载体pcDNA-ZHX2(1-501AA)-HA,pcDNA-ZHX2(242-338AA)-HA,pcDNA-ZHX2(242-501AA)-HA构建成功。②Western blot证实三种ZHX2片段的融合蛋白可以在真核细胞有效表达Western blot结果显示p1ZHX2(1-501AA)-HA,p2ZHX2(242-338AA)-HA和p3ZHX2(242-501AA)-HA转染的HepG2,HepG2.2.15和COS-7细胞中均有明显的特异性条带,而转染空质粒和空细胞的对照组均未见条带,表明重组质粒可以在肝癌细胞系和非洲绿猴肾细胞中有效表达ZXH2不同截短片段。2、不同截短片段的ZHX2蛋白均可有效抑制AFP表达,其中242-501AA抑制作用最强①ZHX2不同截短片段对肝癌细胞中AFP表达的抑制作用ZHX2不同截短片段重组质粒分别转染HepG2,HepG2.2.15细胞48h后,收集上清,ELISA检测上清中AFP的表达。结果显示ZHX2不同截短片段均可以抑制肝癌细胞AFP表达,其中ZHX2(242-501AA)抑制作用最强,转染p3ZHX2(242-501AA)-HA的HepG2和HepG2.2.15细胞中AFP分别为1386.7±193.6ng/ml和1726.2±111.7ng/ml,较全长转染组(1897.4±73.1ng/ml,1896.1±81.1ng/ml)显著降低(p＜0.05),抑制率为31.7%±2%。②.ZHX2不同截短片段对人AFP启动子活性的抑制作用ZHX2不同截短片段真核表达载体与pAFP269报告基因共转染HepG2、HepG2.2.15细胞48h后,收集细胞,双荧光素酶报告基因检测结果显示,不同ZHX2截短片段均可有效抑制AFP核心启动子活性,其中p3ZHX2(242-501AA)-HA作用最强,抑制率为49.9%±0.9%。二、ZHX2及其截短片段蛋白抑制肝癌细胞的体外生长1.ZHX2及其截短片段抑制肝癌细胞的生长ZHX2及其截短片段表达质粒分别转染HepG2.2.15和HepG2细胞后,CCK-8连续监测五天,结果显示ZHX2及其截短片段均可以抑制肝癌细胞生长,其中242-501AA片段抑制作用最强,第5天时,较空载体转染组,抑制率为15%。2.ZHX2及其功能片段抑制肝癌细胞的集落形成ZHX2及其功能片段表达质粒分别瞬时转染HepG2细胞后,集落形成实验结果显示,ZHX2及其功能片段均具有体外抑制肿瘤细胞集落形成的能力,较ZHX2全长及空载体对照组相比,242-501AA片段抑制作用最强,较空载体组抑制率为83%。结论及意义:1.本研究成功构建了三个可以在体外有效表达ZHX2不同截短片段融合蛋白的真核表达载体p1ZHX2(1-501AA)-HA,p2ZHX2(242-338AA)-HA,p3ZHX2(242-501AA)-HA,三种载体的构建为进一步研究ZHX2的功能提供了必要的条件。2.本研究首次证明ZHX2(242-501AA)片段是对AFP有较强抑制作用的功能片段,为进一步深入研究ZHX2结构与功能奠定了基础,同时也为发现肝癌治疗新靶点提供实验依据。3.本研究通过CCK-8检测试剂盒和集落形成实验首次证实ZHX2及其功能片段过表达不仅抑制肝癌细胞的增殖,同时还具有抑制肝癌细胞集落形成的能力,其中,p3ZHX2(242-501AA)的片段抑制作用最强。本实验结果提示ZHX2具有抑癌基因的特性,为探索新型肝癌治疗方案奠定基础。更多还原

【Abstract】 Objective:To identify the functional fragment of ZHX2 repressing AFP expression and explore its inhibition effects on cell growth.Method:PartⅠ.Identification of repressional fragment of ZHX2 on AFP1.Cloning of ZHX2 truncated fragments(1) Construction of ZHX2 truncated fragments expression plasmidsAccording to the sequence of ZHX2 cDNA in PubMed,specific primers for the ZHX2 different truncated fragments using Primer Premier5.0 software.HA tag were included in the reverse primers at its 5 ’end.PCR reaction were used to amplify ZHX2 truncated fragments 1-501AA,242-338AA and 242-501AA.The PCR products were then cloned into the vector pcDNA3.0 to construct the expression plasmids:p1ZHX2(1-501AA)-HA,p2ZHX2 (242-338)-HA and p3ZHX2(242-501AA)-HA.The recombinants were transformed into JM109,and the positive clones were identified by restrictive enzymes assay and DNA sequencing.(2) Western blot analysis for ZHX2 truncated protein expressionIn order to assay the expression of ZHX2 truncated fragments in vitro,p1ZHX2 (1-501AA)-HA,p2ZHX2(242-338AA)-HA and p3ZHX2(242-501AA)-HA were transfected into HepG2,HepG2.2.15and COS-7 cells respectively.Cells were collected 48 h later for western blot.2.The repression effect of ZHX2 truncated fragments on AFP (1) Overexpression of ZHX2 truncated fragments in hepatma cell lines HepG2 and HepG2.2.15Three vectors containing ZHX2 different truncated fragments were transfected into HepG2 and HepG2.2.15 cell lines respectively.48 h later,the medium and the cells were collected seperately to detect the expression of AFP and ZHX2 truncated fragments AFP were analyzed by ELISA and ZHX2 were analyzed by western blot using anti-HA monoclonal antibody as first antibody and anti-mouse IgG antibody labeled with HRP as second antibody.(2) The repression of ZHX2 truncated fragments on AFP core promoter0.6μg phAF269 plasmid was cotransfected with different truncated fragments of ZHX2 plasmids(0.3μg) into HepG2 and HepG2.2.15 cells,and 20ng of pRL-TK plasmid DNA was also involved in each group to normalize transfection efficiency.Cells were harvested 48 h for luciferase assay.(3) Dual-Luciferase reporter assaysFollowing the Promega protocol,dual-Luciferase reporter assays were performed in triplicate,using 20μl of cell extract,100μl of Luciferase Assay ReagentⅡReagent and 100μl of Stop&Glo Reagent by using luminoskan TL plus(Labsystems,Fr ankfurt,Germany).The firefly luciferase activity was normalized with the renilla reniformis luciferase activity of the cotransfected pRL-TK to control for variations in transferction efficiency.All the data shown in this study were obtained from at least three independent experiments,and each experiment was established double-wells.(4)Statistical analysisThe software GraphPad Prism 4 was used in the statistical analysis.Results were expressed as mean value±standard deviation(X~-±1s ).T test was used to evaluate the significance,where a value less than 0.05(p＜0.05) denoted the statistical significance.PartⅡ.The research of ZHX2 and its truncated fragments on liver cancer cells grow in vitro.1.The effect of ZHX2 and its truncated fragments on liver cancer cell grow were detected by CCK-8.HepG2 and HepG2.2.15 cells were inoculated by 1×10~4 cells per well in 96-well plates.The next day the recombinant plasmids of ZHX2 and ZHX2 truncated fragments were transfected into HepG2.2.15 and HepG2 cells respectively.The cell grow were continued detected for five days by CCK-8 every 24h after transfection.The growth curve were drawed to observe the effect of ZHX2 and ZHX2 functional fragment on cell growth.2The colony formation experimentsHepG2 cell were inoculated at 1×10~4 cells per well in 96-well plates.The next day the recombinant plasmids of ZHX2 and its truncated fragments were transfected into HepG2 cell.The cells were digested by trypsin after transfected 24h,the cell were inoculated in the six-well plates at appropriate density,after 2-3 weeks the gentian violet dyed the cells and counted the number of cell clones,calculated cloning formation rate.Result:1 The expression of ZHX2 truncated fragments in vitro(1)The successfully construction of ZHX2 truncated fragment-expression plasmidsEnzymes digestion,PCR and DNA sequencing confirmed that three combinant vectors, p1ZHX2(1-501AA)-HA,p2ZHX2(242-338AA)-HA and p3ZHX2(242-501AA)-HA were successfully constructed.(2) Expression of ZHX2 different truncated fragments in vitroWestern blot results showed that ZHX2 different truncated fragments proteins could all express in HepG2,HepG2.2.15 and COS-7 cells.2.Repression of ZHX2 truncated fragments on AFP in human hepatoma cell lines(1) Overexpression of human ZHX2 truncated fragments repressed AFP expression in hepatoma cell linesELISA results showed that AFP was suppressed by all three ZHX2 truncated fragments, while p3ZHX2(242-501AA)-HA has the stronggest repression function with the inhibition ratio 31.7%±2%(AFP in HepG2 and HepG2.2.15 cells transfected with p3ZHX2(242- 501 AA)-HA was 1386.7±193.6ng/ml and 1726.2±111.7ng/ml;compared with ZHX2-HA: 1897.4±73.1 ng/ml,1896.1±81.1 ng/ml).(2)The repression of ZHX2 truncated fragments on AFP core promoterResults of dual-Luciferase reporter assay showed that all ZHX2 truncated fragments could suppress the activity of AFP core promoter,while p3ZHX2(242-501AA) -HA has the most powful repression with the inhibition rate of 49.9%±0.9%. 3 ZHX2 and its truncated fragments can repress the cancer cell growth in vitro(1) ZHX2 and its truncated fragments can repress the cancer cell growthThe expression plasmids of ZHX2 and its truncated fragments were transfected into HepG2 and HepG2.2.15 cells.The cell growth were continued monitoring by CCK-8.The result shows ZHX2 and ZHX2 truncated fragments can repress the cancer cell growth.,while p3ZHX2(242-501AA) -HA has the most powful repression effects with the inhibition rate of 15%.(2) ZHX2 and its functional fragment can repress the colony forming ability of hepatoma cells in vitroThe expression plasmids of ZHX2 and its functional fragments were transfected into HepG2 cell.The cell colonies were counted by hexamethylpararosaniline dyeing 2 weeks later.The result showed ZHX2 and its functional fragments can repress the colony forming ability of hepatoma cells in vitro,while p3ZHX2(242-501AA) -HA has the most powful repression with the inhibition rate of 83%.Conclusions:1.We successfully constructed three fusion expression vectors p1ZHX2(1-501AA) -HA, p2ZHX2(242-338AA)-HA and p3ZHX2(242-501AA)-HA,which could express ZHX2 truncated fragments fusion proteins in vitro efficiently.This can provide requirement for further study of ZHX2 function.2.This study confirmed that p3ZHX2(242-501AA)-HA is the minimum function fragment owning the repression function on AFP and it has the most powful repression effects.This study provided necessary experimental data for further research on the structure and function of ZHX2.3.This study found that both ZHX2 and its truncated fragments could suppress the cell growth and the colony forming ability in vitro which may have role in HCC therapy.更多还原