HBV X基因转染对HepG2肝癌细胞凋亡的影响及其机制

目的:研究HBV X基因转染对HepG2肝癌细胞凋亡及凋亡相关因子表达的影响.方法:用脂质体转染法将HBx真核表达载体pcDNA3/HBx瞬时转入HepG2细胞,以未转染的HepG2细胞及转染空载体pcDNA3的细胞为对照.RT-PCR法检测HBx基因的表达;MTT法检测各组细胞的增殖活性;TUNEL法检测各组的凋亡情况.β-actin为内参,半定量RT-PCR法检测凋亡相关基因Bax、Bcl-xL、c-myc的表达量变化.结果:pcDNA3-X转染HepG2细胞后,RT-PCR扩增出HBV X片段,空质粒组及正常对照组均未扩增出相应片段.HepG2细胞转染HBx基因后,相对于对照组细胞增殖能力明显下降,凋亡增多,差异有显著性意义(P＜0.01);转染HBx的细胞Bax、Bcl-xL、c-myc mRNA相对表达量较转染空质粒组和未转染质粒组明显增高,差异有显著性意义(P＜0.05).结论:成功将HBx基因转染入HepG2细胞,并在细胞中表达,转染HBx基因可同时上调Bax、Bcl-xL、c-myc mRNA表达,促进HepG2细胞凋亡,抑制增殖.     

丙型肝炎病毒核心区蛋白对 HepG2细胞周期、细胞凋亡和细胞端粒酶活性的影响

目的　研究丙型肝炎病毒核心区 (HCV C)蛋白对肝癌细胞HepG2细胞周期、细胞凋亡和细胞端粒酶活性的影响。方法　首先运用基因重组技术构建含有HCV C基因的真核表达质粒pcDNA3.1( ) ,然后利用脂质体介导将重组真核表达质粒转染HepG2 ,经G4 18筛选获得稳定转染HepG2细胞 (HCV C转染HepG2细胞 ) ,经逆转录 聚合酶链反应技术 (RT PCR)和间接免疫荧光法证实其中有HCV C蛋白表达。然后进行如下实验 :( 1)利用四甲基偶氮唑蓝比色 (MTT)法检测HCV C转染HepG2细胞、空白质粒转染HepG2细胞和未转染HepG2细胞的生长增殖率 ;经流式细胞术(FACS)检测 3组细胞的细胞周期 ;( 2 )经流式细胞术检测细胞凋亡率 ;( 3)经端粒重复扩增 酶联免疫吸附试验 (TRAP ELISA)法检测上述 3组细胞端粒酶活性表达情况。结果　 ( 1)HCV C转染HepG2细胞增殖率显著高于空白质粒转染HepG2和未转染HepG2细胞增殖率 ;HCV C转染HepG2细胞S期所占百分率高于未转染HepG2细胞S期所占百分率 ;( 2 )HCV C转染HepG2细胞凋亡率显著低于无HCV C转染细胞凋亡率 ;( 3)上述 3组细胞端粒酶活性之间差异无显著性。结论　 ( 1)HCV C蛋白具有抑制细胞凋亡的作用 ;( 2 )HCV C蛋白促进HepG2从G0 /1期进入S期 ,从而可能促进细胞生长增殖 ,抑制细胞凋亡 ;( 3)HCV     

AEG1促进肝癌细胞HepG2分泌细胞因子IL-6、IL-1β的实验研究

目的研究AEG1能否促进肝癌细胞HepG2分泌细胞因子IL-6、IL-1β,探讨AEG1是否参与改变肿瘤微环境。方法用脂质体稳定转染法分别转染pcDNA3.1(-)-AEG1(AEG1全长质粒)、psilencer 2.0-shAEG1(AEG1干扰质粒)至HepG2细胞,相对应以转染空质粒pcDNA3.1(-)、psilencer 2.0的HepG2细胞为对照组;采用实时荧光定量RT-PCR(qRT-PCR)、Western blotting检测AEG1 mRNA和蛋白表达变化;采用实时荧光定量RT-PCR和酶联免疫吸附实验(ELISA)检测细胞因子IL-6、IL-1β的表达和分泌。结果与转染相应空载体的阴性对照组和未转染的空白对照组相比,分别转染AEG1全长质粒/AEG1干扰质粒后,HepG2细胞中AEG1的蛋白表达明显增高/降低。转染AEG1全长质粒的HepG2细胞中IL-6、IL-1βmRNA均较对照组和空白组明显增强(P均<0.05),细胞培养介质中IL-6、IL-1β蛋白浓度也显著高于对照组(P均<0.05);转染AEG1 shRNA的HepG2细胞中IL-6、IL-1βmRNA均较对照组和空白组降低(P均<0.05),细胞培养介质中IL-6、IL-1β蛋白浓度也低于对照组(P均<0.05)。结论成功构建稳定过表达和沉默AEG1的HepG2细胞,AEG1能调控IL-6、IL-1β的表达和分泌。     

p33ING1转染HepG2肝癌细胞对p21WAF1/ CIP1表达的影响

目的 探讨p33ING1对pcDNA3.1-p33ING1转染后的HepG2肝癌细胞p21WAF1/CIP1表达的影响.方法 实验分为对照组和转染组;将pcDNA3.1-p33ING1及空载质粒pcDNA3.1转染到HepG2细胞中,最终建立稳定转染的细胞株,RT-PCR检测p33ING1表达;RT-PCR和免疫组化检...     

热休克蛋白70通过Bcl-2抑制氧化应激所致C2C12细胞凋亡

目的 探讨C2C12肌原细胞内热休克蛋白70对Bcl-2表达的影响及Bcl-2对热休克蛋白70抗细胞凋亡作用的影响.方法 应用Western Blotting观察Bcl-2在转染热休克蛋白70真核表达质粒(pcDNA3.1-HSP70)或其反义寡核苷酸C2C12肌原细胞中的表达;采用基因瞬间转染技术使热休克蛋白70过表达的C2C12肌原细胞内Bcl-2表达抑制,应用流式细胞术检测H2O2处理所致细胞凋亡的发生情况.结果 转染热休克蛋白70真核表达质粒的C2C12肌原细胞中,热休克蛋白70和Bcl-2的表达明显高于空载体转染组(P<0.01);而转染热休克蛋白70反义寡核苷酸后,热休克蛋白70和Bcl-2的表达明显低于随机寡核苷酸转染组 (P<0.01);热休克蛋白70过表达的C2C12肌原细胞分别转染Bcl-2反义寡核苷酸及随机寡核苷酸,0.5 mmol/L H2O2处理24 h,Bcl-2反义寡核苷酸转染组的细胞凋亡率明显高于随机寡核苷酸转染细胞组.结论 热休克蛋白70能上调C2C12肌原细胞内Bcl-2的表达;热休克蛋白70的抗细胞凋亡功能可能与其上调Bcl-2表达相关.     

HCV核心基因转染HepG2细胞后基因表达差异筛选

目的将真核表达载体pcDNA3.1(-)HCV core转染到HepG2细胞,在HepG2细胞中表达HCV核心蛋白,并筛选其中的差异表达基因。方法将构建的真核表达载体pCDNA3.1(-)HCVcore转染HepG2细胞后进行蛋白免疫印迹检测;将pcDNA3.1(-)HCVcore和pcDNA3.1(-)载体分别转染HepG2细胞后,提取mRNA并逆转录为cDNA,运用基因表达谱芯片技术分析差异表达基因。结果构建的真核表达载体经双酶切鉴定;转染HepG2细胞后HCV核心蛋白表达经蛋白免疫印迹证实;经基因表达谱芯片分析发现,其中基因表达水平显著上调和下调的分别是181个和48个。结论筛选HCV核心基因转染HepG2细胞后的糖类和脂类物质代谢相关的差异表达基因,从而为丙型肝炎病毒合并糖尿病、脂肪肝等代谢性疾病的分子生物学机制的研究提供了重要依据。     

HCV核心蛋白与截短型HBV表面抗原中蛋白的协同反式激活功能

目的 探讨HCV核心蛋白与截短型HBV表面抗原中蛋白的协同反式激活作用。方法 构建表达HCV核心蛋白的重组质粒pcDNA3.1(-)-core和表达截短型HBV表面抗原中蛋白的重组质粒pcDNA3.1(-)-Mt；转染HepG2细胞，从转录和翻译水平鉴定病毒基因的瞬时表达；与报告质粒pSV-lacZ共转染HepG2细胞，检测β－半乳糖苷酶（β-gal)表达活性，酶的活性反映了表达的肝炎病毒蛋白对SV40病毒早期启动子（增强子）功能的影响。结果 构建成HCV核心蛋白及截短型HBV表面抗原中蛋白的重组表达载体pcDNA3.1(-)-core、pcDNA3.1(-)-Mt；在HepG2细胞均能瞬时表达相应的病毒蛋白；单独的共转染实验中pcDNA3.1(-)-core、pcDNA3.1(-)-Mt组的β-gal的表达分别是对照的4．6和3．2倍，两种质粒共同转染时酶的表达是对照组的8．4倍；表达质粒对β－gal表达的激活作用呈剂量依赖性。结论 HepG2细胞中表达的HCV核心蛋白和截短型HBV表面抗原中蛋白均具有反式激活SV40早期启动子（增强子）的功能，并且两种蛋白的反式激活功能具有协同特性。本实验有助于解释HCV、HBV感染，尤其是共同感染的致病（癌）机制。     

APOBEC3G体外抗乙型肝炎病毒的研究

目的探讨载脂蛋白B mRNA编辑酶催化多肽样蛋白3G（apolipoprotein B mRNA editing enzyme catalytic polypeptide like3G,APOBEC3G）（也称为CEM15）体外抗乙型肝炎病毒（HBV）的作用及其机制。方法脂质体转染pcDNA3.1 Human APOBEC3G-Myc-6Xhis、pcDAN3.1/His-C进入HepG2.2.15细胞,转染后,RT-PCR证实转染基因的表达,Western Blot证实蛋白的表达。通过ELISA方法检测细胞上清液中乙型肝炎表面抗原（HBsAg）及乙型肝炎e抗原（HBeAg）,RT-PCR分析APOBEC3G对HBV mRNA转录的影响。结果APOBEC3G基因与蛋白在HepG2.2.15细胞都有表达,与空质粒转染组相比,pcDNA3.1 Human APOBEC3G-Myc-6Xhis转染组HBsAg含量下降70.38%,HBeAg含量下降62.88%,未转质粒细胞为空白对照组。结论APOBEC3G在体外可以抑制HBV复制,可以作为一种新型的抗病毒制剂治疗乙肝病毒感染。     

HepG2细胞中HCV NS5A蛋白对HCV IRES启动蛋白翻译的影响

目的探讨HepG2细胞中HCV非结构蛋白5A（NS5A）对HCV IRES启动蛋白翻译的影响，以了解HCV的复制调控机制。方法将构建的表达双荧光素酶的双顺反子载体pCMV-Rluc-IRES-Fluc和含HCV NS5A基因的表达质粒pcDNA-NS5A共转染HepG2细胞，用双荧光素酶检测系统检测虫荧光素酶的表达水平，细胞免疫荧光技术检测HCV-NS5A蛋白的表达，RT-PCR检测虫荧光素酶基因mRNA水平，并与相应对照做比较，以观察HCV NS5A对HCV IRES介导虫荧光素酶翻译水平的影响。结果转染pcDNA-NS5A的HepG2细胞中虫荧光素酶活性明显高于转染pCDNA3．I-3flag的对照组，并存在剂量依赖关系；而RT-PCR虫荧光素酶基因mRNA水平在两组间差异无统计学意义。转染pcDNA-NS5A的HepG2细胞质中可见HCV NS5A蛋白的表达。结论HCV NS5A蛋白对HCV IRES介导虫荧光素酶的翻译有正调节作用，并存在剂量依赖关系．     

HCVcore/NS3/NS5A对内源性IFN-β表达影响及其调控机制初步研究

目的探讨丙型肝炎病毒(HCV)core、NS3、NS5A对内源性IFN-β表达的影响及其调控机制。方法将HCVcore、NS3、NS5A表达载体pcDNA3.1/myc-His-core/NS3/NS5A转染至HepG2细胞,验证蛋白表达之后,采用实时荧光定量PCR和Westernblot及ELISA方法观察3种蛋白对IFN-βmRNA及蛋白水平表达的影响。构建IFN-β全长启动子报告基因表达载体,借助双萤虫素酶活性检测,探讨HCVcore、NS3、NS5A对IFN-β转录水平的调控机制。结果 pcDNA3.1/myc-His-core/NS3/NS5A在HepG2细胞中成功表达,与转染pcDNA3.1/myc-His空载体相比,pcDNA3.1/myc-His-NS3/NS5A过表达时,在mRNA及蛋白水平均能抑制HepG2细胞内IFN-β的表达,与转染空载体的对照组相比,差异有统计学意义(P0.05)。双萤虫素酶活性检测显示,转染IFN-β全长启动子报告基因表达质粒后,与对照组相比,双萤虫素酶活性降低,差异有统计学意义(P0.05)。pcDNA3.1/myc-His-core过表达时对IFN-β的表达无明显影响。结论 HCVNS3/NS5A在mRNA及蛋白水平能抑制IFN-β表达,并通过其转录水平影响IFN-β表达,core对IFN-β的表达无明显影响。其具体调控机制有待进一步研究。     

Study of transactivating effect of pre-S2 protein of hepatitis B virus and cloning of genes transactivated by pre-S2 protein with suppression subtractive hybridization

AIM: To investigate the transactivating effect of pre-S2 protein of hepatitis B virus (HBV) and construct a subtractive cDNA library of genes transactivated by pre-S2 protein with suppression subtractive hybridization (SSH) technique, and to pave the way for elucidating the pathogenesis of HBV infection. METHODS: pcDNA3.1(-)-pre-S2 containing pre-S2 region of HBV genome was constructed by routine molecular methods. HepG2 cells were cotransfected with pcDNA3.1 (-)-pre-S2/pSV-lacZ and empty pcDNA3.1(-)/pSV-lacZ. After 48 h, cells were collected and detected for the expression of β-galactosidase (β-gal). SSH and bioinformatics techniques were used, the mRNA of HepG2 cells transfected with pcDNA3.1(-)-pre-S2 and pcDNA3.1(-) empty vector was isolated, respectively, cDNA was synthesized. After digestion with restriction enzyme RsaI, cDNA fragments were obtained. Tester cDNA was then divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library. Amplification of the library was carried out with E.coli strain DH5oα. The cDNA was sequenced and analyzed in GenBank with Blast search after PCR. RESULTS: The pre-S2 mRNA could be detected in HepG2 cells transfected with pcDNA3.1(-)-pre-S2 plasmid. The activity of β-gal in HepG2 cells transfected with pcDNA3.1 (-)-pre-S2/pSV-lacZ was 7.0 times higher than that of control plasmid (P<0.01). The subtractive library of genes transactivated by HBV pre-S2 protein was constructed successfully. The amplified library contains 96 positive clones. Colony PCR showed that 86 clones contained 200-1 000 bp inserts. Sequence analysis was performed in 50 clones randomly, and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank, altogether 25 coding sequences were obtained, these cDNA sequences might be the target genes transactivated by pre-S2 protein. CONCLUSION: The pre-S2 protein of HBV has transactivating effect on SV40 early promoter. The obtained sequences may be target genes transactivated by pre-S2 protein among which some genes coding proteins involved in cell cycle regulation, metabolism, immunity, signal transduction and cell apoptosis.This finding brings some new clues for studying the biological functions of pre-S2 protein and further understanding of HBV hepatocarcinogesis.     

HBx activates FasL and mediates HepG2 cell apoptosis through MLK3-MKK7-JNKs signal module

AIM: To investigate the possible mechanism by which hepatitis B virus X protein (HBx) mediates apoptosis of HepG2 cells.METHODS: HBx expression vector pcDNA3.1-X was transfected into HepG2 cells to establish an HBx high-expression cellular model as pcDNA3.1-X transfected group. The pcDNA3.1-X and pSilencer3.1-shHBX (HBx antagonist) were cotransfected into HepG2 cells to establish an HBx low-expression model as RNAi group. Untransfected HepG2 cells and HepG2 cells transfected with negative control plasmid were used as controls. Apoptosis rate, the expression of Fas/FasL signaling pathway-related proteins and the phosphorylation levels of MLK3, MKK7 and JNKs, which are upstream molecules of death receptor pathways and belong to the family of mitogen-activated protein kinases (MAPKs), were measured in each group.RESULTS: Compared with HepG2 cell group and RNAi group, apoptosis rate, the expression of Fas and FasL proteins, and the activation of MLK3, MKK7 and JNKs were increased in the pcDNA3.1-X transfected group. The activation of JNKs and expression of FasL protein were inhibited in the pcDNA3.1-X transfected group when treated with a known JNK inhibitor, SP600125. When authors treated pcDNA3.1-X transfected group with K252a, a known MLK3 inhibitor, the activation of MLK3, MKK7 and JNKs as well as expression of FasL protein was inhibited. Furthermore, cell apoptosis rate was also significantly declined in the presence of K252a in the pcDNA3.1-X transfected group.CONCLUSION: HBx can induce HepG2 cell apoptosis via a novel active MLK3-MKK7-JNKs signaling module to upregulate FasL protein expression.     

Transactivating effect of hepatitis C virus core protein： A suppression subtractive hybridization study

AIM: To investigate the transactivating effect of hepatitis C virus (HCV) core protein and to screen genes transactivated by HCV core protein. METHODS: pcDNA3.1(-)-core containing full-length HCV core gene was constructed by insertion of HCV core gene into EcoRI/BarnHI site. HepG2 cells were cotransfected with pcDNA3.1(-)-core and pSV-lacZ. After 48 h, cells were collected and detected for the expression of β-gal by an enzyme-linked immunosorbent assay (ELISA) kit. HepG2 cell swere transiently transfected with pcDNA3.1(-)-core using Upofectamine reagent. Cells were collected and total mRNA was isolated. A subtracted cDNA library was generated and constructed into a pGEM-Teasy vector. The library was amplified with E. coil strain JM109. The cDNAs were sequenced and analyzed in GenBank with BLAST search after polymerase chain reaction (PCR). RESULTS: The core mRNA and protein could be detected in HepG2 cell lysate which was transfected by the pcDNA3.1(-)-core. The activity of β-galactosidase in HepG2 cells transfected by the pcDNA3.1(-)-core was 5.4 times higher than that of HepG2 cells transfected by control plasmid. The subtractive library of genes transactivated by HCV core protein was constructed successfully. The amplified library contained 233 positive clones. Colony PCR showed that 213 clones contained 100-1 000 bp inserts. Sequence analysis was performed in 63 clones. Six of the sequences were unknown genes. The full length sequences were obtained with bioinformatics method, accepted by Genl3ank. It was suggested that six novel cDNA sequences might be target genes transactivated by HCV core protein. CONCLUSION: The core protein of HCV has transactivating effects on SV40 early promoter/enhancer. A total of 63 clones from cDNA library were randomly chosen and sequenced. Using the BLAST program at the National Center for Biotechnology Information, six of the sequences were unknown genes. The other 57 sequences were highly similar to known genes.     

丙型肝炎病毒不同基因型核心蛋白对肝母细胞瘤细胞凋亡影响的比较

目的比较HCV不同基因型核心蛋白(core)在肝母细胞瘤细胞凋亡中的作用。方法构建6a基因型HCV core的表达载体pcDNA3.1/myc-His(-)-core(6a)[pCore(6a)];分别将1b、3a和6a基因型HCV core质粒以及空质粒pcDNA3.1/myc-His(-)(pNC)瞬时转染HepG2细胞,48小时后利用流式细胞术检测细胞凋亡发生的情况;利用Real-time PCR检测胱冬肽酶-3的mRNA水平变化;利用化学发光法检测胱冬肽酶-3的活性变化,比较HCV不同基因型core对细胞凋亡作用的差异。结果成功构建了6a基因型HCV core的表达载体pcDNA3.1/myc-His(-)-core(6a)[pCore(6a)];与pNC组相比,表达1b、3a和6a基因型HCV core的HepG2细胞中,Annexin V+细胞数均显著减少,其中6a基因型core较1b和3a基因型core作用显著(P=0.000、0.001);同时实验组中凋亡效应分子胱冬肽酶-3的mRNA水平及其活性较对照组均降低;其中与1b和3a基因型core相比较,6a基因型core显著降低胱冬肽酶-3的mRNA水平,而在抑制胱冬肽酶-3活性方面无显著差别。结论 HCV不同基因型core对细胞凋亡的作用不同;基因6a型core对细胞凋亡的抑制作用更为显著;HCV基因6型核心蛋白可能更易导致肝癌的发生,有待进一步研究。     

乙型肝炎病毒/P22蛋白对HepG2细胞凋亡的影响

目的研究HBV／P22蛋白对肿瘤坏死因子（TNF）α诱导的HepG2细胞凋亡的影响。方法用放线菌素D和TNFo【分别诱导表达HBV／P22蛋白的HepG2细胞（实验组）、表达空载体的HepG2细胞（阴性对照组）和HepG2细胞（空白对照组）凋亡，雅培试剂检测细胞上清液中HBeAg的表达，Westernblot及免疫细胞化学法检测HBV／P22蛋白表达，流式细胞仪和末端脱氧核苷酸转移酶介导的脱氧三磷酸尿苷缺口末端标记（TUNEL）法检测细胞凋亡。体内实验：将前述三种细胞分别注射入裸鼠皮下，放线菌素D、TNF仅注射瘤体后，免疫组织化学法检测组织HBV／P22蛋白的表达，TUNEL法检测组织细胞凋亡率。结果实验组细胞培养上清液中HBeAg表达阳性，两对照组阴性；Westernblot及免疫细胞化学法检测均显示实验组细胞有HBV／P22蛋白表达，两对照组均为阴性；流式细胞仪和TUNEL结果均显示实验组细胞凋亡率明显低于两对照组（P〈0．05）。体内实验：实验组瘤体组织免疫组织化学检测结果显示HBV／P22蛋白表达阳性，TUNEL检测结果显示接种表达HBV／P22蛋白的HepG2细胞裸鼠瘤组织细胞凋亡率明显低于两对照组（P〈0．05）。结论HBV／P22蛋白在体内外均可抑制TNFα诱导的HepG2细胞凋亡。     

乙型肝炎病毒表面抗原基因启动子Ⅰ结合蛋白1反式调节基因的筛选

目的应用表达谱基因芯片技术，研究乙型肝炎病毒(HBV)表面抗原基因启动子DNA结合蛋白1(SBP1)反式调节基因，阐明SBP1蛋白可能的分子生物学功能。方法设计并合成SBP1基因序列特异性的引物，应用聚合酶链反应(PCR)技术扩增SBP1基因片段，以常规的分子生物学技术将获得的SBP1编码基因片段克隆到TA载体中进行核苷酸序列测定，构建真核表达载体pcDNA3．1(-)-SBP1。以脂质体转染肝母细胞瘤细胞系HepG2，提取mRNA，逆转录为cDNA，与转染空表达载体pcDNA3．1(-)的HepG2细胞进行cDNA芯片分析。结果构建的表达载体经过限制性内切酶分析和DNA序列测定，证实准确无误。提取高质量的mRNA，逆转录为cDNA，进行cDNA芯片分析。在1152个基因容量的表达谱芯片的筛选中，发现有12个基因表达水平显著上调，6个基因表达水平显著下调。结论应用基因表达谱芯片成功筛选了SBP1转染细胞后差异表达基因，为进一步阐明SBP1蛋白可能的生物学功能提供依据。     

Inhibition of HBV targeted ribonuclease enhanced by introduction of linker

AIM: To construct human eosinophil-derived neurotoxin(hEDN) and HBV core protein (HBVc) eukaryotic fusion expression vector with a linker (Gly(4)Ser) (3) between them to optimize the molecule folding, which will be used to inhibit HBV replication in vitro. METHODS: Previously constructed pcDNA3.1(-)/TR was used as a template. Linker sequence was synthesized and annealed to form dslinker, and cloned into pcDNA3.1(-)/TR to produce plasmid pcDNA3.1(-)/HBc-linker. Then the hEDN fragment was PCR amplified and inserted into pcDNA3.1(-)/HBc-linker to form pcDNA3.1(-)/TNL in which the effector molecule and the target molecule were separated by a linker sequence. pcDNA3.1(-)/TNL expression was identified by indirect immunofluorescence staining. Radioimmunoassay was used to analyse anti-HBV activity of pcDNA3.1(-)/TNL. Meanwhile, metabolism of cells was evaluated by MTT colorimetry. RESULTS: hEDN and HBVc eukaryotic fusion expression vector with a linker (Gly(4)Ser)(3) between them was successfully constructed. pcDNA3.1(-)/TNL was expressed in HepG2.2.15 cells efficiently. A significant decrease of HBsAg concentration from pcDNA3.1(-)/TNL transfectant was observed compared to pcDNA3.1(-)/TR (P=0.036, P<0.05). MTT assay suggested that there were no significant differences between groups (P=0.08, P>0.05). CONCLUSION: Linker introduction enhances the inhibitory effect of HBV targeted ribonuclease significantly.     

基因表达谱芯片筛选NS5ATP2剪切体NS5ATP2-512转染细胞差异表达基因

目的应用基因芯片技术检测丙型肝炎病毒(HCV)非结构蛋白5A(NS5A)反式激活基因NS5ATP2剪切体512的表达对肝母细胞瘤细胞HepG2基因表达谱的影响,进一步阐明NS5ATP2剪切体512蛋白可能的分子生物学功能。方法设计并合成NS5ATP2基因序列特异性的引物,应用聚合酶链反应(PCR)技术扩增NS5ATP2蛋白编码基因片段,以常规的分子生物学技术将获得的NS5ATP2编码基因片段克隆到TA载体中进行核苷酸序列的测定,在此过程中发现其剪切体NS5ATP2512并构建真核表达载体pcDNA3.1(-)NS5ATP2512。以脂质体转染肝母细胞瘤细胞系HepG2,提取mRNA,逆转录为cDNA,与转染空白表达载体pcDNA3.1(-)的HepG2细胞进行cDNA芯片分析。结果构建的表达载体经过限制性内切酶分析和DNA序列测定,证实准确无误。提取高质量的mRNA,逆转录为cDNA,进行DNA芯片技术分析。在1152个基因表达谱的筛选中,发现有14个基因表达水平显著上调,15个基因表达水平显著下调。结论应用基因表达谱芯片技术成功筛选了NS5ATP2剪切体NS5ATP2512转染细胞后差异表达基因,为进一步阐明NS5ATP2512蛋白可能的生物学功能提供依据。     

应用基因表达谱芯片技术筛选XTP6基因转染细胞差异表达基因

目的应用基因芯片技术,检测乙型肝炎病毒(HBV)X蛋白(HBxAg)反式激活基因XTP6的表达对肝母细胞瘤系HepG2基因表达谱的影响,进一步阐明XTP6蛋白可能的分子生物学功能.方法设计并合成XTP6基因序列特异性的引物,应用聚合酶链反应(PCR)技术扩增XTP6基因片段,以常规的分子生物学技术将获得的XTP6编码基因片段克隆到TA载体中进行核苷酸序列测定,构建真核表达载体pcDNA3.1(-)-XTP6.以脂质体转染肝母细胞瘤细胞系HepG2,提取mRNA,逆转录为cDNA,与转染空表达载体pcDNA3.1(-)的HepG2细胞进行cDNA芯片分析.结果构建的表达载体经过限制性内切酶分析的DNA序列测定,证实准确无误.提取高质量的mRNA,逆转录为cDNA,进行cDNA芯片分析.在1 152个基因表达谱的筛选中,发现21个基因表达水平显著上调,18个基因表达水平显著下调.结论应用基因表达谱芯片成功筛选了XTP6转染细胞后差异表达基因,为进一步阐明XTP6蛋白可能的生物学功能提供依据.     

表达HBeAg和HBsAg对HepG2细胞系细胞因子分泌及ACE活性的影响

目的:观察HBeAg和HBsAg对人肝癌细胞系 HepG2细胞中血管紧张素转化酶(ACE)分泌的影响．方法:利用本所既往构建的表达乙肝病毒 (HBV)表面抗原和E抗原的真核表达载体 pcDNA3．1(-)-HBsAg、pcDNA3．1(-)-HBeAg 及空载体pcDNA3．1(-)转染HepG2细胞,观察转染24,48 h后细胞上清中ACE活性和炎性细胞因子IL-6、IL-8的变化．培养细胞上清液内 ACE活性采用底物裂解法检测;IL-6、IL-8浓度采用流式细胞仪检测．结果:转染48 h的HepG2细胞裂解液中检测到了HBsAg和HBeAg的表达,但二者细胞上清中的ACE与转染空载体对照pcDNA3．1(-)细胞上清中的ACE无显著差异;同样,炎性因子 IL-6和IL-8也没有显著差异．结论:HBV HBsAg和HBeAg对ACE活性、 IL-6和IL-8不具有显著的上调或下调作用; HBsAg和HBeAg在肝纤维化发生过程中的作用机制尚待进一步阐明．