HepG2.2.15细胞中siRNA与拉米夫定抗乙型肝炎病毒复制作用的比较

目的：对HepG2.2.15细胞中siRNA与拉米夫定的抗乙型肝炎病毒（HBV）作用进行比较。方法：构建HBV siRNA的表达载体并转染入HepG2.2.15细胞。分别于48、72、96h收获细胞,与拉米夫定治疗组比较抗HBV作用。用ELISA方法检测HBsAg浓度;HBV DNA水平用实时定量PCR测定;用逆转录PCR检测HBV mRNA水平。结果：拉米夫定能明显降低HBV DNA水平,对HBsAg和mRNA抑制率较低;siRNA能全面降低HBsAg、HBV DNA和mRNA水平（P〈0.05）。结论：HepG2.2.15细胞中,siRNA与拉米夫定的抗病毒作用完全不同,siRNA抗HBV作用明显优于拉米夫定。     

以HepG2.2.15细胞为对象的RNAi实验研究

目的观察针对乙型肝炎病毒(HBV)X区设计的siRNA表达载体pGenesil-HBVX对HepG2.2.15细胞相应蛋白表达的影响。方法筛选出最佳转染效率的剂量配比,将阳离子脂质体METAFECTENE与pGen-esil-HBVX的复合物转染HepG2.2.15细胞,于转染后24、48、72h采用时间分辨荧光免疫测定技术(TRFIA)检测上清液中乙型肝炎表面抗原(HBsAg)和乙型肝炎e抗原(HBeAg)。结果具有最佳转染效率而且不显著影响细胞活性的剂量配比为8μl∶2.5μg。pGenesil-HBVX转染HepG2.2.15细胞后24h,细胞上清液中HBsAg、HBeAg含量尚无显著变化(P>0.05);48h后HBsAg、HBeAg表达均有明显下降(P<0.01),并且持续至72h。结论pGenesil-HBVX可以在获得一定转染效率的基础上有效抑制HepG2.2.15细胞中HBV的表达。     

乳糖化赖氨酸与反义x基因质粒复合物体外对HBV表达的抑制

目的研制一种能把基因药物定向导入肝脏组织的小分子肝靶向药物载体。方法用还原氨化法合成乳糖化赖氨酸(Lac-Lys)共价结合物作为肝靶向药物载体。将含HBV反义x基因的质粒在一定条件下与Lac-Lys制成复合物后转染HepG2.2.15。用酶联免疫吸附试验(ELISA)检测细胞培养上清中的HBsAg和HBeAg。结果经红外光谱定性,成功制备出了Lac-Lys的共价结合物;此共价结合物能与质粒DNA形成复合物,经细胞培养观察可将质粒DNA载入细胞;抗原检测结果显示,含有反义xDNA的质粒在细胞内能有效抑制病毒的复制。xDNA与Lac-Lys的最佳比例是1∶5,最大抑制率是73.2%。结论合成的Lac-Lys可将反义x基因质粒定向载入细胞内,并有效抑制HBsAg和HBeAg的表达。     

Molecular design and delivery of siRNA

Double stranded short interfering RNAs (siRNAs) mediate gene silencing in a sequence specific manner. By virtue of their specific gene silencing activity and owing to the recent discoveries on their plasmid and virus driven expression, siRNAs are being widely adopted in research and therapeutics. Efforts were made to optimize the siRNA expression system for the application in therapy. One major obstacle in developing RNA interference (RNAi) therapy is the delivery of siRNAs to the target cells. Combination of novel molecular targeting technologies, such as recombinant protein technology and ribosome display technology, will enable to deliver gene silencing agents to target cells specifically and efficiently.     

Molecular design and delivery of siRNA

Double stranded short interfering RNAs (siRNAs) mediate gene silencing in a sequence specific manner. By virtue of their specific gene silencing activity and owing to the recent discoveries on their plasmid and virus driven expression, siRNAs are being widely adopted in research and therapeutics. Efforts were made to optimize the siRNA expression system for the application in therapy. One major obstacle in developing RNA interference (RNAi) therapy is the delivery of siRNAs to the target cells. Combination of novel molecular targeting technologies, such as recombinant protein technology and ribosome display technology, will enable to deliver gene silencing agents to target cells specifically and efficiently.     

甘草甜素联合单磷酸阿糖腺苷对HepG 2.2.15细胞及其HBsAg表达的影响

目的探讨甘草甜素联合单磷酸阿糖腺苷对HepG2.2.15细胞株存活率及其HB-sAg表达的影响,评价甘草甜素(GL)与单磷酸阿糖腺苷(Ara-AMP)合用体外抗HBV效果。方法利用乙型肝炎病毒基因转染的人肝癌细胞系HepG2.2.15细胞检测细胞培养上清液中的乙肝病毒表面抗原(HBsAg),作为这两种药物联合在体外抗HBV效果的观察指标(以抑制率来表示)。用MTT法检测HepG2.2.15细胞的存活率。结果(1)各药对HepG2.2.15细胞分泌HBsAg的抑制率呈药物浓度和时间依赖性;联合组与两个单药组分别比较,抑制HBsAg差异有显著意义(P<0.05或P<0.01)。(2)随着浓度的增加,各药对细胞的存活率均显示抑制作用,尤其联合组高于其它两个单药组。结论GL联合Ara-AMP在体外HepG2.2.15细胞中具有协同抗HBV的作用。     

靶向Ccnd1基因的siRNA对大鼠晶状体上皮细胞增殖的抑制作用

目的探讨靶向Ccnd1基因小干扰RNA(si RNA)对培养的大鼠晶状体上皮细胞(LECs)Ccnd1 mRNA和周期蛋白D1表达及细胞增殖的抑制作用。方法合成3条靶向大鼠Ccnd1基因的siRNA(si-Ccnd1-1,si-Ccnd1-2,si-Ccnd1-3),LipofectamineTM2000转染体外培养的大鼠LECs,应用RT-PCR和Western blot方法分别检测Ccnd1 mRNA和周期蛋白D1表达变化,筛选最有效si RNA序列。MTT法连续测定转染最有效si RNA序列后24、48、72、96和120 h细胞增殖情况。结果与对照组相比,3条si RNA均能不同程度地抑制Ccnd1 mRNA和周期蛋白D1的表达,以si-Ccnd1-3最为有效(P<0.05)。与对照组比较,si-Ccnd1-3转染后24 h开始明显发挥对细胞增殖的抑制作用,并持续至120 h以后(P<0.05)。结论靶向Ccnd1的si RNA能有效降低大鼠LECsCcnd1 mRNA和周期蛋白D1蛋白表达,抑制LECs增殖。     

亚硒酸钠对乙型肝炎病毒的体外抑制作用

目的探讨亚硒酸钠在体外对乙型肝炎病毒(HBV)蛋白合成、DNA复制的影响及机制。方法将不同浓度的亚硒酸钠作用于HepG2.2.15细胞系,通过检测细胞培养上清液中乙型肝炎表面抗原(HBsAg)、乙型肝炎e抗原(HBeAg)水平、乙型肝炎核心抗原(HBcAg)和HBV DNA水平来评价HBV复制情况;免疫组织化学检测HepG2.2.15细胞p53蛋白的表达情况。结果亚硒酸钠对HBV复制具有抑制作用,随着亚硒酸钠浓度的升高,对HBsAg和HBeAg的抑制率逐渐上升,但对HBsAg的抑制作用要小于HBeAg。细胞内HBV DNA复制水平也逐渐下降(P<0.01)。p53蛋白的分布也发生了改变。结论亚硒酸钠对HepG2.2.15细胞HBsAg、HBeAg和HBcAg表达、HBV DNA复制均有抑制作用,作用机制与其干扰p53蛋白的表达有关。     

SiRNA沉默 HBx对 HepG2.2.15细胞人端粒酶逆转录酶基因表达的影响

目的探讨应用RNA干扰技术沉默HBx对HepG2．2．15细胞中hTERT基因表达的影响。方法（1）将pSIHBV／X质粒转染HepG2．2．15细胞,RT-PCR法评估沉默效率。（2）MTT法检测转染24h、48h、72h后细胞增殖情况。（3）Real-timePCR和Westernblot检测hTERT表达情况。结果测序结果显示pSIHBV／X质粒构建正确;RT-PCR检测HBVX基因的沉默效率为53．6％;MTT检测结果显示转染24h、48h、72h后H印G2．2．15细胞的增殖受抑制,与对照组比较差异有统计学意义（P〈0．05）;Real-timePCR和Western blot检测结果均显示,转染pSIHBV／X质粒后HepG2．2．15细胞中hTERT基因的mRNA水平和蛋白水平表达分别有不同程度的下调,与对照组比较差异有统计学意义（P〈0．05）。结论siRNA沉默HBx基因可抑制HepG2．2．15细胞中癌症标志基因hTERT的表达。     

短发夹RNA介导的RNA干扰技术在HepG2.2.15细胞中的应用研究

目的探讨shRNA真核表达质粒介导的RNA干扰技术对HepG2.2.15肝癌细胞中乙型肝炎病毒(HBV)复制及甲胎蛋白(AFP)表达的特异性抑制作用。方法采用时间分辨荧光免疫测定技术(TRFIA)检测乙型肝炎表面抗原(HBsAg)和乙型肝炎e抗原(HBeAg)含量;采用微粒子化学发光免疫分析法(MLFA)检测甲胎蛋白含量。采用免疫细胞化学技术检测细胞中HBsAg及甲胎蛋白的表达。结果 pGenesil-shHBV X对HepG2.2.15细胞HBsAg、HBeAg的特异性抑制作用随时间而递增(P<0.05),pGenesil-shAFP对HepG2.2.15细胞甲胎蛋白的特异性抑制作用同样随时间而递增(P<0.05),且二者之间无交叉抑制作用。结论 shRNA真核表达质粒pGene-sil-shHBV X介导的RNA干扰作用可特异性抑制HepG2.2.15细胞HBV的复制表达,pGenesil-shAFP可特异性抑制HepG2.2.15细胞甲胎蛋白的表达,为RNA干扰技术抗病毒及抑制肿瘤的进一步研究奠定基础。     

山芝麻水提液对HepG2.2.15细胞HBsAg和HBeAg的抑制作用

目的观察山芝麻水提液（water extracts from Helicteres angustifolia,WHA）的体外抗乙型肝炎病毒的作用。方法采用HepG2．2．15细胞模型进行体外培养,给予不同浓度山芝麻,以拉米夫定作阳性对照,作用72h后检测上清液中HBsAg、HBeAg的分泌,观察药物对HepG2．2．15细胞分泌HBV病毒抗原的影响,同时以MTT法检测药物在体外对HepG2．2．15细胞的生长抑制作用,从而评价药物的抗HBV作用。结果山芝麻水提液对HepG2．2．15细胞的半数细胞毒浓度（TC50）为482．1mg·L-1;对HepG2．2．15细胞分泌HBsAg的半数抑制浓度（IC50）为7．3mg。L-1,其治疗指数（TI）为66;对HBeAg,其Ic,n为14．6mg·L-1,TI为33。结论山芝麻水提液在体外有显著的抗HBV的作用,且毒性较低。Tel：（0771）5358342E—mail：gxLx60@163．COll]     

Three new shRNA expression vectors targeting the CYP3A4 coding sequence to inhibit its expression

RNA interference (RNAi) is useful for selective gene silencing. Cytochrome P450 3A4 (CYP3A4), which metabolizes approximately 50% of drugs in clinical use, plays an important role in drug metabolism. In this study, we aimed to develop a short hairpin RNA (shRNA) to modulate CYP3A4 expression. Three new shRNAs (S1, S2 and S3) were designed to target the coding sequence (CDS) of CYP3A4, cloned into a shRNA expression vector, and tested in different cells. The mixture of three shRNAs produced optimal reduction (55%) in CYP3A4 CDS-luciferase activity in both CHL and HEK293 cells. Endogenous CYP3A4 expression in HepG2 cells was decreased about 50% at both mRNA and protein level after transfection of the mixture of three shRNAs. In contrast, CYP3A5 gene expression was not altered by the shRNAs, supporting the selectivity of CYP3A4 shRNAs. In addition, HepG2 cells transfected with CYP3A4 shRNAs were less sensitive to Ginkgolic acids, whose toxic metabolites are produced by CYP3A4. These results demonstrate that vector-based shRNAs could modulate CYP3A4 expression in cells through their actions on CYP3A4 CDS, and CYP3A4 shRNAs may be utilized to define the role of CYP3A4 in drug metabolism and toxicity.KEY WORDS: RNAi, Cytochrome P450, CYP3A4, shRNA, Chemosensitivity     

更昔洛韦的体外抗乙肝病毒活性

目的：评价更昔洛韦（ＧＣＶ）体外抗乙肝病毒（ＨＢＶ）活性。方法：应用ＨＢＶ ＤＮＡ转染的人肝癌细胞株（ＨｅｐＧ２２．２．１５）,进行ＧＣＶ体外抗ＨＢＶ活性的研究。结果：ＧＣＶ能明显减少ＨｅｐＧ２２．２．１５细胞培养上清液中ＨＢｓＡｇ、ＨＢＶ ＤＮＡ的产生。Ｓｏｕｔｈｅｒｎ印迹法分析：ＧＣＶ能显著抑制ＨＢＶ复制中间体（包括共价闭合环状态ＤＮＡ）,而同时该药物对细胞形态、细胞计数及总细胞ＤＮＡ无明显的     

Protocatechuic aldehyde inhibits hepatitis B virus replication both in vitro and in vivo

Natural compounds provide a large reservoir of potentially active anti-hepatitis B virus (HBV) agents. We examined the direct effects of protocatechuic aldehyde (PA; derived from the Chinese herb, Salvia miltiorrhiza) on HBV replication in HepG2 2.2.15 cell line and duck hepatitis B virus (DHBV) replication in ducklings in vivo. The extracellular HBV DNA, hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) concentrations in cell culture medium were determined by quantitative real-time PCR and ELISA, respectively. DHBV in duck serum was analyzed by dot blot. PA appeared to downregulate the secretion of HBsAg and HBeAg as well as the release of HBV DNA from HepG2 2.2.15 in a dose- and time-dependent manner at concentrations between 24 and 48 microg/mL. PA (25, 50, or 100 mg/kg, intraperitoneally, twice daily) also reduced viremia in DHBV-infected ducks. We provide the first evidence that PA, a novel anti-HBV substance derived from traditional Chinese herb S. miltiorrhiza, can efficiently inhibits HBV replication in HepG2 2.2.15 cell line in vitro and inhibit DHBV replication in ducks in vivo. PA therefore warrants further investigation as a potential therapeutic agent for HBV infections.     

Evaluation of hepatitis B virus replication and proteomic analysis of HepG2.2.15 cell line after cyclosporine A treatment

Aim： The effect of cyclosporine A （CsA） on hepatitis B virus （HBV） replication was investigated, and proteomics expression differentiation after CsA treatment was studied in order to provide clues to explore the effect of CsA on HBV replication. Methods： Methyl thiazolyl tetrazolium （MTT） assay was used to evaluate the cytotoxicity of CsA. The HBV replication level in the HBV genomic DNA transfected HepG2.2.15 cell line was determined by an ELISA analysis of hepatitis B surface antigens （HBsAg） and Hepatitis B e antigens （HBeAg） in culture supernatant, while the intracellular HBV DNA replication level was analyzed by slot blot hybridization. Two-dimensional electrophoresis was used to investigate the alteration of protein expression in HepG2.2.15 after CsA treatment in vitro. The differentially-expressed proteins were identified by Matrix-assisted laser desorption/ionization-time of flight mass spectrometry combined with an online database search. Results： CsA was able to inhibit the expression of HBsAg, HBeAg, and HBV DNA replication in vitro in a dose-dependent manner. A proteomics analysis indicated that the expression of 17 proteins changed significantly in the CsA treatment group compared to the control group. Eleven of the 17 proteins were identified, including the overexpression of eukaryotic translation initiation factors （eIF） 3k, otubain 1, 14.3.3 protein, eIF2-1α, eIF5A, and the tyrosine 3/tryptophan 5-mono-oxygenase activation protein in CsA-treated HepG2.2.15 cells. The downregulation of the ferritin light subunit, erythrocyte cytosolic protein of 51 kDa （ECP-51）, stathmin 1/oncoprotein, adenine phosphoribosyl-transferase, and the position of a tumor protein, translationally- controlled 1, was shifted, suggesting it had undergone posttranslational modifications. Conclusion： Our study identified the inhibitory effect of CsA on HBV replication, and found that a group of proteins may be responsible for this inhibitory effect.     

应用 RNA 干扰技术下调 ANXA3表达的 MDA-MB-231乳腺癌细胞株的建立

目的：建立针对膜联蛋白A3（Annexin A3, ANXA3）的shRNA稳定转染的乳腺癌细胞株MDA-MB-231,为后续进一步研究奠定基础。方法荧光定量RT-PCR及western blot方法检测两种乳腺癌细胞株（ MDA-MB-231及MCF-7）中ANXA3 mRNA及蛋白表达水平。构建沉默ANXA3基因的shRNA质粒3个（ ANXA3-sh1-3）及阴性对照质粒,脂质体法转染人乳腺癌细胞MDA-MB-231;选出ANXA3沉默效果最好的干扰质粒做后续实验。嘌呤霉素筛选出稳定转染细胞;Western blot 方法检测转染后细胞中ANXA3蛋白表达水平。结果 MDA-MB-231细胞中 ANXA3 mRNA及蛋白表达水平显著高于MCF-7中的表达（ P ＜0 k．05）;成功构建3个针对ANXA3基因的shRNA质粒;转染ANXA3-sh1～3及阴性对照质粒后MDA-MB-231细胞中ANXA3 mRNA表达水平分别为（0．0196±0．0002）、（0．0085±0．0002）、（0．0220±0．0035）、（0．0661±0．0057）,未转染的MDA-MB-231细胞中ANXA3 mRNA表达水平为（0．0692±0．0050）,脂质体组MDA-MB-231细胞中ANXA3 mRNA表达水平为（0．0652±0．0118）,ANXA3-sh2对MDA-MB-231细胞中ANXA3 mRNA沉默效率最高,达87．72％,因此后续实验选择ANXA3-sh2;嘌呤霉素筛选出稳定转染细胞分别命名为MDA-MB-231-Sh及MDA-MB-231-NC细胞。 Western blot检测结果显示,MDA-MB-231-Sh细胞中ANXA3蛋白显著低于MDA-MB-231及MDA-MB-231-NC细胞中的表达（ P ＜0．01）。结论 MDA-MB-231细胞较MCF-7细胞高表达ANXA3,成功的建立了稳定下调ANXA3表达的乳腺癌细胞株MDA-MB-231,为后续进一步研究ANXA3的表达及特性打下基础。     

核酸类似物PNA在HepG2．2．15细胞中对乙型肝炎病毒复制的抑制作用

目的评价核酸类似物PNA在HepG2．2．15细胞中对乙型肝炎病毒复制的抑制作用。方法以HepG2．2．15细胞作为细胞模型,拉米夫定作为阳性对照药物。HepG2．2．15细胞于药物处理14d后,收集上清液及细胞。采用ELISA检测上清液中HBsAg和HBeAg含量,HBV荧光定量检测上清液和细胞内HBV DNA水平,CCK-8试剂盒检测药物对细胞的毒性作用。结果核酸类似物PNA与拉米夫定在浓度1．6-1000μmol·L^-1内对HepG2．2．15细胞的毒性均较小。与药物未处理组比较,PNA和拉米夫定均可有效抑制细胞上清HBV DNA,半数抑制浓度（IC50）分别为0．05-0．10μmol·L^-1和0．09-0．18μmol·L^-1,两者之间差异无统计学意义。结论在体外细胞实验中,PNA对细胞的毒性小,与拉米夫定的安全指数相当;PNA对乙型肝炎病毒复制有较强的抑制作用,且具有一定时效量效关系,与拉米夫定的抑制强度相当。