变异IκBα抑制肝癌细胞株SMMC-7721中NF-κB活性及细胞生长

目的 了解变异IκBα(mIκBα)转染到肝癌细胞株SMMC-7721细胞中是否抑制NF-κB向核内转位活性及细胞的生长。 方法 电泳迁移率分析检测32p标记的寡核苷酸探针与NF-κB结合情况,westernblot检测核内NF-κB表达情况,细胞生长曲线分析和细胞增殖实验分析肝癌细胞生长情况。 结果 转染mIκBα质粒肝癌细胞在0、24、48、96 h未见核内蛋白与κ B探针结合转染,而转染对照PcDNA3质粒肝癌细胞始终可见核内蛋白与κB探针强结合;Western blot结果也显示0、24、48、96 h未见核内NF-κB表达,而对照PcDNA3质粒核内NF-κB高水平表达。细胞增殖实验分析发现转染mIκBα质粒肝癌细胞生长受到抑制,而转染对照PcDNA3质粒肝癌细胞生长未受影响,第2天开始转染mIκBα质粒肝癌细胞与其它两种细胞比较差异有非常显著性,增殖效率值分别是5 092.63±541.41、7 851.87±72.76、8 240.88±603.26,t值分别是14.29、10.99,P<0.01。 结论 转染mIκBα质粒肝癌细胞可以持续表达mIκBα,抑制NF-κB向核内转位,从而抑制肿瘤细胞的生长。     

亚砷酸联合重组腺病毒Ad-IκBαM对肝细胞癌的治疗作用

目的:观察感染Ad-IκBαM对NF-κB激活的抑制及对亚砷酸诱导肝癌细胞凋亡的增强效应,探讨亚砷酸对肝癌细胞的治疗作用.方法:选择人肝癌细胞系BEL-7402和SMMC-7721,以不同浓度亚砷酸处理.制备重组腺病毒Ad-IκBαM,用来转染经和未经亚砷酸处理的肝癌细胞.MTT和TUNEL方法观察各组细胞生长及凋亡情况;应用EMSA及Western blot研究肝癌细胞核内NF-κB的激活情况和感染Ad-IκBαM对NF-κB激活的抑制效果.结果:MTT结果表明各浓度亚砷酸对肝癌细胞的作用较正常肝细胞显著(P＜0.01);Western blot和EMSA结果提示亚砷酸可明显抑制肝癌细胞生长,使细胞内NF-κB系统活化;感染重组腺病毒Ad-IκBαM的肝癌细胞后,亚砷酸引起的NF-κB的激活受到明显抑制.亚砷酸联合Ad-IκBαM或Ad-IκBα作用于肝癌细胞SMMC-7721的凋亡指数分别为66.47%和36.67%;亚砷酸联合Ad-IκBαM或Ad-IκBα作用于肝癌细胞Bel-7402的凋亡指数分别为74.5%和32.37%.结论:亚砷酸对肝癌细胞有明显的杀灭作用,同时激活了肝癌细胞内的NF-κB;应用重组腺病毒Ad-IκBαM可以有效抑制NF-κB的激活,并可明显增强亚砷酸对肝癌细胞的作用.     

约氏疟原虫环子孢子蛋白对TNF-α刺激人肝癌细胞株核转录因子活化的抑制作用

目的 观察约氏疟原虫环子孢子蛋白（CSP）对肿瘤坏死因子α（TNF-α）刺激人肝癌细胞株HepG2核转录因子-κB （NF-κB）活化的影响。 方法 以约氏疟原虫BY265株子孢子总RNA为模板,用RT-PCR扩增CSP基因的编码区序列并克隆至pFLAG-CMV8载体,构建重组质粒pFLAG-CMV8-CSP。以兔抗CSP多克隆抗体间接免疫荧光法观察pFLAG-CMV8-CSP能否在HepG2细胞中正确表达,及其在细胞中的分布。实验分为3组,A组（阴性对照组）为转染质粒pFLAG-CMV8的HepG2细胞,B组以100 ng/ml TNF-α刺激转染质粒pFLAG-CMV8的HepG2细胞,C组以100 ng/ml TNF-α刺激转染质粒pFLAG-CMV8-CSP的HepG2细胞。采用双荧光素酶试验和凝胶迁移试验（EMSA）检测NF-κB 的核转位及其活化,观察pFLAG-CMV8?鄄CSP对于TNF-α刺激HepG2细胞活化NF-κB是否具有抑制作用。 结果 　质粒pFLAG-CMV8-CSP主要在HepG2细胞胞浆中表达。 检测HepG2细胞浆中NF-κB活性,C组萤火虫荧光素酶活性与海肾荧光素酶活性比值为0.228±0.029,明显低于B组（0.571±0.030）和A组（0.438±0.085）（P＜0.05）。EMSA结果显示,C组的条带明显弱于B组。　结论 位于细胞浆中的疟原虫CSP蛋白通过抑制NF-κB核转位, 从而抑制TNF-α刺激HepG2细胞活化NF-κB。     

亚砷酸联合重组腺病毒Ad-IκBaM对肝细胞癌的治疗作用

目的:观察感染Ad-IκBaM对NF-κB激活的抑制及对亚砷酸诱导肝癌细胞凋亡的增强效应,探讨亚砷酸对肝癌细胞的治疗作用.方法:选择人肝癌细胞系BEL-7402和SMMC-7721,以不同浓度亚砷酸处理.制备重组腺病毒Ad-IκBaM,用来转染经和未经亚砷酸处理的肝癌细胞.MTT和TUNEL方法观察各组细胞生长及凋亡情况;应用EMSA及Westernblot研究肝癌细胞核内NF-κB的激活情况和感染Ad-IκBaM对NF-κB激活的抑制效果.结果:MTT结果表明各浓度亚砷酸对肝癌细胞的作用较正常肝细胞显著(P<0.01);Westernblot和EMSA结果提示亚砷酸可明显抑制肝癌细胞生长,使细胞内NF-κB系统活化;感染重组腺病毒Ad-IκBaM的肝癌细胞后,亚砷酸引起的NF-κB的激活受到明显抑制.亚砷酸联合Ad-IκBaM或Ad-IκBa作用于肝癌细胞SMMC-7721的凋亡指数分别为66.47%和36.67%;亚砷酸联合Ad-IκBaM或Ad-IκBa作用于肝癌细胞Bel-7402的凋亡指数分别为74.5%和32.37%.结论:亚砷酸对肝癌细胞有明显的杀灭作用,同时激活了肝癌细胞内的NF-κB;应用重组腺病毒Ad-IκBaM可以有效抑制NF-κB的激活,并可明显增强亚砷酸对肝癌细胞的作用.     

神经节苷脂GD3增加阿霉素的抗肝癌敏感性

阿霉素能诱导肝癌细胞发生凋亡，亦能激活肝癌细胞的核因子kappa B（NF—κB）活性，从而抑制肝癌细胞的凋亡，故而影响了其抗癌疗效。为此，在我们过去的研究中通过转染变异IKBQ到肝癌细胞内抑制阿霉素对NF—κB的激活，进而抑制肝癌细胞的生长。神经节苷脂GD3是一类含有唾液酸的糖神经鞘酯，可以抑制NF—κB从细胞质内向细胞核内移位，抑制了NF—κB的活性，达到诱导细胞凋亡的目的，而且它是一类脂溶性药物，容易透过细胞膜而发生作用。本实验旨在研究神经节苷脂GD3能否抑制肝癌细胞中被阿霉素激活的NF-κB活性、促进肝癌细胞的凋亡和抑制细胞的生长，从而增加阿霉素的抗肝癌敏感性。     

乙型肝炎病毒P22e蛋白经核因子-κB抑制HepG2细胞凋亡

目的 探讨核因子-κB(NF-κB)在乙型肝炎病毒P22e蛋白抑制HepG2细胞凋亡中的作用.方法 用含HBV P22e基因的重组pEGFP-C2HBVP22e质粒的肝癌细胞HepG2,以放线菌素-D(Act-D)、肿瘤坏死因子(TNF)α诱导该细胞凋亡,采用激光共聚焦显微镜、核蛋白电泳迁移率等技术,观察在HBV P22e抑制TNFα诱导HepG2EGFP-C2HBVP22e细胞的凋亡过程中NF-κB的核转移、活化等情况.用NF-κB抑制剂ALLN抑制其信号通路,检测以Act-D、TNFα诱导的HepG2、HepG2EGFP-C2HBVP22e细胞凋亡率的变化.对实验结果的数据分析用秩和检验和t检验. 结果激光共聚焦显微镜及电泳迁移率实验观察到HepG2EGFP-C2HBVP22e细胞在发生凋亡前后,有明显的NF-κB向核内迁移活化现象.NF-κB抑制剂ALLN可使以Act-D、TNF α诱导HepG2EGFP-C2HBVP22e细胞的凋亡率明显升高(6.19%±1.58%与39.99%±7.620/0,t=7.515,P<0.01).结论 在HBV P22e蛋白抑制肝癌细胞凋亡过程中,NF-κB信号途径起着重要作用.     

N-乙酰半胱氨酸对肝星状细胞核因子κB的影响

目的　阐明N乙酰半胱氨酸(NAC)对肝星状细胞(HSC)核因子κB(NF-κB)结合活性和环氧合酶 2(COX-2)表达的影响机制。方法　体外培养大鼠 HSC-T6 细胞株,MTT法检测 NAC对HSC增殖的抑制作用。分别予NAC(1 mmol/L)处理 1 h;NAC和肿瘤坏死因子(TNF)α联合干预(先予 NAC处理1 h,再予TNFα干预1 h);TNFα100 ng/ml处理1 h。凝胶电泳移动抑制实验检测NF κB的结合活性。免疫蛋白质印迹检测相应的胞质内NF-κB抑制蛋白(IκBα)表达。免疫组化观察 HSC-T6NF-κB表达的核转移。激光共聚焦检测NAC对 HSC-T6 中 COX-2 表达的影响。结果　NAC对 HSC具有明显的抑制作用。TNFα可诱导 NF-κB结合活性,而 NAC可显著抑制 TNFα诱导的 NF-κB结合活性。TNFα处理后 IκBα表达减弱,NAC处理后 IκBα表达增强。TNFα刺激 1 h后,NF κB表达从细胞质转移至细胞核内。NAC预处理后再予TNFα刺激,NF κB表达主要位于细胞质,很少发生核转移。HSC T6经TNFα处理后细胞内COX 2表达明显高于NAC和TNFα联合处理组以及正常对照组(P<0.05);NAC和TNFα联合处理组与正常对照组差异无统计学意义(P> 0. 05)。结论　NAC可抑制HSC增殖,抑制HSC-NF κB结合活性和COX-2表达。     

氧化苦参碱对胰腺星状细胞中脂多糖诱导的NF-κB表达的影响

目的:观察氧化苦参碱(oxymatrine,OM)对脂多糖(lipopolysaccharide,LPS)诱导胰腺星状细胞(LTC-14细胞株)中核因子-κB(nuclear factorκB,NF-κB)表达和核易位的影响.方法:体外培养LTC-14细胞,分别用相应浓度的LPS和/或OM刺激后,检测NF-κB的表达.用MTT法检测细胞增殖活性,免疫细胞化学技术检测LTC-14细胞胞浆和胞核内NF-κB的表达,实时荧光定量PCR检测细胞内NF-κB mRNA的表达,Western blot法检测NF-κB蛋白含量.结果:OM呈时间-剂量依赖性地抑制LTC-14细胞增殖,LPS(10μg/mL)刺激LTC-14细胞引起NF-κB mRNA及其蛋白表达量增高,NF-κB核内易位量明显增加,OM干预后可以下调NF-κB mRNA和蛋白表达,抑制NF-κB核内易位.结论:对LPS诱导的LTC-14细胞NF-κB mRNA、蛋白表达及NF-κB向核内易位抑制作用可能是OM治疗胰腺纤维化的机制之一.     

分泌性内皮抑制素真核质粒治疗小鼠肝癌的初步研究

目的 构建并表达分泌性内皮抑制素真核表达质粒,以此对肝癌进行基因治疗。方法 人工合成Ig κ信号肽序列,和内皮抑制素编码序列一起克隆入pcDNA3.1质粒。重组质粒转染上清液作用于ECV304内皮细胞,MTT法检测内皮细胞的增殖。局部注射重组质粒治疗接种在小鼠腿部肌肉内的H_(22)肝癌瘤株,疗程结束后解剖称取瘤重。结果 构建的内皮抑制素真核表达质粒转染上清液可以抑制内皮细胞的增殖抑制率为29.2％。经质粒裸DNA注射治疗后,治疗组瘤重[(1.34±0.96)g]比空载体组[(2.70±0.82)g]和生理盐水组[(3.73±1.41)g]明显减小(P<0.05)。结论 分泌性内皮抑制素真核表达质粒用于H_(22)肝癌的基因治疗有一定效果。     

骨桥蛋白促进人肝癌细胞株SMMC-7721恶性表型的实验研究

目的研究骨桥蛋白(OPN)对低侵袭性人肝癌细胞株SMMC-7721恶性表型的影响。方法pcDNA 3,1(-)/OPN重组质粒转染SMMC-7721细胞,以空质粒转染作对照,用RT- PCR反应、Western blot检测OPN表达水平,用ELISA检测细胞培养上清液OPN、MMP-2、-9、尿激酶纤溶酶原活化因子(uPA)水平,并用体外功能试验观察转染前后恶性表型的变化。结果重组质粒转染SMMC-7721后OPN表达明显升高,细胞培养上清液OPN为(3.02±0.12)ng/ml,对照组为(1.43±0.07)ng/ml,MMP-2重组质粒转染组为(43.04±3.06)ng/ml,对照组为(22.15±4.34)ng/ml、uPA重组质粒转染组水平明显高于空质粒转染组,分别为(4.78±0.70)ng/ml和(1.61±0.34)ng/ml,两组差异均有统计学意义,t值分别为19.89、6.81和7.03,P值均<0.01。MMP-9水平分别为(7.82±2.25)ng/ml和(7.70±1.92)ng/ml,两组差异无统计学意义。体外功能试验提示SMMC-7721转染OPN重组质粒后细胞黏附、运动和侵袭能力明显增强,细胞黏附率为75.33%±10.59%,对照组为57.34%±2.52%,t=2.86,P<0.05。运动试验透膜细胞数分别为(14.3±2.5)个和(6.3±1.5)个,t=4.70,P<0.05。侵袭试验透膜细胞数分别为(8.2±1.5)个和(4.1±1.3)个,t=4.11,P<0.05。而细胞增殖能力无明显改变。结论OPN可能是通过增加MMP-2、uPA分泌促进人肝癌细胞株SMMC-7721的恶性表型。     

Expression of angiostatin cDNA in human hepatocellular carcinoma cell line SMMC-7721 and its effect on implanted carcinoma in nude mice

AIM: To transfect murine angiostatin cDNA into human hepatocellular carcinoma cell line SMMC-7721 and to investigate its effects on implanted carcinoma in nude mice. METHODS: A eukaryotic expression vector of pcDNA3.1-mAST containing murine angiostatin was constructed. Then pcDNA3.1-mAST plasmid was transfected into cell line SMMC-7721 by Lipofectamine. The resistant clone was screened by G418 filtration and identified by RT-PCR and Western blotting. Nude mice were divided into three groups of 10 each. Mice in blank control group were only injected with SMMC-7721 cells. Mice in vector control group were injected with SMMC-7721 cells transfected with pcDNA3.1 (+) vector, whereas mice in angiostatin group were injected with SMMC-7721 cells transfected with pcDNA3.1-mAST plasmid. Volume, mass and microvessel density (MVD) of the tumors in different groups were measured and compared. RESULTS: Murine angiostatin cDNA was successfully cloned into the eukaryotic expression vector pcDNA3.1 (+). pcDNA3.1-mAST was successfully transfected into SMMC-7721 cell line and showed stable expression in this cell line. No significant difference was observed in the growth speed of SMMC-7721 cells between groups transfected with and without angiostatin cDNA. Tumor volume, mass and MVD in the angiostatin group were significantly lower than those in the blank control group and vector control group (P<0.01). The inhibitory rate of tumor reached 78.6%. Mass and MVD of the tumors only accounted for 34.6% and 48.9% respectively of those in the blank control group. CONCLUSION: Angiostatin cDNA could be stably expressed in human hepatocellular carcinoma cell line SMMC-7721 without obvious inhibitory effects on the growth of SMMC-7721 cells. When implanted into nude mice, SMMC-7721 cells transfected with angiostatin cDNA show a decreased tumorigenic capability. It suggests that angiostatin can inhibit tumor growth through its inhibition on angiogenesis in tumors.     

缺氧环境中黏着斑激酶对SMMC-7721细胞侵袭能力的影响

目的 研究缺氧对人肝癌细胞SMMC-7721黏着斑激酶(FAK)表达的影响以及FAK表达对SMMC-7721细胞侵袭能力的影响.方法 通过1%体积分数O2的低氧培养建立人肝癌细胞SMMC-7721物理缺氧模型,Western blot检测FAK的表达.构建针对FAK mRNA的干扰质粒pshRNA-FAK及阴性对照质粒pGensil-2,并将其转染至SMMC-7721细胞,G418筛选稳定转染细胞株.Western blot检测FAK蛋白表达的变化,细胞迁移和侵袭实验检测缺氧条件下细胞迁移和侵袭能力的改变.在正常条件下将FAK真核表达质粒pcDNA3-FAK转染至SMMC-7721细胞,观测其侵袭能力的改变.根据数所资料的不同分别采用t检验、单因素方差分析,LSD法及Dunnett法进行统计学处理. 结果低氧培养的SMMC-7721细胞FAK蛋白表达水平逐渐升高,24 h后较0 h时明显升高(P<0.01).SMMC-7721细胞稳定转染pshRNA-FAK后,FAK蛋白表达显著下降,抑制率达74.6%±5.1%,在正常及缺氧条件下都对FAK表达有显著抑制作用.细胞迁移实验结果显示,缺氧显著促进SMMC-7721细胞迁移能力(t=18.66,P<0.01),侵袭实验结果与迁移实验结果一致.转染pshRNA-FAK对促进SMMC-7721细胞在缺氧环境中的迁移能力有显著抑制作用,透膜细胞数(353±36)个较对照组(392±31)个明显降低(F=173.983,P<0.05);细胞侵袭实验显示,转染pshRNA-FAK对促进SMMC-7721细胞侵袭能力有显著抑制作用,透膜细胞数(160±12)个较对照组(194±13)个明显降低(F=59.674,P<0.05).同时转染真核表达质粒pcDNA3-FAK显著促进SMMC-7721细胞侵袭能力.结论 缺氧促进SMMC-7721细胞侵袭可能与FAK表达水平升高相关,FAK表达的上调可能是缺氧促进肝癌细胞侵袭转移的机制之一.     

低表达Smad4对肝癌细胞SMMC-7721上皮间质转化的影响

目的:探讨低表达Smad4调控上皮间质转化影响肝癌细胞(hepatocellular carcinoma cells,HCC)侵袭转移的相关机制.方法:采用Western blot法观察低表达Smad4对β-catenin和Vimentin总蛋白表达水平的影响;逆转录PCR法测定低表达Smad4后,β-catenin和VimentinmRNA表达的变化;细胞免疫荧光法检测Smad4、β-catenin和Vimentin在肝癌细胞SMMC-7721及其转染组中的定位及荧光表达强度.结果:低表达Smad4后,相较于CON组和RNAi-NC组,在转染组RNAi-Smad4-2和RNAi-Smad4-12细胞中,β-cateninmRNA和相应总蛋白的表达水平明显增加(P<0.05),并促使其发生了核转位的改变;另一方面,在RNAi-Smad4-2和RNAi-Smad4-12两转染组细胞中,Vimentin的蛋白表达水平和相应胞浆荧光的表达强度比对照组明显下调(P<0.05),相应mRNA的表达水平在肝癌SMMC-7721细胞及其转染组无显著差异.结论:低表达Smad4可调控上皮间质转化相关标志物β-catenin和Vimentin的表达,发挥抑制肝癌SMMC-7721细胞上皮间质转化的作用.     

血管内皮细胞生长因子反义核糖核酸对肝癌细胞生长的抑制作用

目的 探讨血管内皮细胞生长因子(VEGF)反义RNA转染人肝癌细胞后对细胞体内外生物学性状的影响。方法 将含正义、反义VEGFcDNA序列的质粒PCMV—VEGF、PCMV—FGEV及空载体质粒pcDNA3.1,在脂质体介导下导入SMMC—7721肝癌细胞,分别称为正义、反义及对照组,并通过G418筛选获得阳性克隆。细胞原位杂交和免疫组织化学方法检测转染后VEGF在肝癌细胞内的表达情况;MTT法和FCM检测转染后细胞在体外的增殖和凋亡情况;并制备裸鼠动物模型,观察转染后细胞的体内生长情况。结果 转染PCMV—FGEV后肝癌细胞内VEGF的转录及其蛋白的表达水平显著下降,但转染后体外细胞的增殖与凋亡情况均无明显变化。转染PCMV—FGEV后细胞在裸鼠体内的生长缓慢,反义组成瘤时间为(25.0±1.8)d,明显长于正义组(15.7±2.5)d和对照组(18.5±2.1)d,F=19.455,P<0.01;而平均瘤重以反义组最轻,为(0.96±0.28)g,F=21.501,P<0.01;同时反义组裸鼠肿瘤细胞发生明显的凋亡。结论 VEGF反义RNA转染人肝癌细胞可抑制肿瘤细胞VEGF的表达,在体外对细胞增殖和凋亡无影响,而体内可显著诱导细胞凋亡并抑制肿瘤生长。     

Inhibitory effect of antisense vascular endothelial growth factor RNA on the profile of hepatocellular carcinoma cell line in vitro and in vivo

AIM:To evaluate the effect of antisense vascularendothelial growth factor(VEGF)RNA(PCMV-FGEV)transfection on the profile of hepatocellular carcinoma(HCC)SMMC-7721 cells in vitro and in vivo.METHODS:SMMC-7721 cells were transfectedwith PCMV-FGEV antisense,PCMV-VEGF sense andempty vector plasmid encapsulated by lipofectamineas antisense group,sense group and control grouprespectively.The positive cell clones were selectedwith G418.The stable transfection and expressionof VEGF in the cells were determined by RT-PCR andimmunohistochemistry.Cell proliferation was observedby MTT assay.FACS analysis was used to determine theeffect of PCMV-FGEV transfection on cell apoptosis.Thegrowth of transfected cells in Wvo was also observed innude mice.RESULTS:VEGF expression was reduced in SMMC-7721transfected with PCMV-FGEV,which was confirmed byRT-PCR and immunohistochemistry.No effect of PCMV-FGEV transfection was found on cell proliferation andcell apoptosis of SMMC-7721 in vitro.The growth of cellstransfected with PCMV-FGEV was slow in nude miceand accompanied with obvious apoptosis.The latenttime of tumors in the antisense group was 25.0±1.8d,which was longer than that in sense and controlgroups(F=19.455,P<0.01).The average tumor weightin antisense group(0.96 g±0.28 g)was the smallestamong the three groups(F=21.501,P<0.01).CONCLUSION:The expression of VEGF can be inhibitedby antisense PCMV-FGEV.Antisense PCMV-FGEV has no effect on cell proliferation and apoptosis of SMMC-7721in vitro but can inhibit tumor growth and induce cellapoptosis in vivo.     

Downregulation of alpha-fetoprotein siRNA inhibits proliferation of SMMC-7721 cells

AIM: To study the function of α-fetoprotein (AFP) in SMMC-7721 hepatoma cells. METHODS: A hairpin siRNA expressing plasmid pSilencer3.0-H1-afp was constructed and transfected into SMMC-7721 cells with Lipofectamine 2000. The expression of AFP was monitored by real-time RT-PCR and immunoassays, its effect on SMMC-7721 cell proliferation and cell death was detected by MTT and fluorescenceactivated cell sorter (FACS). RESULTS: The AFP-siRNA expressing plasmid downregulated the expression of AFP obviously (about 34%), and inhibited SMMC-7721 cell proliferation, but did not induce apoptosis. CONCLUSION: Downregulation of AFP siRNA inhibits proliferation of SMMC-7721 cells, but cannot cause apoptosis.     

人胰岛素样生长因子Ⅱ反义RNA对肝癌细胞恶性表型的抑制作用

研究人胰岛素样生长因子Ⅱ（ＩＧＦ－Ⅱ）反义ＲＮＡ对肝癌细胞株ＳＭＭＣ－７７２１的抑制效应。方法人ＩＧＦ－ⅡｃＤＮＡ０．１ｋｂ反向插人真核细胞表达载体ｐｃＮＡ３,获得ＩＧＦ－Ⅱ反义ＲＮＡ表达载体ｐＩＧＦ－ⅡＡＳ,将其导人人肝癌细胞株ＳＭＭＣ-７７２１,观察软琼脂培养集落形成的能力。流式细胞仪（ＦＣＭ）测定ｐＩＧＦ－ⅡＡｓ表达ＩＧＦ－Ⅱ反义ＲＮＡ对ＳＭＭＣ-７７２１细胞生长的影响。结果转导人ｐＩＧＦ－ⅡＡｓ的ＳＭＭＣ－７７２１细胞,不能在软琼脂上形成集落,ＦＣＭＭ实导人ＰＩＧＦ－ⅡＡｓ细胞Ｓ期增多,而对照组ＳＭＭＣ－７７２１细胞和带ＤＣＤＮＡ３空载体的ＳＭＭＣ－７７２１细胞则无明显变化．结论ｐＩＧＦ-ⅡＡＳ＆义ＲＮＡ体外可抑制肝癌细胞株ＳＭＭＣ－７７２１的致瘤性。     

Effects of insulin-like growth factors-IR and -IIR antisense gene transfection on the biological behaviors of SMMC-7721 human hepatoma cells

BACKGROUND AND AIMS: Insulin-like growth factors (IGFs) are closely related to hepatocellular carcinoma growth. The study aim was to investigate the effects of IGF-IR and IGF-IIR antisense gene transfection on the biological behaviors of SMMC-7721 human hepatoma cells. METHODS: 7721-IGF-IR-AS cells (human hepatoma SMMC-7721 cells transfected with IGF-IR antisense gene in our previous study) were transfected with a plasmid vector expressing IGF-IIR cDNA in the antisense orientation by DOTAP liposome.7721-IGF-R-AS cells were obtained by selection with G418 and hygromycin. Morphological changes of the cells were observed with optic and electron microscopes. In vitro growth of the 7721-IGF-R-AS cells was observed with a soft agar test, MTT test and with naked mice inoculation test in vivo. RESULTS: The following changes were found in the SMMC-7721 cells after being transfected with the IGF-IR and IGF-IIR antisense genes: (i) the degree of malignancy of the tumor cells as revealed by cell morphology was ameliorated; (ii) the growth capability of the tumor cells in soft agar and their tumorigenicity in naked mice were significantly depressed. However, in the control groups, the SMMC-7721 cells transfected both with IGF-IR and IGF-IIR sense cDNA and SMMC-7721 cells transfected without any external genes, had no such changes. However, the cell growth curves had no significant differences among these three groups. CONCLUSION: IGF-IR and IGF-IIR antisense genes could significantly restrain the malignant behavior of human hepatoma cells and might be useful in investigating a potential route for hepatocellular carcinoma gene therapy.     

核转录因子-κB圈套寡核苷酸对环格列酮诱导肺癌细胞分化作用的影响

目的 探讨核转录因子-κB(NF-κB)圈套寡核苷酸在环格列酮对肺癌细胞A549增殖抑制和分化调控过程中的影响。方法 运用基因转染技术将NF-κB圈套寡核苷酸转入人肺癌细胞A549中,凝胶阻滞分析实验(EMSA)检测转染前后NF-κB活性的变化,免疫印迹观察转染前后多药耐药蛋白(mdrl)的变化。进而以。100μmol/L环格列酮处理转染和未转染的细胞1-4d,生长曲线观察A549细胞生长,流式细胞仪进行细胞周期分析,免疫印迹观察处理前后细胞周期素D1的变化。结果 (1)EMSA显示转染后NF-κB活性明显下降,mdrl蛋白表达水平降低。(2)环格列酮能抑制A549细胞增殖。(3)转染NF-κB圈套寡核苷酸增强环格列酮对A549细胞增殖的抑制作用,更多的细胞被阻滞于G1／G0期,细胞中细胞周期素D1的水平更低。结论NF-κB圈套寡核苷酸能增加肺癌细胞A549对环格列酮的敏感性,即NF-κB圈套寡核苷酸能协同环格列酮抑制肺癌细胞A549的恶性增殖及诱导其分化。