EPA诱导人肝癌细胞SMMC-7721凋亡及端粒酶活性调控机制研究

目的:探究二十碳五烯酸(Eicosa Pentaenoic Acid.EPA)对SMMC-7721人肝癌细胞的凋亡、端粒逆转录酶h TERT的调控作用及端粒酶表达活性的影响。方法:体外培养SMMC-7721人肝癌细胞,用不同浓度的EPA(0μM、25μM、50μM、100μM、200μM)作用于SMMC-7721肝癌细胞(24 h、48 h、72 h)后,显微镜下观察其形态学变化;应用MTT法检测SMMC-7721肝癌细胞细胞增殖变化情况;Western-blot法检测h TERT、Bax、Bcl-2蛋白表达水平变化;Real Time-PCR检测h TERTm RNA的表达变化;ELISA法检测SMMC-7721肝癌细胞端粒酶活性的表达水平。结果:EPA可诱导肝癌细胞SMMC-7721发生细胞凋亡,具有明显的时间计量依赖关系。在此过程中Bcl-2蛋白表达的降低和Bax蛋白表达上调,同时端粒酶逆转录酶h TERT蛋白及其m RNA的表达水平和端粒酶活性均明显降低。结论:抑制端粒酶逆转录酶基因(h TERTm RNA)表达而抑制端粒酶的活性、诱导癌细胞凋亡,可能是EPA的抗癌作用机制之一。     

端粒酶RNA反义基因对肝癌细胞的影响

用RT-PCR的方法钓取端粒酶RNA基因的cDNA,并将其反向插入到逆转录病毒载体pLNCX上,构建hTR基因的反义表达质粒。将质粒经脂质体介导转染人肝癌SMMG-7721细胞中表达。结果表明hTR反义基因的表达有效地封闭或抑制肝癌细胞的端粒酶活性,抑制细胞的生长和增殖,延长细胞的倍增时间并促进细胞凋亡。     

怀化医学高等专科学校医学形态学实验中心

目的:探讨抑癌基因p16对肝癌细胞生长的抑制作用及其机制。方法:将p16 cDNA亚克隆至pcDNA3.1真核表达载体上,并经脂质体介导转染至人肝癌细胞株SMMC-7721。用MTT法和Western blot分析转染细胞的生长情况。结果:成功构建重组表达质粒pcDNA3.1-p16,转染pcDNA3.1-p16的SMMC-7721细胞生长速度受到明显抑制;转染后有外源p16蛋白的表达,且伴随Bax上调,Bcl-2和cIAP2的下调。结论:重组pcDNA3.1-p16质粒能在人肝癌细胞SMMC-7721内表达,且能抑制SMMC-7721的生长,其机理与诱导肿瘤细胞凋亡相关。     

抑癌基因p16对人肝癌细胞生长抑制的机制研究

目的：探讨抑癌基因p16对肝癌细胞生长的抑制作用及其机制。方法：将p16cDNA亚克隆至pcDNA3．1真核表达载体上,并经脂质体介导转染至人肝癌细胞株SMMC-7721：用MTT法和Western blot分析转染细胞的生长情况。结果：成功构建重组表达质粒pcDNA3．1-p16,转染pcDNA3．1-p16的SMMC-7721细胞生长速度受到明显抑制;转染后有外源p16蛋白的表达,且伴随Bax上调,Bcl-2和cIAP2的下调。结论：重组pcDNA3．1-p16质粒能在人肝癌细胞SMMC-7721内表达,且能抑制SMMC-7721的生长,其机理与诱导肿瘤细胞凋亡相关。     

The inhibitory effect of transthyretin gene on growth of human hepatoma cells

Transthyretin(TTR) gene was highly expressed in normal liver and it has been found to be deleted in part of DNA samples from human hepatic cancer.Its mRNA expression was suppressed in most hepatoma samples.In order to study the biological effect of TTR gene on the growth of hepatoma cells,a recombinant vector containing TTR cDNA was constructed by pCMV,then it was transfected into hepatoma cell lines SMMC-7721 and Q3.It has been demonstrated that the inhibition of growth rate of TTR cDNA transfected hepatoma cells was about 50% in strength compared with that of the control.This inhibition was further enhanced when the transfected hepatoma cells were treated with all-trans retinoic acid.Hepatoma cells of cell lines PLC/PRF/5,SMMC-7721 and Q3 as well as hepatoma cells SMMC-7721 transfected with pCMV or pCMV-TTR were analyzed for TTR expression by Northern hybridization.The low level of TTR expression was found in both hepatoma cell lines and in SMMC-7721 cells transfected with pCMV alone.However,a remarkable TTR mRNA expression was observed in hepatoma SMMV-7721 cells transfected with pCMV-TTR.It seems possible that TTR gene might be a candidate of cancer suppressor gene for human hepatic cancer.     

Effects of matrine against the growth of human lung cancer and hepatoma cells as well as lung cancer cell migration

The purpose of this study is to investigate in vitro and ex vivo effects of matrine on the growth of human lung cancer and hepatoma cells and the cancer cell migration as well as the expressions of related proteins in the cancer cells. Matrine significantly inhibited the in vitro and ex vivo growth of human non-small cell lung cancer A549 and hepatoma SMMC-7721 cells. Matrine induced the apoptosis in A549 and SMMC-7721 cells. Western blot analysis indicated that matrine dose-dependently down-regulated the expression of anti-apoptotic protein Bcl-2 and up-regulated the level of pro-apoptotic protein bax, eventually leading the reduction of ratios of Bcl-2/Bax proteins in A549 and SMMC-7721 cells. Furthermore, matrine significantly suppressed the A549 cell migration without reducing the cell viability. In addition, matrine dramatically reduced the secretion of vascular endothelial growth factor A in A549 cells. More importantly, matrine markedly enhanced the anticancer activity of anticancer agent trichostatin A (the histone deacetylase inhibitor) by strongly reducing the viability and/or the ratio of Bcl-2/Bax protein in A549 cells. Our findings suggest that matrine may have the broad therapeutic and/or adjuvant therapeutic application in the treatment of human non-small cell lung cancer and hepatoma.     

乙型肝炎病毒X基因真核表达载体的构建及表达

目的:构建含有天然完整的乙型肝炎病毒(HBV)X基因序列的真核表达载体,观察其在肝癌细胞株中的表达。方法:设计并合成HBV X基因的引物,用PCR方法从含完整HBV全基因的HepG2细胞中扩增X基因序列,并将其连接到真核表达载体pVAX-1上,酶切、PCR鉴定;用Triton X-114去除质粒内毒素后,采用电穿孔法将重组质粒pVAX-HBV X和空质粒pVAX-1分别转染SMMC-7721细胞,RT-PCR法检测HBV X基因mRNA的表达,Western印迹鉴定HBV X蛋白(HBx)的表达。结果:酶切和PCR鉴定证实pVAX-HBV X载体中包含完整的HBVX基因片段,该重组质粒转染的SMMC-7721细胞中HBV X基因mRNA及HBx蛋白的表达稳定。结论:构建了HBV X基因的真核表达载体,为X基因及其编码蛋白的生物学功能的研究提供了可靠的基因材料。     

壳寡糖对肝癌细胞SMMC-7721中Bcl-2和Caspase-3表达的影响

目的检测壳寡糖对人肝癌SMMC-7721细胞的抑制效果及对凋亡调控蛋白Bcl-2和Caspase-3的影响。方法采用噻唑蓝（MTT）法检测不同浓度壳寡糖对肝癌细胞SMMC-7721细胞增殖的抑制作用,并利用荧光Hoechst33258染色法检测细胞凋亡状况。最后通过免疫细胞化学方法研究壳寡糖对肝癌细胞SMMC-7721中Bcl-2和Caspase-3表达的影响。结果壳寡糖能够抑制SMMC-7721细胞增殖,并且促进SMMC-7721细胞的凋亡,并且壳寡糖能够上调促凋亡蛋白Caspase-3的表达和降低抑制凋亡蛋白Bcl-2的表达。结论壳寡糖对人肝癌SMMC-7721细胞的增殖有抑制作用,此作用可能是通过促进Caspase-3的表达和抑制Bcl-2的表达来实现的。     

SeO(2) induces apoptosis with down-regulation of Bcl-2 and up-regulation of P53 expression in both immortal human hepatic cell line and hepatoma cell line

An immortal human hepatic cell line HL-7702 and human hepatoma cell line SMMC-7721 were treated with 3-30 microM SeO(2). SeO(2) at 30 microM markedly inhibited cell proliferation and viability, and prompted apoptosis of both normal hepatic and hepatoma cells after 48h treatment. SeO(2) could also down-regulate the Bcl-2 level, greatly in HL-7702 and slightly in SMMC-7721 cells, but up-regulate wild type P53 level a little in HL-7702 and significantly in SMMC-7721 cells. The Bcl-2/P53 value was closely correlated with the apoptotic rate as well as SeO(2) concentrations.     

蛋白激酶PRKX对人肝癌细胞粘附和迁移能力的影响

目的观察蛋白激酶PRKX对人肝癌细胞SMMC-7721粘附和迁移能力的影响。方法采用脂质体转染的方法,将PRKX表达质粒转染到SMMC-7721细胞中,蛋白印迹方法鉴定转染前后PRKX蛋白的表达。细胞-基质粘附实验测定对照组和PRKX转染组SMMC-7721细胞的粘附能力。细胞迁移实验测定对照组和PRKX转染组SMMC-7721细胞的迁移能力。结果 SMMC-7721细胞转染组PRKX蛋白的表达增加,SMMC-7721细胞转染组的粘附能力和迁移能力均较对照组增加。结论 PRKX可增加人肝癌细胞SMMC-7721的粘附和迁移能力。     

Gambogic acid induces apoptosis in hepatocellular carcinoma SMMC-7721 cells by targeting cytosolic thioredoxin reductase

The thioredoxin reductase (TrxR) isoenzymes, TrxR1 in cytosol or nucleus and TrxR2 in mitochondria, are essential mammalian selenocysteine (Sec)-containing flavoenzymes with a unique C-terminal -Gly-Cys-Sec-Gly active site. TrxRs are often overexpressed in a number of human tumors, and the reduction of their expression in malignant cells reverses tumor growth, making the enzymes attractive targets for anticancer drug development. Gambogic acid (GA), a natural product that has been used in traditional Chinese medicine for centuries, demonstrates potent anticancer activity in numerous types of human cancer cells and has entered phase II clinical trials. We discovered that GA may interact with TrxR1 to elicit oxidative stress and eventually induce apoptosis in human hepatocellular carcinoma SMMC-7721 cells. GA primarily targets the Sec residue in the antioxidant enzyme TrxR1 to inhibit its Trx-reduction activity, leading to accumulation of reactive oxygen species and collapse of the intracellular redox balance. Importantly, overexpression of functional TrxR1 in cells attenuates the cytotoxicity of GA, whereas knockdown of TrxR1 sensitizes cells to GA. Targeting of TrxR1 by GA thus discloses a previously unrecognized mechanism underlying the biological action of GA and provides useful information for further development of GA as a potential agent in the treatment of cancer.     

FOXQ1在肝癌中的临床意义及其对SMMC-7721细胞体外血管形成的影响

探讨叉头框蛋白Q1(forkhead box Q1, FOXQ1)基因在肝癌中的临床意义及对肝癌细胞体外血管生成作用.利用 qRT-PCR法及Western印迹法,检测24例肝癌、癌旁组织、正常肝细胞L02及肝癌细胞SMMC-7721中FOXQ1的mRNA和蛋白质的表达;利用免疫组织化学法检测68例肝癌及癌旁组织中FOXQ1的蛋白质表达.合成shRNA-FOXQ1及shRNA-NC慢病毒,转染到SMMC-7721细胞.用体外血管生成实验检测转染shRNA-FOXQ1的肝癌细胞血管生成能力. 用qRT-PCR和Western印迹法检测细胞间FOXQ1、VEGF基因和蛋白质的表达.结果显示,癌组织和SMMC-7721细胞中FOXQ1 mRNA和蛋白质的表达均高于癌旁组织和正常肝细胞(P<0.05),FOXQ1蛋白的表达与TNM分期、肿瘤分化程度、肿瘤数目、肿瘤大小等参数差异显著(P<0.05).shRNA-FOXQ1组血管生成能力明显低于shRNA-NC组和空白组(P<0.05),FOXQ1、VEGF基因和蛋白质的表达也明显低于shRNA-NC组和空白组(P<0.05).研究结果证实,FOXQ1在肝癌中高表达,如果沉默FOXQ1的表达可抑制肝癌细胞血管生成,与肝癌的临床病理特征密切相关.     

重组新城疫病毒Anhinga株与TRAIL蛋白协同杀伤肿瘤细胞

将新城疫病毒（Newscastle disease virus, NDV）Anhinga株的全 长基因组cDNA克隆质粒、pTM1-L、pTM1-P、pTM1-NP表达质粒共转染稳定表 达T7 RNA聚合酶的BSRT7/5细胞,得到拯救NDV病毒．通过PCR、酶切法、测 序证明拯救病毒中存在引入的分子标签.通过血凝实验、蚀斑测定证明成功 拯救病毒.并研究了该重组病毒对4种不同人肿瘤细胞的体外杀伤效果．首 次证明重组Anhinga株对SMMC-7721细胞、A549细胞、HepG2细胞和SH-SY5Y 细胞均有杀伤作用．该重组病毒主要诱导SMMC-7721细胞和A549细胞凋亡, 诱导HepG2细胞和SH-SY5Y细胞坏死．TNF家族成员TRAIL蛋白可以显著增强 NDV杀伤肿瘤细胞的效果.本实验为进一步研究重组NDV用于肿瘤治疗奠定基 础．     

外源性神经节苷脂GM_3影响人肝癌细胞生长的可能机制的初步研究

通过苔酚蓝染色细胞发现,外源性GM3（10μg/ml）能明显抑制人肝癌细胞株SMMC-7721细胞生长,在GM3处理3d时,出现明显差异．通过NorthernBlot分析发现,外源性GM3可明显影响人肝癌细胞株SMMC-7721细胞中c-fos、c-jun、c-myc和N-ras这四种癌基因的mRAN表达．未经GM3处理的细胞中没有检测到c-fosmRNA,但c-jun微量表达,并有c-myc和N-rasmRNA的高水平表达而GM3可在短时间内快速大量地诱导c-fos、c-junmRNA的生成．GM3处理的细胞,c-myc和N-rasmRNA的表达均明显减少．GM3处理45min时,c-myc基因表达只为对照组的39．55％;GM3处理24h时,N-ras基因表达为对照组的30.48％．以上结果提示：GM3抑制SMMC-7721细胞生长很可能是通过改变癌基因表达来实现的．