Overexpression of cyclooxygenase-2 in human HepG2, Bel-7402 and SMMC-7721 hepatoma cell lines and mechanism of cyclooxygenase-2 selective inhibitor celecoxib-induced cell growth inhibition and apoptosis

AIM: To investigate the cyclooxygenase-2 (COX-2) expression level in human HepG2, Bel-7402 and SMMC-7721 hepatoma cell lines and the molecular mechanism of COX-2 selective inhibitor celecoxib-induced cell growth inhibition and cell apoptosis. METHODS: Hepatoma cells were cultured and treated with celecoxib. Cell In situ hybridization (ISH) and immunocytochemistry were used to detect COX-2 mRNA and protein expression. Proliferating cell nuclear antigen and phosphorylated Akt were also detected by immunocytochemistry assay. Cell growth rates were assessed by 3-(4, 5-dimethylthiazol-2-yl-2, 5-diphenylte-trazolium (MTT) bromide colorimetric assay. Celecoxib-induced cell apoptosis was measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and flow cytometry (FCM). The phosphorylated Akt and activated fragments of caspase-9, caspase-3 were examined by Western blotting analysis. RESULTS: Increased COX-2 mRNA and protein expression were detected in all three hepatoma cell lines. Celecoxib could significantly inhibit cell growth and the inhibitory effect was in a dose- and time-dependent manner evidenced by MTT assays and morphological changes. The apoptotic index measured by TUNEL increased correspondingly with the increased concentration of celecoxib and the reaction time. With 50 μmol/L celecoxib treatment for 24 h, the apoptotic index of HepG2, BEL-7402 and SMMC-7721 cells was 25.01±3.08%, 26.40±3.05%, and 30.60±2.89%, respectively. Western blotting analysis showed remarkable activation of caspase-9, caspase-3 and dephosphorylation of Akt (Thr308). Immunocytochemistry also showed the reduction of PCNA expression and phosphorylation Akt (Thr308) after treatment with celecoxib. CONCLUSION: COX-2 mRNA and protein overexpression in HepG2, Bel-7402 and SMMC-7721 cell lines correlate with the increased cell growth rate. Celecoxib can inhibit proliferation and induce apoptosis of hepatoma cell strains in a dose- and time-dependent manner.     

Melatonin and Doxorubicin synergistically induce cell apoptosis in human hepatoma cell lines

AIM:To investigate whether Melatonin has synergistic effects with Doxorubicin in the growth-inhibition and apoptosis-induction of human hepatoma cell lines HepG2 and Bel-7402.METHODS:The synergism of Melatonin and Doxorubicin inhibited the cell growth and induced cell apoptosis in human hepatoma cell lines HepG2 and Bel-7402.Cell viability was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide(MTT)assay.Cell apoptosis was evaluated using TUNEL method and flow cytometry.Apoptosis-r...     

腺病毒介导的环氧合酶-2反义RNA对肝癌细胞株生长的影响

目的探讨环氧合酶-2(COX-2)的表达与肝癌的关系,并构建表达人COX-2反义RNA的腺病毒载体,研究其对人肝癌细胞生长的抑制作用。方法采用免疫组织化学法探讨34例肝癌组织COX-2 的表达与肝癌病理特征的关系。采用基因重组法把人COX-2的cDNA片段反向克隆于穿梭质粒pHCMVSP1A,获得pAd-AShcox-2,通过脂质体与pJM17共转染293细胞,经同源重组产生编码COX-2反义RNA的重组腺病毒--Ad-AShcox-2。经聚合酶链反应法鉴定为阳性克隆者大量扩增、纯化,转染人肝癌细胞株SMMC-7402和SMMC-7721,采用免疫细胞化学、细胞集落形成率及流式细胞术检测其对肝癌细胞生长、凋亡及细胞周期分布的影响。结果34例肝癌组织中有28例COX-2高度表达,阳性率达82.4%; COX-2的表达水平与肝癌的病理分级有关,与甲胎蛋白、细胞类型、有无肝内转移无关。成功构建、扩增、纯化得到编码COX-2反义RNA的重组腺病毒Ad-AShcox-2,滴度达1.06×1012PFU/ml;Ad-AShcox- 2转染两种肝癌细胞株后,发现高度表达COX-2的SMMC-7402 COX-2表达水平明显降低,细胞凋亡率明显增加,出现G1期阻滞,与Ad-LacZ组及空白对照组比较差异有统计学意义(P<0.05);而不表达COX- 2的SMMC-7721变化不明显。细胞集落形成实验显示SMMC-7402细胞集落形成率较低(2.7%±0.94%); 而SMMC-7721     

黑色素瘤相关抗原A1在肝癌细胞株中的表达与基因甲基化程度的相关性

目的 探讨人肝癌细胞株中黑色素瘤相关抗原A1(MAGE-A1)表达状况与肝癌细胞基因甲基化的关系。方法 提取10种人肝癌细胞株的总RNA,用逆转录聚合酶链反应对细胞株的MAGE-A1基因的表达进行检测;提取10种人肝癌细胞株的基因组DNA,用限制性酶切和Southern印迹杂交对每种细胞的基因组甲基化程度进行定量分析。另外对基因组DNA行HpaⅡ酶切后,用引物CDS21、EDP4和CDS20、EDP4进行聚合酶链反应扩增,然后用特异性探针杂交来检测肝癌细胞株MAGE-A1启动子的甲基化状态。用特异性序列聚合酶链反应方法检测每一种细胞株的人白细胞抗原-A位点型别。结果 QGY-7703、SMMC-7721、HLE、BEL-7402、BEL-7404、BEL-7405肝癌细胞株的MAGE-A1基因表达阳性,且细胞分化程度均为中低分化;HepG2 2．2．15、HepG2、QGY-7701和Huh7肝癌细胞株的MAGE-A1基因表达阴性,且细胞分化程度均为高中分化。通过定量分析表明,MAGE-A1表达的肝癌细胞株与MAGE-A1不表达的肝癌细胞株比较,前者基因组甲基化程度较低(t=2．896,P=0．02)。肝癌细胞株MAGE-A1基因启动子区甲基化分析结果表明HepG2 2．2．15、HepG2、QGY-7701和Huh7为高甲基化,SMMC-7721、HLE、BEL-7402、BEL-7404、BEL-7405为低甲基化。结论 肝癌细胞株MAGE-A1 mRNA表达与其基因甲基化程度有关。     

三氧化二砷对肝癌细胞表达PTTG1和VEGF的影响及其意义

目的 观察三氧化二砷(As2O3)对人肝癌细胞株BEL-7402和SMMC-7721的作用及其可能机制.方法 采用四甲基偶氮唑蓝(MTT)法检测As2O3对两种细胞的生长活性;用流式细胞仪测定经不同浓度As2O3溶液处理后的两种细胞的周期变化,并用Annexin V-FITC与PI双染法检测细胞凋亡;用实时逆转录-聚合酶链反应(RT-PCR)检测As2O3对两种细胞垂体肿瘤转化基因1(PTTG1)和血管内皮生长因子(VEGF)mRNA表达的影响;用蛋白免疫印迹法(western-blotting)检测As2O3对两种细胞PTTG1和VEGF蛋白表达的影响.结果 As2O3可显著抑制BEL-7402和SMMC-7721细胞的生长,并呈量效关系(r=0.973,P<0.01;r=0.985,P<0.01),半数抑制浓度(IC50)分别为4.38 μmol/L和5.16 μmol/L.BEL-7402和SMMC-7721经As2O3处理后均发生显著凋亡(P<0.01).As2O3能显著下调两种细胞PTTG1和VEGF mRNA及蛋白的表达(P<0.01),并使细胞周期阻滞在G2/M期.结论 As2O3可有效抑制人肝癌细胞的生长增殖,其机制可能与诱导细胞凋亡、阻滞细胞周期及下调PTTG1和VEGF的表达有关.     

干细胞相关基因在肝癌细胞系中的表达

目的 了解Oct4、Sox2、Nanog、SMO、β-Catenin、Wnt5b等干细胞相关基因在4株人肝癌细胞系SMMC-7721、Bel-7402、HepG2、MHCC-97和正常人肝脏细胞系L02中的表达情况,并比较不同细胞系中各基因量的差异和对全反式维甲酸(tRA)的反应.方法 逆转录聚合酶链反应测定4株人肝癌细胞系中Oct4、Sox2、Nanog、SMO、β-Catenin、Wnt5b mRNA的表达,实时荧光定量PCR比较不同细胞中各基因量的差异以及对tRA的反应.单因素方差分析比较各基因在不同细胞系中的表达差异.结果 干细胞相关基因Oct4、Sox2、Nanog、SMO、β-Catenin和Wnt5b在4株人肝癌细胞系SMMC-7721、Bel-7402、HepG2、MHCC-97和正常人肝脏细胞系L02中有不同程度的表达(P<0.05);人肝癌细胞系HepG2和正常人肝脏细胞系L02对tRA的反应也存在明显差异(P<0.05).结论 干细胞相关基因在人肝癌细胞系中不同程度的表达,不同肝癌细胞系中这些基因表达有一定的差异,可能与其生物学特性不同有关,人肝癌细胞系HepG2中Oct4和Sox2的表达调控不同于胚胎干细胞.在肝癌细胞中可能存在着Oct4对于Wnt/β-catenin信号转导通路的调节作用.     

5-脱氧杂氮胞苷抑制肝癌细胞株生物学行为的机制

目的 DNA甲基化模式的改变伴随着永生性细胞系的确立，生长凋控基因CpG岛的重新甲基化可能导致了其转录的不活跃，在体外培养的情况下，使得肿瘤细胞选择了有利于生长的条件．我们用DNA甲基转移酶抑制剂5-脱氧杂氮胞苷处理两株肝癌细胞，研究其对肝癌细胞的影响，探讨DNA甲基化异常与肝细胞癌间的相关性及5-脱氧杂氮胞苷对肝癌细胞株恶性生物学行为的影响及其机制。方法 用5-脱氧杂氮胞苷处理肝癌细胞株SMMC-7721和HePG2，然后采用相差显微镜观察药物处理前后细胞的形态变化，采用MTT法观察细胞的生长速度变化，采用流式细胞仪检测细胞周期、细胞凋亡率、P16蛋白表达的变化．采用RT-PCR法比较p16和甲基转移酶mRNA表达量的变化，比较裸鼠致瘤性的大小。结果 5-脱氧杂氨胞苷处理后细胞形态趋于规则，生长速度减慢．SMMC-7721细胞，G1期细胞增加了8.5％，而s期和G2／M期细胞分别减少了47．8％和10.4％．HePG2细胞，G1期细胞增加了3．5％，S期减少了46．2％．G2／M期增加了23．7％．两细胞凋亡率分别增加了91．6％和133．3％．P16蛋白表达分别增加了23．4％和20.9％，p16mRNA表达增高，而DNA甲基转移酶mRNA表达明显降低，裸鼠皮下移植瘤生长减慢。结论 肝细胞癌的发生与DNA甲基化异常有关，甲基化酶抑制剂5-脱氧杂氮胞苷可能通过抑制甲基转移酶抑制抑癌基因启动子区域的高甲基化，恢复其生长调控功能，从而使肝癌细胞株的恶性生物学行为发生逆转。     

Effects of adenovirus-mediated human cyclooxygenase-2 antisense RNA on the growth of hepatocellular carcinoma

AIM: To investigate the relation between the expression of cyclooxygenase-2 (COX-2) and liver cancer, to construct the recombinant adenovirus encoding human COX-2 antisense RNA, and to explore its effects on liver cancer cell proliferation. METHODS: We studied the expression of COX-2 in 34 cases of hepatocellular carcinoma (HCC) and SMMC7402 and SMMC7721 by immunohistochemical technique. Recombinant adenovirus Ad-AShcox-2 was constructed and transfected into human HCC cell lines SMMC7402 and SMMC7721, and its effects on COX-2 expression, cell apoptosis and cell cycle were analyzed by flow cytometry. Cell proliferation was determined by colony-forming efficiency. RESULTS: We observed COX-2 expression in 82.4% of HCC and SMMC7402 cells, but no COX-2 expression in SMMC7721 cells. In addition, recombinant adenovirus encoding antisense COX-2 fragment Ad-AShcox-2 was obtained with the titer of 1.06×1012PFU/mL. Ad-AShcox-2 could reduce the expression of COX-2 and enhance the percentage of cells in G1/G0 phase in SMMC7402 cell line. The difference of apoptotic index between the Ad-AShcox-2 group and control group was statistically significant (tcontrol group=32.62 and tAd-Lacz = 10.93, P<0.001) in SMMC7402 but not in SMMC7721. Similarly, colony-forming rates of SMMC7402 and SMMC7721 cell lines, after the transfer of Ad-AShcox-2, were (2.7±0.94)% and (33.6±4.24)%, respectively. CONCLUSION: Reduction in the expression of COX-2 can inhibit COX-2 expressing HCC cells.     

Cyclooxygenase-2 promotes hepatocellular carcinoma cell growth through Akt activation: evidence for Akt inhibition in celecoxib-induced apoptosis

Cyclooxygenase-2 (COX-2)-controlled prostaglandin (PG) metabolism recently has been implicated in the pathogenesis of hepatocellular carcinoma (HCC). However, the biologic role and molecular mechanism of COX-2-mediated PGs in the control of liver cancer growth have not been established. This study was designed to examine the direct effect of COX-2 and its inhibitor celecoxib on the growth control of liver cancer cells. Human HCC cell lines Hep3B and HepG2 transfected with COX-2 expression vector showed increased cell growth and enhanced phosphorylation of serine/threonine protein kinase B (Akt). The level of COX-2 expression and Akt phosphorylation is correlated positively in cultured HCC cells and human liver cancer tissues. Inhibition of Akt activation by phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY294002 significantly decreased the viability of Hep3B and HepG2 cells (P <.01). These results reveal a novel role of Akt activation in COX-2-induced HCC cell survival. Furthermore, HCC cells treated with the COX-2 inhibitor celecoxib showed significant reduction of Akt phosphorylation and marked morphologic and biochemical characteristics of apoptosis. Overexpression of COX-2 or addition of exogenous PGE(2) partially prevented celecoxib-induced apoptosis (P <.01). In conclusion, our results suggest the involvement of COX-2-dependent and -independent mechanisms in celecoxib-mediated HCC cell apoptosis.     

siRNA沉默Cyclin E基因对肝癌HepG2、SMMC-7721和BEL-7402细胞增殖和侵袭能力的影响

目的:观察siRNA沉默Cyclin E基因表达对肝癌HepG2、SMMC-7721和BEL-7402细胞增殖和侵袭能力的影响.方法:构建2个靶向Cyclin E基因siRNA载体,转染人肝癌HepG2、SMMC-7721和BEL-7402细胞.RT-PCR、Western blot检测转染后HepG2、SMMC-7721和BEL-7402细胞Cyclin E基因mRNA和蛋白表达水平.CCK-8试验、软琼脂克隆形成实验检测HepG2、SMMC-7721和BEL-7402细胞增殖、克隆形成能力.流式细胞术、transwell试验分别检测HepG2、SMMC-7721和BEL-7402细胞周期和侵袭能力.结果:构建的2个Cyclin E基因siRNA载体插入序列与所设计序列均一致;转染HepG2、SMMC-7721和BEL-7402细胞后,干扰1组、干扰2组与空白对照组和阴性对照组比较,C y c l i n E m R N A和蛋白表达量均显著降低(P<0.05),细胞生长速度延缓,软琼脂细胞集落形成数、穿透细胞数均显著降低(P<0.05),S和G2/M期细胞比例减少,G0/G1期细胞比例增加.结论:沉默肝癌细胞Cyclin E表达水平,可有效抑制细胞生长、增殖和侵袭能力.     

Specific COX-2 inhibitor, meloxicam, suppresses proliferation and induces apoptosis in human HepG2 hepatocellular carcinoma cells

BACKGROUND AND AIMS: Cyclooxygenase-2 (COX-2) is associated with carcinogenesis. The aim of this study was to investigate the expression of COX-2 in four hepatocellular carcinoma (HCC) cell lines, and evaluate the effect of a selective COX-2 inhibitor, meloxicam, in HepG2, a high COX-2 expressing cell line. METHODS: Expression of COX-2 was detected using RT-PCR, Western blotting and immunohistochemical analysis. Cell proliferation was measured using MTT assay. Cell cycle distribution was determined by flow cytometry. Apoptosis was detected with TUNEL method. Expression of proliferating cell nuclear antigen (PCNA), cell cycle regulatory proteins including cyclins A, B1, D1 and E, and apoptosis-related proteins including Fas, Fas ligand and Bcl-2 were examined using Western blotting. RESULTS: Cyclooxygenase-2 was intensely expressed in HepG2, HLE and BEL7402 cells, but weakly expressed in SMMC-7402 cells. Meloxicam suppressed proliferation of HepG2 cells in a dose- and time-dependent manner, resulting in cell cycle arrest in S phase and cell accumulation in G0/G1 phase. Expression of PCNA, cyclin A but not cyclin B1, cyclin D1 or cyclin E was down-regulated by meloxicam. Meloxicam also induced apoptosis of HepG2 cells, with increased expression of Fas ligand, but the expression of Fas and Bcl-2 was not affected by meloxicam treatment. CONCLUSIONS: The present study demonstrates that the specific COX-2 inhibitor meloxicam suppresses proliferation and induces apoptosis in HCC cells that express COX-2, suggesting that COX-2 inhibition may offer a novel chemopreventive and therapeutic approach for HCC.     

塞来昔布联合 NK 细胞对裸鼠人肝癌皮下移植瘤生长的影响

目的：观察塞来昔布联合NK细胞对裸鼠人肝癌细胞SMMC-7721皮下移植瘤生长的影响,并探讨其可能的作用机制。方法选择裸鼠40只,制备人肝癌细胞SMMC-7721移植瘤模型,随机分为对照组、NK细胞组、塞来昔布组及联合干预组各10只。 NK细胞组瘤内注射NK细胞悬液0．6 mL,每7 d注射1次,共注射5次;塞来昔布组从接种后第3天起给予塞来昔布100 mg/kg灌胃,每天1次,连续35 d;联合干预组同时给予塞来昔布和NK细胞,给药剂量及途径与单用组相同;对照组灌胃和瘤内注射等量生理盐水。35 d后切取移植瘤组织计算体积,称取瘤质量,并计算抑瘤率;TUNEL法评价肿瘤细胞凋亡情况,免疫组化法检测肿瘤内VEGF、Bax、Bcl-2、caspase-3、Ki-67、NF-κB的阳性表达。结果联合干预组移植瘤体积、肿瘤质量均明显低于单用组（ P均＜0．05）,抑瘤率、凋亡指数明显高于单用组（ P均＜0．05）。联合干预组移植瘤中VEGF、Bcl-2、NF-κB、Ki-67表达明显低于单用组（ P均＜0．05）,Bax、caspase-3表达明显高于单用组（P均＜0．05）。结论塞来昔布联合NK细胞可明显抑制裸鼠人肝癌细胞SMMC-7721皮下移植瘤的生长;其作用机制可能是通过促进凋亡级联通路上Bax、caspase-3的表达,抑制VEGF、Bcl-2、NF-κB、Ki-67的表达而实现。     

Celecoxib-induced apoptosis in rat cholangiocarcinoma cells mediated by Akt inactivation and Bax translocation

Recently, we demonstrated that the cyclooxygenase-2 (COX-2) inhibitor celecoxib acts to significantly suppress the growth of rat C611B cholangiocarcinoma (ChC) cells in vitro. To establish a molecular mechanism for this growth suppression, we investigated the effects of celecoxib on apoptotic signaling pathways in cultured rat C611B ChC cells. Celecoxib and another COX-2 inhibitor, rofecoxib, at 5 microM were almost equally effective in inhibiting prostaglandin E(2) (PGE(2)) production by these cells, but at this low concentration, neither inhibitor suppressed growth or induced apoptosis. Celecoxib at 50 microM induced prominent apoptosis in these cells, whereas rofecoxib at 50 microM was without effect in either suppressing growth or inducing apoptosis. Celecoxib (50 microM) did not alter Bcl-2, Bcl-x(L), or COX-2 protein levels, nor did it inhibit p42/44 mitogen-activated protein kinase (MAPK) phosphorylation; however, it significantly suppressed serine/threonine kinase Akt/PKB (Akt) phosphorylation and kinase activity in cultured C611B cells. This effect, in turn, directly correlated with Bax translocation to mitochondria, cytochrome c release into cytosol, activation of caspase-9 and caspase-3, and cleavage of poly (ADP-ribose) polymerase (PARP). Addition of 25 microM PGE(2) to C611B cell cultures blocked the apoptotic actions of celecoxib. Rofecoxib (50 microM) was without effect in suppressing Akt phosphorylation and caspase-3 activation. In vivo, celecoxib partially suppressed tumorigenic growth of C611B ChC cells. In conclusion, our results indicate that celecoxib preferentially acts in vitro to induce apoptosis in ChC cells through a mechanism involving Akt inactivation, Bax translocation, and cytochrome c release. Our in vivo results further suggest celecoxib might have potential therapeutic or chemopreventive value against ChC.     

MMP-2和ICAM-1在裸鼠体内塞来昔布抑制肝癌组织中的表达

目的:研究机体内部选择性环氧合酶-2(COX-2)抑制剂对肝细胞癌的抑制作用.方法:将三种肝癌细胞株HepG2、BEL-7402和SMMC-7721分别接种于6周龄裸鼠肝脏被膜下;将接种了不同肝癌细胞株的裸鼠分别分为3组,阴性对照组给予生理盐水灌胃,实验组给予塞来昔布灌胃,阳性对照组进行生理盐水灌胃的同时使用阿霉素腹腔注射;3 wk后对裸鼠肝脏肿瘤取材、免疫组织化学法观察肿瘤组织中基质金属蛋白酶-2(MMP-2)及其抑制剂(TIMP-2)以及细胞间黏附因子-1(ICAM-1)的表达.结果:在肝脏被膜下接种了HepG2、BEL-7402和SMMC-7721肝癌细胞株的裸鼠中,应用塞来昔布的裸鼠肿瘤组织中MMP-2的表达下降(P<0.05),MMP-2的表达增加(P<0.05),TIMP-2/MMP-2比值增加.在肝脏被膜下接种了BEL-7402和SMMC-7721肝癌细胞株的裸鼠中,应用塞来昔布的裸鼠肿瘤组织中ICAM-1表达下降.结论:塞来昔布在机体内部可能具有抑制肝癌细胞转移和改善预后的作用.     

Mechanism of apoptotic effects induced selectively by ursodeoxycholic acid on human hepatoma cell lines

AIM: To investigate the effects of ursodeoxycholic acid (UDCA) on apoptosis and proliferation of hepatoma cell lines. METHODS: Human hepatoma cell lines HepG2 and BEL 7402 were cultured in medium supplemented with different concentrations of UDCA, normal human hepatic line L-02 was used as control. Cell proliferation, apoptosis and gene expression were detected using methyl thiazolyl tetrazolium (MTT) assay, flow cytometry, Western blot, DNA ladder assay, electron microscopy, and immunocytochemistry. RESULTS: Ursodeoxycholic acid inhibited the proli- feration of HepG2 and BEL7402 cell lines in a dose- dependent manner. Ursodeoxycholic acid can change cell cycle distribution of HepG2 and BEL7402, the proportion of cells in G0-G1 phase increased whereas the proportion of S phase cells and G2-M phase cells decreased. Ursodeoxycholic acid arrested the cell cycle in G0-G1 phase by down-regulating the cell cycle related proteins cyclin D1, D3 and retinoblastoma protein (pRb). The apoptotic rates of HepG2 and BEL7402 treated with UDCA (1.0 mmol/L) were significantly higher than those of control. In the HepG2 and BEL7402 treated with UDCA, expression of bcl-2 decreased whereas expression of Bax increased, the nuclear fragmentation and chromosomal condensed, cells shrank and lost attachment, apoptotic bodies and DNA ladders appeared. UDCA had no effect in inducing apoptosis on L-02 cell lines. CONCLUSION: UDCA can selectively inhibit proliferation and induce apoptosis of HepG2 and BEL7402 cell lines by blocking cell cycle and regulating the expression of Bax/bcl-2 genes.     

肝癌细胞放射敏感性与survivin蛋白表达的关系

目的 探讨肝癌细胞放射敏感性与survivin表达的关系.方法 肝癌细胞HepG2和SMMC-7721在接受不同剂量γ射线照射后,分别采用克隆形成法、免疫细胞化学法、流式细胞术、比色法等检测细胞存活率、survivin蛋白表达、细胞周期变化和Caspase-3活性.结果 在2Gy照射下HepG2和SMMC-7721细胞的存活分数分别为0.43±0.01与0.70±0.02,SMMC-7721较HepG2放射抗拒.γ射线对SMMC-7721细胞的G2/M期阻滞时间较HepG2细胞长(48 h对24 h),在阻滞峰各剂量点SMMC-7721细胞的G2/M期比例也更高.γ射线可上调两株肝癌细胞survivin蛋白的表达,照射后48～72 h,SMMC-7721细胞的survivin蛋白表达水平显著高于HepG2细胞(t值为2.81～5.20,P值均＜0.05).而Caspase-3的活化水平在放射敏感的HepG2细胞中更高(t值为6.05～6.72,P值均＜0.01).结论 射线诱导的survivin表达上调及survivin对Caspase-3的负调控可能是SMMC-7721细胞较HepG2细胞放射抗拒的原因之一.     

靶向甲硫氨酸腺苷转移酶2A基因小干扰RNA抑制肝癌细胞生长的研究

目的研究靶向甲硫氨酸腺苷转移酶(MAT)2A基因的小干扰RNA(siRNA)对肝癌细胞生长和细胞凋亡的影响。方法以MAT 2A为目的基因,以产生siRNA质粒载体pSilence-2.1-U6为表达模板,细胞内转录合成4条siRNA;并构建携带荧光素酶报告基因的重组质粒载体plucA-MAT 2A。脂质体转染法将重组质粒载体plucA-MAT 2A与产生siRNA的质粒pSilence-2.1-U6共转染293 T细胞,定量检测荧光素酶活性,初步筛选出抑制荧光素酶表达的有效siRNA,然后将有效的siRNA转染Bel-7402肝癌细胞,半定量逆转录聚合酶链反应检测MAT 2A mRNA表达,并检测转染后肝癌细胞MAT的活性,进一步采用四甲基偶氮唑盐法观察siRNA对肝癌细胞生长的抑制率,用流式细胞仪检测siRNA对肝癌细胞凋亡的影响。结果所合成的4条siRNA中有2条抑制荧光素酶表达,抑制效率分别为81%和89%,并特异性抑制肝癌细胞MAT 2A表达,降低了肝癌细胞中MAT活性,抑制肝癌细胞的生长,诱导肝癌细胞凋亡。结论靶向MAT 2A基因的siRNA抑制肝癌细胞生长,诱导肝癌细胞凋亡;MAT 2A是肝癌基因治疗的一个很有希望的靶位点。     

Very low density lipoprotein receptor subtype II silencing by RNA interference inhibits cell proliferation in hepatoma cell lines

Very low density lipoprotein receptor (VLDLR) belongs to the low density lipoprotein receptor family, it is divided into two subtypes according to forms with an absence (type II) or a presence (type I) of the O-linked sugar domain. VLDLR have been detected in kinds of cancers so far; however, the subtype of VLDLR in hepatocellular carcinoma (HCC) tissues and hepatoma cell lines has yet to be reported. We detected the VLDLR expression in 39 cases of hepatocellular carcinoma and in three kinds of hepatoma cell lines: HepG2, HBV transfected HepG2.2.15, SMMC-7721 and normal human fetal liver cell line LO2 using RT-PCR and western blotting. The results showed that both type I and type II VLDLR were detected in HCC tissues and hepatoma cell lines, and the type II VLDLR expression was significantly higher than that of type I in cell lines. We inhibited the type II VLDLR expression by shRNA-mediated RNA interference in HepG2, SMMC-7721 cell and then subsequently found the cell proliferation slowed down. The cyclinD1 expression confirmed the cell cycle was arrested at the G0/G1 phase, suggesting that inhibiting the type II VLDLR expression may have a positive impact on carcinogenesis of HCC.     

环氧合酶-2对香烟提取物诱导的内皮细胞凋亡的作用

目的 探讨环氧合酶-2在香烟提取物(CSE)所诱导的内皮细胞凋亡中的作用.方法 体外培养人血管内皮细胞株ECV304,按3个步骤完成实验:(1)用0.0%、0.5%、1.0%和5.0%CSE分别F预ECV-304细胞12 h;(2)用5.0%CSE分别干预ECV304细胞0、3、6、9、12和24 h;(3)用5.0% CSE 及0.0、2.5、5.0、10.0、20.0、50.0μmol/L选择性环氧合酶-2抑制剂赛来昔布联合干预ECV304细胞9 h.采用Hoechst染色法和流式细胞术检测内皮细胞凋亡,免疫细胞化学和Western blot 法检测环氧合酶-2蛋白的表达.采用Bartlett法进行方差齐性检验,多组间均数比较采用单因素方差分析和LSD-t检验.结果 随着CSE干预浓度的升高,内皮细胞凋亡率和环氧合酶-2蛋白表达水平均逐渐升高,5.0%CSE干预后内皮细胞凋亡率最高[(5.40±0.39)%],环氧合酶-2蛋白表达也最高(206.1±15.5),差异均有统计学意义(F值分别为90.03和159.94,均P<0.05).5.0%CSE干预内皮细胞3 h,环氧合酶-2蛋白的表达开始增加,9 h达峰值,12 h开始减弱,24 h恢复止常.随着干预时间的延长,内皮细胞凋亡率逐渐升高,5.0%CSE干预24 h内皮细胞凋亡率最高[(8.87±0.41)%],差异均有统汁学意义(F=155.65,均P<0.05).5.0%CSE与不同浓度的选择件环氧合酶-2抑制剂赛来昔布共同作用于内皮细胞9 h后,随着赛来昔布干预浓度升高,环氧合酶-2的表达逐渐减弱;内皮细胞凋亡率首先出现小幅的下降,随后明显增加,以5.0%CSE+50μmol/L赛来昔布组的内皮细胞凋亡率最高[(32.60±5.51)%],差异均有统计学意义(F=81.28,均P<0.05).结论 CSE能够诱导内皮细胞凋亡和环氧合酶-2蛋白表达.选择性环氧合酶-2抑制剂赛来昔布能够抑制CSE诱导内皮细胞表达环氧合酶-2蛋白,并促进内皮细胞凋亡,甚至诱发其死亡.环氧合酶-2有助于缓解CSE诱导的内皮细胞凋亡,对内皮细胞有一定程度的保护作用.