survivin反义寡核苷酸对SMMC-7721细胞增殖和凋亡的影响

目的 观察survivin反义寡核苷酸(ASODN)对人肝癌细胞株SMMC-7721增殖和凋亡的影响.方法 人工合成survivin基因ASODN和正义ODN(SODN),并行硫代磷酸化修饰,通过脂质体途径转染SMMC-7721;分别用RT-PCR和Western blot检测survivin mRNA和蛋白表达;用MTT法检测ASODN对SMMC-7721增殖的影响;流式细胞仪检测细胞周期变化及细胞凋亡率;倒置显微镜观察细胞形态变化.结果 SMMC-7721可强表达survivin mRNA和蛋白;ASODN呈浓度依赖性抑制survivin mRNA和蛋白表达及SMMC-7721增殖,诱导细胞凋亡,使细胞阻滞于G2/M期.SODN对survivin mRNA和蛋白及SMMC-7721的增殖、细胞周期无明显抑制作用.结论 脂质体介导转染survivin ASODN可抑制细胞增殖、使细胞阻滞于G2/M期,从而促进细胞凋亡.     

肝癌细胞系放射诱导的细胞凋亡和细胞周期的变化

目的：探讨肝癌细胞系放射诱导的细胞周期和细胞凋亡的变化特点．方法：研究细胞系为肝癌细胞系HepG2和SMMC-7721．对照细胞系为正常肝细胞系HL-7702、肺小细胞癌HCI-H460和肺腺癌A549．常规培养48h后接受4Gy射线照射,收获受照前(0h)和受照后6,12,24,36和48h的细胞,采用流式细胞术(FCM)检测各细胞系细胞周期和细胞凋亡．结果：4GyX线照射后,HepG2在照射后12h出现细胞凋亡高峰,射线诱导的细胞凋亡比率为45．16%(t=8．864,P＜0．0025),而SMMC-7721在24h达高峰,诱导的细胞凋亡比率为24．94％,HepG2较SMMC-7721射线诱导的细胞凋亡高峰出现早、比率高：HepG2和SMMC-7721与HCI-H460和A549变化较一致,凋亡变化的走势和峰值均与S期的相反,两株肝癌细胞可能均发生了射线诱导的有丝分裂前S期细胞凋亡．HepG2在照射后12h有明显的G_2/M期阻滞,可能有射线诱导的G_2/M期细胞损伤,发生了延迟的间期死亡．结论：两株肝癌细胞可能均发生了射线诱导的有丝分裂前S期细胞凋亡,HepG2可能伴有射线诱导的G_2/M期细胞损伤,发生了延迟的间期死亡．     

毛花苷C促进肝癌SMMC-7721细胞凋亡及抑制survivin的表达

目的:通过毛花苷C作用于人肝癌SMMC-7721细胞,研究其对SMMC-7721细胞增殖的影响初步探讨其作用机制.方法:使用不同浓度毛花苷C干预S M M C-7721细胞,通过细胞增殖实验及克隆形成实验,检测毛花苷C对SMMC-7721细胞增殖的作用;通过流式细胞仪检测毛花苷C对SMMC-7721细胞周期和凋亡的影响;采用Western blot技术分析细胞凋亡抑制基因survivin的表达.结果:毛花苷C对SMMC-7721细胞增殖有明显的抑制作用,各加药组与对照组相比差异有统计学意义(P0.01),并呈现剂量-效应相关关系;流式细胞术显示,毛花苷C将S M M C-7721细胞阻滞于S期,并诱导其凋亡;Western blot检测结果显示毛花苷C下调SMMC-7721细胞内survivin蛋白的表达.结论:毛花苷C明显抑制SMMC-7721细胞增殖,将细胞阻滞在S期并诱导其凋亡.该机制可能与下调survivin的蛋白表达有关.     

LIGHT、IFN-γ基因转染人肝癌HepG2细胞凋亡及其Caspase-3和survivin表达

目的 观察LIGHT、IFN-γ基因转染人肝癌细胞HepG2的凋亡及其Caspaae-3和survivin的表达变化. 方法 用LIGHT、IFN-γ真核细胞表达质粒pcDNA4C-LIGHT-cDNA、pcDNA4C-IFN-γ-cDNA及其转化的E.coli JM-109感受态大肠杆菌,经质粒提取后,转染人肝癌细胞HepG2,同时设对照组(未转染).分别于转染后12、24、48 h收集HepG2细胞.流式细胞仪检测细胞凋亡和Caspase-3、survivin. 结果 转染后细胞凋亡及Caspase-3表达量在对照组、LIGHT单转组、联合转染组呈逐渐递增趋势,survivin表达量呈递减趋势. 结论 LIGHT转染后能通过调节HepG2细胞Caspase-3及survivin的表达来发挥促细胞凋亡作用,INF-γ能增强LIGHT诱导的HepG2细胞凋亡,且显著上调其Caspase-3的表达.     

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.     

Egr-1基因表达及其在射线诱导的肝癌细胞凋亡中的作用

目的:研究Egr-1基因的表达在放射诱导的细胞凋亡中的作用．方法:选择肝癌细胞系HepG2,SMMC-7721和正常肝细胞系HL-7702培养;培养细胞接受4Gy X射线照射;收获受照前和受照后1,2,4, 6,12和24 h的细胞,采用荧光定量PCR(FQ- PCR)检测0,1,2和4 h Egr-1基因的表达,采用流式细胞术(FCM)检测0,6,12和24 h细胞周期和细胞凋亡．结果:随HepG2,SMMC-7721和HL-7702在4GY X射线照射后1 h即诱导了Egr-1基因表达增高,4 h均未达峰值,分别为ΔEgrHepG2(12．9629±1．0649)、ΔEgr7702(0．0096±0．0008)和ΔEgr7721(0．0017±0．0003),HepG2显著高于HL-7702和SMMC-7721(P<0．01)．照射后6 h射线诱导的3株细胞凋亡均不明显,但在12 h均诱导了明显的细胞凋亡,而且HepG2(41．16％)和HL-7702(27．45％)已达峰值; SMMC-7721诱导的细胞凋亡水平较低,24 h仅为24．94％,且未达峰值．在射线诱导的细胞周期变化中,HepG2和SMMC-7721 S期的变化与细胞凋亡变化在6-12 h走势相反．结论:在HepG2,SMMC-7721和HL-7702细胞中,射线通过诱导Egr-1基因表达而诱导了细胞周期和细胞凋亡的变化;射线诱导的Egr-1基因表达水平可能与射线诱导的细胞凋亡成正相关;S期肿瘤细胞可能易发生射线诱导的细胞凋亡．     

冬凌草甲素通过激活ATM蛋白诱导人肝癌HepG2细胞G_2/M细胞周期阻滞

目的研究冬凌草甲素诱导肝癌HepG2细胞G2/M细胞周期阻滞的机制。方法不同浓度的冬凌草甲素(16、32、64μmol/L)处理HepG2细胞48 h后,流式细胞仪检测冬凌草甲素诱导肝癌HepG2细胞的细胞周期。Western印迹检测不同浓度冬凌草甲素作用肝癌HepG2细胞后H2AX蛋白的磷酸化。免疫荧光实验检测Phos-S1981ATM和γ-H2AX焦点。结果彗星实验结果表明不同浓度的冬凌草甲素(16、32、64μmol/L)处理HepG2细胞48 h后,可发生G2/M细胞周期阻滞,并且随浓度增加细胞周期阻滞增加。Western印迹检测不同浓度冬凌草甲素作用肝癌HepG2细胞后,H2AX蛋白发生磷酸化,γ-H2AX随着浓度增加表达增大。免疫荧光实验发现Phos-S1981ATM和γ-H2AX焦点随着浓度增加表达量增加。结论冬凌草甲素可以诱导肝癌HepG2细胞G2/M细胞周期阻滞,H2AX蛋白发生磷酸化,诱导DNA损伤的发生。     

丙戊酸钠对肝癌SMMC-7721细胞增殖和细胞周期的影响及机制

目的:探讨丙戊酸钠(VPA)对人肝癌SMMC-7721细胞增殖、细胞周期及对p21WAF1/CIP1mRNA表达的影响.方法:实验分为空白对照组、PBS组、VPA0.2mmol/L组、VPA1.0mmol/L组和VPA5.0mmol/L组.不同浓度VPA干预人肝癌SMMC-7721细胞24h、48h和72h,采用MTT法检测细胞存活率,流式细胞仪检测细胞周期;干预72h后,用Real-timePCR法检测VPA干预72h后p21WAF1/CIP1mRNA的表达情况.结果:与空白对照组及PBS组比较,不同浓度的VPA作用24h,48h及72h时组肝癌SMMC-7721细胞增殖均出现了不同程度抑制(请将具体数据列出来P<0.05),随着VPA药物浓度升高,细胞增殖抑制作用逐渐增强,随作用时间延长,抑制程度逐渐增强(P<0.05).随药物浓度升高,G1期细胞比例逐渐增多,S期细胞比例逐渐减少,细胞发生G0/G1期阻滞.VPA干预肝癌SMMC-7721细胞72h后,VPA组p21WAF/CIP1mRNA表达较空白对照组及PBS组表达明显升高(请将具体数据列出来P<0.01).结论:VPA可抑制人肝癌SMMC-7721细胞的增殖,且呈时间及剂量依赖性,并诱导出现G0/G1细胞周期阻滞,同时上调p21WAF1/CIP1mRNA的表达.     

羽扇豆醇影响肝癌细胞株SMMC-7721增殖与凋亡的机制

目的:讨论羽扇豆醇影响肝癌细胞株SMMC-7721增殖及促进其凋亡作用机制.方法:使用不同浓度(0、2、5、10、20μg/m L)的羽扇豆醇在不同处理时间(24、36、48 h)下,处理S M M C-7721细胞株后,通过MTT法检测SMMC-7721细胞增殖情况;对使用不同浓度的羽扇豆醇处理48 h后的SMMC-7721细胞,利用流式细胞仪检测S M M C-7721细胞的细胞周期以及细胞凋亡情况,还使用Western blot检测细胞细胞增殖细胞核抗原(proliferating cell nuclear a n t i g e n,P C N A)、B淋巴细胞瘤-2(B-c e l l lymphoma-2,Bcl-2)基因及Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)蛋白的表达情况,最后我们通过免疫组织化学的方式检测在裸鼠体内接种的SMMC-7721细胞瘤体的微血管密度(microvessel density,M V D)值,检测羽扇豆醇对抑制肿瘤血管生成的作用.结果:相较于对照组,通过不同浓度的羽扇豆醇处理后的SMMC-7721细胞增殖水平均受到不同程度的抑制,而且既显示出量效关系又显示出时效关系;受到羽扇豆醇体外诱导处理后的SMMC-7721细胞能够使G0/G1期受到阻滞,甚至出现凋亡现象,SMMC-7721细胞的PCNA和Bcl-2蛋白表达情况下调,Bax蛋白的表达情况上调(P0.05).肿瘤瘤体MVD值下降(P0.01).结论:SMMC-7721细胞的增殖会因为羽扇豆醇的处理而受到抑制,并且还会因为羽扇豆醇的处理而出现凋亡.羽扇豆醇还可以抑制肿瘤瘤体血管生成.     

三磷酸腺苷结合盒G2调控肝癌细胞多药耐药的作用

目的探讨三磷酸腺苷结合盒(ABC)G2在肝癌耐药细胞中的表达及其在肝癌细胞耐药中的作用。方法 MTT法检测阿霉素(ADM)作用肝癌SMMC-7721细胞24 h后半数生长抑制浓度(IC50)。利用药物浓度递增细胞培养方法建立肝癌耐药细胞,在SMMC-7721细胞培养液中加入ADM,逐渐提高ADM浓度,浓度从0.001μg/ml提高至0.100μg/ml,使细胞在0.100μg/ml ADM中稳定生长,命名为SMMC-7721/ADM细胞。倒置显微镜下观察SMMC-7721/ADM及其亲代SMMC-7721细胞形态。MTT法检测ADM作用肝癌耐药细胞SMMC-7721/ADM 24 h后的IC50,计算耐药指数。流式细胞术(FCM)检测SMMC-7721/ADM及其亲代SMMC-7721细胞凋亡、周期及细胞中ABCG2蛋白表达水平。结果 ADM对SMMC-7721细胞的IC50=(1.11±0.09)μg/ml。历时3个月时间成功使SMMC-7721/ADM细胞在含0.100μg/ml ADM的细胞培养液中稳定生长,细胞命名为SMMC-7721/ADM。倒置显微镜下观察,SMMC-7721/ADM细胞体积增大,细胞形态变得更不规则。SMMC-7721/ADM与SMMC-7721细胞相比细胞凋亡率无显著差异(P0.05),细胞G0/G1期显著降低(P0.05),细胞S及G2/M期无显著差异(P0.05)。SMMC-7721/ADM与SMMC-7721细胞相比,细胞中ABCG2蛋白表达水平显著增高(P0.05)。结论 ABCG2在肝癌多药耐药细胞中异常增高,高表达的ABCG2参与了肝癌多药耐药的形成。     

Survivin反义寡核苷酸诱导肝癌细胞凋亡的作用

目的 用反义寡核苷酸封闭肝癌细胞中survivin基因的表达,研究其诱导细胞凋亡的作用及其机制。方法 用脂质体介导survivin反义寡核苷酸转染人肝癌细胞株SMMC-7721细胞,流式细胞仪检测细胞周期变化及细胞凋亡比率,激光共聚焦显微镜观察细胞骨架微丝系统变化,激酶活性检测方法测定细胞内caspase-3活性变化,免疫沉淀法测定细胞内丝裂原活化蛋白激酶p38(p38MAPK)活性的变化。结果 脂质体介导survivin反义寡核苷酸转染肝癌细胞后Ⅰ～Ⅵ组(空白对照、正义对照、400、600、800、1000ng/ml反义转染组)细胞内p38MAPK活性分别为7.03、7.07、13.47、16.37、43.97及47.87;caspase-3活性分别为0.015±0.010、0.014±0.002、0.026±0.003、0.042±0.001、0.093±0.001及0.100±0.001;Ⅳ～Ⅵ组细胞内p38MAPK及caspase-3活性较对照组明显升高。转染后细胞发生G_2/M期阻滞,Ⅰ～Ⅵ组细胞凋亡率分别为0.70％、0.76％、2.43％、7.82％、23.11％及31.35％,各实验组细胞凋亡较对照组明显增加。细胞内微丝形态结构破坏,Ⅰ～Ⅵ组细胞肌动蛋白平均荧光强度分别为189.69±6.68、184.23±8.76、173.14±8.15、99.48±6.57、76.69±10.05及63.80±6.79,Ⅳ～Ⅵ组细胞肌动蛋白平均荧光强度较对照组明显降低。结论 脂质体介导转染survivi     

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。     

短发夹状RNA抑制survivin基因在肝癌细胞中的表达

目的 构建抗凋亡因子survivin的短发夹状RNA(shRNA)，观察其在肝癌细胞株HepG2、SMMC—7721中对survivin基因表达的抑制作用。方法 设计，合成两对survivin编码基因的反向重复序列，中间分别间隔4和8个nt，分别定向克隆至载体pTZU6 1的U6转录启动子下，构建pshRNA—survivin1和pshRNA—survivin2重组质粒，与表达survivin基因的质粒pEGFP—Cl—survivin共转染肝癌细胞株HepG2和SMMC—7721，荧光显微镜下观察融合蛋白中GFP的表达以分析pshRNA—survivin对survivin基因表达的抑制作用。结果 酶切分析和测序证实pshRNA—survivin1和pshRNA—survivin2构建成功；两组shRNA对survivin的表达均有明显的抑制作用，抑制率达80％以上；两重组质粒在HepG2和SMMC—7721两种细胞中抑制目的基因的作用无明显差异；反向重复序列中间隔4或8个nt构建的shRNA，对抑制目的基因的表达无显著差异。结论 构建的pshRNA—survivin重组质粒能有效抑制survivin基因在肝癌细胞株HepG2、SMMC—7721中的表达。     

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.     

Prediction value of radiosensitivity of hepatocarcinoma cells for apoptosis and micronucleus assay

AIM: To investigate the prediction value of radiosensitivity of hepatocarcinoma cells for apoptosis and micronucleus assay. METHODS: Clonogenic assay, flow cytometry, and CB micronuclei assay were used to survey the cell survival rate, radiation-induced apoptosis and micronucleus frequency of hepatocarcinoma cell lines SMMC-7721, HL-7702, and HepG2 after being irradiated by X-ray at the dosage ranging 0-8 Gy. RESULTS: After irradiation, there was a dose-effect relationship between micronucleus frequency and radiation dosage among the three cell lines (P<0.05). A positive relationship was observed between apoptosis and radiation dosage among the three cell lines. The HepG2 cells had a significant correlation (P<0.05) but apoptosis incidence had a negative relationship with micronucleus frequency. There was a positive relationship between apoptosis and radiation dosage and the correlation between SMMC-7721 and HL-7702 cell lines had a significant difference (P<0.01). After irradiation, a negative relationship between cell survival rate and radiation dosages was found among the three cell lines (P<0.01). There was a positive relationship between cell survival rate and micronucleus frequency (P<0.01). No correlation was observed between apoptosis and cell survival rate. CONCLUSION: The radiosensitivity of hepatocarcinoma cells can be reflected by apoptosis and micronuclei. Detection of apoptosis and micronuclei could enhance the accuracy for predicting radiosensitivity.     

Knockdown of survivin gene expression by RNAi induces apoptosis in human hepatocellular carcinoma cell line SMMC-7721

AIM: To investigate the survivin gene expression in human hepatocellular carcinoma cell line SMMC-7721 and the effects of survivin gene RNA interference (RNAi) on cell apoptosis and biological behaviors of SMMC-7721 cells. METHODS: Eukaryotic expression vector of survivin gene RNAi and recombinant plasmid pSuppressorNeo-survivin (pSuNeo-SW), were constructed by ligating into the vector, pSupperssorNeo (pSuNeo) digested with restriction enzymes Xba I and Sail and the designed double-chain RNAi primers. A cell model of SMMC-7721 after treatment with RNAi was prepared by transfecting SMMC-7721 cells with the lipofectin transfection method. Strept-avidin-biotin-complex (SABC) immunohistochemical staining and RT-PCR were used to detect survivin gene expressions in SMMC-7721 cells. Flow cytometry was used for the cell cycle analysis. Transmission electron microscopy was performed to determine whether RNAi induced cell apoptosis, and the method of measuring the cell growth curve was utilized to study the growth of SMMC-7721 cells before and after treatment with RNAi. RESULTS: The eukaryotic expression vector of survivin gene RNAi and pSuNeo-SW, were constructed successfully. The expression level of survivin gene in SMMC-7721 cells was observed. After the treatment of RNAi, the expression of survivin gene in SMMC-7721 cells was almost absent, apoptosis index was increased by 15.6%, and the number of cells was decreased in G2/M phase and the cell growth was inhibited. CONCLUSION: RNAi can exert a knockdown of survivin gene expression in SMMC-7721 cells, and induce apoptosis and inhibit the growth of carcinoma cells.     

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.     

变异IkBα抑制肝癌细胞株SMMC—7721中NF—kB活性及细胞生长

目的了解变异I κBα(mI κBα)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mI κB α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PcDNA 3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PcDNA 3质粒核内NF-κB高水平表达.细胞增殖实验分析发现转染mI κB α质粒肝癌细胞生长受到抑制,而转染对照P cDNA 3质粒肝癌细胞生长未受影响,第2天开始转染mI κB α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mI κBα质粒肝癌细胞可以持续表达mI κBα,抑制NF-κB向核内转位,从而抑制肿瘤细胞的生长.     

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-7721hepatoma 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-diphenyltetrazolium (MTT) bromide colorimetric assay. Celecoxibinduced 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 MTr 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-7402and 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-3and 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.     

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