AEG-1对裸鼠肝癌模型中肝癌细胞生长及肺转移的影响

摘要:目的 研究过表达和沉默 AEG-1基因对肝癌细胞生长的影响,以及 AEG-1基因在调节肝癌细胞定向肺转移 中的作用。方法 分别以携带过表达 AEG-1序列及对照基因序列的慢病毒(lentivirus)转染 SMMC-7721肝癌细胞株 (SMMC-7721-AEG-1-L;SMMC-7721-control-L);以携带shRNAAEG-1及对照shRNA 质粒(plasmid)转染SMMC-7721 细胞株(SMMC-7721-shAEG-1-P;SMMC-7721-control-P);使用实时定量 PCR 和 Westenblot检测 AEG-1的表达;随后 使用荧光素酶基因慢病毒包装颗粒转染上述4种稳定转染细胞株。使用上述细胞株分别建立3种裸鼠肝癌模型:皮下 移植瘤模型,原位移植瘤模型和血行播散模型;每种细胞株每一模型5只 Balb-c裸鼠,共60只。建立皮下移植瘤模型, 观测肿瘤的生长情况并绘制肝细胞肿瘤生长曲线;建立肝癌原位移植瘤和血行播散模型,采用生物发光活体成像技术及 组织病理学方法监测造模成功率及肿瘤在肝内、肝外转移情况。结果 通过肝癌皮下移植瘤模型可观察到 AEG-1过表 达组肿瘤体积显著高于对照组,且裸鼠肝脏组织出现弥漫性侵袭转移灶;在肝癌原位移植瘤和血行播散模型中可观察到 AEG-1过表达/沉默可以导致肝癌细胞肝内转移、肺转移率和转移灶数量显著增高/降低。结论 过表达 AEG-1基因可 促进肝癌细胞生长以及定向肺转移,沉默 AEG-1基因抑制肝癌细胞生长及定向肺转移。     

多罗清泰颗粒对人肝癌细胞株信号转导基因表达的影响

目的观察多罗清泰颗粒对人肝癌细胞株SMMC-7721信号转导基因表达的影响。方法体内抑瘤实验研究多罗清泰颗粒对小鼠肝癌移植瘤生长的影响;采用中药血清药理学方法和基因芯片技术,分析多罗清泰含药血清对人肝癌细胞株SMMC-7721信号转导基因表达的影响。结果多罗清泰颗粒能显著抑制小鼠肝癌移植瘤生长,抑制率为63%~67.8%。多罗清泰颗粒对所检测的99条信号转导基因中的68条基因表达具有显著影响,占所测基因的69%。其中,上调的基因为42条,下调的基因为36条。在多罗清泰颗粒小剂量处理组和大剂量处理组之间,SMMC-7721细胞信号转导基因表达则存在相当大的差异。结论多罗清泰颗粒能显著抑制肝癌细胞的生长,显著影响肝癌细胞信号转导基因的表达。     

靶向肝癌表达p16基因的腺病毒载体对裸鼠移植瘤的抗癌活性

目的:利用已构建好的在肝癌细胞中定向表达p16基因的腺病毒载体AdAFP-p16,研究腺病毒介导p16基因的表达对肝癌裸鼠移植瘤的抗癌活性.方法:培养肝癌细胞SMMC-7721,于裸鼠右侧腹部近腋下皮肤注射1×106细胞数细胞悬液,建立裸鼠肝癌移植瘤模型.成瘤后给于瘤体内AdAFP-p16病毒注射治疗,病毒总量1×109 pfu/只,观察AdAFP-p16对肝癌模型的抗肿瘤疗效以及p16基因表达引起的肝癌细胞病理变化.结果:AdAFP-p16能够介导p16基因在肝癌细胞中特异性表达,具有明显的肿瘤生长抑制作用,抑制率可达60.12%(P＜0.01),引起肝癌细胞的坏死.结论:AFP启动子控制的p16基因在肝癌细胞中定向表达,能够抑制肝癌模型的生长,对肝癌综合治疗具有重要意义.     

野生型p53基因抑制胆囊癌细胞裸鼠体内生长的实验研究

目的：研究转染野生型p53（wtp53）基因对胆囊癌细胞裸鼠体内生长的影响。方法：在脂质体介导下将含有wtp53的真核表达质粒pCMV—p53，导入GBC—SD细胞中。用G418筛选，建立转染克隆细胞系。以PCR、RT—PCR和蛋白质印迹法证实外源p53基因的整合与表达。用裸鼠移植瘤试验检测体内致瘤性的影响。结果：野生型p53基因成功的导入胆囊癌细胞系，转染细胞中存在外源p53基因及蛋白的稳定表达。表达外源wtp53的GBC—SD—wtp53细胞，裸鼠体内致瘤性受到显著抑制。结论：野生型p53基因可有效抑制胆囊癌细胞在裸鼠体内的生长。     

人Survivin基因真核表达载体的构建及在肝癌细胞中的表达

目的:研究Survivin在肝癌细胞中的表达定位。方法:用RT-PCR的方法从肝癌组织中获取Survivin基因,并克隆到真核表达载体pEGFP-N2中,获得重组质粒pEGFP-Survivin,再转染到肝癌细胞SMMC-7721中,利用载体上的GFP跟踪其细胞内定位。结果:成功克隆了Survivin基因,经DNA测序与人Survivin基因完全一致。重组质粒pEGFP-Survivin成功转染到人肝癌细胞SMMC-7721中,并观察到GFP表达于肝癌细胞的胞浆中。结论:重组质粒pEGFP-Survivin能成功构建。     

荧光素酶标的人肝癌细胞裸鼠异种移植瘤模型的生物发光成像和PET-CT成像比较

目的利用荧光素酶基因标记的人肝癌细胞株BEL-7402建立裸鼠肝原位移植模型,及小鼠肝原位移植模型的生物发光和小动物PET-CT成像的比较。方法构建表达荧光素酶基因的真核表达载体并将其转入人肝癌细胞BEL-7402,经梯度浓度G418筛选获得稳定表达荧光素酶基因的细胞克隆并扩大培养。BALB/cA-nu裸鼠肝门静脉接种5×105个发光细胞使其成瘤,活体荧光成像和小动物PET-CT成像系统观察肿瘤的生长情况。结果获得了稳定表达Luc的人肝癌细胞株,将其接种到裸鼠体内,活体荧光成像系统观察发现能够成瘤,小动物PET-CT影像观察发现小鼠肝脏边缘对18 F-FDG有高摄取区域。结论利用荧光素酶基因标记的人肝癌细胞BEL-7402成功建立了原位肝癌裸鼠模型,小动物活体成像结合小动物PET-CT技术为原位肿瘤模型的建立提供了一种新的可靠的技术,为进一步研究肝癌生长转移机制和药物开发提供了新的有用工具。     

人胰岛素样生长因子Ⅱ型受体反义基因对肝癌细胞恶性表型的影响

目的 研究人胰岛素样生长因子Ⅱ型受体（IGF-ⅡR）反义基因对SMMC-7721人肝癌细胞恶性表型的逆转作用。方法 构建IGF-ⅡR正、反义基因真核表达质粒,用磷酸钙-DNA共沉淀的方法,导入SMMC-7721人肝癌细胞株,经G418培养基筛选稳定的抗性细胞。采用Northern杂交检测对转染细胞内源性IGF-ⅡR基因转录的抑制,观察转染细胞克隆形成率,细胞生长曲线,软琼脂生长及裸鼠致瘤能力的变化。结果 IGF-ⅡR反义基因转染肝癌细胞的内源性IGF-ⅡR基因转录受到有效抑制,转染细胞克隆形成率明显降低,并失去在软琼脂上生长的能力,裸鼠致瘤性明显降低,但细胞生长曲线无明显变化。结论 IGF-ⅡR反义基因可以有效阻断IGF-Ⅱ至IGF-ⅡR的自泌/旁泌生长刺激信号环路,一定程度地抑制肝癌细胞的恶性表型。     

hTERT基因反义cDNA转染对卵巢癌细胞系SKOV3生物学行为的影响

目的: 研究反义hTERT基因对卵巢癌细胞系SKOV3生物学行为的影响.方法: 构建反义hTERT基因的真核表达载体,采用LIPOFCTAMINETM Reagent转染卵巢癌细胞系SKOV3,通过RT-PCR、Trap-ELASA、生长曲线、裸鼠体内致瘤能力等方法比较转染前后细胞hTERT基因表达、端粒酶活性、细胞生长、致瘤性等方面的变化.结果:转化细胞系细胞hTERT基因的表达受到抑制,端粒酶活性下调,肿瘤细胞的增殖与生长速度明显降低,细胞的克隆形成能力及裸鼠体内的成瘤能力下降,肿瘤的恶性表型部分逆转.结论:应用反义技术下调hTERT基因表达可有效降低端粒酶活性并部分逆转卵巢癌细胞的恶性表型,hTERT基因可望成为卵巢癌基因治疗的新靶点.     

Nrf3基因对肝癌SMMC7721细胞系的体外研究

目的在体外检测Nrf3基因对肝癌SMMC7721的生长影响作用。方法构建其融合蛋白真核表达载体,脂质体介导该真核表达载体转染肝癌SMMC7721细胞系,FCM观察其在体外对肝癌SMMC7721细胞系细胞周期和凋亡的影响;荧光显微镜观察候选基因亚细胞定位。结果Nrf3在肝癌SMMC7721细胞中表达,荧光显微镜下观察Nrf3定位于细胞核内。FCM分析显示Nrf3影响下肝癌SMMC7721G2/M＋S期细胞所占比例较对照组明显减少,G0/G1细胞所占比例明显增加。证实Nrf3可抑制肝癌SMMC7721的DNA合成和有丝分裂,促使细胞阻滞于G0/G1期,抑制肝癌SMMC7721细胞的体外生长。FCM分析,未观察到明显的＂亚G1＂峰（即凋亡峰）,提示Nrf3在体外对肝癌SMMC7721细胞的凋亡无影响。结论Nrf3在体外具有抑制肝癌细胞增殖的功能,对肝癌细胞的凋亡无影响,为肿瘤抑制基因。     

人BNIP3真核表达载体和shRNA表达载体的构建及其对肝癌细胞自噬的影响

目的利用人BNIP3的真核表达载体和靶向BNIP3的shRNA表达载体,研究BNIP3过表达和封闭时对肝癌细胞自噬的影响。方法从人肝癌细胞系中,通过RT-PCR扩增人BNIP3基因编码区序列,酶切后插入pcDNA3.1-His-C载体,以此构建重组pcDNA3.1-BNIP3真核表达载体。同时构建靶向人BNIP3基因的shRNA表达载体pSilencer-BNIP3。分别经测序、酶切、RT-PCR和Western blot等方法对重组载体是否构建成功进行验证。并通过采用Western blot检测自噬标志物LC3蛋白的剪切,研究BNIP3过表达和抑制时对肝癌细胞自噬的影响。结果pcDNA3.1-BNIP3和pSilencer-BNIP3分别过表达BNIP3和抑制BNIP3的表达。BNIP3过表达能够显著增加肝癌细胞的自噬;BNIP3表达受到抑制时,肝癌细胞的自噬明显减少。结论成功构建了人BNIP3的真核表达载体pcDNA3.1-BNIP3和shRNA表达载体pSilencer-BNIP3,并在人肝癌细胞系中证实BNIP3对肝癌细胞自噬的促进作用。     

肿瘤相关线粒体蛋白12对移植瘤细胞增殖的影响

目的探讨肿瘤相关线粒体蛋白12(TAMP12)对小鼠移植瘤细胞增殖的影响。方法采用脂质体法将TAMP12基因及空载体转染肝癌细胞系SMMC-7721,经G418筛选获得稳定高表达TAMP12细胞株(SMMC-7721-TAMP12)及对照细胞(SMMC-7721-pcDNA3.1),实时定量PCR和蛋白印迹检测TAMP12在这两组细胞中的表达。将SMMC-7721-TAMP12和对照SMMC-7721、SMMC-7721-pcDNA3.1三组细胞分别接种于BALB/cnu/nu小鼠的前肢腋下,连续观察荷瘤小鼠中肿瘤的生长状况。接种4周后处死小鼠,取瘤块分别测量瘤体质量和体积,应用实时定量PCR和免疫组化方法检测三组移植瘤中TAMP12的表达;透射电镜观察移植瘤细胞中亚细胞器形态;并应用基因芯片技术检测三组移植瘤肿瘤细胞中的基因差异表达。结果将经实时定量PCR和蛋白印迹检测显示高表达TAMP12的SMMC-7721和对照细胞分别接种裸鼠后,三组荷瘤小鼠中肿瘤的生长曲线呈现明显差异,转染TAMP12的荷瘤小鼠中肿瘤生长速度明显快于对照组荷瘤小鼠。肉眼观察可见实验组肿瘤组织中血管较对照组丰富;电镜观察发现实验组的细胞分裂增殖明显。基因芯片显示,该基因的高表达可促进多个与细胞增殖相关的基因表达上调。结论TAMP12基因具有促进细胞增殖的作用,其机制可能与调节细胞增殖的胰岛素样受体介导的信号转导通路有关。     

Differential expression of βig-h3 in human hepatoma cell lines

Objective：To observe the differential expression of TGF-β-induced gene human clone 3 （βig- h3） in human hepatoma cell lines. Methods：Human hepatoma cells HHCC, 7721, T7721 constructed by stably transfectiug HAb18G/CD147 cDNA into 7721 and normal human liver cell QZG were cultured as previously. RT-PCR and western blot were used to investigate the differential expression of βig-h3 in human hepatoma cell lines and normal liver cell. Results ：The results of RT-PCR suggested that the expression of βig-h3 mRNA in human hepatoma cells was higher than that in normal human liver cell QZG（P〈0.01）, and its expression level in human hepatoma cells in turn was HHCC〉T7721〉7721. Moreover, the similar results of βig-h3 protein expression were testified by western blot. Conclusion：This study demonstrates that the expression of βig-h3 in hepatoma cell lines is higher than that in normal liver cell QZG, which provides a sound basis for exploring the function of βig-h3 in processes of adhesion and metastasis of human hepatoma cells.     

Down-regulation of lung resistance related protein by RNA interference targeting survivin induces the reversal of chemoresistances in hepatocellular carcinoma

Background Both survivin and lung resistance related protein （LRP） are hepatocellular carcinoma （HCC）. But the relationship between survivin and LRP is investigate the effects of down-regulation of survivin on LRP expressions and the both in vitro and in vivo. related to the chemoresistances in ndefinite. The aim of this study was to reversal of chemoresistances in HCC Methods The expressions of survivin were detected by RT-PCR and Western blotting in HCC cell line SMMC-7721 and SMMC-7721/ADM. The sensitivities of these two cell lines to ADM were evaluated by MTT assays. SiRNA which targeted survivin was transfected into SMMC-7721/ADM cells, then the sensitivity of SMMC-7721/ADM cells to ADM and the expressions of survivin and LRP were detected respectively. SMMC-7721/ADM cells were transplanted subcutaneously into nude mice to establish xenograft tumors. Antitumor activities of RNA interference （RNAi） targeting survivin, various doses of ADM and combination therapies were observed respectively. Possible toxicities were evaluated. LRP expression changes were tested. Student＇s ttest was used for evaluating statistical significance. Results The expressions of survivin in SMMC-7721/ADM cell line showed significant elevation compared to those in SMMC-7721 cell line （P 〈0.05）. Positive siRNA down-regulated the expressions of survivin significantly （P 〈0.05）. SiRNA targeting survivin could sensitize SMMC-7721/ADM cells to ADM and down-regulate the expressions of LRP significantly （P 〈0.05）. Growths of the tumors were significantly inhibited in positive siRNA group as compared with those in the control group from the 8th day （P 〈0.05）. Combination therapies caused significant tumor inhibitions compared with tumors of nude mice in the other three groups respectively （P 〈0.05）. No toxicities were found in nude mice treated by siRNA and combination therapies. The expressions of LRP were markedly reduced in tumors treated with siRNA targeting survivin （P 〈0.05）. Conclusions Down regulation of survivin gene by RNAi can increase chemosensitivity of HCC both in vitro and in vivo. The reversal of drug resistance may be reduced through the inhibitions of LRP.     

Gankyrin在肝癌细胞系中的表达

目的检测肝癌细胞系中癌基因Gankyrin的表达。为在肝癌细胞中对Gankyrin基因进行RNA干涉研究的体外实验筛选靶细胞。方法对肝癌细胞系HepG2、SMMC-7721、HHCC进行逆转录聚合酶链反应（RT—PCR）及Western blotting检测。结果Gankyrin基因在3种肝癌细胞系中均有表达。结论多种肝癌细胞系中均有Gankyrin基因的表达，HepG2、SMMC-7721、HHCC均可作为阳性靶细胞，进行Gankyrin基因的RNA干涉研究。     

全反式维甲酸对肝癌细胞连接蛋白基因表达及细胞间隙连接通讯功能的影响

目的 观察肝癌细胞株SMMC-7721和BEL-7404经全反式维甲酸(ATRA)诱导前后CX26、CX32、CX43基因在转录水平的改变以及对细胞间隙连接功能的影响。方法 分别用常规培养基和含ATRA浓度为10～mol／L的培养基培养SMMC-7721和BEL-7404两种肝癌细胞株细胞,于药物诱导细胞24、48、72h时,收集各组细胞并提取总mRNA,RT-PCR法检测CX26、CX32、CX43基因表达的情况。通过染料传输实验,观察细胞间隙连接通讯功能的变化。结果 SMMC-7721细胞和BEL-7404细胞在ATRA处理前没有CX26 mRNA和CX32 mRNA的表达,但均有CX43 mRNA的表达。经ATRA处理后出现CX26 mRNA和CX32 mRNA的表达。经ATRA处理后SMMC-7721细胞间隙连接通讯功能增强,而且随ATRA作用时间延长,细胞间隙连接通讯功能增强越明显。BEL-7404细胞经ATRA处理24、48、72h后细胞间隙连接通讯功能没有明显的改变。结论 全反式维甲酸能够在转录水平上调CX26、CX32基因在肝癌细胞SMMC-7721和BEL-7404中的表达。肝癌细胞SMMC-7721在CX26、CX32基因转录水平上调的同时伴有细胞间隙连接通讯功能的增强。肝癌细胞BEL-7404在CX26、CX32基因转录水平上调的同时不伴有细胞间隙连接通讯功能的增强。这说明两种细胞的间隙连接通讯功能调节机制不同。     

Inhibiting effect of antisense hTRT on telomerase activity of human liver cancer cell line SMMC-7721

Objective: To induce changes in biological character of human liver cancer cell line SMMC-7721 by blocking the expression of telomerase genes hTRT and to explore its value in cancer gene therapy. Methods: The vehicle for eukaryotic expression of antisense hTRT was constructed and then transfected into SMMC-7721 cells. The effects of antisense hTRT gene on telomerase activity, cancer cell growth and malignant phenotypes were analyzed. Results: The obtained transfectants that could express antisense hTRT gene stably showed marked decrease in telomerase activity;the shortening of telomere was obvious; cells presented contact growth inhibition; in nude mice transplantation, the rate of tumor induction dramatically decreased. Conclusion : Antisense hTRT gene expression can significantly inhibit telomerase activity of cancer cells and decrease malignant phenotypes in vitro and in vivo. Therefore, as a telomerase inhibitor, antisense hTRT gene may be a new pathway for cancer therapy.     

SATB1在不同侵袭潜能肝癌细胞株的表达

目的研究SATB1在不同侵袭潜能的人肝癌细胞株表达状况。方法分别用荧光定量PCR,RT-PCR,Western blotting及免
疫荧光方法,检测人永生化肝细胞株HL-7702及肝癌细胞株HepG2、SMMC-7721、MHCC97L、MHCC97H、HCCLM3 中SATB1
的表达。结果相对于HL-7702,SATB1 mRNA在其余5 种肝癌细胞株中都有较高程度的表达,其中高侵袭性HCCLM3、
MHCC97H 表达最高,MHCC97L 次之,SMMC-7721、HepG2 最低（P<0.001）;Western blotting 分析HepG2、SMMC-7721、
MHCC97L、MHCC97H、HCCLM3肝癌细胞株的蛋白相对表达量分别为0.271±0.002;0.351±0.023;0.621±0.026;0.878±0.026;
1.236±0.006,高侵袭性HCCLM3相当于HepG2的4.6倍（P<0.001）;免疫荧光显示SATB1在5种肝癌细胞株的胞浆和胞核内均
有分布,高侵袭性细胞株HCCLM3、MHCC97H表现强阳性染色。结论SATB1在不同侵袭潜能肝癌细胞株中的表达有差异,
与肝癌转移密切相关。     

Effect of TRAF6 gene silencing on hepatocellular carcinoma cell line SMCC7721 and its possible mechanism

Objective: To explore the effect of TRAF6 gene silencing on the function of hepatocellular carcinoma SMCC7721 and its possible mechanism. Method: Cell lines were constructed by cell transfection technology and verified by quantitative real-time PCR. Cell functional changes were observed by CCK8 method, Transwell test and Method of EdU. Western blotting was used to explore the possible mechanism of action. Result: TRAF6 RNA was abnormally up-regulated in HCC, and TRAF6 levels were detected in both HCC cell lines and L02 cells. SMCC7721 was selected as TRAF6 high expression cell. The results of CCK8 assay and EdU method showed that the decrease of TRAF6 expression significantly inhibited the proliferation of SMCC7721 cells. The results of CCK8 assay and EdU method showed that the decrease of TRAF6 expression significantly inhibited the proliferation of SMCC7721 cells. Overexpression of TRAF6 in TRAF6 knockdown cells can restore and enhance cell proliferation. Transwell assay confirmed that the invasiveness of SMCC 7721 cells treated with siRNA was significantly reduced. After treatment with LV-Rescue plasmid, the cell invasion was restored and enhanced. Western blotting showed that the protein levels of YB-1, Wnt, β-catenin, c-myc and Cyclin D1 were significantly down-regulated in siRNA group. On the contrary, the expression level of CYLD protein increased. Conclusion: As an important intracellular junctive protein in tumor cells, TRAF6 may improve the expression of pro-cancer factors C-myc and Cyclin D1 by modifying (ubiquitination) YB-1, thus improving the proliferation ability of cells. This process may be closely positively correlated with the Wnt/β-catenin pathway, and negatively correlated with the expression of CYLD protein.     

顺铂对肝癌细胞株HepG2增殖和SATB1表达的影响

目的:观察特异AT序列结合蛋白1(special AT-rich sequence-binding protein 1,SATB1)在不同肝癌细胞株中的表达情况,并探讨顺铂对肝癌细胞株HepG2的细胞形态及SATB1表达的影响。方法:半定量反转录聚合酶链反应(RT-PCR)检测HepG2、BEL-7402、SMMC-7721三种肝癌细胞株中SATB1 mRNA的表达情况;HepG2细胞株中加入终浓度为2.5、5.0和10.0μg/ml顺铂培养24 h,倒置显微镜下观察细胞形态变化。结果:SATB1在肝癌细胞株BEL-7402、SMMC-7721、HepG2中均有表达,差异无统计学意义(P0.05)。HepG2细胞与顺铂共培养24 h后,倒置显微镜下可见细胞形态明显改变,细胞数减少,损伤、死亡的细胞增多。SATB1 mRNA的表达随着顺铂浓度的增加而减少,其中5.0、10.0μg/ml顺铂组SATB1 mRNA的表达量与对照组差异均有统计学意义(P0.05)。结论:SATB1在三种肝癌细胞株中均有表达,顺铂可抑制HepG2细胞增殖和SATB1的表达,从而达到抑制肝癌细胞生长的目的。     

腺病毒介导的THANK基因在SMMC-7721细胞中的表达

目的:构建THANK腺病毒载体,观察腺病毒感染后THANK在SMMC-7721细胞中的表达情况.方法:把THANK基因克隆入腺病毒穿梭载体pAdTrack-CMV中,与腺病毒骨架质粒pAdEasy-1共转染大肠杆菌BJ5183,获得腺病毒重组质粒,再将获取的腺病毒重组质粒转染入293细胞进行包装,获取THANK重组腺病毒,以不同感染复数(MOI)腺病毒感染SMMC-7721细胞,观察腺病毒对SMMC-7721细胞的感染情况.结果:成功地构建了THANK腺病毒,该腺病毒可高效介导THANK在SMMC-7721细胞中表达,基因转染后第5天,THANK表达高于35 ng/ml,且随时间延长增加.结论:腺病毒可诱导THANK基因在SMMC-7721细胞中高表达.