线粒体分裂蛋白FIS1通过诱导上皮间质转化促进肝癌侵袭与迁移

目的:研究线粒体分裂蛋白1(Mitochondrial fission protein 1,FIS1)介导的线粒体分裂对肝癌细胞侵袭与迁移的调控作用与机制。方法:采用免疫组化实验比较10对肝癌原发灶与转移灶组织中FIS1表达,以明确FIS1与肝癌转移的关系。通过si RNA干扰FIS1的表达后,用Transwell实验检测肝癌细胞迁移与侵袭能力的变化,q PCR与Western Blot检测上皮间质转化标志分子上皮型钙黏蛋白(epithelia cadherin,E-cadherin)、紧密连接蛋白(zonula occludens-1,ZO-1)、神经型钙黏蛋白(neural cadherin,N-cadherin)、波形蛋白Vimentin的表达。结果:肝癌转移灶组织中FIS1的表达显著高于原发灶组织。干扰FIS1表达后,肝癌细胞迁移和侵袭能力均明显下降,细胞上皮间质转化标志蛋白E-cadherin和ZO-1的表达上调,而N-cadherin和Vimentin的表达下调。结论:线粒体分裂蛋白FIS1在肝癌转移灶组织中高表达,并可能通过调节细胞上皮间质转化促进肝癌细胞转移。     

骨桥蛋白通过调控MMP-2和VEGF参与肝癌细胞侵袭的机制研究

目的：骨桥蛋白（Osteopontin,OPN）在肝癌细胞侵袭中的作用机制。方法：采用siRNA干涉的方法处理人肝癌细胞,用PCR和Western-blot法检测OPN的表达;用transwell小室检测不同处理后的HepG2和MHCC97H细胞的侵袭能力;采用Western．b1．ot和ELISA方法检测基质金属蛋白酶-2（matrixmetalloproteinase-2,MMP-2）和血管内皮生长因子（vascularendothelialgrowthfactor,VEGF）蛋白表达和活力的变化情况。结果：在不同肝癌细胞系中,随着肝癌细胞系侵袭能力的增强,OPN的表达逐渐增高。siRNA可以降低HepG2和MHCC97H细胞中OPN的表达,并且能够降低HepG2和MHCC97H细胞的侵袭能力;抑制OPN的表达能够降低MMP-2和VEGF蛋白表达和蛋白活性。结论：OPN在肝癌侵袭过程中起着重要作用,其作用机制可能是通过调控MMP-2和VEGF蛋白表达和活性来参与肝癌的侵袭,OPN可作为肝癌侵袭转移治疗的新靶点。     

白藜芦醇苷通过抑制长链非编码RNA HULC表达抑制肝癌SMMC-7721和HepG2细胞的增殖和侵袭

原发性肝癌是临床上最常见的恶性肿瘤之一,目前仍在寻找有效的治疗手段。我们之前的研究证实白藜芦醇苷能够抑制肝癌细胞的增殖和侵袭,但其具体的分子生物学机制仍不清楚。本文主要探讨白藜芦醇苷调控肝癌细胞系SMMC-7721和HepG2肝癌细胞系增殖能力的分子生物学机制。首先,我们构建大鼠原发性肝癌模型,发现模型组肝组织中长链非编码RNA HULC的表达较正常组明显升高;同时检测白藜芦醇苷预防组（分为低剂量组,中剂量组和高剂量组）肝组织中肝癌细胞中出现异常的高表达(HULC)的表达情况。结果显示,在中、高剂量组中HULC的表达明显降低。在体外实验中,SMMC7721和HepG2中HULC的表达明显较正常肝组织显著增高,然而白藜芦醇苷高剂量组中HULC的表达发生明显降低,同时在SMMC7721和HepG2中加入白藜芦醇苷后,高剂量组中细胞增殖能力明显下降。为了进一步探究HULC在白藜芦醇苷预防肝癌中的功能,我们构建了HULC过表达质粒以及针对HULC的siRNA片段,并验证了过表达和敲低的效率。在使用高剂量白藜芦醇苷处理SMMC7721和HepG2的同时,过表达HULC能够逆转白藜芦醇苷引起的对细胞增殖的抑制,然而敲低HULC则能够更加有效地降低白藜芦醇苷对细胞增殖的抑制效果。这提示我们白藜芦醇苷能够可能通过调控HULC的表达抑制肝癌细胞的增殖和侵袭,二者具有协同作用。本文结果为预防原发性肝癌提供了新的理论依据但其临床疗效还需要进一步验证。     

PER1对人肝癌细胞BEL-7402辐射敏感性的影响

本文通过X射线照射SMMC-7721、BEL-7402和HepG2三种肝癌细胞后,以克隆形成试验检测其存活分数,结果显示在梯度剂量X射线0、2、4、6、8、10 Gy照射下SMMC-7721、BEL-7402、HepG2三种细胞克隆存活分数逐渐下降,其中SMMC-7721在三种肝癌细胞系中对辐射最敏感,BEL-7402辐射抗性在三种肝癌细胞系中最高。Western blot检测发现PER1在SMMC-7721中的表达水平明显显著高于BEL-7402和HepG2(P<0.05)。过表达PER1蛋白以后,BEL-7402接受5 Gy X射线照射后凋亡明显增多,同时,western blot和RT-qPCR试验结果发现,X射线照射过表达PER1的BEL-7402细胞,抗凋亡蛋白Bcl-2表达明显降低,凋亡执行蛋白Caspase-3断裂明显增多。研究结果表明PER1蛋白的高水平表达可以促进X射线诱导的凋亡,增强肝癌细胞的辐射敏感性。     

白花蛇舌草豆甾醇对肝癌细胞的体内外抑制作用及对其增殖周期、凋亡的影响

目的:研究白花蛇舌草豆甾醇(stigmasterol from Hedyotis diffusa willd.,SHD)对人肝癌细胞SMMC-7721、BEL-7402的体外抑制作用,对肝癌H22的体内抑制作用及对其增殖周期、凋亡的影响。方法:MTT法评价SHD对人肝癌细胞SMMC-7721、BEL-7402的抑制率变化规律。昆明雄性小鼠60只,随机取10只为正常对照组,余接种H22瘤株,随机分为模型对照组、5-FU阳性对照组(30mg/kg)和高中低剂量SHD给药组(剂量分别为15、30、60mg/kg),腹腔给药10 d后,比较各组瘤重抑制率、H22细胞周期分布、凋亡率。结果:SHD对SMMC-7721、BEL-7402细胞具有体外抑制作用;SHD显著抑制H22肿瘤,增加G0-G1期细胞比例,降低G2/M期细胞比例,促进肿瘤细胞凋亡。结论:SHD在体外、体内均具有抑制肝癌细胞的作用,此作用与阻滞肿瘤细胞增殖周期,促进肿瘤细胞凋亡有关。     

转hGM-CSF基因HepG2细胞疫苗侵袭性和生长活性初步检测

目的：探讨经60Co处理的转hGM-CSF基因的HepG2肝癌疫苗的侵袭性和生长活性变化。方法：体外培养三种肝癌细胞（①野生型HepG2肝癌细胞②转染hGM-CSF基因的HepG2肝癌细胞③60Co射线处理的转hGM-CSF基因的HepG2肝癌疫苗）采用MTT方法检测三种细胞在24h、48h、72h的OD值并绘出生长曲线;利用transwell小室进行体外侵袭实验来观察上述三种细胞侵袭性;用RT-PCR技术检测上述三种细胞基质金属蛋白酶2（MMP-2）在mRNA水平上表达的变化;结果：经60Co照射处理的转hGM-CSF基因的HepG2肝癌疫苗组OD值在相同培养时间点较其他两组明显变小且差异有显著性（P〈0.05）。三种细胞（上述①②③种细胞）transwell侵袭试验显示：③组穿过人造基底膜的细胞数量明显少于前两组;PT-PCR示：③组细胞的MMP-2的mRNA的表达明显低于①②。结论：经过60Co处理过的转hGM-CSF基因的HepG2肝癌疫苗的侵袭性和生长活性均明显降低。     

黄芩甙对人肝癌BEL-7402细胞增殖和侵袭转移的影响及机制

目的探讨黄芩甙对人肝癌BEL-7402细胞系增殖、侵袭转移的影响及其机制。方法应用细胞培养技术培养人肝癌BEL-7402细胞,MTT实验、软琼脂克隆形成实验检测黄芩甙对肝癌细胞增殖的影响。通过Boyaen小室模型测定其侵袭力,细胞迁移实验测定细胞运动能力,同时观察细胞形态。流式细胞术测定肝癌细胞MMP2、TIMP2表达,免疫组化测定VEGF表达。结果黄芩甙能明显抑制肝癌细胞增殖,细胞侵袭力及运动能力明显下降,且呈量效关系（P〈0．05）。形态学观察发现,黄芩甙处理组细胞形态较圆,伪足数目较少;MMP2阳性表达细胞减少,TIMP2阳性表达细胞增多,MMP2／TIMP2比值下降;VEGF表达减少。结论黄芩甙能抑制肝癌BEL-7402增殖、侵袭与转移,其机制可能与直接抑制细胞迁移运动,抑制细胞基质溶解相关基因蛋白MMP2表达,促进TIMP2表达;VEGF表达减少有关。     

S100A9促进肝癌细胞HepG2的存活与侵袭依赖于RAGE

目的:探讨S100A9对人肝癌细胞系HepG2生物学行为的影响及可能机制。方法:采用免疫组织化学法与Western blot方法检测人肝癌组织与癌旁组织中S100A9蛋白表达水平;原核表达重组蛋白的方法构建重组蛋白GST-S100A9,用GST-S100A9处理肝癌细胞HepG2和肝正常细胞L02,然后用MTT法检测细胞存活能力,Transwell侵袭实验检测细胞侵袭力;Western blot方法检测肝癌细胞HepG2与肝正常细胞L02中晚期糖基化终末产物受体(RAGE)的表达水平。结果:S100A9在人肝癌组织中的表达较癌旁组织显著增高;GST-S100A9可以促进肝癌细胞HepG2的存活与侵袭,但对肝正常细胞L02无作用;RAGE的表达在HepG2细胞中较在L02细胞中显著升高;RAGE阻断抗体可阻断GST-S100A9对HepG2细胞的促存活与促侵袭作用,表明这些作用是通过RAGE介导的。结论:S100A9促进肝癌细胞HepG2的存活与侵袭依赖于RAGE。     

糖基因ST6GAL家族在肝癌转移中的作用

目的通过研究糖基因ST6GAL家族在人肝癌高转移细胞株MHCC97-H和人肝癌低转移细胞株MHCC97-L中的差异表达,明确糖基因ST6GAL家族与肝癌转移的相关性,从而确证肝癌转移诊断及抗肿瘤治疗新靶点。方法采用Real-time PCR、Western Blot分析糖基因ST6GAL家族在人肝癌高、低转移细胞株的差异表达;通过RNA干扰技术干预差异表达的糖基因,检测干扰前后MHCC97-H细胞的体外侵袭能力及体内成瘤性。结果糖基因ST6GAL1在人肝癌高、低转移细胞株中表达差异具有统计学意义,而ST6GAL2的表达差异具无统计学意义;当通过RNA干扰技术特异性使MHCC97-H细胞中ST6GAL1表达下调时,该细胞在体外的侵袭能力下降及体内成瘤性受到抑制(P﹤0.05)。结论人肝癌细胞中糖基因ST6GAL1的差异表达与肿瘤细胞的侵袭、成瘤性密切相关,为肿瘤的化学治疗提供新靶点。     

人参皂苷IH901抗肝癌生长及其侵袭转移的作用机理

人参皂苷IH901是一种天然二醇组人参皂苷肠道细菌最终代谢产物.目前研究表明,IH901具有抗肝癌活性,然而其作用机理尚不清楚.采用人肝癌细胞株SMMC-7721,HepG2,MHCC97-H来建立体内外的抗肿瘤模型,以探讨人参皂苷IH901的抗肝癌活性及其作用机理.结果发现,人参皂苷IH901能够通过细胞周期中亚二倍体峰的增加、线粒体跨膜电位（ΔΨm）崩溃、DNA梯形条带的形成和提高Caspase-9/Caspase-3的表达量、促进凋亡来抑制肝癌细胞的生长.通过抑制细胞的迁移和降低VEGF/bFGF的表达来抑制肝癌细胞的侵袭转移.裸鼠体内实验均表明了人参皂苷在体内外的抗肝癌活性,并且在分子、细胞、动物水平初步揭示了IH901抗肝癌生长及其侵袭转移的作用机理,为其作为抗肝癌药物的开发提供理论支持.     

Inhibition of the growth of human hepatoma cell line both in vitro and in vivo by transducing CKI gene p21~(WAF-1) with GE7 targeting gene delivery system

The EGF receptor-mediated targeting gene delivery system GE7 was used to transduce exogenous gene pCEP-p21WAF-1 into human hepatocellular carcinoma cell both in vitro and in vivo. After in vitro transduction of the exogenous gene, the growth of the cell lines SMMC-7721 and BEL-7402 was significantly inhibited compared with the control. On day 8 the inhibition rates of the above cell lines reached 56.0% and 66.7%, respectively. The in vivo experiment showed that the growth of human hepatoma transplanted in nude mice injected with GE7 gene delivery system subcutaneously once a week for 3 weeks was remarkably inhibited compared with that of untrans-fected control. The average tumor weight of the experiment group was (0.083 ?0.043) g, while that of the control group was (0.28110.173) g. The difference is significant (P<0.05). It was indicated that GE7 gene delivery system could efficiently transduce exogenous gene pCEP-p21WAF-1 into hepatoma cell with high EGF receptor expression, and inhibit the cell growt     

In vitro panning of a targeting peptide to hepatocarcinoma from a phage display peptide library

Phage display technology has been used as a powerful tool in the discovery of ligands specific to receptor(s) on the surface of a cancer cell and could also impact clinical issues including functional diagnosis and cell-specific drug delivery. After three rounds of in vitro panning and two rounds of reverse absorption, a group of phages capable of addressing BEL-7402 enormously were obtained for further analysis. Through a cell-based ELISA, immunofluorescence, FACS, and in vivo binding study, WP05 (sequence TACHQHVRMVRP) was demonstrated to be the most effective peptide in targeting four kinds of liver cancer cell lines (BEL-7402, BEL-7404, SMMC-7721, and HepG2), but not the normal liver cell line HL-7702. In conclusion, the peptide WP05 which was screened by in vitro phage display technology was proved to be a targeting peptide to several common hepatocellular carcinoma cell lines.     

Inhibition of the growth of human hepatoma cell line both in vitro and in vivo by transducing CKI gene p21WAF-1 with GE7 targeting gene delivery system

The EGF receptor-mediated targeting gene delivery system GE7 was used to transduce exogenous gene pCEP-p21WAF-1 into human hepatocellular carcinoma cell both in vitro and in vivo. After in vitro transduction of the exogenous gene, the growth of the cell lines SMMC-7721 and BEL-7402 was significantly inhibited compared with the control. On day 8 the inhibition rates of the above cell lines reached 56.0% and 66.7%, respectively. The in vivo experiment showed that the growth of human hepatoma transplanted in nude mice injected with GE7 gene delivery system subcutaneously once a week for 3 weeks was remarkably inhibited compared with that of untransfected control. The average tumor weight of the experiment group was (0.083 ± 0.043) g, while that of the control group was (0.281± 0.173) g. The difference is significant (P<0.05). It was indicated that GE7 gene delivery system could efficiently transduce exogenous gene pCEP-p21WAF-1 into hepatoma cell with high EGF receptor expression, and inhibit the cell growth with high efficacy both in vivo and in vitro.     

BIX-01294对肝癌细胞周期、凋亡及移植瘤的影响

目的:探究组蛋白甲基转移酶G9a抑制剂(BIX-01294)对肝癌细胞周期、凋亡及移植瘤的影响.方法:将SMMC-7721、BEL-7402、HL-7702原始细胞株传代培养后,分为空白对照组和不同浓度(1 μM、5 μM、10 μM、20 μM)BIX-01294处理组.应用Western-blot法检测G9a及肝癌...     

Osteopontin promotes hepatocellular carcinoma invasion by up-regulating MMP-2 and uPA expression

Osteopontin (OPN) is over-expressed in a variety of cancers, but its role in hepatocellular carcinoma (HCC) progression has not been clarified. In this study, weakly tumorigenic, non-metastastic human HCC cell line SMMC-7721 cells were forced to over-express OPN via stable transfection. A series of functional assays were performed to assess the effects of OPN on tumor cell behaviors and cDNA microarray was used to identify the genes regulated by OPN. The results showed that OPN significantly enhanced the migration and invasion of SMMC-7721 cells in vitro. In addition, CD44v6 antibody could significantly inhibit the invasion of OPN over-expressing SMMC-7721 cells. Moreover, MMP-2 and uPA expressions were significantly up-regulated in OPN over-expressing SMMC-7721 cells. Together, these findings indicate that OPN enhanced HCC cells invasion through interaction with its receptor CD44v6 and increased MMP-2 and uPA expressions, providing at least one mechanism for OPN-mediated HCC progression and metastasis.     

Reversal effect of 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone on multi-drug resistance in resistant human hepatocellular carcinoma cell line BEL-7402/5-FU

Multi drug resistance (MDR) is a major obstacle in the chemotherapeutic treatment of many human cancers. 2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethylchalcone (DMC), a chalcone, isolated from the buds of Cleistocalyx operculatus, has been shown to have antitumor effects on human carcinoma SMMC-7721 cells in vitro and in vivo. In this paper, we studied the reversal effect and the mechanism of DMC on human hepatocellular carcinoma drug-resistant cells BEL-7402/5-FU in vitro. Administration of DMC reversed the multi-drug resistance of human hepatocellular carcinoma BEL-7402/5-FU cells significantly. DMC enhanced the sensitivity of BEL-7402/5-FU cells to 5-fluorouracil (5-FU) and doxorubicin (DOX). Staining with Hoechst 33258 and flow cytometric analysis showed that DMC has apoptosis-inducing effect on BEL-7402/5-FU cells. It could also increase the concentration of 5-FU in the resistant multi-drug-resistant cells. We also observed that over-expression of the multi-drug resistance-associated protein (MRP1) and of the glutathione S-transferase π (GST-π) contributed to MDR in BEL-7402/5-FU cells. The mRNA expressions of MRP1 and GST-π and the protein expression of MRP1 were decreased by DMC. These data demonstrated that DMC could effectively reverse MDR in BEL-7402/5-FU cells.     

沉默EVL表达抑制肝癌细胞SMMC-7721的增殖和迁移能力

Ena/VASP 样蛋白（Ena/VASP like protein,EVL）是Ena/VASP家族成员之一,它参与肌动蛋白细胞骨架重组,以及细胞迁移、收缩环形成和细胞间附着.EVL在肝癌SMMC-7721细胞中高表达. 抑制EVL蛋白表达后,SMMC-7721细胞的增殖与迁移能力降低.为研究EVL在肝癌细胞的功能,构建了靶向shRNA干扰表达载体,稳定转染肝癌SMMC-7721细胞. MTT实验和细胞集落形成实验显示,与转染对照比较,沉默EVL蛋白表达可明显抑制SMMC-7721肝癌细胞的增殖、集落形成能力. Transwell实验证明,沉默EVL表达导致SMMC-7721细胞迁移能力降低. 进而,流式细胞术揭示,沉默EVL表达的SMMC-7721细胞G0/G1期细胞比例增多.研究结果提示,EVL蛋白可促进肝癌细胞的增殖与迁移;该结果可解释EVL在肝癌细胞中高表达的意义.