新基因pp3774的亚细胞定位以及在肝癌细胞系和组织中的表达谱研究

目的探讨新基因pp3774在肝癌细胞系、人肝癌及癌旁肝、肝硬化以及正常肝组织中的表达差异及其生物学功能.方法脂质体转染-荧光观察pp3774-GFP融合蛋白的亚细胞定位;合成新基因pp3774特异性多肽,制备兔源性多克隆抗体;用RT-PCR、蛋白印迹、免疫细胞化学及免疫组化方法检测pp3774基因在7个肝癌细胞系及214例肝细胞癌、18例肝硬化及10例正常肝组织中的表达差异.流式细胞术检测转染pp3774基因的SMMC-7721细胞的凋亡率.结果 pp3774蛋白主要定位于细胞质(内质网为主);RT-PCR检测结果表明pp3774 mRNA在肝癌细胞系BEL-7402、Huh-7、MHCC-97L及HepG2中高表达,SMMC-7721、MHCC-LM3及人永生化肝细胞系L-02中度表达,而在Hep3B中低表达;肝癌及癌旁组织中pp3774 mRNA的表达表现为癌旁高于癌(P<0.05);免疫组化结果显示pp3774蛋白表达程度依次为正常肝≥肝硬化≥癌旁肝组织>肝癌.转染pp3774基因的SMMC-7721细胞的凋亡发生率高于对照组约10%.结论 pp3774基因是一个具有抑制肝癌细胞生长功能的新基因.     

干扰PRMT2基因对肝癌细胞系生物学行为的影响

目的:研究抑制蛋白质精氨酸甲基转移酶2(protein arginine methyltransferase 2,PRMT2)基因的表达对肝癌细胞系生物学行为的影响。方法:qRT-PCR检测人肝癌细胞系(HepG2和SMMC-7721)和人正常肝细胞(LO2)中PRMT2的表达情况;设计并合成针对PRMT2基因的3对小干扰RNA,体外通过脂质体Lipofectamine 3000转染到相对高表达PRMT2的人肝癌细胞系HepG2和SMMC-7721中抑制PRMT2基因的表达,并设置阴性对照组（NC组）和空白对照组。qRT-PCR、Western blot检测转染后细胞PRMT2 mRNA水平和蛋白水平的变化。CCK-8法检测转染后细胞增殖能力,Transwell小室检测细胞体外迁移及侵袭能力。结果:与人正常肝细胞LO2相比,PRMT2基因在人肝癌细胞系HepG2和SMMC-7721中相对高表达。将siPRMT2转染HepG2和SMMC-7721细胞后,与NC组相比,siPRMT2-1和siPRMT2-2组的PRMT2 mRNA和蛋白表达均明显降低（P均<0.05）,NC组与空白对照组无明显差异。与NC组相比,将siPRMT2-1和siPRMT2-2转染进HepG2和SMMC-7721细胞后,细胞增殖速度减慢、迁移和侵袭能力显著减弱（P均<0.05）,NC组与空白对照组无明显差异。结论:RNA干扰抑制PRMT2表达后,抑制肝癌细胞的增殖、迁移和侵袭,PRMT2可能影响肝癌的预后。     

hAph2β和c-abl在肝细胞癌中的表达及其相互作用

目的:探讨hAph2β蛋白与c-abl蛋白在肝细胞癌(hepatocellular carcinoma,HCC)中的相互作用.方法:RT-PCR和Western 印迹法分析c-abl基因和hAph2基因在肝癌细胞株及永生化人肝细胞株中的表达.脂质体转染、免疫共沉淀和Western印迹法检测hAph2β与c-abl在SMMC-7721细胞中的相互作用.激光共聚焦-荧光显微镜观察hAph2β和c-abl在SMMC-7721细胞中的定位.用组织芯片结合免疫组织化学法检测hAph2β和c-abl蛋白在肝癌和相应癌旁组织中的表达差异,统计学分析其表达差异的临床意义.结果:RT-PCR检测结果表明,c-abl mRNA在HepG2、L-02及MHCC-LM3细胞中高表达,在SMMC-7721、BEL-7402、Hep3B和Chang's liver细胞中中度表达,而在Huh-7、MHCC-97L细胞中低表达.Western印迹法检测结果发现,hAph2总蛋白在HepG2、Huh-7、MHCC-LM3和MHCC-97L细胞中高表达,在BEL-7402、L-02细胞中低表达;c-abl蛋白在Huh-7细胞中低表达.免疫共沉淀观察发现SMMC-7721细胞中过表达的hAph2β-GFP和内源性c-abl相互作用.荧光免疫细胞化学检测发现hAph2β和c-abl在SMMC-7721细胞中部分共定位.免疫组织化学检测结果表明,hAph2β/c-abl蛋白在人正常肝组织、硬化肝组织、癌旁肝及原发性HCC癌组织中的强阳性率分别为:100%/50%、91.7%/100%、100%/89.5%和50.9%/19.3%,其中hAph2β和c-abl在癌旁肝组织中的表达均明显高于肝癌组织(均P<0.001),2者在56.1%(32/57)的肝癌组织中同时高或低表达.结论:hAph2β和c-abl在SMMC-7721细胞中相互作用,在原发性HCC及相关组织中共表达.     

肝细胞癌高表达基因TPT1转染对肝细胞系生物学行为的影响

目的 将TPT1转染肝癌细胞系SMMC-7721和肝细胞系L-02,观察肝细胞系生物学行为的变化.方法 将连有TPT1的、带有绿色荧光蛋白标签的真核报告表达载体pEGFP-N3TPT1,通过Lipofectamine分别转染SMMC-7721和L-02细胞,同时设pEGFP-N3对照.采用逆转录聚合酶链反应(RT-PCR)检测TPT1 mRNA水平,四甲基偶氮唑蓝(MTT)法、软琼脂克隆形成实验、细胞周期分析检测细胞增殖和致瘤性.结果 pEGFP-N3TPT1转染后,SMMC-7721细胞TPT1 mRNA相对表达水平与对照组的比值为1.78,L-02细胞TPT1 mRNA相对表达水平与对照组的比值为2.59.pEGFP-N3TPT1转染可明显增强肝细胞的增殖能力,转染SMMC-7721后24、48、72、96h的吸光度(A)值分别为0.80、1.56、2.69和2.94,均高于pEGFP-N3(分别为0.71、1.23、1.92和2.75)和脂质体对照(分别为0.76、1.27、1.89和2.78),差异均有统计学意义(均P＜0.05).在接种500个和1000个细胞时,pEGFP-N3TPT1组软琼脂克隆形成数分别为(6.00±1.73)个和(12.00±2.65)个,pEGFP-N3组软琼脂克隆形成数分别为(1.00±1.00)个和(7.00±1.00)个(P＜0.05).pEGFP-N3TPT1转染后的SMMC-7721细胞增殖指数(P1)为40.9%,高于pEGFP-N3组(26.4%).EGFP-N3TPT1转染正常肝细胞L-02后,24、48、72、96、120 h测得的A值分别为0.87、1.11、1.55、2.12和2.42,均高于pEGFP-N3组(分别为0.65、0.79、1.02、1.49和1.96)和脂质体组(分别为0.67、0.82、0.89、1.56和1.85),差异有统计学意义(P＜0.05);pEGFP-N3TPT1组平板克隆数为(18.00±2.00)个,pEGFP-N3组为(7.00±2.65)个(P＜0.05),但L-02细胞不能在软琼脂中形成克隆.pEGFP-N3TPT1转染后L-02细胞的PI为35.9%,高于pEGFP-N3组(26.1%).结论 TPT1转染可增强肝癌细胞SMMC-7721的恶性表型,促进正常肝细胞L-02的增殖能力,但不能使L-02发生恶性转化.

Abstract：

Objective The aim of this study was to transfect TPT1 into cell lines SMMC-7721 and L-02, seperately, and to observe the changes of biological behaviors of the cell lines. Methods Through lipofectamine, the eukaryotic report expression vector containing TPT1 ORF( open reading frame), pEGFP-N3TPT1, were transducted into hepatocarcinoma cell line SMMC-7721 cells and normal liver cell line L-02 cells, seperately. The transduction was repeated three times in 24 hrs. The differences of biological behaviors between the pEGFP-N3TPT1 and pEGFP-N3 groups were studied by RT-PCR, MTT assay, soft agar colony formation assay and cell cycle analysis. Results The pEGFP-N3TPT1 transfected cells had a high mRNA level in the two cell lines ( P ＜ 0. 05 ) compared with the pEGFP-N3 controls. The ability of proliferation and the soft agar colony formation were enhanced in the SMMC-7721 transducted cells with pEGFP-N3TPT1 compared with that transducted with pEGFP-N3 ( P ＜ 0.05 ), and the cell cycle analysis showed that the cells in the phase G2 + S/M increased after pEGFP-N3TPT1 transduction. In the L-02 cell line, we obtained similar results, pEGFP-N3TPT1 enhanced the colony formation in plate( P ＜0.05 ), but not make it form colony in soft agar. Conclusions TPT1 can enhance malignant phenotype of SMMC-7721 cells and promote the growth of L-02 cells, but not transform L-02 into malignant phenotype.     

PDCD4对肝癌细胞生物学功能的影响机制及其在化疗敏感性中的作用

目的 探讨程序性细胞死亡-4(PDCD4)对肝癌细胞生物学功能的影响机制及其在化疗敏感性中的作用.方法 应用qRT-PCR和Western Blot检测肝癌细胞HepG2、SMMC-7721、MHCC97H和正常的肝细胞L02中PDCD4的水平.采用肝癌细胞HepG2为研究对象,将转染PDCD4过表达载体(pDsRed2-N1-PDCD4)的HepG2细胞设置为PDCD4过表达组;将60μmol/L替莫唑胺作用24 h的HepG2细胞设置为替莫唑胺组;将正常肝癌细胞HepG2(不加替莫唑胺)设置为对照组;将转染pDsRed2-N1-PDCD4载体后经60μmol/L替莫唑胺作用24 h的HepG2细胞设置为联合组.采用噻唑蓝(MTT)法检测各组细胞的存活率,流式细胞仪检测细胞凋亡情况,West-ern Blot检测细胞中Bax、Bcl-2、MMP-9表达水平.结果 PDCD4在肝癌细胞HepG2、SMMC-7721、MHCC97H中的表达水平均低于正常的肝细胞L02(P<0.01),肝癌细胞HepG2中的PDCD4表达水平最低;PDCD4过表达组、替莫唑胺组、联合组的肝癌细胞存活率均明显低于对照组(P<0.01);PDCD4过表达组、替莫唑胺组、联合组的肝癌细胞凋亡率均明显高于对照组(P<0.01);PDCD4过表达组、替莫唑胺组、联合组肝癌细胞中Bax的表达水平均明显高于对照组(P<0.01);Bcl-2、MMP-9的表达水平均明显低于对照组(P<0.01);联合组肝癌细胞中Bax的表达水平明显高于PDCD4过表达组和替莫唑胺组(P<0.01).结论 PDCD4在肝癌细胞中低表达,PDCD4可抑制肝癌细胞的增殖,促进肝癌细胞的凋亡,增加肝癌细胞的化疗敏感性,其作用机制可能与Bax、Bcl-2、MMP-9有关.     

lncRNA PTPRG-AS1通过靶向miR-124-3p调控肝癌细胞凋亡和放射敏感性的研究机制

目的:探讨lncRNA PTPRG-AS1对肝癌细胞凋亡和放射敏感性的影响及作用机制。方法:培养正常肝细胞L02和肝癌细胞HepG2、SMMC-7721和BEL-7402,qRT-PCR检测细胞中PTPRG-AS1和miR-124-3p表达水平。转染si-PTPRG-AS1、miR-124-3p mimics至HepG2细胞,抑制HepG2细胞中PTPRG-AS1表达或过表达miR-124-3p,流式细胞术检测细胞凋亡,克隆形成实验检测细胞放射敏感性,Western Blot法检测Bcl-2和Bax蛋白表达。生物信息学软件预测PTPRG-AS1与miR-124-3p存在互补的核苷酸序列,双荧光素酶报告基因实验验证PTPRG-AS1与miR-124-3p之间的调控关系。结果:与正常肝细胞L02相比,肝癌细胞HepG2、SMMC-7721和BEL-7402中PTPRG-AS1表达显著升高（P＜0.05）,miR-124-3p表达显著降低（P＜0.05）。抑制PTPRG-AS1或过表达miR-124-3p均可促进肝癌HepG2细胞的凋亡,增强肝癌HepG2细胞的放射敏感性,抑制Bcl-2蛋白表达,促进Bax蛋白表达。PTPRG-AS1负调控miR-124-3p表达。抑制miR-124-3p表达可部分逆转抑制PTPRG-AS1表达对肝癌HepG2细胞凋亡的促进作用以及放射敏感性的增强作用。结论:抑制PTPRG-AS1表达可能通过上调miR-124-3p表达促进肝癌细胞的凋亡并提高其放射敏感性,是肝癌治疗的潜在作用靶点。     

miR-526b-3p通过靶基因TNKS2调控肝癌细胞增殖、侵袭、迁移的分子机制探讨

目的:探讨miR-526b-3p对肝癌细胞增殖、迁移及侵袭的影响及其潜在的作用机制。方法:运用qRT-PCR检测人肝癌细胞HepG2、SMMC-7721、BEL-7402和正常肝细胞L02中miR-526b-3p和TNKS2的mRNA表达水平。建立miR-526b-3p过表达和TNKS2抑制表达的HepG2细胞株,采用MTT法检测细胞增殖活力,Transwell小室检测细胞的迁移及侵袭能力,Western blot检测TNKS2蛋白的表达水平;双荧光素酶报告基因分析法验证miR-526b-3p可能的靶基因。结果:与正常肝细胞L02相比,肝癌细胞HepG2、SMMC-7721及BEL-7402中miR-526b-3p的表达显著降低（P<0.05）,TNKS2的表达显著升高（P<0.05）。过表达miR-526b-3p或抑制表达TNKS2均可抑制HepG2细胞的增殖、迁移及侵袭（P<0.05）。TNKS2是miR-526b-3p的靶基因。过表达TNKS2可部分逆转过表达miR-526b-3p对HepG2细胞增殖、迁移及侵袭的抑制作用。结论:miR-526b-3p可通过下调TNKS2的表达,从而抑制肝癌细胞的增殖、迁移及侵袭能力。     

5/35嵌合型溶瘤腺病毒SG635对肝癌细胞的抑制作用

目的:研究5/35嵌合型溶瘤腺病毒SG635在体外对肝癌HepG2和SMMC-7721细胞的特异性杀伤作用。方法:将SG600载体中5型腺病毒(Ad5)纤毛蛋白的knob和shaft结构域替换为35型腺病毒(Ad35)纤毛蛋白的相应结构域,构建成5/35嵌合型溶瘤腺病毒SG635。流式细胞术检测5/35嵌合型腺病毒Ad5/35-EGFP对HepG2和SMMC-7721细胞的感染效率,体外病毒增殖实验观察溶瘤腺病毒SG635的增殖能力,Western blotting检测SG635感染后肝癌细胞中E1A蛋白的表达,CCK-8实验检测SG635对肝癌HepG2和SMMC-7721细胞的杀伤作用。结果:在肝癌HepG2和SMMC-7721细胞中,Ad5/35-EGFP的感染效率明显强于5型腺病毒Ad5-EGFP;5/35嵌合型溶瘤腺病毒SG635在HepG2和SMMC-7721细胞中72 h的增殖倍数高于5型溶瘤腺病毒SG600(15 848.93vs6 309.57,6 309.57vs5 011.87,均P<0.01),而在人正常成纤维细胞BJ中几乎不增殖。SG635感染后,HepG2和SMMC-7721细胞中E1A蛋白表达高于SG600感染,在BJ中则无E1A表达。在一定MOI范围内,SG635对于HepG2细胞和SMMC-7721细胞的杀伤作用逐渐增强,且杀伤率明显强于SG600(MOI为1时,90%vs60%;MOI为10时,90%vs50%),对BJ无杀伤作用。结论:5/35嵌合型溶瘤腺病毒SG635能够高效感染并特异性杀伤肝癌细胞,具有较好的靶向性和安全性。     

死亡受体5与肝癌细胞凋亡的相关性

目的探讨人死亡受体5(DR5)及其配体(TRAIL)和竞争性单克隆抗体(DR5mAb)对肝癌细胞凋亡的影响。方法采用半定量RT-PCR及Western blot法检测肝癌细胞株HepG2、SMMC7721和正常人肝细胞株LO2的DR5在mRNA和蛋白水平的表达含量。应用四甲基偶氮唑盐法测试细胞的生长曲线,以观察TRAIL和DR5mAb对肝癌细胞生长的抑制作用,并用流式细胞仪测定细胞的凋亡率。结果肝癌细胞株HepG2、SMMC7721的DR5高表达,正常人肝细胞株LO2的DR5低表达,差异有统计学意义(P<0.05)。肝癌细胞的增殖率随着TRAIL浓度的增加而逐渐降低,但当TRAIL超过1000ng/ml时,肝癌细胞株的增殖率就不再下降;而肝癌细胞的增殖率随着DR5mAb浓度的增加而逐渐降低,呈现明显的剂量依赖关系。1000ng/ml TRAIL处理24h时,HepG2的凋亡率在14.74%±0.48%,继续增加TRAIL浓度,其凋亡率无显著改变;而同样剂量的DR5mAb处理24h时,HepG2的凋亡率高达52.45%±0.57%,明显高于前者(P<0.05)。同时发现,正常人肝细胞株LO2的增殖率随着TRAIL浓度的增加呈下降趋势,与PBS阴性对照组差异有统计学意义(P<0.05);而DR5mAb的浓度变化对人正常肝细胞株LO2无明显影响。结论死亡受体DR5在诱导肝癌细胞凋亡中起重要的作用;DR5mAb选择性地诱导肝癌细胞凋亡,对人正常肝细胞无诱导凋亡作用。     

TPX2 knockdown suppressed hepatocellular carcinoma cell invasion via inactivating AKT signaling and inhibiting MMP2 and MMP9 expression

Objective： Targeting protein for Xenopus kinesin-like protein 2 （TPX2） is a nuclear proliferation-related protein that plays a critical role in the formation of mitotic spindle. High expression of TPX2 has been observed in several types of tumors. However, the role of TPX2 in hepatocellular carcinoma （HCC） remains unclear. Our study aimed to investigate the effect of TPX2 on HCC cell invasion. Methods： The immortalized normal human liver cell line L02 and six HCC cell lines including SMMC- 7721, BEL-7402, Huh-7, HepG2, Hep3B and SKHepl were subjected to qRT-PCR and western blot for TPX2 mRNA and protein, respectively. Furthermore, TPX2 small interfering RNA （siRNA） was used to knock down TPX2 expression in SMMC-7721 and HepG2 cells. Cell proliferation and invasion were determined by MTT and transwell assays. Otherwise, expression of p-AKT, MMP2 and MMP9 were evaluated by western blot in SMMC-7721 cells. Results： The expression of TPX2 in HCC cell lines was markedly higher than that in normal human liver cell line. TPX2 knockdown using a specific TPX2-siP, NA reduced the number of invaded cells and inhibited cell proliferation in SMMC-7721 and HepG2 cells. Furthermore, TPX2 knockdown resulted in inactivation of AKT signaling and down-regulation of MMP2 and MMP9 expression in SMMC-7721 cells. Conclusions： Our study identified that TPX2 might contribute to tumor cell invasion through activating AKT signaling and subsequently increasing MMP2 and MMP9 in HCC.     

可溶性肿瘤坏死因子相关凋亡诱导配体诱导肝癌细胞凋亡的研究

方法 采用原位杂交方法检测肝癌组织、肝癌细胞株以及正常肝组织中TRAILR的表达。采用不同浓度TRAIL蛋白处理肝癌细胞株Hep2和SMMC7721,应用流式细胞仪和原位末端标记,观察经药物处理前后该细胞株的凋亡发生率。结果 60例肝癌组织及20例正常肝组织均表达死亡受体DR5和DR4,但肝癌组织DR表达量显著强于正常肝组织。54例(90．0％)肝癌组织不表达诱捕受体DcR1,25例(41．7％)肝癌组织不表达DcR2,而20例正常肝组织均表达DcR。肝癌组织中DR的高表达及DcR的低表达,不同于正常肝组织中DR的低表达及DcR的高表达,两者间差异有显著性。两种肝癌细胞株中均可检测到DR5、DR4、DcR2的表达,但DcR1表达缺失。肝癌组织中DR的表达与肿瘤的分化、肿瘤分期有关,低分化的肿瘤DR表达减少(P＜0．01),Ⅲ、Ⅳ期肿瘤DR表达显著低于I、Ⅱ期(P＜0．05)。DR表达与患者的性别、年龄、HBsAg阳性与否、AFP水平、肿瘤大小以及是否转移无关。经TRAIL(100ng/ml)处理24h,肝癌细胞凋亡发生率约10％,而Jurkat细胞凋亡率达70％以上,胆管癌细胞QBC939凋亡发生率约50％。结论 肝细胞肝癌普遍存在TRAILR的表达,并存在受体类型的表达差异。但单一的TRAIL治疗只能有限的诱导肝癌细胞HepG2、SMMC7721发生凋亡,HCC对TRAIL诱导的凋亡存在耐药现象。     

Increased expression of IL-6 mRNA in hepatocellular carcinoma cell lines correlates with biological characteristics

Background & aims: IL-6 has been implicated in both virus-associated and diethylnitrosamine-induced hepatocellular carcinomas (HCCs). Generally it is produced by immune cells such as Kupffer cells inthe liver. To understand mechanisms by which IL-6 might participate in the genesis of HCCs, the production of IL-6 by cell lines under different conditions was examined to determine inducing factors Methods: Expression of IL-6 mRNA in both hepatoma cell lines and a normal liver cell line L-02 was measured by quantitative RT-PCR. Biological molecules including liposome, dsRNA and cell debris were used to stimulate IL-6 mRNA expression in HepG2 cells and inhibition was effected by RNAi. Proliferation was assessed by MTT and clone formation and migration was determined by scratch assay. Results: All of the HCC cell lines observed expressed IL-6 mRNA, including HepG2, Bel-7402(7402), MHCC-97H and SMMC-7721.Normal liver cell line L-02 also expressed IL-6 mRNA. SiRNA to IL-6 specifically knockdowned IL-6 mRNA expression in HepG2, and liposome, dsRNA and cell debris increased it. Both proliferation and migration of HepG2 cells were related to the level of IL-6 HepG2 expressed. Conclusion: Both normal liver cell line and HCC cell lines can produce IL-6 so that Kupffer cells are noit the only source of the cytokine in the liver well as other immune cells. That the fact that HCC cells reacted to stimulation of biological molecules such as liposome, dsRNA or cell debris with increasing production of IL-6 indicates that the cytokine might play an important role not only in the period of tumor initiation but progression and recurrence as well.     

RNA干扰SMYD3基因表达对诱导肝癌细胞凋亡的影响

背景与目的:SMYD3(SETandMYNDdomain-containingprotein3)基因的表达蛋白是一种组蛋白甲基转移酶,参与肿瘤细胞增殖与凋亡的调控。本研究旨在探讨利用RNA干扰(RNAinterference,RNAi)抑制SMYD3基因表达对肝癌细胞增殖与凋亡的影响。方法:RT-PCR、免疫组织化学法分别检测SMYD3在肝癌细胞和肝癌组织中的表达。构建小发夹状RNA(smallhairpinRNA,shRNA)干扰质粒Pgenesil-1-s1、Pgenesil-1-s2及无干扰效应质粒Pgenesil-1-hk并转染入肝癌细胞HepG2,阻抑其表达SMYD3,以空质粒Pgenesil-1转染组为对照。Westernblot检测阻抑效应;MTT检测细胞增殖抑制率,流式细胞术及TUNEL检测细胞凋亡。结果:SMYD3在肝癌组织和多种肝癌细胞中表达明显增强。shRNA转染HepG2细胞后:SMYD3蛋白表达下调75%～85%;细胞增殖明显受抑制,抑制率高达60.95%～72.14%;流式细胞术结果显示Pgenesil-1-s1组HepG2细胞凋亡率(17.68±2.36)%、Pgenesil-1-s2组(19.07±1.78)%,均显著高于Pgenesil-1-hk组[(1.44±0.28)%]及Pgenesil-1组[(0.47±0.12)%](P<0.01);TUNEL检测的凋亡指数结果与流式细胞术检测结果类似。结论:SMYD3高表达于多种肝癌细胞及肝癌组织;RNAi能特异性下调SMYD3的表达,抑制肝癌细胞增殖并促进细胞凋亡,提示其可能为治疗肝癌提供新的途径。     

缺氧诱导肝癌细胞获得干细胞特性的初步实验研究

目的:观察缺氧对肝癌干细胞特性的影响.方法:用肝癌细胞系HepG2、SMMC-7721建立细胞缺氧培养模型,利用倒置显微镜观察细胞形态学变化;克隆形成实验检测肿瘤细胞克隆形成能力;"肿瘤球"形成实验检测肿瘤细胞自我更新能力;实时定量RT-PCR和Western blot检测干细胞相关基因Oct-4、Sox-2、Nanog mRNA和蛋白表达;采用流式细胞术检测干细胞表面标志物CD133表达.结果:缺氧条件下HepG2、SMMC-7721细胞克隆形成率显著高于常氧组(P<0.05).缺氧条件下HepG2、SMMC-7721细胞肿瘤球形成率显著高于常氧组(P<0.05).常氧条件下HepG2、SMMC-7721细胞中干细胞相关基因Oct-4、Sox-2、Nanog mRNA及蛋白表达水平极低,缺氧处理48 h后,其mRNA及蛋白表达水平明显增高(P<0.05).缺氧条件下HepG2、SMMC-7721细胞CD133阳性表达率表达量显著增加(P<0.05).在缺氧36 h和48 h,CD133蛋白表达水平显著增强(P<0.05).结论:缺氧能诱导肝癌细胞干细胞特性的获得.     

CISD2在肝细胞肝癌组织和细胞中的表达及其临床意义

目的:探究CDGSH铁硫结构域2(CISD2)在肝细胞肝癌（hepatocellular carcinoma,HCC）组织和细胞中的表达及临床病理意义。方法:采用免疫组织化学染色方法检测100例HCC肿瘤组织标本及46例癌旁肝组织标本中CISD2的蛋白表达水平,分析CISD2与临床病理特征、增殖相关蛋白Ki-67、患者生存率的相关性;运用Western blot法检测人HCC细胞株BEL7404、HepG2、SMMC-7721以及人正常肝细胞株L02中CISD2的表达,分析其表达差异。结果:CISD2在HCC肿瘤组织中的表达水平明显高于癌旁肝组织,CISD2在肝癌细胞株中的表达水平也明显高于正常肝细胞,差异有统计学意义(P＜0.01)。CISD2的表达与HCC患者的肿瘤大小（P＜0.05）以及Ki-67指数相关(P＜0.001),CISD2高表达患者的总生存率低于低表达者。结论:CISD2在HCC组织和细胞中高表达,其表达水平与患者的预后有关。CISD2可能成为HCC预后判断的标志和治疗的潜在靶点。     

Role of the pituitary tumor transforming gene 1 in the progression of hepatocellular carcinoma

In order to demonstrate the role of the pituitary tumor transforming gene 1 (PTTG1) in the development of hepatocellular carcinoma (HCC) and its value as a molecular target for cancer therapy, we analyzed the expression of PTTG1 mRNA and protein, and their relation to clinicopathological characteristics and basic fibroblast growth factor (bFGF) expression in HCC. It was observed that the level of PTTG1 mRNA and the positive rate of PTTG1 protein in cancerous tissues were significantly higher than that in adjacent non-cancerous tissues (both P< 0.001). The PTTG1 protein levels were correlated with several clinicopathological parameters, including alpha-fetoprotein level, portal vein tumor thrombosis, tumor stage, and bFGF protein level (P< 0.05). The proliferation indices were significantly less and the apoptotic rates were significantly higher in the HepG2 and SMMC-7721 cells treated with PTTG1 siRNA transfection than their untransfected counterparts. The expressions of Caspase-3, Bax, p21 and p53 in HepG2 and SMMC-7721 cells were significantly increased after siRNA knockdown of PTTG1 expression. In conclusion, the PTTG1 gene is up-regulated in the cancerous tissue from patients with HCC and involved in the progression of HCC. Inhibiting PTTG1 expression decreases cell proliferation and induces apoptosis in hepatic cancer cell lines, indicating that PTTG1 may be a new therapeutic target for HCC treatment.     

长链非编码RNA CCAT1在肝细胞癌组织及细胞株中的表达及临床意义

目的 探讨长链非编RNA 结肠癌相关转录子1（LncRNA-CCAT1）在肝细胞癌（HCC）组织及细胞株中的表达及临床意义。方法 采用QPCR法检测CCAT1在4种肝癌细胞株（SMMC-7721、Hep3B、Hub7和HepG2）、正常肝细胞株LO2和42例HCC组织及其癌旁组织中的差异性表达,分析CCAT1表达与HCC临床病理特征的关系（性别、年龄、甲胎蛋白、肿瘤大小、肿瘤数量、淋巴结转移、分化程度和TNM分期）。结果 与正常肝细胞株LO2相比,CCAT1在SMMC-7721、Hep3B、Hub7和HepG2细胞株中均呈高表达（P＜0.05）,其中在HepG2细胞中表达水平最高。CCAT1在HCC组织中的表达水平高于其癌旁组织,差异有统计学意义（P=0.016）;CCAT1在肝癌组织中的表达水平与肿瘤大小、甲胎蛋白、分化程度、TNM分期和淋巴结转移有关（P＜0.05）,而与性别、年龄、肿瘤数量无关（P＞0.05）。结论 LncRNA-CCAT1与HCC发生、发展及预后有关,其作用机制值得进一步研究。