沉默HOXA13基因对肝癌HepG2和QGY-7703细胞恶性表型的影响

目的：探讨沉默HOXA13基因对肝癌细胞株HepG₂与QGY-7703恶性表型的影响,为肝癌的诊断及治疗提供新的分子靶点。方法：构建HOXA13基因的干扰重组质粒plent-U6-GFP/si-HOXA13,将其稳定转染至HepG₂与QGY-7703细胞;采用RT-PCR和Western blotting法鉴定干扰效果;以HOXA13基因沉默细胞为实验组,以转染空质粒的细胞为对照组,采用四甲基偶氮唑蓝（MTT）法、细胞倍增时间测定、平板克隆形成实验和流式细胞术等分析HOXA13基因沉默后HepG₂与QGY-7703细胞生长速度、细胞倍增时间、克隆形成能力和细胞周期等的改变。结果：MTT实验,与对照组比较,实验组细胞的生长速度明显下降;其细胞周期也发生改变,G₁期细胞增多、S期细胞减少。对照组HepG₂和QGY-7703细胞平均倍增时间分别为（4.59±0.27）和（4.93±0.17） h,实验组HepG₂和QGY-7703细胞平均倍增时间分别为（6.02±0.86）和（6.43±0.66） h,2组间比较差异有统计学意义（P<0.05）。对照组HepG₂和QGY-7703细胞平均细胞克隆形成数分别为（264.00±12.62）和（269.00±4.55）个,实验组HepG₂和QGY-7703细胞平均细胞克隆形成数目分别为（165.00±10.61）和（215.00±4.43）个,2组比较差异有统计学意义（P<0.01）。结论：HOXA13基因可以使肝癌细胞HepG₂与QGY-7703的增殖加快、克隆形成能力增强、G₁期细胞减少、S期细胞增多,可以成为肝癌诊断和治疗新的分子靶点。     

纤连蛋白对人肝癌细胞系QGY-7703细胞分化及细胞周期的影响

用流式细胞光度术等细胞生物学技术,研究纤连蛋白(FN)对人肝癌细胞系QGY-7703细胞分化及细胞周期的影响。结果表明,FN抑制其细胞增殖和有丝分裂,对照组和实验组间差异有显著性(P<0.05);细胞形态向正常细胞逆转,细胞被阻断在S期。作者认为FN可能是一种具有潜在治疗肝癌的药物。     

MiR-105抑制肝癌细胞增殖能力的实验研究

目的 探讨miR-105对人肝癌细胞增殖的影响及可能机制.方法 改变QGY-7703及HepG2两种细胞的miR-105表达,细胞克隆形成实验和软琼脂增殖试验检测细胞增殖能力的改变;流式细胞仪检测细胞周期改变.结果 细胞克隆形成和软琼脂增殖实验显示,过表达miR-105可抑制QGY-7703及HepG2细胞增殖,反之抑制miR-105表达则促进细胞增殖;细胞流式检测显示miR-105过表达能促进两种癌细胞G0/G1期阻滞.结论 miR-105抑制肝癌细胞增殖,其机制与引起细胞周期阻滞有关.     

肝癌细胞铁蛋白磁共振成像的实验研究

目的 研究肝癌细胞中内源性铁蛋白的表达情况,并进一步分析铁蛋白的表达与磁共振成像（MRI）的信号关系如何。方法 对3种肝癌细胞株QGY-7703、HepG2、SK-Hep-1以及正常肝细胞L02,4种细胞株内源性铁蛋白表达情况进行检测,用RT-PCR方法 检测基因表达,Western blot检测细胞株中铁蛋白表达,同时测定细胞培养液上清中铁蛋白的分泌量。最后检测4种细胞磁共振信号的情况,分析细胞铁蛋白表达与磁共振信号的关系。结果 RT-PCR与Western Blot均显示铁蛋白的基因表达与蛋白表达,HepG2与QGY-7703表达最强,SK-Hep-1表达中等,L02表达很弱。4种细胞培养液上清中HepG2与QGY-7703分泌铁蛋白量较高,SK-Hep-1分泌较低,L02分泌量很低。磁共振细胞显像,T1WI、T2WI及PD均显示HepG2与QGY-7703磁共振信号强度低,而SK-Hep-1与L02信号强度较高。结论 各种肝癌细胞中铁蛋白表达水平不同,铁蛋白表达水平与磁共振细胞显像信号强度呈负相关。     

RASSF1A基因表达对肝癌细胞化疗药物敏感性的影响

目的:研究RASSF1A提高肝癌细胞化疗药物敏感性的作用。方法:通过脂质体介导的基因转染法将RASSF1A基因的真核表达载体及空载体转染肝癌细胞株QGY-7703,G418筛选稳定表达株,并以RT-PCR和Western Blot进行鉴定目的基因的表达。化疗药物阿霉素(ADM)、丝裂霉素(MMC)、鬼臼乙叉甙(VP-16)、5-氟尿嘧啶(5-FU)、顺铂(DDP)分别作用各细胞株后,检测各细胞株的生长抑制率。结果:经800μg/ml的G418最佳筛选浓度筛选QGY-7703细胞株,成功建立稳定表达RASSF1A基因的肝癌细胞株。以QGY-7703细胞为对照,MMC对表达RASSF1A基因的肝癌细胞的生长抑制率最大为21%。结论:RASSF1A基因的表达可明显提高肝癌细胞对MMC的敏感性。RASSF1A的表达不影响肝癌细胞QGY-7703对ADM、VP-16、5-FU和DDP敏感性。     

塞来昔布诱导人肝癌细胞株QGY-7701凋亡的研究

目的探讨塞来昔布体外诱导人肝癌细胞株QGY-7701凋亡的作用。方法设立空白对照组(培养液中不加任何药物)及塞来昔布5、25、50、100、150μmol.L-1 5个剂量组,作用时间分为24、48、72 h,应用四甲基偶氮唑盐(MTT)法观察塞来昔布不同浓度和作用时间对QGY-7701细胞生存率的影响,选择合适的作用时间和3个剂量再做其他凋亡方法的检测;采用Hoechst 33258染色荧光显微镜观察凋亡细胞的形态学变化;采用双荧光染色(AnnexinV-EGFP/PI)流式细胞检测、末端脱氧核苷酰基转移酶介导性dUTP切口末端标记(TUNEL)检测、DNA含量分析(检测细胞周期)等方法多指标检测细胞凋亡率。结果通过Hoechst 33258染色可以从细胞形态上看出塞来昔布诱导QGY-7701细胞凋亡,而Annexin V-EGFP/PI流式细胞检测、TUNEL检测、细胞周期及MTT检测结果显示随着塞来昔布浓度升高和作用时间延长,凋亡率升高(P<0.05,P<0.01)。结论塞来昔布有诱导肝癌细胞QGY-7701凋亡,抑制其生长的作用,具有治疗肝癌的潜在功能。     

红景天对肝癌细胞增殖的抑制作用

目的:研究红景天(Rhodiola)对体外培养肝癌QGY-7703的直接抑制作用及可能机制。方法:通过液体培养法、克隆形成法、3H-TdR掺入法与MTT比色法分别检测给药后肿瘤细胞的增殖情况、克隆形成率、DNA合成抑制率及生存率来探讨红景天的抑瘤效应。结果:与红景天共育24h的细胞伸展不良,胞体回缩,贴附型细胞不贴壁,胞质粗糙,有大量颗粒状物堆积,而且药物浓度越大,形态学改变越明显。给药组肿瘤细胞增殖缓慢甚至停滞,出现细胞脱落、胞浆内颗粒状物堆积等形态学改变,克隆形成数明显少于对照组,cpm和A值明显降低即3H-TdR掺入率减少,生存率下降。结论:红景天对体外培养肝癌细胞均具有直接杀伤作用。     

抗人肝癌单克隆抗体的制备及鉴定

The hepatocellular carcinoma (HCC) cell line, QGY-7703, derived from a Chinese patient, was used to immunize the BALB/c mice. Fifteen hybridomas producing McAb that reacted with QGY-7703 cells were isolated from 858 hybridomas created in three cell fusions. In further studies two McAb, namely, AQGY1 and A-QGY2, were selected which specifically stained HCC cells grown in vitro. The reactivity of these McAb was not removed by the absorption by homogenates of the normal liver, but was by homogenates of HCC cells. A-QGY1 and A-QGY2 also reacted definitely with HCC cells in liver tissues of HCC patients, but neither with other cells in the tissues nor with nontransformed liver tissues of the same patients. Furthermore these two McAb stained the adult or fetal liver tissues, nor all of the other normal or tumor tissues that had been tested. Blocking and absorbing experiments revealed that A-QGY1 and AQGY2 antigens had no immunohomogenicity with antigens such as HBsAg, HBcAg, HBeAg, AFP and CEA. The specificity of these two McAb may be used potentially for the sero diagnosis, histologic identification, radioimmunoimaging and destruction of human hepatocellular carcinoma.     

Internalization of monoclonal antibodies against human hepatoma in the target cells]

The monoclonal antibodies against human hepatoma, HAb23, HAb18 and HAb8, were linked with 5 nm colloid gold. They were used to incubate with the target cells, QGY-7703 and SMMC-7721 human hepatoma cell lines, at 4 degree C for 1 hour. The incubated hepatoma cells were divided into 6 groups and then were put into 37 degree C water incubator for 0, 5, 10, 30, 60 and 120 minutes respectively. After washing, the target cells were fixed with Karnovsky's fixative and embedded in Epon 812. There were four intracellular routings for HAb 23, HAb18 and HAb8 to enter the QGY-7703 and SMMC-7721 hepatoma cells: (1) Coated pits coated: The colloid gold-antibodies which clustered in the specialised regions of the surface membrane were invaginated rapidly into the cells to form coated vesicles. (2) Enclosed invagination: One or two colloid gold-antibodies which were attached on the surface membrane were invaginated into the cell by endocytosis. (3) Microvilli involved routing: The microvilli which were adsorbed by the colloid gold-antibodies were broken and then were phagocytized by the cells. (4) Routing via glycocalyx-like material: The glycocalyx-like material which was clustered by the colloid gold-antibodies was invaginated during endocytosis.     

抗人肝癌单克隆抗体的制备及鉴定

用国人肝癌细胞免疫BALB/c鼠,经三次细胞融合、反复筛选及克隆化,得到了15株杂交瘤;又经正常肝组织及肝癌细胞吸收试验,进一步选出两株杂交瘤。它们分别能持续稳定地分泌特异性抗人肝癌单克隆抗体A-QGY_1和A-QGY_2。此两株单克隆抗体只与体外培养或患者组织中的肝癌细胞产生反应,而不与正常成人和胎儿肝组织及其他多种正常或肿瘤组织发生反应,与乙肝病毒抗原及甲胎蛋白、癌胚抗原等也无免疫交叉反应。