Role of VEGF-A/C and CCR-7 in the enhanced metastasis of A549 cells induced by 2 and 4 Gy X-rays in vitro and in vivo

Radiotherapy is a common strategy in treating lung cancer.Accumulating evidence suggested that radiotherapy has the potential to promote the metastasis and invasion of carcinoma cells.In this study,we aimed to testify the role of vascular endothelial growth factor(VEGF)and C-C chemokine receptor-7(CCR-7)in the metastasis of human adenocarcinoma A549 cells in vivo and in vitro.Nude mice were injected with A549 cells irradiated by 0,2 and 4 Gy X-rays,respectively.Quantitative detections of VEGF-A/C and CCR-7 mRNA from lung sample were performed by real-time RT-PCR.Small interfering RNA(siRNA)transfection technology was further used to testify the role of the genes in the metastasis of A549 cells.VEGF and CCR-7mRNA could only be detected 10 weeks post injection in vivo when visible tumor foci scattered in lung.In addition,VEGF-A/C mRNA expressed significantly higher in mice injected with A549 cells irradiated by 2 and 4 X-rays than those with 0 Gy X-rays irradiation.On the other hand,A549 cells with or without X-rays irradiation transfected with VEGF siRAN and CCR-7 siRNA showed a dramatic decrease of invasiveness compared to normal A549 cells with or without irradiation.The migration indexes were?0.7,?0.48,?0.34 and?0.32 for A549 cells with CCR-7 siRNA,VEGF siRNA,X-rays combined CCR-7 siRNA and X-rays combined VEGF-siRNA respectively.These results demonstrated that X-rays could promote the metastasis of A549 cells,and VEGF-A/C and CCR-7 mRNA were closely related to the metastasis of A549 cells in vivo and in vitro.     

TGF-β1对肝癌细胞系Hep3B中干性相关基因表达的影响

有研究表明TGF-β1可以诱导诸多上皮来源的癌细胞和正常细胞发生EMT并使其功能发生改变。实验就TGF-β1对肝癌细胞系Hep3B的作用展开研究:通过CCK8检测TGF-β1对Hep3B细胞增殖的影响,RT-PCR实验检测TGF-β1处理后细胞中EMT及干性相关基因的表达变化。结果表明:TGF-β1对Hep3B细胞的增殖无抑制作用;TGF-β1处理Hep3B细胞6d后EMT相关基因的mRNA表达水平并无显著改变,但TGF-β1可上调Hep3B细胞干性基因Oct-4,Klf-4,Nanog,C-myc的表达,并下调分化基因albumin的表达。结果提示TGF-β1一定程度上影响肝癌细胞系的干性基因表达,但并不一定是以发生EMT为前提的。     

TGF—β1对肝癌细胞系Hep3B中干性相关基因表达的影响

有研究表明TGF-β1可以诱导诸多上皮来源的癌细胞和正常细胞发生EMT并使其功能发生改变。实验就TGF-β1对肝癌细胞系Hep3B的作用展开研究：通过CCK8检测TGF-β1对Hep3B细胞增殖的影响,RT-PCR实验检测TGF-β1处理后细胞中EMT及干性相关基因的表达变化。结果表明：TGF-β1对Hep3B细胞的增殖无抑制作用;TGF-β1处理Hep3B细胞6d后EMT相关基因的mRNA表达水平并无显著改变,但TGF-β1可上调Hep3B细胞干性基因Oct-4,Klf-4,Nanog,C-myc的表达,并下调分化基因albumin的表达。结果提示TGF-β1一定程度上影响肝癌细胞系的干性基因表达,但并不一定是以发生EMT为前提的。     

聚酰基硫脲基因输送体系的构建与评价

基于新型树枝状大分子聚酰基硫脲(PATU),通过巯基-烯Michael加成反应得到表面N, N-二甲基半胱胺修饰的聚酰基硫脲(PATU-DMCA). 核磁结果表明：PATU-DMCA树枝状结构正确且完整. 琼脂糖凝胶电泳及动态光散射粒径测定结果表明：PATU-DMCA在低代数、低氮磷摩尔比条件下可以有效包载DNA,形成粒径为60~100 nm、Zeta电位为10~20 mV的纳米复合物. 在人宫颈癌细胞HeLa和人肺癌细胞A549上,低氮磷摩尔比的PATU-DMCA的无血清转染效率优于经典的树枝状大分子基因载体聚酰胺胺,而两者细胞毒性相当. HeLa上的亚细胞分布结果表明：纳米复合物在细胞水平的转运过程基本包括黏附细胞膜、内吞入胞、进入溶酶体,通过“质子海绵”效应从溶酶体逃逸后进入细胞质及细胞核进行转染. PATU-DMCA以及PATU本身在基因输送中均具有很大的潜力.     

The influence of nano-apatite on c -myc and p 53 gene in the hepatocellular carcinoma

The influence mechanism of the nano-apatite on the human hepatocellular carcinoma in vitro was investigated. Using the homogeneous precipitation method, the nano-apatite was synthesized at room temperature, and it was characterized with transmission electron microscopy (TEM) and the Zataplus. The influence on the expression of the c-myc and p53 gene in the human hepatocellular carcinoma cell lines were tested with the TEM and hybridization in situ. The TEM and the Zataplus analyses show that the nano-apatite is distributed homogenously in size and needle-shaped sizes, which ranges from 67.5 nm to 88.3 nm. It is found that the nanoapatitet increases the volume of the human hepatocellular carcinoma cells, makes extensive cytoplasmic vacuolization, the mitochondria swelling, chromatin, in nucleus dispersed partially and condensed around the nuclear membranes. The interspace in nuclear membranes were separated and even the cytoplasm dissolved. It is also found that the expression of the c-myc gene is inhibited, but the p53 is enhanced. The experimental results demonstrate that the nano-apatite enables the oncosis of the human hepatocellular carcinoma cells by down-regulation of the expression of the c-myc and up-regulation of the expression of the p53 in vitro. Funded by the State Key Laboratory of Advanced Technology for Materials Synthesis and Processing (Wuhan University of Technology, WUT2004Z02), and the Science and Technology Brain-storm Key Project of Hubei Province (2002AA105A)     

The Influence of Nano-apatite on c-myc and p53 Gene in the Hepatocellular Carcinoma

The influence mechanism of the nano-apatite on the human hepatocellular carcinoma in vitro was investigated. Using the homogeneous precipitation method, the nano-apatite was synthesized at room temperature, and it was characterized with transmission electron microscopy (TEM) and the Zataplus. The influence on the expression of the c-myc and p53 gene in the human hepatocellalar carcinoma cell lines were tested with the TEM and hybridization in situ. The TEM and the Zataplus analyses show that the nano-apatite is distributed homogenously in size and needle-shaped sizes, which ranges from 67.5 nm to 88.3 nm. It is found that the nanoapatitet increases the volume of the human hepatocellalar carcinoma cells, makes extensive cytoplasmic vacuolization, the mitochondria swelling, chromatin in nucleus dispersed partially and condensed around the nuclear membranes. The interspace in nuclear membranes were separated and even the cytoplasm dissolved. It is also found that the expression of the c-myc gene is inhibited, but the p53 /s enhanced. The experimental results demonstrate that the nano-apatite enables the oneosis of the human hepatocellular carcinoma cells by down-regulation of the expression of the c-myc and up-regulation of the expression of the p53 in vitro.     

纳米磷灰石对肝癌癌基因表达的影响

研究纳米磷灰石体外对人肝癌细胞系Bel-7402的作用机理。应用均相共沉淀法室温下合成纳米磷灰石，用透射电镜和电位粒度仪进行表征；利用透射电镜、原位杂交细胞化学检测其对人肝癌细胞系Bel-7402 c-myc和p53基因表达的影响。纳米磷灰石呈均匀分散的针状颗粒．粒径范围在67．5～88．3nm之间。可使肝癌细胞体积增大，胞质空泡化，线粒体肿胀；部分细胞核内染色质分散，凝集在核膜周围，呈团块状，核膜间隙不等，胞浆溶解。抑制c-myc和增强p53基因的表达。纳米磷灰石在体外通过下调c-myc和上调p53基因的表达，致肝癌细胞胀亡。     

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.     

肝癌抑制基因-1转运功能区的研究

肝癌抑制基因-1(hepatocellular carcinorna suppressor 1,HCCS1)是一种潜在的肝癌抑制基因,在细胞内发挥蛋白分拣运输的作用,其抑癌作用有可能与蛋白转运的功能有一定的关系.本研究旨在寻找HCCS1序列中与转运功能相关的序列区域.首先通过亚克隆技术,构建以pEGFP-C2为载体,对不同长度HCCS1-cDNA片段进行亚克隆,将构建的亚克隆转染到HeLa细胞,通过免疫荧光显微镜观察不同长度HCCS1蛋白在细胞内的分布,及其与6-磷酸甘露糖受体(M6PR)的共定位.经过PCR及亚克隆后,目的片段成功插入载体质粒,构建了以pEGFPC2为载体的含有4个不同长度HCCS1片段的亚克隆.分别是:pEGFP-C2-N1836,pEGFP-C2-N1572,pEGFP-C2-1707和pEGFP-C2-1506,其中pEGFP-C2-N1836和pEGFP-C2-N1572编码的HCCS1蛋白,长度不同均呈颗粒状、极性分布于核周的胞质内,且与M6PR有共定位;而pEGFP-C2-1707片段编码的HCCS1蛋白虽然也呈颗粒状分布于核周,但极性分布消失,且与M6PR共定位消失;pEGFP-C2-1506与pEGFP-C2-1707结果相似.结果显示了HCCS1-cDNA的5'端的129 bp序列为与HCCS1的极性及M6PR共定位相关的区域.     

纳米羟基磷灰石对肝癌细胞端粒酶基因表达的影响

研究羟基磷灰石（HAP）纳米粒子对Bel-7402人肝癌细胞端粒酶基因表达的影响。采用均相沉淀法制备出稳定单分散的HAP纳米粒子,应用透射电镜、电位粒度仪对其进行表征。HAP纳米粒子作用Bel-7402肝癌细胞4 h后,采用原位杂交技术检测Bel-7402肝癌细胞的端粒酶基因表达。结果表明HAP纳米粒子作用组的Bel-7402肝癌细胞的端粒酶阳性细胞比例为61.38%,而对照组的端粒酶阳性细胞比例为87.89%,2组有显著性差异（P〈0.01）。HAP纳米粒子可使Bel-7402肝癌细胞的端粒酶基因表达下调。     

羟基磷灰石纳米粒子对肝癌细胞PCNA表达的影响

为了研究羟基磷灰石(HAP)纳米粒子对肝癌细胞增殖细胞核抗原表达的影响,采用均匀沉淀法制备了均匀分散的纳米尺度的HAP纳米粒子。以0.56 mmol/L的HAP纳米粒子与Bel-7402肝癌细胞作用后,采用流式细胞技术检测细胞周期时相的变化,行免疫细胞化学法PCNA染色,形态学观察和定量分析细胞的PCNA表达。结果显示HAP纳米粒子能使Bel-7402细胞的细胞增殖周期阻滞于G1期,PCNA表达降低,与对照组比较有显著性差异(P<0.01)。HAP纳米粒子可能通过抑制PCNA的表达,起到抑制肝癌细胞增殖的作用。     

癌基因DEK蛋白表达判断肺鳞状细胞癌预后的临床价值

为了探讨癌基因DEK蛋白在肺鳞癌中的表达及预后评估价值,应用免疫组化染色检测96例肺鳞癌及其癌旁正常组织中DEK蛋白的表达,结合临床生物学指标及生存时间进行统计学分析。结果发现,肺鳞癌组织中DEK蛋白阳性表达率为45.83%,明显高于癌旁正常组织（15.73%,P=0.001）,DEK蛋白在肺鳞癌中的表达与肿瘤大小（P=0.008）、组织分化程度（P=0.004）及病理学分期（P=0.011）密切相关;癌组织中DEK蛋白的阳性率与患者总生存期明显相关,且与病理分期、CEA水平和转移共同影响患者预后;DEK是肺鳞癌不良预后的独立风险因子。得出结论：检测肺鳞癌组织中DEK蛋白的表达对其预后有一定的临床评估价值,有望成为肺鳞癌新的预后生物标志物。     

Ezrin低表达与肺鳞状细胞癌患者预后之间的关系

为了探讨癌基因Ezrin蛋白表达在肺鳞癌中的预后意义,应用免疫组化染色检测了96例肺鳞癌及其癌旁正常肺组织中Ezrin蛋白的表达,并结合临床生物学指标及生存时间进行统计学分析。结果显示:肺鳞癌、癌旁正常组织中Ezrin蛋白阳性表达率分别为41.67%和91.67%,癌组织中Ezrin蛋白阳性表达率明显低于癌旁正常组织(P<0.01),Ezrin蛋白在肺鳞癌中的阴性表达与肿瘤大小(P<0.05)、组织分化程度(P<0.05)、病理学分期(P<0.01)、是否有转移(P<0.01)密切相关,且癌组织中Ezrin蛋白的阴性率与患者5年生存率明显相关(P<0.01);Ezrin蛋白阴性表达(HR:0.292,95%CI:0.141～0.606,P=0.001)、病理学分期和转移是肺鳞癌预后不良的独立风险因素。得出结论:检测肺鳞癌组织中Ezrin蛋白的表达对其诊断及预后评估有重要价值,有望成为肺鳞癌新的生物标志物及靶向治疗的分子靶点。     

以干细胞特异性转录因子诱导小鼠肝癌细胞Hepa1—6重新编程的研究

利用逆转录病毒携带小鼠Oct4、Klf4、SOx2和c-Myc基因介导Hepa1-6小鼠肝癌细胞系重新编程,得到一团自律性跳动的细胞团,及一群形态学上明显类似于小鼠胚胎干细胞的克隆样细胞团。经长期传代,克隆样细胞能保持其形态。检测证实,这些克隆样细胞团表达Oct4、Sox2蛋白。证实,Hepa1-6小鼠肝癌细胞可以被携带Oct4、KK4、SOX2和cMyc基因的逆转录病毒感染的方法诱导重新编程。     

外显子跳跃模式中组蛋白修饰的组合模式分析

可变剪接是基因表达调控的重要过程。组蛋白修饰参与了可变剪接,但具体调控机制尚不清楚。为了研究组蛋白修饰与可变剪接的关系,首先基于人胚肺成纤维细胞系的RNA-Seq数据,获得了该细胞系外显子跳跃模式的可变剪接数据。在此基础上,分析了外显子跳跃模式中排除和包含外显子中组蛋白修饰间的相关性。通过构建组蛋白修饰间的贝叶斯网络,推断了人胚肺成纤维细胞系外显子跳跃可变剪接事件中组蛋白修饰之间的因果关系。     

T C-1-HPV16 L1细胞模型与动物模型的建立与评价

为了解决人乳头瘤病毒16型L1蛋白(HPV16L1)为主要靶点的疫苗缺乏肿瘤模型细胞来验证疫苗的免疫效果的问题,构建了一个细胞模型并可以利用此细胞在实验动物体内形成肿瘤,用于以HPV16L1为主要靶点疫苗的验证实验。利用这个模型细胞或肿瘤模型可以在体内外检测疫苗的免疫学性质和保护功效。首先,利用聚合酶链式反应(polymerase chain reaction,PCR)的方法获得目标基因HPV16L1的基因序列并连接到载体质粒中,将质粒转染到细胞TC-1后在杀稻瘟菌素抗性压力筛选下获得稳定表达HPV16L1的细胞株。对TC-1和TC-1-HPV16L1两种细胞的生长增殖和成瘤特性进行比较发现,外源基因的加入对细胞特性没有影响。通过体外杀伤实验证实细胞TC-1-HPV16L1能够被HPV16L1靶点疫苗免疫过的小鼠脾淋巴细胞杀伤。实验动物被疫苗免疫后,进行攻瘤实验发现疫苗只对TC-1-HPV16L1组具有保护作用。综上,在本研究中成功地构建了可以检测HPV16L1疫苗的抑瘤效果的模型细胞TC-1-HPV16L1,为HPV疫苗的研究提供了一个模型细胞。     

桦木醇吡啶盐衍生物的合成及其生理活性研究

以桦木醇为原料合成了4种桦木醇吡啶盐,运用¹H-NMR和IR确认了化合物的结构.用噻唑蓝(MTT)比色法对这4种化合物对肝星状细胞、人肺癌细胞和人肝癌细胞的抑制作用进行了活性分析,结果表明,化合物3、4a和4c对上述3种癌细胞有明显的抑制作用,并且对肝星状细胞的IC₅₀值均明显低于桦木醇.     

Hsp90抑制剂BIIB021对人急性T细胞白血病Molt-4细胞周期和凋亡的影响

为了研究Hsp90抑制剂BIIB021对人急性T淋巴细胞白血病细胞株Moh-4增殖和凋亡的影响,采用MTT法检测了BILB021对Molt-4细胞增殖的影响,Hoechst33258荧光染色法检测细胞凋亡形态学变化,流式细胞术检测药物作用前后细胞周期和凋亡的改变,Westernblotting检测BIIB02l对细胞周期和凋亡相关蛋白的影响。结果显示：BIIB021以时间和刺量依赖性方式抑制Molt-4细胞增殖,48、72h的IC50分别为384．6nmol／L和301．8nmol／L;Hoechst33258染色可观察到细胞内出现凋亡小体,且随着药物浓度增大,凋亡小体的数目也逐渐增多;流式结果同样显示,随着药物浓度升高,细胞凋亡率逐渐上升,400nmol／LBILB021可诱导细胞凋亡率达34．4％;另外,BIIB021可显著阻滞细胞于GO／G1期;Westernblotting显示BILB021激活Caspase-8、-9、-3和PARP,下调CDK4／6,上调p21和p18,并能明显抑制Akt和p65。由此表明,BIIB021能激活死亡受体途径和线粒体途径诱导Molt-4细胞凋亡,并通过影响CDK4／6和p21、p18使得Molt-4细胞被阻滞于GO／G1期,抑制细胞增殖;BIIB021对Mott-4的Akt和NF-kB也有抑制作用。