顺铂和放疗联合应用在人喉鳞癌细胞中相互作用的研究

 目的 研究CDDP和放疗联合应用于人喉鳞状细胞癌细胞的相互作用，以探讨放、化疗两种处理方式应用的顺序及与端粒酶活性的关系。方法 Hep-2细胞分别经放化疗不同剂量及顺序处理．实验设对照组、CDDP组、放疗组、CDDP+放疗组及放疗+CDDP组，分别用MTT法、端粒酶活性检测PCR-ELISA法及聚丙烯酰胺凝胶电泳及银染法分析各种处理后的细胞存活率及端粒酶活性的变化。结果 CDDP+放疗组比同等处理剂量的放疗+CDDP组的细胞存活率低。且有显著差异。CDDP及放疗均能使端粒酶活性明显增高，并表明为浓度依赖性。但在加入另一种处理后，端粒酶活性降低，其降低的程度与细胞存活率的变化一致(r=0.9445，b=13．61)。结论 对Hep-2细胞先化疗后放疗的治疗效果优于先放疗后化疗，联合治疗后的端粒酶活性可反映治疗效果。     

RNA干扰抑制Hep-2细胞人端粒酶逆转录酶基因表达对放射敏感性的影响

目的构建人端粒酶逆转录酶(hTERT)基因特异性短发夹样RNA(shRNA)真核表达载体,观察其联合射线对人喉癌Hep-2细胞端粒酶活性和细胞存活的影响,研究hTERT基因在放射增敏中的作用。方法根据hTERT mRNA编码序列设计RNA干扰(RNAi)靶点,构建重组表达质粒pshRNA-hTERT,构建成功后,转染Hep-2细胞并作射线处理,用端粒重复序列扩增方法(TRAP-PCR- EHSA)检测端粒酶活性的动态变化,采用克隆形成分析方法观察质粒pshRNA-hTERT对Hep-2细胞放射敏感性的影响,计算放射增敏比SER_(SF2)。结果转染质粒pshRNA-hTERT后,Hep-2细胞的hTERT mRNA表达抑制率为60.8%,质粒pshRNA-hTERT不仅能够抑制Hep-2细胞的端粒酶活性(P<0.05),而且还可以抑制辐射诱导的端粒酶活性上升(P<0.05)。照射前经pshRNA-hTERT处理24h,明显降低了2Gy照射后Hep-2细胞的存活分数(85.7%),与单纯放射组(67.7%)相比,差异有统计学意义(P<0.05),pshRNA-hTERT的放射增敏比SER~(SF2)为1.27。结论RNAi显著抑制了靶基因hTERT的表达,质粒pshRNA-hTERT能明显抑制2Gy照射诱导的Hep-2细胞端粒酶活性升高,并显著提高其体外放射敏感性;质粒pshRNA-hTERT的放射增敏作用可能与端粒酶活性受抑制有关。     

顺铂对人鼻咽癌CNE-2Z细胞端粒酶活性和细胞周期及凋亡的影响

目的研究顺铂对人鼻咽癌CNE-2Z细胞端粒酶活性、细胞周期及凋亡的影响,以探讨顺铂诱导CNE-2Z细胞凋亡与端粒酶活性水平的关系以及顺铂诱导鼻咽癌细胞凋亡的可能机制。方法分别以不同的药物浓度作用于体外培养的CNE-2Z细胞不同的时间,用TRAP-ELISA的方法定量检测CNE-2Z细胞在顺铂处理前后的端粒酶活性水平,同步进行细胞形态观察,流式细胞仪分析细胞周期的改变并检测凋亡。结果CNE-2Z细胞端粒酶呈阳性。用不同浓度的顺铂不同的时间作用于CNE-2Z细胞,结果细胞周期被阻滞在G1期,出现细胞凋亡,下调端粒酶活性,且呈时间依赖性及剂量依赖性。结论化疗药物顺铂可能是通过改变细胞周期分布(G1期阻滞)并同时诱导细胞凋亡、下调其端粒酶活性而发挥抗癌作用的,故可将化疗前后细胞端粒酶活性的变化作为鼻咽癌化疗敏感性指标之一。     

顺铂对人肺腺癌细胞SLC-89作用的机理探讨

目的 观察顺铂对SLC-89细胞增殖生长、端粒酶活性、细胞周期、p53、bcl-2及增殖细胞核抗原(PCNA)表达的影响，探讨顺铂抗癌作用的机理。方法 以不同浓度的顺铂作用于培养的SLC-89细胞72h，运用MTT法测定细胞增殖，端粒酶重复序列扩增酶联免疫吸附法(TRAP-ELISA)检测细胞端粒酶活性以及流式细胞术观察细胞周期的变化和p53、bcl-2及PCNA的表达。结果 顺铂能显著抑制SLC-89细胞的生长，IC50为18．47mg／L。顺铂能以浓度依赖方式下调SLC-89细胞端粒酶活性，减少S期细胞而阻滞细胞于G0／G1期，降低bcl-2和PCNA的表达，同时诱导p53的表达。结论 顺铂能显著抑制SLC-89细胞生长增殖、改变细胞周期分布、降低端粒酶活性及bcl-2与PCNA的表达，并诱导p53的表达，这可能是顺铂抗癌作用的重要机理。     

雌、孕激素对子宫内膜癌细胞端粒酶活性及细胞周期的影响

 目的　研究雌、孕激素对子宫内膜癌HHUA细胞端粒酶活性及细胞周期的影响。方法　用TRAP ELISA法和实时荧光定量PCR技术检测雌、孕激素作用前后HHUA子宫内膜腺癌细胞系端粒酶活性及hTERT RNA的表达 ,流式细胞仪检测细胞周期及细胞凋亡的变化。结果　雌激素对HHUA细胞端粒酶活性和hTERT的表达有明显增强作用 (P <0 .0 5 ) ,而孕激素对其影响不显著。孕激素作用下 ,G1期细胞比例明显增高 ,S期及G2 ～M期细胞比例明显降低 (P <0 .0 5 ) ;细胞的分裂、增殖受到明显抑制 ;凋亡细胞比例明显增加。雌激素的作用则相反。结论　雌激素能增强子宫内膜癌细胞端粒酶活性和hTERT的表达 ,促进其分裂和增殖 ,降低凋亡细胞比例 ,孕激素的作用相反     

以hTERT基因反义核酸抑制端粒酶活性增加白血病细胞对CDDP诱导凋亡的敏感性(英文)

目的以 hTERT 反义核酸抑制白血病细胞(HL-60和 K562)端粒酶活性,研究 CDDP 诱导凋亡敏感性的变化。方法全硫代反义核酸由上海生物化学研究所合成和纯化;端粒酶活性用试剂合测定(宝灵曼公司产品);用形态学方法和流式细胞仪检测细胞调亡。结果实验结果显示,hTERT 全硫代反义核酸,通过下调 hTERT 基因表达,显著地抑制端粒酶活性;端粒酶活性下降以后,白血病细胞对 CDDP 诱导调亡的敏感性显著升高。结论以 hTERT 基因反义核酸抑制端粒酶活性增加白血病细胞对 CDDP 诱导凋亡的敏感性。     

以hTERT基因反义核酸抑制端粒酶活性增加白血病细胞对CDDP诱导凋亡的敏感性

目的 以hTERT反义核酸抑制白血病细胞(HL-60和K562)端粒酶活性,研究CDDP诱导凋亡敏感性的变化。方法 全硫代反义核酸由上海生物化学研究所合成和纯化;端粒酶活性用试剂合测定(宝灵曼公司产品);用形态学方法和流式细胞仪检测细胞调亡。结果实验结果显示,hTERT全硫代反义核酸,通过下调hTERT基因表达,显著地抑制端粒酶活性;端粒酶活性下降以后,白血病细胞对CDDP诱导调亡的敏感性显著升高。结论 以hTERT基因反义核酸抑制端粒酶活性增加白血病细胞对CDDP诱导凋亡的敏感性。     

虫草素诱导人肝癌HepG-2细胞凋亡及对端粒酶活性影响的研究

目的:研究虫草素(Cordycepin)对人肝癌细胞HepG-2诱导凋亡作用及对端粒酶活性的影响.方法:虫草素处理人肝癌HepG-2细胞株后,用四甲基偶氮唑蓝(MTT)法测定细胞生长抑制效应;倒置相差显微镜下观察肝癌细胞形态变化;用透射电镜观察超微结构的变化及荧光显微镜观察细胞凋亡效应;应用流式细胞仪(FCM)检测细胞周期变化及凋亡率;免疫组化方法测定NF-κBp65的表达;用TRAP-PCR-银染法对肝癌细胞端粒酶活性进行检测.结果:不同浓度的虫草素对HepG-2有明显抑制作用,P= 0.002,且呈剂量依赖性;形态学观察也发现,大量细胞的染色质边集、凝聚、核碎裂及凋亡小体产生;免疫组化示,NF-κBp65的表达下调,P=0.000 1;FCM检测结果显示虫草素作用后,使细胞阻滞于S期,P=0.000 1,并出现典型的"亚二倍体"凋亡峰,同时伴随端粒酶活性下调,P=0.000 1.结论:虫草素对人肝癌细胞具有明显抑制作用,其诱导细胞凋亡的机制可能与下调NF-κB的表达并抑制端粒酶活性有关;虫草素与5-FU 联合对肝癌细胞无明显协同抑制作用.     

冬凌草甲素对K562细胞端粒酶活性调控和细胞周期的影响

目的　目的 :研究冬凌草甲素 (ORI)对K5 62细胞端粒酶活性及其细胞周期的影响。方法　采用MTT法观察不同浓度ORI对K5 62细胞增殖的影响 :采用端粒重复序列扩增法 (TRAP) 酶联免疫吸附试验 (ELISA)法检测了端粒酶活性变化 ;采用流式细胞仪测定细胞周期各时相百分比。结果　ORI可明显抑制K5 62细胞增殖 ;在一定的浓度范围内 ,ORI可下调K5 62细胞端粒酶活性。流式细胞仪结果显示 ,经ORI处理的K5 62细胞 ,细胞周期各时相分布发生变化 ,G2 /M期细胞增多 ,S期细胞减少。结论　ORI可下调K5 62细胞的端粒酶活性 ,其机制可能与其细胞周期阻滞作用有关。     

γ射线照射后HeLa细胞端粒酶活性变化及机制探讨

[目的]观察射线照射后细胞端粒酶活性变化，并探讨其机制。[方法]HeLa细胞体外培养。照射2、4、8和20Gv后，分别于0、2、4、8、12、24、72和168h收集细胞．测定端粒酶活性、凋亡指数和’H掺人量。另取定量细胞照射0，1．2，3，4，6，8和10Gv．培养7d后。计算细胞存活率。[结果]2Gy和4Gy γ射线照射后．HeLa细胞端粒酶活性在短时间内上升，然后下降；8Gy和20Gy γ射线照射后酶活性迅速下降，并保持低水平。[结论]辐射后细胞端粒酶活性呈现规律性变化，这种变化是细胞死亡、亚致死损伤修复和存活细胞调节的综合结果。     

Egf/r3单克隆抗体治疗肺腺癌的实验研究

目的非小细胞肺癌(NSCLC)存在表皮生长因子受体(EGFR)过度表达。Egf/r3为EGFRMcAb,属IgC2a亚型,具有一定的治疗肺癌作用,但其机理尚不完全清楚。为更进一步全面了解Egf/r3对肺癌的治疗作用,本文以高表达EGFR的SPC-A-1和A549肺腺癌细胞系做为靶细胞,观察Egf/r3对肺腺癌细胞周期的影响、Egf/r3与CDDP和8-Mop是否存在协同抗肺癌作用。方法细胞周期检测采用流式细胞仪PI染色法分析,协同作用采用MTT法。结果SPC-A-1和A549G1期细胞分别增加5·0%和7·8%,S期分别减少5·6%和8·6%,G2-M期变化不明显。Egf/r3分别与CDDP和8-Mop联用,对SPC-A-1和A549杀伤率分别达到35%、39%和36%、39·5%。结论Egf/r3阻抑G1期癌细胞进入S期,使细胞同步化,增加了靶细胞对化疗药物的敏感性,减少癌细胞化疗后损伤修复,与CDDP、8-Mop存在协同抗肺腺癌作用。     

Interferon-alpha repressed telomerase along with G1-accumulation of Daudi cells.

The implications of telomerase on senescence and human carcinogenesis are widely accepted, but the changes of telomerase activity along with cell cycle modulation by anticancer treatment still remain obscure. In this paper, we issued whether the telomerase activity fluctuated along with cell cycle of cultured cancer cells using the antiproliferative effect of interferon-alpha (IFN-alpha). Daudi Burkitt lymphoma cells, treated with IFN-alpha, showed proliferation inhibition and cell cycle arrest at G1. The telomerase activity at 72 h was repressed to about 20% of control cells. Furthermore, after 72 h IFN-alpha treatment, the cells in G1 phase showed the marked decrease of telomerase activity, while cells in S and G2/M still possessed it. Among expressions of telomerase-related genes, only the catalytic subunit of telomerase (hTERT) decreased from 48 h, while the template RNA component (hTERC) and telomerase-associated protein 1 (TEP-1) were not affected. The downregulation of c-Myc preceded the change of hTERT. Moreover, the analysis of cells treated with IFN-alpha for 24 h revealed that cells in G1-to-S transition mainly expressed high hTERT, while S and G2/M cells had higher level of telomerase activity than that of G1 cells. These results indicate that (i) the expression of hTERT precedes the telomerase activity which is higher in S and G2/M phases than G1 phase, (ii) IFN-alpha repressed the telomerase activity in a cell cycle-dependent manner with the downregulation of hTERT.     

CoCl2诱导缺氧对Eca109细胞细胞周期及放射敏感性的影响

Objective:To investigate the change of the cell cycle,apoptosis and radiosensitivity effect by CoCl2 induced hypoxia in esophageal cancer line Eca109 cells in vitro.Methods:The hypoxia culture model induced by 150 microM CoCl2 was established.The cell cycle and apoptosis were measured with flow cytometry (FCM).The radiosensitivity was analysized with clonogenic assay after irradiation alone or combined with hypoxia in Eca109 cells in vitro.Results:Eca109 cells were treated with 150 microM CoCl2 for 24 h,cell cycle arrest in G0/G1 phase increase and decreasing arrest in S phase with longer of hypoxiac time (0-24 h),the other rate of cell cycle and apoptosis did not change obviously.The G2/M phase block was arrested obviously in radiation alone comparing with the hypoxia plus irradiated group,apoptosis did not occur in Eca109 cell line following irradiation.The DO value and cell surviving fraction of Eca109 cell was 2.48 Gy,2.44 Gy and 97.33%,96.33% in hypoxia and control group,respectively;the Dq value of Eca109 cell was 2.89 Gy,0.52 Gy,the cell surviving fraction after radiation with 4 Gy was 48.3%,21.7% in hypoxia and control group,respectively.The hypoxia decreased the radiosensitivity in esophageal cancer Eca109 cells with clonogenic assay.Conclusion:Hypoxia induced by CoCl2 influences radiosensitivity of Eca109 cell through regulating cellular proliferation rates.     

Analysis of cell proliferation kinetics and the effects of cisplatin on the cell cycle of human gastric cancer cells by autostage cytofluorometry]

Analysis of both cell proliferation kinetics and effects of cis-diamminedichloroplatinum (CDDP) on cell cycle in human gastric cancer cell line (HGC-Y2) by measuring the contents of nuclear DNA, RNA and the Ki-67 antigen using autostage cytofluorometry system was described. In HGC-Y2 cells, RNA content increased during the cell cycle and reached to the maximum at G2/M phase. The results of pulse treatment with CDDP on these cells demonstrated a prolongation of S phase and G2 arrest with increasing of RNA content of these cells. We classified the cells by intranuclear distribution pattern of Ki-67 antigen and thus could identified the cells at G0 and M phases from these classification. The content of Ki-67 antigen was moderate grade at G1 phase and it decreased in the early S phase, then increased gradually during S phase and at the late S phase. It increased rapidly, reaching to the maximum at G2/M phase. After CDDP treatment, the content of Ki-67 antigen increased in the cells in prolonged S phase and in the cells arrested at G2 phase. It was also found that the syntheses of both Ki-67 antigen and RNA were not inhibited by CDDP. These results suggest that the method using autostage cytofluorometry system was useful for the research, on the mechanism of cancer therapy because of making possible to analyze precisely the cell cycle and the influence of anticancer drugs.     

人喉鳞癌细胞端粒长度、端粒酶活性与放射敏感性的关系

背景与目的:肿瘤细胞的端粒长度、端粒酶活性与其增殖能力和恶性程度关系密切,而且端粒和端粒酶还可能参与放射诱导的DNA损伤的修复;由此推测肿瘤细胞的端粒长度、端粒酶活性与放射敏感性之间可能存在联系。本研究旨在探讨人喉鳞癌细胞端粒长度、端粒酶活性与放射敏感性的关系。方法:体外长期传代的人喉鳞癌细胞系Hep鄄2经0、2、4、8、12Gy剂量照射3次后的存活后代体外培养20代,以克隆形成实验测定其放射敏感性参数SF2,Southernblot法测定其端粒长度(meanlengthoftelomererestrictionfragments,TRF),TRAP鄄ELISA法测定其端粒酶活性(telomeraseactivity,TA)。结果:人喉鳞癌细胞不同放射剂量存活后代的SF2:0.47～0.64,TRF:3.76～9.43kb,TA:2.606～1.761,且它们各自的SF2、TRF、TA存在差异(P均<0.05);而且,SF2与TRF呈现明显的正相关(r=0.921,P<0.01),SF2与TA呈现明显的负相关(r=-0.929,P<0.01),TRF与TA呈现明显的负相关(r=-0.944,P<0.01)。结论:人喉鳞癌细胞接受不同放射剂量存活后代的放射敏感性与其端粒长度和端粒酶活性具有一定的相关性,提示端粒长度与端粒酶活性检测对预测肿瘤细胞放射敏感性有一定意义。     

Immunohistochemical estimation of cell cycle phase in laryngeal neoplasia

We previously developed an immunohistochemical method for estimating cell cycle state and phase in tissue samples, including biopsies that are too small for flow cytometry. We have used our technique to examine whether primary abnormalities of the cell cycle exist in laryngeal neoplasia. Antibodies against the markers of cell cycle entry, minichromosome maintenance protein-2 (Mcm-2) and Ki67, and putative markers of cell cycle phase, cyclin D1 (G1-phase), cyclin A (S-phase), cyclin B1 (G2-phase) and phosphohistone H3 (Mitosis) were applied to paraffin-embedded sections of normal larynx (n = 8), laryngeal dysplasia (n = 10) and laryngeal squamous cell carcinoma (n = 10). Cells expressing each marker were determined as a percentage of total cells, termed the labelling index (LI), and as a percentage of Mcm-2-positive cells, termed the labelling fraction (LF). The frequency of coexpression of each putative phase marker was investigated by confocal microscopy. There was a correlation between Mcm-2 and Ki67 LIs (rho = 0.93) but Mcm-2 LIs were consistently higher. All cells expressing a phase marker coexpressed Mcm-2, whereas Ki67 was not expressed in a proportion of these cells. The putative phase markers showed little coexpression. Labelling index values increased on progression from normal larynx through laryngeal dysplasia to squamous cell carcinoma for Mcm-2 (P = 0.001), Ki67 (P = 0.0002), cyclin D1 (P = 0.015), cyclin A (P = 0.0001) and cyclin B1 (P = 0.0004). There was no evidence of an increase in the LF for any phase marker. Immunohistochemistry can be used to estimate cell cycle state and phase in laryngeal biopsies. Our data argues against primary cell cycle phase abnormalities in laryngeal neoplasia.