对MOLT-4细胞用cyclin E+A/DNA流式细胞术分析 - 细胞周期六分法的建立

目的　越来越多的资料表明肿瘤是一类细胞周期性疾病 ,从而使得细胞周期分析在细胞生物学 ,肿瘤生物学领域显得格外重要。迄今为止 ,有很多方法应用于细胞周期分析中 ,但这些方法因为这样或者那样的缺点已无法适应今天的细胞周期分析。本研究的目的是建立一种新的 ,更为合理的细胞周期分析方法。方法　将cyclinE以及cyclinA的单克隆抗体按一定的比例混合后与固定了的MOLT 4细胞孵育 ,后用流式细胞仪检测。结果　我们发明的CyclinE A/DNA多参数流式细胞术能将细胞分为六个细胞群体 :G0 、早G1、晚G1、S、G2 、M期细胞。而在DNA含量直方图仅能区分三个细胞群体 :G0 /G1、S、G2 /M期细胞。结论　CyclinE A/DNA多参数流式细胞术能同时在同一样本中将细胞分为六个细胞群体 :G0 、早G1、晚G1、S、G2 、M期细胞。其对细胞周期分析明显优于目前采用的细胞周期分析方法 ,并且有明显的生物学基础。     

Standard and Quantitative Analysis of Cyclin E Threshold by Cyclin E／DNA Multiparameter Flow Cytometry

E-type cyclins (cyclin E1 and cyclin E2) areexpressed during the late G1 phase of the cell cycleuntil the end of the S-phase . The activity of cyclinE canli mit the passage of cells throughthe restric-tion point“R”which marks a“point of no return”for cells entering the division cycle from a restingstate or passing from G1 into S-phase[1].In manyhuman tumors ,the expressionlevels of cyclin Eissignificantly higher than physiological ones .Cyclins/DNA multiparameter flow cytometrydetects…     

复发与缓解期急性白血病的细胞周期分析

应用流式细胞术对初治、复发及缓解期急性白血病骨髓细胞的周期分布进行测定。复发期及缓解期患者骨髓增殖期细胞(s+G_2M％)高于初治病例。缓解期骨髓s+G_2M％存在个体差异性,多数接近正常骨髓s+G_2M％均值。3例同种异体骨髓移植后造血重建及1例胎肝移植后患者的骨髓s+G_2M％均达到或超过正常水平。     

流式细胞术检测细胞周期分析软件的比较

目的探讨流式细胞术中不同细胞周期分析软件在细胞周期分析中的特点和差异。方法采用碘化丙啶染色,在Epics XL流式细胞仪上检测细胞周期,分别应用MultiCycle和Modfit LT软件对检测数据进行分析,并比较分析结果。结果 MultiCycle和Modfit LT软件分析细胞周期各时相比例、DNA倍体分析、细胞增殖及细胞凋亡数值,比较其结果基本一致。仅人肺癌细胞系A549细胞G2/G1值应用2种不同软件分析其数据差异有统计学意义(P<0.01);Jurkat T细胞聚集体的比例应用2种不同软件分析其数据差异有统计学意义(P<0.01)。结论 MultiCycle和Modfit LT软件分析同一样本所得拟合结果有较高的一致性;对G2/G1值进行限制的操作可能更适用于组成成分较复杂的样本;虽然不同采集软件区分粘连细胞和细胞集落的方法不同,导致聚集体的比例差异显著,但并未对细胞周期分析结果的判定产生影响。     

造血组织周期细胞增殖特性的流式细胞术分析

研究造血组织细胞的动力学和增殖特性可帮助了解肿瘤的发生和病理分型,并为早期诊断,放射治疗和化疗,予后估计提供有价值的生物信息。本文报告用流式细胞术(Flow Cytometry),对健康自愿献髓者的外周血,骨髓及非血液系统肿瘤患者的骨髓单个核细胞和3—7月的胎儿肝,牌,骨髓细胞进行细胞周期测定,并对增殖细胞作了比较和分析。     

辣椒素诱导人膀胱癌RT4细胞细胞周期阻滞的实验观察

目的 了解辣椒素对膀胱癌RT4细胞生长的影响及可能机制.方法 采用细胞计数kit-8(CCK-8)试剂盒观察辣椒素(50、100、150、200、250μmol/L)对膀胱癌RT4细胞生长的影响,以辣椒素(0 μmol/L)为对照组;流式细胞术检测细胞周期和凋亡;逆转录-聚合酶链式反应(RT-PCR)和免疫荧光检测瞬时受体电位香草酸亚型1(TRPV1)的表达;Western印迹检测细胞周期蛋白P53、P21、细胞周期依赖性激酶(CDK)2表达水平.结果 100 μmol/L辣椒素明显抑制RT4细胞生长,细胞成活率为82.0%±6.2%,低于对照组(100.0%±12.4%,P=0.036),且生长抑制呈剂量依赖性,250μmol/L时细胞成活率仅为7.8%±2.9%(P=0.000).辣椒素能诱导RT4细胞G0/G1期阻滞,同样呈剂量依赖性,对照组G0/G1期细胞比例为37.4%±5.6%,而250 μmol/L时达到72.4%±5.3%(P=0.000).RT4细胞表达TRPV1受体mRNA和蛋白.与对照组比较,辣椒素处理后48 h,P53、P21表达上调,而CDK2表达下调.结论 辣椒素可以通过TRPV1受体调节P53、P21、CDK2表达以诱导人膀胱癌RT4细胞G0/G1期阻滞而抑制其生长.     

Cyclin D1调控U87胶质瘤细胞周期研究

目的探讨细胞周期蛋白D1（Cyclin D1,CCND1）在U87胶质瘤细胞周期调控中的作用。方法根据NCBI GenBank中cyclin D1序列设计双链siRNA,利用Western blotting技术验证设计的siRNA序列的有效性,并通过流式细胞术检测抑制CCND1对U87胶质瘤细胞周期进程的影响。结果经Western blotting检测,成功抑制了CCND1的表达。并且抑制CCND1后,U87细胞周期停滞在G1期,抑制了U87细胞的G1／S期转换。结论U87细胞中抑制CCND1可以显著抑制细胞周期进程,使细胞周期停滞在G0／G1期,为进一步研究胶质瘤的靶向治疗奠定了理论基础。     

Analysis of DNA Ploidy, Cell Cycle and Ki67 Antigen in Nasopharyngeal Carcinoma by Flow Cytometry

The expression of DNA ploidy, the cell cycle and Ki67 antigen in nasopharyngeal carcinoma (NPC) were studied and their relationship with the clinical biological behaviors and prognosis of NPC was evaluated. Biopsied specimens of NPC were made into cell suspension. By using cytometric double labeling Ki67 and DNA method, the expression of DNA ploidy, the cell cycle and Ki67 antigen were analyzed. The patients were followed up for about 3 years and the relationship between the above mentioned parameters and the clinical biological behavior and prognosis of NPC were evaluated. Of the 62 cases of NPC, the DNA aneuploid accounted for 29.03 %. The S phase cells accounted for 0 to 54 % in the cell cycle and the positive expression of Ki67 ranged from 0 to 52%. There were 40 cases of LPI (64.5%) including 15 negative cases and 22 cases of HPI (35.5%) respectively. The DNA anneploid content was positively related to the S phase cells. The patients having a low expression of Ki67 or DNA aneuploid in tumor cells were not sensitive to chemotherapy, liable to metastasis to distant organs and had a poor prognosis, while Ki67 showed no correlation with DNA ploidy and the cell cycle. It was suggested that DNA ploidy and Ki67 could be used as an independent and objective marker to evaluate the radiosensitivity and prognosis of NPC.     

Cyclin D1在胶质瘤细胞系中表达分布模式的初步研究

目的：探讨cyclin D1在胶质瘤细胞系中的细胞周期表达模式，将为从生物学功能上阐明cyclin D1在胶质瘤细胞周期演进中的作用．方法：利用流式细胞仪双参数法和细胞同步化，确定cyclin D1在胶质瘤细胞系SHG-44和BT-325中的细胞周期表达分布模式．结果：在胶质瘤细胞系SHG-44和BT-325中，Cyclin Dl的表达呈细胞周期依赖性：在SHG-44中，cyclin D1在G1期表达最高，4h左右达到峰值，S期及G2／M期下降，但仍可检测到；在BT-325中，cyclin D1在G1期表达最高，6h左右达到峰值，S期及G2／M期下降，但仍可检测到．结论：cyclin D1蛋白质的表达呈细胞周期依赖性，且与细胞周期行进有关；cyclin D1在SHG-44和BT-325胶质瘤细胞进入G1期的早期发挥着重要的生物学作用。     

流式细胞仪测定成人急性白血病患者DNA含量的临床意义

为探讨急性白血病患者骨髓细胞的周期分布，非整倍体，及其与临床疗效的关系。作者应用流式细胞仪测定了５７例ＡＬ患者的骨髓细胞ＤＮＡ含量。不同类型ＡＬ患者骨髓细胞Ｓ％均明显低于正常对照和缓解后患者。急性早幼粒细胞白血病Ｓ％最低；ＡＬ患者骨髓细胞ＤＮＡ非整倍体检出率２９．８％，４例完全缓解的患者仍检出ＤＮＡ非整倍体细胞，随化疗进展而消失。     

VES体外抑制Raji细胞增殖及机制的初步研究

目的：观察维生素E琥珀酸酯（VES）体外对人单核细胞白血病Raji细胞的抗增殖作用及对细胞周期和细胞周期素依赖性激酶2（CDK2）的影响。方法：应用荧光显微镜、流式细胞仪、免疫细胞化学染色等技术方法，体外观察VES对Raji细胞的作用。结果：VES对Raji细胞的生长抑制作用与诱发细胞凋亡的明显增加同步发生，具有剂量疚和时间效应关系，同时VES作用后，细胞内CDK2蛋白表达下降，细胞周期发生变化，细胞阻滞于C1期。结论：VES通过影响细胞周期调控因子CDK2的表达从而干扰细胞周期可能是其抗肿瘤的途径之一。     

放射敏感性不同的鼻咽癌细胞DNA倍体及细胞周期分布

目的　研究不同放射敏感潜能的人鼻咽癌细胞亚克隆株F1、S1的DNA倍体及细胞周期分布,并探讨与放射敏感性异质性的关系。方法　采用细胞培养及裸鼠体内实验,用Feulgen染色法和图像分析方法观察F1、S1细胞及其裸鼠移植瘤的DNA倍体及细胞周期的分布情况。结果　F1、S1细胞及其裸鼠移植瘤均为异倍体细胞或肿瘤。F1细胞及裸鼠移植瘤DNA含量、5C、>5C比S1组高(P<0.01),F1细胞及裸鼠移植瘤3-4C比例低于相应S1组(P<0.01)。F1细胞及其裸鼠移植瘤G2M比例均高于相应S1组,G0G1期低于S1组(P<0.05-0.01),F1细胞的S期比例低于S1细胞(P<0.01)。结论　F1DNA含量较高、倍体分布较广而右移,较多细胞处于对放射线敏感的G2M期,S1DNA含量较低、倍体分布较窄,较多细胞处于G0G1期和S期。DNA倍体及细胞周期分布地不同可能是F1、S1存在放射敏感性差异的原因之一。     

Vitamin C Inhibits Benzo[a]pyrene-lnduced Cell Cycle Changes Partly via Cyclin D1/ E2F Pathway in Human Embryo Lung Fibroblasts

Objective To study the molecular mechanism of the inhibitory effects of vitamin C on benzo[a]pyrene （B[a]P）-induced changes of cell cycle in human embryo lung fibroblast （HELF） cells. Methods The stable transfectants, HELF transfected with antisense cyclin D1 and antisense CDK4, were established. Cells were cultured and pretreated with vitamin C before stimulation with B[a]P for 24 h. The expression levels of cyclin DI, CDK4, E2FI, and E2F4 were determined by Western blot. Flow cytometric analysis was employed to detect the distributions of cell cycle. Results B[a]P significantly elevated the expression levels of cyclin D 1, E2F1, and E2F4 in HELF cells. Vitamin C decreased the expression levels of cyclin D 1, E2F1, and E2F4 in B [a]P-stimulated HELF cells. Dose-dependent relationships were not found between the different concentrations of vitamin C （10, 100, 500, 1000, and 5000 lamol/L） and the expression levels of cyclin D 1, E2F1, and E2F4 in HELF cells. The expression levels of cyclin D1, E2FI, and E2F4 in B[a]P-treated transfectants were lower than those in B[a]P-treated HELF cells. The expression levels of cyclin DI and E2F4 treated with vitamin C and antisense cyclin D1 were decreased compared with those treated with antisense cyclin DI alone. The effects of vitamin C combined with antisense CDK4 on the expression levels of cyclin DI and E2FI/E2F4 were similar to those of antisense CDK4 alone. B[a]P progressed HELF cells from GI to S phase. Both vitamin C and antisense cyclin DI suppressed the changes of cell cycle progressed by B[a]P. However, antisense CDK4 did not attenuate the above changes. Vitamin C combined with antisense CDK4 markedly suppressed B[a]P-induced changes of cell cycle as compared with antisense CDK4. But the inhibitory effects of vitamin C combined with antisense cyclin DI on B[a]P-induced changes of cell cycle were similar to those of vitamin C alone or antisense cyclin DI alone. Conclusions B[a]P progressed HELF cells from G1 to S phase via intracellular signaling pathway of cyclin D I/E2F. Vitamin C may modulate this signaling pathway to protect cells from injury caused by B[a]P.     

重组人生长激素对肾癌细胞株细胞周期动力学影响

目的　本实验试图阐明生长激素对于肾癌细胞有无促增殖和分化作用。方法　取对数生长期的人肾癌细胞株GRC -1,以氟尿嘧啶和 /或不同浓度的重组人生长激素 (rhGH)体外培养 ,2 4小时以后流式细胞仪测定细胞增殖周期和细胞凋亡等指标。结果　在空白对照组 ,G0 ～G1期的细胞数约占 5 3%,S期细胞数次之 ,G2 ～M期占不足 4%。在生长激素组 ,G0 ～G1期细胞数率降低 (P <0 .0 5 ) ,S期细胞百分率增高 (P <0 .0 1)。在单纯氟尿嘧啶组 ,G0 ～G1期细胞数增高 ,S期细胞数降低 ;同时加氟尿嘧啶和生长激素组与单纯氟尿嘧啶组相比 ,G0 ～G1期细胞数进一步增加 ,S期细胞数进一步降。结论　rhGH在体外作用于GRC -1细胞 ,能诱导细胞分化 ,促使静止细胞群进入增殖周期 ,并使处于DNA合成期的细胞增多 ,说明rhGH在体外有促进肾癌细胞生长增殖的作用 ,所以 ,我们推断肾癌细胞膜表面可能存在生长激素受体。氟尿嘧啶能使S期细胞数降低即抑制细胞的增殖 ,与氟尿嘧啶的作用机理相符。生长激素和氟尿嘧啶合用 ,S期细胞数进一步降低 ,说明生长激素能增强氟尿嘧啶抗肿瘤细胞生长增殖作用。     

细胞周期素A、E在胃癌组织中的表达及与细胞周期的关系

目的　探讨cyclinA和CyclinE在胃癌组织中的表达及与细胞周期的关系。方法　采用免疫组织化学方法和流式细胞仪分别检测cyclinA和cyclinE在胃癌组织中的表达和测定胃癌组织中S期细胞所占百分比。结果　cyclinA的阳性率为 71 8% (2 8/ 39) ;cyclinE的阳性率为 4 8 7% (19/ 39) ;cyclinA的阴阳性S期百分数所占的比例无显著性差异 ;cyclinE的阴阳性S期百分数所占的比例有显著性差异。它们的阴阳性与DNA的二倍体或异倍体无关 ;cyclinA和cyclinE 2者在胃癌组织中的表达有关联。结论　cyclinA在胃癌组织中的表达阳性率明显高于cyclinE ,2者在胃癌组织中的表达相关联 ,cyclinE阳性者的S期细胞比例明显高于阴性者     

Different patterns of cyclin D1/CDK4-E2F-1/4 pathways in human embryo lung fibroblasts treated by benzo[a]pyrene at different doses

Objective To investigate the roles of the cyclin D1/CDK4 and E2F-1/4 pathways and compare their work patterns in cell cycle changes induced by different doses of B[a]E Methods Human embryo lung fibroblasts （HELFs） were treated with 2 μmol/L or 100 μmol/L B[a]P which were provided with some characteristics of transformed cells （T-HELFs）. Cyclin D l, CDK4 and E2F-1/4 expressions were determined by Western blotting. Flow cytometry was used to detect the distribution of cell cycle. Results After B[a]P treatment, the proportion of the first gap （G 1） phase cells decreased. CDK4 and E2F-4 expression did not change significantly. In 2 μmol/L treated cells, a marked overexpression of cyclin D1 and E2F-1 was observed. However, in T-HELFs overexpression was limited to cyclin D1 only, and no overexpression of E2F-1 was observed. The decreases of G1 phase in response to B[a]P treatment were blocked in antisense cyclin D1 and antisense CDK4 transfected HELFs （A-D1 and A-K4） and T-HELFs （T-A-D1 and T-A-K4）. After 2 μmol/L B[a]P treatment, overexpression of E2F-1 was attenuated in A-D1, and E2F-4 expression was decreased significantly in A-K4. In T-A-D1 and T-A-K4, E2F-4 expression was increased significantly, compared with T-HELFs. The E2F-1 expression remained unchanged in T-A-D1 and T-A-K4. Condusions Cyclin DI/CDK4-E2F-1/4 pathways work in different patterns in response to low dose and high dose B[a]P treatment. In HELFs treated with 2 μmol/L B[a]P, cyclin D1 positively regulates the E2F-1 expression while CDK4 negatively regulates the E2F-4 expression; however, in HELFs treated with 100 μmol/L B[a]P, both cyclin D1 and CDK4 negatively regulate the E2F-4 expression.     

XPD/P44亚复合物对人肝癌细胞周期的调控

目的 探讨着色性干皮病互补基因D(XPD)/P44亚复合物对人肝癌细胞周期的调控机制.方法 重组质粒增强型绿色荧光蛋白(pEGFP)-N2/XPD,空载质粒pEGFP-N2分别通过Lipofectamine 2000转染SMMC-7721细胞,构建稳定表达的细胞株,再应用P44反义寡核苷酸阻断SMMC-7721-pEGFP-N2/XPD中P44的表达.实验分为6组:①空白对照组;②SMMC-7721-pEGFP-N2组;③SMMC-7721-pEGFP-N2/XPD组;④反义SMMC-7721-pEGFP-N2/XPD翻译起始部位组;⑤反义SMMC-7721-pEGFP-N2/XPD翻译终止部位组;⑥反义SMMC-7721-pEGFP-N2/XPD外显子5组.用逆转录聚合酶链反应(RT-PCR)、Western印迹法检测转染各组细胞内P44、XPD以及cdk7、cdk2、c-myc和cdc25A的表达量,并用四甲基偶氮唑盐(MTr)和流式细胞仪检测细胞增殖及其细胞周期的变化.结果 ①、②组中P44、XPD的mRNA表达量均明显低于③组(均P<0.01).P44、XPD的蛋白变化趋势与其mRNA变化趋势一致;而细胞周期调控基因cdk7、cdk2、c-myc和cdc25A的mRNA及蛋白的表达量下调,细胞增殖力减弱,③组与①组、②组相比,停滞在G1期细胞多,进入S期细胞少.阻断P44后XPD的表达量下调,④、⑤、⑥组中XPD的mRNA表达量分别是③组的(0.55±0.09)、(0.65±0.05)、(0.61±0.11)倍,差异均有统计学意义(均P<0.01);④组、⑤组、⑥组中XPD蛋白的表达量分别为③组的(0.75±0.06)、(O.79±0.02)、(0.88±0.07)倍,差异均有统计学意义(均P<0.01).cdk7、cdk2、c-myc和cdc25A的mRNA以及蛋白的表达量上调;细胞增殖明显;与③组相比,④组、⑤组、⑥组的细胞进入S期细胞增多,停滞在G1期细胞减少.结论 XPD基因具有抑制癌细胞生长、促进癌细胞凋亡的功能;XPD的表达受其分子伴侣P44的调节,XPD/P44亚复合物可能是通过DNA损伤检控点来调控细胞周期的.     

An analysis of DNA content in 405 cases of human tumor by flow cytometry]

This study was intended to evaluate the relationship between the DNA content of cell cycle and the histology in human tumors. We detected the DNA content in 405 cases of fresh human tumor tissue by means of flow cytometry and observed the histology of tumor with light microscopy. The occurrences of aneuploidy in 22 cases of benign tumor and 383 cases of malignant tumor were 27% and 52% respectively. There were differences in aneuploidy in different histologic types of tumor. The aneuploidy in adenocarcinoma was about 50%, that in sarcoma was more than 37%, and that in squamous carcinoma less than 17%. S > or = 10, G2/m > or = 10 or S > or = 20, G2/m > or = 5 were present in malignant tumors, but were not found in benign tumors. The results suggest that there is obvious difference in the occurrences of aneuploidy in benign and malignant tumors and in different histologic type of tumor(P < 0.05). Benign and malignant tumor may be distinguished when SPF and G2/m reach a higher level (P < 0.01).     

miR-150靶向调控FOXO4促进宫颈癌C 33A细胞生长与生存

【摘要】 目的 检测miR 150在宫颈癌细胞C 33A中的表达,并探讨其是否通过调控靶向基因FOXO4促进宫颈癌细胞的增殖与凋亡。方法 将携带miR 150 mimic（模拟物）和miR 150 inhibitor（抑制物）、阴性对照siRNA经 LipofectamineTM 2000 转染至C 33A细胞中,将细胞分为 NC组( 阴性对照组) 、miR 150 模拟物组( 转染miR 150 mimic细胞组) 及 miR 150 in 抑制物组( 转染miR 150 inhibitor细胞组)。采用流式细胞仪、TUNEL检测法、荧光定量PCR、Western bolt 分别检测miR 150对宫颈癌C 33A细胞周期、凋亡以及周期和凋亡相关基因的影响。3’ 端非翻译区（UTR）荧光素酶活性分析证实miR 150的直接靶向作用。结果 miR 150模拟物促进宫颈癌细胞C 33A的增殖与凋亡,抑制物作用相反,miR 150模拟物促进细胞由G1/G0期进展到S期,从而促进宫颈癌细胞增殖,抑制物相反,miR 150调控几个细胞周期、凋亡相关基因CyclinD1、 p27、 BIM和FASL的表达; miR 150通过靶向结合FOXO4的3’UTR区从而抑制FOXO4的表达。结论 miR 150靶向作用于FOXO4从而调节多种基因的表达以致宫颈癌细胞C 33A的增殖与凋亡。     

用流式细胞光度术测定冬凌草甲素和博莱霉素A_5合用对艾氏腹水癌细胞周期移行的影响

用流式细胞光度术(FcM)测定了冬凌草甲素和博莱霉素A_5联合应用对艾氏腹水癌细胞增殖周期的影响。发现冬凌草甲素可阻滞细胞M→G_1或M→G_1和G_2→M的进程,导致G_2→M%的增加,博莱霉素A_2阻断G_2→M的进行,使G_2%增加,合用后G_2 M%的构成比随时延长而大于单用组,这些现象有利于博莱霉素A_5等对M,G_2敏感时相的细胞杀伤。