急性白血病血管内皮生长因子表达的研究

目的:研究血管内皮生长因子(VEGF)表达与白血病细胞增殖的相关性.方法:应用流式细胞术检测骨髓单个核细胞VEGF蛋白的表达,PI染色测定白血病细胞的增殖指数.结果:急性白血病细胞VEGF表达量比对照组高(P＜0.01),与细胞增殖指数呈正相关(P＜0.05).结论:VEGF在急性白血病的发病过程中可能起重要作用.     

和厚朴酚对人白血病细胞系U937细胞增殖和凋亡的影响

 【目的】 探讨和厚朴酚(HNK)对白血病细胞系U937细胞增殖和凋亡的影响｡【方法】 将U937细胞和正常外周血单核细胞分别与HNK共培养,Hoechst33342荧光染色检测细胞凋亡形态,用噻唑蓝比色法(MTT)检测HNK对细胞增殖的抑制作用;流式细胞仪检测凋亡;罗丹明123检测线粒体膜电位;Caspase3/7活性检测试剂盒检测Caspase3/7活性;荧光定量PCR(FQ-PCR)检测Caspase-3､Caspase-7 mRNA水平变化｡【结果】 HNK对U937的体外增殖有显著抑制作用,48 h半数抑制浓度(IC50)为11.8 μg/mL,而对正常外周血单核细胞的IC50为40.3 μg/mL,Annexin V/PI双标染色显示,细胞凋亡与和厚朴酚呈浓度和时间依赖相关｡HNK明显降低U937细胞线粒体膜电位,增加Caspase3/7活性及mRNA表达水平,但对正常外周血单核细胞的增殖抑制和凋亡作用不明显｡【结论】 HNK可能通过激活caspase-3/7抑制U937细胞增殖､诱导U937细胞凋亡｡     

甘露聚糖结合凝集素诱导人白血病细胞系U937细胞凋亡

目的研究甘露聚糖结合凝集素(MBL)对白血病细胞系U937凋亡的影响。方法不同浓度MBL处理U937细胞后,应用CCK-8法分析细胞增殖情况,Hoechst33258染色观察细胞核及染色质,AnnexinV/PI双染流式细胞术分析细胞凋亡率,real-timePCR和免疫印迹分析凋亡相关基因及蛋白表达。结果 30～50μg/mlMBL培养72h,U937细胞增殖明显受抑,出现不同程度核固缩、核碎裂;随MBL浓度升高或作用时间延长,凋亡细胞数逐渐增多;Fas、Caspase-3mRNA、Fas和FasL蛋白表达量升高,Caspase-3和多腺苷二磷酸多聚酶(PARP)蛋白被剪切激活或失活。结论 MBL可诱导白血病细胞U937细胞凋亡,其机制与上调Fas表达、剪切Caspase-3和PAPR有关。     

Antitumor Effect of Interferon-α on U937 Human Acute Leukemia Cells in vitro and Its Molecular Mechanism

In order to investigate the antitumor effect and molecular mechanism of interferon-α(IFN-α) on human acute myeloid leukemia cell line U937 cells in vitro,the proliferation of U937 cells was determined by MTT assay,the apoptosis rate was analyzed by flow cytometry(FCM),and the mRNA expression of cell cycle regulatory protein cyclin E was detected by RT-PCR.The results showed that IFN-α could inhibit the proliferation of U937 cells significantly in a dose-and time-dependent way(P<0.01),and induce the apoptosis of U937 cells also in a dose-and time-dependent manner at the concentration of 1000-4000 U/L(P<0.01).The apoptosis rate of U937 cells was even over 50% when cultured with IFN-α for 36-48 h at the concentration of 2000-4000 U/L.Moreover,the expression of cyclin E mRNA was markedly inhibited by the addition of IFN-α,and the inhibition was time-dependent(P<0.01).It was concluded that the anti-leukemia mechanism of IFN-α might be correlated with its antiproliferative and apoptotic inducing effects,and the down-regulation of the cyclin E expression might be one of its molecular mechanisms.     

白藜芦醇诱导胃癌细胞脱落凋亡

目的 研究白藜芦醇诱导胃癌细胞脱落凋亡的作用。方法 采用细胞形态学观察、Annxin-V和PI细胞染色和流式细胞仪检测，观察白藜芦醇(0．1、0．2、0．5 mmol／L)对脱落培养胃癌细胞BGC823、SGC7901和HGC27的生长状态、细胞周期的影响及诱导脱落凋亡的作用。结果 倒置显微镜下发现脱落培养的胃癌细胞聚集成团生长，0．1mmol／L白藜芦醇作用细胞后可抑制抗脱落凋亡的胃癌细胞聚集成团；流式细胞仪检测和Annxin-V和PI细胞染色表明0．5mmol／L白藜芦醇可分别阻滞脱落培养的BGc823、sGc7901和HGc27细胞于G0／G1、S、G2／M期，并可诱导这3株胃癌细胞发生脱落凋亡，其中自藜芦醇诱导HGC27细胞脱落凋亡比例达19．3％，远高于其他两株胃癌细胞。结论自藜芦醇可诱导胃癌细胞发生脱落凋亡。     

白藜芦醇诱导急性T淋巴母细胞白血病Jurkat细胞凋亡

目的：探讨白藜芦醇抑制急性T淋巴母细胞白血病Jurkat细胞增殖、引发S期阻滞和诱导凋亡的作用。方法：白藜芦醇体外处理培养的Jurkat细胞后,采用MTT法测定细胞活力,Wrigh-Giemsa染色、Hoechest 33258/PI荧光染色和透射电镜观察Jurkat细胞的形态学改变;流式细胞术分析细胞周期。结果：白藜芦醇0.2 mmol·L^-1处理组的抑制Jurkat细胞增殖率达64.01%,并具有剂量和时间依赖性。白藜芦醇处理细胞24 h后可见凋亡形态学改变,Hoechest 33258/PI染色显示白藜芦醇处理组细胞部分细胞核呈亮蓝色,随培养时间延长,亮蓝色荧光染色细胞数量逐渐增多。Wrigh-Giemsa染色和透射电镜观察发现部分细胞体积缩小、染色质浓缩及边集等现象的出现。流式细胞术检测表明,0.05 mmol·L^-1白黎芦醇处理48 h组62.57％的细胞被阻滞于细胞周期S期,亚二倍体细胞数量12.01%,未加药对照组细胞亚二倍体细胞数量2.05%。结论：白藜芦醇可以抑制Jurkat细胞的增殖,引起细胞周期的S期阻滞,并诱导其发生凋亡     

白藜芦醇抑制HepG2细胞生长和对细胞间隙连接通讯的影响

目的研究白藜芦醇对人肝癌细胞HepG2的生长抑制作用以及对细胞间隙连接通讯（GJIC）的影响。方法利用MTT法观察白藜芦醇对HepG2细胞的生长抑制作用。流式细胞仪检测细胞周期的时相分布，使用荧光探针5＇-CFDA／AM标记细胞．采用荧光漂白后重恢复技术（FRAP）和共聚焦显微镜技术观察白藜芦醇对HepG2细胞间隙连接通讯的影响。结果不同浓度的白藜芦醇处理细胞不同时间后，HepG2细胞的生长增殖明显受到抑制，抑制率最高达92．1％，各处理组间比较均有显著性差异（F=18．532，P〈0．05）；细胞周期分析显示，白藜芦醇能诱导HepG2细胞在S期停滞．抑制细胞DNA的合成并可诱导细胞凋亡：另外，白藜芦醇有恢复HepG2细胞间隙连接通讯的功能，且随浓度增加而增加。结论白藜芦醇可抑制HepG2细胞增殖，使细胞停滞于S期，并能诱导细胞凋亡；白藜芦醇还有恢复HepG2细胞间隙连接通讯功能的作用，提示此作用可能是白藜芦醇抑制HepG2细胞增殖和抗肿瘤作用的机制之一。     

Effect of Cytokines Secreted by Human Adipose Stromal Cells on Endothelial Cells

Recently, several observations have pointed to the presence of a population named adipose-derived adult stem cells (ADAS) or adipose stromal cells (ASCs) in human adipose tissue. Subsequent studies revealed that ASCs were multipotent, differentiating along the cardiac myocyte, endothelial, neuronal, and other cells lineages[1, 2], and could secrete cytokines such as HGF and VEGF[3]. Since adipose tissue is an abundant, accessible, and re- plenishable source of adult stem cells that can b…     

胡桃醌对白血病细胞增殖的抑制作用

目的: 探讨胡桃醌对白血病细胞增殖的抑制作用,阐明胡桃醌抗白血病的机制。方法: 体外培养人白血病K562、Jurkat、U937、U266及NB4细胞株,按不同处理因素分组：调零组,含有培养基、MTT和三联溶解液;空白对照组,含有各种人白血病细胞、药物溶解介质、培养液、MTT 和三联溶解液;实验组,分别采用不同浓度（终浓度为0.25、0.50、1.00、2.00、4.00、6.00、8.00和16.00 mg/L）的胡桃醌作用各种细胞株。MTT比色法检测胡桃醌对白血病细胞的增殖抑制作用。流式细胞术测定不同浓度胡桃醌对K562细胞株凋亡的影响。结果: 胡桃醌对白血病K562、Jurkat、U937、U266及NB4细胞株增殖均有较强的抑制作用,其半数抑制浓度(IC50)(48 h)分别为3.87、1.13、4.48、1.87和4.67 mg/L。其中胡桃醌对Jurkat和U266细胞的抑制效果最为明显（P<0.05）,胡桃醌对其他3个白血病细胞株的IC50值均<40 mg/L,且胡桃醌对这5种细胞株增殖的抑制作用呈时间和剂量依赖性。流式细胞术检测显示,胡桃醌可诱导K562细胞凋亡,1、2、4、8和16 mg/L胡桃醌作用6 h,K562细胞凋亡率分别为(4.54±0.6) %、(11.5 4±0.6) %、(28.7±0.3) % 、(45.0±1.2) %和(76.1±1.4) %。结论: 胡桃醌可抑制多种白血病细胞株增殖,其机制可能与促进白血病细胞凋亡相关。     

B7H3分子在人髓性白血病细胞株U937和K562上的表达及生物学意义

目的:研究共刺激分子B7H3分子在人髓性白血病细胞株U937和K562上的表达及其生物学意义?方法:应用流式细胞术及RT-PCR法检测B7H3分子在人髓性白血病细胞株U937和K562上的表达;并用鼠抗人B7H3单克隆抗体(mAb)作用于U937和K562细胞株,细胞计数法检测鼠抗人mAb B7H3对U937和K562细胞株生长的影响?结果:流式细胞术分析显示U937和K562细胞株均表达B7H3膜蛋白,RT-PCR检测到U937和K562细胞有B7H3 mRNA产物;鼠抗人mAb B7H3对U937和K562细胞株有抑制生长的作用?结论:U937和K562细胞株组成性表达B7H3分子;鼠抗人mAb B7H3与U937和K562细胞表面的B7H3分子交联后可以抑制白血病细胞的生长?     

人脐带间充质干细胞培养上清液对甲状腺乳头状癌细胞诱导人脐静脉内皮细胞血管形成的影响

目的: 探讨人脐带间充质干细胞（human umbilical cord mesenchymal stem cells,hUCMSCs）对甲状腺乳头状癌细胞TPC-1诱导人脐静脉内皮细胞（human umbilical vein endothelial cells, HUVEC）血管形成的作用。方法: 使用不同浓度的hUCMSCs培养上清液（hUCMSC-CM）刺激甲状腺乳头状癌细胞系TPC-1;采用基质胶小管形成实验检测经hUCMSC-CM作用后的TPC-1细胞对HUVEC血管形成的影响;CCK-8法检测hUCMSC-CM对TPC-1细胞增殖能力的影响;实时荧光定量PCR、蛋白质印迹法、酶联免疫吸附实验（ELISA）分别检测经hUCMSC-CM作用后的TPC-1细胞中血管内皮生长因子（vascular endothelial growth factor, VEGF）的mRNA和蛋白表达及其培养上清液中VEGF的含量。结果： 与对照组相比,hUCMSC-CM可显著抑制TPC-1细胞的增殖（P<0.05和P<0.01）;经hUCMSC-CM刺激后的TPC-1细胞诱导HUVEC血管形成能力显著下调（P<0.001）,且TPC-1细胞中VEGF的mRNA和蛋白表达水平均显著降低（P<0.001）,其培养上清液中VEGF含量显著降低（P<0.05）。结论： hUCMSC-CM可显著下调TPC-1细胞中VEGF的表达及分泌,抑制TPC-1细胞诱导HUVEC血管形成的能力。     

白藜芦醇对高糖高脂处理的原代人脐静脉血管内皮细胞E-选择素表达及NO分泌的影响

目的：探讨白藜芦醇对高糖、高脂培养人原代脐静脉血管内皮细胞E-选择素mRNA表达及胰岛素刺激的一氧化氮（NO）分泌的影响。方法：原代培养人脐静脉内皮细胞,以0．1μmol／L白藜芦醇预处理4h后,以高糖（33．3mmol／L葡萄糖）＋高脂（棕榈酸0．5mmol／L）DMEM培养基培养24h,以RT—PCR法检测培养细胞E-选择素的表达。部分细胞经100nmol／L胰岛素刺激25min后,以硝酸还原酶法检测培养上清NO的含量。结果：与正常对照组相比．高糖高脂培养使内皮细胞E-选择素的表达升高,同时胰岛素刺激的NO分泌降低。而白藜芦醇预处理可以明显降低E-选择素的表达,并明显促进胰岛素对内皮细胞NO分泌的刺激作用。结论：白藜芦醇可以改善高糖、高脂诱导的血管内皮细胞胰岛素刺激的NO分泌作用,并可降低E-选择素表达,保护内皮细胞的功能,从而可能在糖尿病动脉粥样硬化的防治中具有广阔的前景。     

Effects of TNF-α and curcumin on the expression of VEGF in Raji and U937 cells and on angiogenesis in ECV304 cells

Background To better understand the possibilities of antiangiogenic tumor therapy and to assess possible side effects, we investigated the effect of tumour necrosis factor （TNF）-α and curcumin on the expression of vascular endothelial growth factor （VEGF） in U937 and Raji cell lines and their effect on angiogenesis in a human umbilical vein endothelial cell （HUVECs）-derived cell line （ECV304）, and also the relationship between Notchl and VEGF. The aim of this study was to elucidate potential mechanisms controlling tumor neovascularization. Methods VEGF secreted by U937 and Raji cell lines was determined by ELISA. Angiogenesis was tested by network formation of endothelial cells on Matrigel. Levels of VEGF mRNA in U937 and Raji cells and Notchl mRNA levels in EV304 cells were determined by RT-PCR. Results Secretion of VEGF by U937 and Raji cells was increased by TNF-α treatment and suppressed by curcumin （P 〈 0. 01 ）. The mRNA expression of VEGF165 and VEGF121 （containing 165 and 121 amino acid residues, respectively） were detected in any fractions. TNF-α augmented the expression of VEGF165 and VEGF121 mRNA and curcumin reduced the expression （P 〈0. 01 ）. No networks or cords formed in control and curcumin groups. There was tube formation on matrigel in the supernatants of the Raji culture group and the supernatants groups treated by VEGF group and TNF-α in Raji cell. Notch1 mRNA was detected but there was no significant change in the VEGF group compared with control （P 〉 0. 05）. Conclusions Expressions of VEGF mRNA in U937 and Raji cells were increased by TNF-α and suppressed by curcumin. VEGF and TNF-α can induce angiogenesis, and curcumin can inhibit angiogenesis in ECV304 cells.     

miRNA-195抑制剂对高糖培养血管内皮细胞生物学行为的影响及机制

目的：探讨高糖环境下miRNA-195 抑制剂对血管内皮细胞行为的影响及机制。方法：培养人脐静脉血管内皮细胞（HUVECs）ECV304,分别用高糖（HG）、miRNA-195模拟质粒（mimic）、miRNA-195抑制质粒（inhibitor）、Sirt1激动剂白藜芦醇（RES）处理细胞株,CCK-8试剂盒检测增殖活力,RT-PCR检测miRNA-195及Sirt1 mRNA水平,Western blot检测Sirt1、p-FoxO1、FoxO1、Caveolin-1、血管内皮生长因子（VEGF）蛋白表达水平,划痕实验检测HUVECs的迁移能力,transwell小室实验检测细胞侵袭能力,Matrigel胶检测HUVECs的成管能力。结果：HG促进HUVECs的增殖、迁移、侵袭,增强成管能力（P ＜0.05）;而miRNA-195inhibitor则明显抑制HUVECs的增殖、迁移,降低成管能力（P ＜0.05）。结论：miRNA-195 inhibitor或许通过miRNA-195/Sirt1/FoxO1/Caveolin-1/VEGF通路影响HG环境下HUVECs的生物学功能。     

Antitumor Effect of Interferon-α on U937 Human Acute Leukemia Cells in vitro and Its Molecular Mechanism

In order to investigate the antitumor effect and molecular mechanism of interferon-α(IFN-α) on human acute myeloid leukemia cell line U937 cells in vitro, the proliferation of U937 cells was determined by MTT assay, the apoptosis rate was analyzed by flow cytometry (FCM), and the mRNA expression of cell cycle regulatory protein cyclin E was detected by RT-PCR. The results showed that IFN-α could inhibit the proliferation of U937 cells significantly in a dose- and time-dependent way (P<0.01), and induce the apoptosis of U937 cells also in a dose- and time-dependent manner at the concentration of 1000-4000 U/L (P<0.01). The apoptosis rate of U937 cells was even over 50% when cultured with IFN-α for 36-48 h at the concentration of 2000-4000 U/L. Moreover, the expression of cyclin E mRNA was markedly inhibited by the addition of IFN-α, and the inhibition was time-dependent (P<0.01). It was concluded that the anti-leukemia mechanism of IFN-α might be correlated with its antiproliferative and apoptotic inducing effects, and the down-regnlation of the cyclin E expression might be one of its molecular mechanisms.     

反义血管内皮细胞生长因子基因转染在调节人肺巨细胞癌血管生成及肿瘤生长和转移中的作用

目的　探讨反义血管内皮细胞生长因子 (VEGF)基因转染在减少肿瘤细胞内源性血管内皮细胞生长因子分泌 ,调节肿瘤血管生成和肿瘤生长转移中的作用。方法　利用脂质体法将反义基因转染到高转移性人肺癌细胞 (PG) ,以转基因肿瘤细胞培养上清刺激人脐静脉内皮细胞 ,进行噻唑蓝 (MTT)和3H胸腺嘧啶 (3HTdR)掺入实验 ;并将转基因肿瘤细胞接种于裸鼠体内 ,应用免疫组织化学法检测肿瘤内微血管密度 (MVD) ,探讨MVD与肿瘤生长转移的关系。结果　反义VEGF基因的转染 ,使肿瘤细胞VEGF分泌减少 ,其培养上清刺激人脐静脉内皮细胞 (HUVEC)生长活性较空载体组减弱 ,HUVEC之DNA合成减少 ,裸鼠移植瘤内MVD为 41个± 9个 ,空载体组为 5 8个± 10个 ,两组比较t=2 715 ,P <0 .0 5。结论　反义VEGF基因的转染使肿瘤细胞分泌VEGF能力下降 ,肿瘤血管生成能力降低 ,这在肿瘤的生长和转移调节中有重要意义。     

反义血管内皮细胞生长因子基因转染在调节人肺巨细胞癌血管生成及肿瘤生长

OBJECTIVE: To study the regulatory effect of antisense VEGF121 cDNA transfection on endogenous VEGF secretion and angiogenesis of human metastatic lung carcinoma cell line PG and explore the significance of microvessel density (MVD) in tumor growth and metastasis. METHODS: The eukaryotic expression vectors bearing antisense VEGF121 cDNA was transfected into PG cells. Human umbilical vein endothelial cells (HUVEC) were cultured in conditioned mediums from transfected cells, and proliferation was determined by methyl thiazolyl tetrazolium (MTT) and 3H thymidine incorporation (3H TdR) assays in vitro. Microvessel density (MVD) in xenografted tumors in nude mice was analyzed by immunohistochemistry. RESULTS: The transfectant of antisense VEGF121 cDNA exhibited a reduction in VEGF secretion. HUVEC grown in conditioned medium from the antisense VEGF transfected cells exhibited a decrease in capacities of DNA syntheses and cell proliferation. MVD of tumor with transfected antisense VEGF gene was significantly lower than that in control vector. CONCLUSION: Antisense VEGF gene transfection can inhibit vascular endothelial cell proliferation in vitro and tumor angiogenesis in vivo, which may explain its inhibitory effects on tumor growth and metastasis.     

血管内皮生长因子及其Flt-1受体在白血病细胞中的表达

目的探讨人白血病细胞系血管内皮生长因子(VEGF)及其Fh-1受体的表达,进一步阐明VEGF在白血病细胞异常增殖中的作用机制.方法以人脐静脉血管内皮细胞系ECV304作为对照,采用RT-PCR半定量技术和免疫组化检测体外培养的人白血病细胞系K562和HL-60中VEGF及Flt-1的表达.采用MTT试验观察VEGF反义寡核苷酸(VEGF-AS)对白血病细胞体外增殖的影响.结果K562和HL-60细胞同时表达VEGF和Fh-1 mRNA和蛋白.VEGF-AS能够抑制白血病细胞体外增殖.结论在白血病细胞异常增殖中自分泌VEGF可能起着重要作用.