三氧化二砷对肝癌细胞株BEL-7404、SMMC-7721 和肝癌裸鼠移植瘤模型的作用

砒霜主要成分为三氧化二砷(Ａｓ2 Ｏ3) ,用于治疗急性早幼粒细胞白血病效果良好 ,且毒副作用较低[1] 。本研究旨在观察Ａｓ2 Ｏ3对人肝癌细胞株ＢＥＬ 740 4,ＳＭＭＣ 772 1和肝癌裸鼠移植瘤模型的影响 ,探讨Ａｓ2 Ｏ3对原发性肝癌的作用及其机制 ,现将结果报道如下。一、材料和方法1.药物和试剂 :0 .1%Ａｓ2 Ｏ3溶液购自哈尔滨医科大学第一附属医院 ;噻唑蓝 (ＭＴＴ)购自美国Ｓｉｇｍａ公司 ;细胞凋亡原位检测试剂盒为德国ＢｏｅｈｒｉｎｇｅｒＭａｎｎｈｅｉｍ公司产品。小鼠抗增殖细胞核抗原多克隆抗体为美国Ｃａｌｂｉｏｃｈｅｍ公司产…     

抗人VEGF发卡状核酶对肝癌细胞SMMC-7721生物学特性的影响

我们设计了针对VEGF的发卡状核酶，观察其对肝癌细胞生物学特性的影响。     

淫羊藿苷对肝癌细胞株SMMC-7721增殖与凋亡的影响

目的:探讨淫羊藿苷对肝癌细胞株SMMC-7721的抑增殖和促凋亡作用及其机制。方法:以不同浓度(0,6.25,12.5,25,50,100,200,400,800μg/mL)的淫羊藿苷作用于SMMC-7721细胞株不同时间(24,48,72 h)后,用MTT法检测药物对SMMC-7721细胞增殖状态;以不同浓度淫羊藿苷作用SMMC-7721细胞48 h后,用流式细胞仪检测细胞周期及凋亡情况,用Western blot检测细胞PCNA,Bcl-2,Bax蛋白的表达。结果:与对照组(0μg/mL)比较,各浓度的淫羊藿苷均明显抑制SMMC-7721细胞的增殖,并呈明显的时间与浓度依赖性(均P<0.05);淫羊藿苷呈浓度依赖性诱导SMMC-7721细胞发生G0/G1期阻滞和凋亡,下调SMMC-7721细胞PCNA蛋白和Bcl-2蛋白,上调Bax蛋白的表达(均P<0.05)。结论:淫羊藿苷可抑制SMMC-7721细胞增殖并诱导其凋亡。     

PTD-BCR/ABL SH3融合蛋白对肝癌细胞株SMMC-7721的杀伤作用

目的：探讨PTD-BCR／ABL SH3融合蛋白对肝癌细胞株SMMC-7721的体外杀伤活性。方法：通过将PTD-BCR／ABL SH3融合蛋白与SMMC-7721细胞共培养，用免疫细胞化学染色的方法观察和检测PTD-BCR／ABL SH3融合蛋白进入细胞后的定位，MTT法检测蛋白对肝癌细胞的杀伤活性，电镜观察SMMC-7721肝癌细胞与PTD-BCR／ABL SH3融合蛋白共培养后超微结构变化。结果：PTD-BCR／ABL SH3融合蛋白进入SMMC-7721细胞株后主要分布在细胞核，少量分布于胞浆。融合蛋白对SMMC-7721细胞株的杀伤作用呈浓度依赖性，在所选蛋白浓度和观察时限内．当蛋白浓度≥0．14mg／ml、共培养4h后，SMMC-7721细胞形态就出现改变，8h后出现死亡细胞。电镜显示，PTD-BCR／ABL SH3融合蛋白促使SMMC-7721细胞株凋亡。结论：PTD-BCR／ABL SH3融合蛋白能抑制SMMC-7721细胞株增殖，促进其凋亡，并改变其骨架结构。     

短发夹RNA对SMMC-7721肝癌细胞株ICAM-1基因表达的影响

目的探讨细胞间黏附分子-1(ICAM-1,CD54)基因的短发夹结构RNA(shRNA)表达载体pGenesil-1/CD54对SMMC-7721肝癌细胞株CD54表达的抑制作用。方法设计CD54基因shRNA片段和阴性对照shRNA片段,构建pGenesil-1/CD54和阴性对照表达载体,载体在coli菌扩增、鉴定后,转入SMMC-7721细胞。应用免疫组织化学技术检测肝癌细胞中CD54基因的表达。结果阳性和阴性表达载体酶切,电泳有65 bp和4.9 kb条带,载体插入片段测序,与设计序列相同,表达载体构建正确。免疫组织化学检测表明,阴性对照表达载体转染SMMC-7721细胞,细胞膜和细胞质着黄色和棕色,pGenesil-1/CD54转染SMMC-7721细胞,不着色或着淡黄色。结论CD54的shRNA能特异性地抑制CD54在肝癌细胞SMMC-7721中的表达。     

CD147-shRNA对SMMC-7721细胞增殖和运动能力的影响

目的：探讨CD147-shRNA重组质粒对肝癌细胞SMMC-7721增殖和运动能力的影响。方法：将前期合成并筛选的重组质粒pBS/U6/CD147-shRNA3转染至SMMC-7721细胞中,采用MTT法检测细胞增殖;采用划痕愈合实验测定细胞迁移能力。结果：MTT实验结果显示0.3 μg和1 μg的pBS/U6/CD147-shRNA3转染后,SMMC-7721细胞的增殖受到明显抑制,且呈浓度依赖性,最大抑制率为32.2%;转染对照载体的SMMC-7721细胞在划痕24 h后趋于愈合,相比之下,转染pBS/U6/CD147-shRNA3的SMMC-7721细胞划痕愈合能力明显减弱。结论：基因沉默CD147基因对肝癌细胞SMMC-7721的增殖和运动有抑制作用。     

三氧化二砷对膀胱癌细胞株BIU-87影响的研究

三氧化二砷 (As2 O3 )用于白血病的治疗疗效确定 ,目前亦有诱导实体肿瘤细胞凋亡的报道[1] 。我们采用免疫组化和流式细胞术观察As2 O3 对人膀胱癌细胞系BIU 87的诱导凋亡作用。现报告如下。材料与方法　膀胱癌细胞株BIU 87由北京大学泌尿外科研究所提供 ,As2 O3 由伊达药业有限公司提供。Bcl 2、Bax、p5 3抗体及免疫组化试剂盒购自迈新公司。将指数生长期BIU 87细胞随机分4组 ,A组为对照组 ,B组含 2 0 μg/ml丝裂霉素 (MMC) ,C组含 18.8μg/mlAs2 O3 ,D组含 37.6 μg/mlAs2 O3 。 2h后换 10 %培养液 3次。去除药物后第1、2、3…     

三氧化二砷及6种抗癌药物对肝癌细胞株作用的对比研究

目的研究三氧化二砷(As2O3)对肝癌细胞的抑制作用.方法应用MTT法检测不同浓度的As2O3对人肝癌细胞株BEL-7404和SMMC-7721的抑制率,并与6种常用抗癌药新生霉素、阿霉素、丝裂霉素、5-氟脲嘧啶、顺铂及环磷酰胺的抑瘤率进行比较. 结果与6种抗癌药相比,As2O3抑瘤作用最强(P<0.01或P<0.05),但并非As2O3浓度越高对细胞株的抑制率就越高.在6个浓度梯度中,1.0 μg/ml以上的As2O3对BEL-7404的抑瘤作用间差异无显著性意义(P>0.05),对于SMMC-7721,各浓度的As2O3的抑瘤作用间差异也无显著性意义(P>0.05).结论 As2O3体外试验能有效抑制肝癌细胞株BEL-7404和SMMC-7721的生长; 如果应用As2O3治疗肝癌,应做不同浓度As2O3的药敏试验,以选择浓度最低而且有效的剂量,以减少As2O3的毒副作用.     

NS-398对肝癌SMMC-7721细胞裸鼠移植瘤凋亡和血管生成的影响

为观察COX-2对肝细胞癌生长和血管生成的影响，我们通过构建肝癌SMMC-7721细胞株荷瘤裸鼠，检测NS-398对血管内皮生长因子（VEGF）、基质金属蛋白酶-9（MMP-9）、肿瘤微血管密度（MVD）和凋亡的影响，探讨环氧合酶2（COX-2）在肝细胞癌生长和血管生成过程中的作用及其机制。     

SMMC-7721与Bel-7402人肝癌细胞株生长激素受体的表达及意义

目的了解生长激素受体（GHR）在SMMC-7721与Bel-7402人肝癌细胞株的表达情况,以获得肝癌GHR在细胞水平上的定量表达。方法分别采用放射性配体分析与逆转录-多聚酶链式反应（RT-PCR法）检测GHR在SMMC-7721与Bel-7402肝癌细胞株的表达情况,使用双脱氧末端终止法进行基因测序。结果SMMC-7721与Bel-7402肝癌细胞均可表达GHRmRNA;在蛋白质表达水平发现GHR在7721肝癌细胞的位点数量（Site,103/cell）为3.462±0.632,平衡解离常数（Kd）为0.52±0.05942nmol/L;7402肝癌细胞位点数量为7.348±0.891,Kd为0.63±0.04583nmol/L。结论由于上述两种肝癌细胞株均可表达GHR,因此可为研究rhGH与肝细胞肝癌的生长关系奠定一定的实验基础。     

Adrenergic regulation of the intracellular [Ca2+] and voltage-operated Ca2+ channel currents in the rat prostate neuroendocrine cells

BACKGROUND: The prostate gland contains numerous neuroendocrine cells (PNECs) innervated by adrenergic neurons. PNECs are believed to influence the growth and physiological function of the prostate gland via paracrine release of hormones. MATERIALS AND METHODS: Using fura-2 fluorescence measurement and patch-clamp techniques, we investigated the effects of adrenergic stimulation on cytosolic concentration of Ca2+ ([Ca2+]c) and high voltage-activated Ca2+ channel currents (HVA-I(Ca)) of the putative rat prostate neuroendocrine cells (RPNECs) freshly isolated by an enzymic digestion. RESULTS: Noradrenaline (NA, 1 microM) induced a sharp, transient increase of [Ca2+]c measured by the fura-2 fluorescence. Pharmacological studies showed that alpha1-adrenoceptors (alpha1-ARs) coupled with PLC/IP3 signaling pathway induce the release of stored Ca2+, which subsequently recruits store-operated Ca2+ entry pathways. In the whole-cell voltage clamp experiment, NA decreased the amplitude of HVA-I(Ca) by 40%, which was mimicked by an alpha2-AR agonist (UK14304) but not by an alpha1-AR agonist (phenyleprine). After selective blockade of N-type Ca2+ channels by omega-conotoxin GVIA, the addition of NA showed no further inhibition on the remaining L-type Ca2+ channel currents. The adrenergic inhibition of HVA-I(Ca) was partially prevented by the pretreatment with pertussis toxin (PTX) (5 microg/ml, 4 hr, 37 degrees C). CONCLUSIONS: RPNECs express both alpha1- and alpha2-ARs, signaling the release of stored Ca2+ and the inhibition of N-type Ca2+ channels, respectively.     

Intracellular Ca2+ regulation in a human prostate stromal cell culture

AIMS: Prostate stromal cell cultures are used in vitro to study the cellular pathophysiology of benign prostatic hyperplasia (BPH), but their functional properties are poorly understood. This study characterized intracellular Ca2+ ([Ca2+]i) regulation in a cultured cell line in comparison to freshly isolated cells, as a background to understanding contractile regulation and cellular proliferation in this tissue. METHODS: Prostate stromal cells were isolated from either PrS6 cell cultures, with an extended life span by transfection with the SV40 T-antigen, tsA58-U19, or freshly obtained transition zone prostate samples, primary cells. [Ca2+]i was measured in vitro with the indicator Fura-2 by epifluorescence microscopy. RESULTS: Phenylephrine, high-K+, and caffeine induced Ca2+-transients in primary cells (resting [Ca2+]i 94 +/- 8 nM, n = 29; peak 193 +/- 26 nM, n = 19). In PrS6 cells resting [Ca2+]i was 96 +/- 8 nM (n = 78) and in 34 of these 78 cells, 30 microM phenylephrine increased [Ca2+]i to 296 +/- 28 nM. 5-methyl-urapidil (10-30 microM) inhibited this response in 10 of 16 cells. Spontaneous Ca2+-transients were also observed in 91% of phenylephrine-responsive cells, but in only 20% of non-responsive cells (P < 0.01). Ca2+-transients were also induced by high-K+ solution, and 20 mM caffeine. The latter abolished the response to subsequent phenylephrine application. Depletion of intracellular Ca2+ stores by caffeine or restoration from a Ca2+-free superfusate caused a substantial rise of [Ca2+]i. CONCLUSIONS: PrS6 prostate stromal cells express functional alpha1-adrenoceptors associated with spontaneous intracellular Ca2+-transients. They exhibit functional Ca2+ channels, intracellular Ca2+ stores, and Ca2+ entry induced by store depletion. Stromal cultures can therefore be used to characterize the cellular physiology of prostate stromal cell contraction and proliferation.     

The role of Ca2+ influx and intracellular Ca2+ release in the muscarinic-mediated contraction of mammalian urinary bladder smooth muscle

OBJECTIVE To study the involvement of extracellular Ca2+ and the properties of the intracellular Ca2+ ([Ca2+]i) stores on the carbachol-induced contraction of mammalian urinary bladder smooth muscle strips under polarized and depolarized conditions. MATERIALS AND METHODS: Strips of bladder were suspended between platinum ring electrodes in a cylindrical organ bath (0.2 mL) and continuously superfused with Krebs' solution at 1 mL/min. The effect of nifedipine, cyclopiazonic acid (CPA), thapsigargin, procaine, ryanodine and caffeine before and during a 10-s application of 100 microm carbachol under polarized conditions were studied. The effect of these drugs was also assessed under depolarized conditions using a protocol that allowed a more detailed assessment of the role of [Ca2+]i stores, consisting of emptying the stores by exposure to Ca2+-free solution, rapidly refilling them by a 10-s application of 81.5 mm Ca2+ (priming), returning to the Ca2+-free solution for 3 min and then applying 100 microm carbachol (10 s) in Ca2+-free solution (store release). RESULTS: Under polarized conditions, nifedipine and Ca2+ removal almost completely inhibited the carbachol-induced contractions. CPA increased the amplitude and duration of both carbachol- and electrical field stimulation-induced contractions. Although ryanodine had no inhibitory effect, caffeine and procaine significantly inhibited the carbachol-induced contraction. Under depolarized conditions nifedipine blocked both priming and store release contractions. CPA, thapsigargin, procaine and ryanodine significantly increased the priming and inhibited the store release contractions. However, caffeine virtually abolished both priming and store release contractions. CONCLUSION: These results suggest that in guinea-pig urinary bladder smooth muscle the Ca2+ necessary for contraction enters the cell through voltage-dependent dihydropyridine-sensitive Ca2+ channels and is pumped into an intracellular store that is released by carbachol. Under polarized conditions, the blockade of sarco-endoplasmic reticulum calcium ATP-ase (SERCA) with CPA increases [Ca2+]i and carbachol-induced contractions. The effects of caffeine and procaine suggest that store release involves ryanodine receptors and calcium-induced calcium release. Under depolarized conditions, Ca2+ entry is blocked by nifedipine and the stores diminish. Stored Ca2+ is also greatly reduced by the blockade of SERCA with either CPA or thapsigargin. Procaine, ryanodine and caffeine blocked the store release contractions, suggesting that this involves ryanodine receptors and calcium-induced calcium release.     

microRNA-126靶向PIK3R2调控肝癌细胞SMMC-7721的增殖和凋亡

【摘要】 目的 研究microRNA-126(miR-126)对肝癌细胞SMMC-7721迁移能力的影响以及对靶基因PIK3R2表达的影响。方法 通过慢病毒转染肝癌细胞株SMMC-7721使其过表达miR-126,利用CCK-8法检测细胞增殖能力。流式细胞术检测细胞凋亡;采用实时定量PCR和蛋白印迹检测细胞PIK3R2表达水平的变化,并通过荧光素酶实验验证miR-126与PIK3R2基因的直接调控关系。结果 与对照组相比,转染miR-126组的SMMC-7721细胞的增殖能力减弱,凋亡增加。过表达miR-126后,SMMC-7721细胞PIK3R2的蛋白表达下调(P=0.0135)。荧光素酶报告基因实验显示,miR-126能明显抑制PIK3R2-3’UTR的荧光素酶活性(P=0.0016)。结论〓过表达miR-126可能通过靶向降低PIK3R2基因的蛋白表达,抑制肝癌细胞的迁移。     

肝癌SMMC-7721细胞中侧群细胞干细胞标记的表达

目的 分选肝癌细胞株SMMC-7721中的侧群(SP)细胞,并分析其干细胞标记的表达.方法 采用流式细胞荧光激活分选(FACS)技术将SMMC-7721细胞分为SP细胞和非侧群(NSP)细胞两个亚群,以实时荧光定量聚合酶链反应(real-time PCR)技术和流式细胞术对两个亚群细胞干细胞标记mRNA和蛋白表达进行分析.结果 SMMC-7721细胞株中分选出的SP细胞比例为(9.2±0.2)%.SP细胞ABCG2、CD133、Oct4、Sox2和NANOG等干细胞标记mRNA的表达水平分别是NSP细胞的7.132倍、4.985倍、8.642倍、5.095倍和5.164倍,差异均有统计学意义(P＜0.01);ABCG2、CD133、Oct4、Sox2和NANOG蛋白在肝癌SP细胞中的含量分别为(92.65±3.92)%、(12.75±1.62)%、(17.35±2.31)%、(9.57±1.71)%和(28.39±5.28)%,在NSP细胞中的含量分别为(0.26±0.06)%、(2.51±0.17)%、(1.74±0.38)%、(1.52±0.41)%和(3.37±1.02)%,差异有统计学意义(P＜0.01).结论 肝癌SMMC-7721细胞中的SP细胞可能富集了肝癌干细胞,联合应用多种干细胞标记筛选肝癌SP细胞可能会获得纯化的肝癌干细胞.

Abstract：

Objective To study the expression of stem cell markers in side population cells sorted from SMMC-7721 cell line. Methods Fluorescence-activated cell sorting (FACS) was used to sort side population (SP) cells and non-SP (NSP) cells from SMMC-7721 cell line. Real-time polymerase chain reaction (PCR) and flow cytometry (FCM) were used to evaluate the expression of several stem cell markers such as ABCG2, CD133, Oct4, Sox2 and NANOG in SP cells and NSP cells. Results FACS analysis indicated that (9.2 ±0. 2)% of the SMMC-7721 cells were SP cells. Real-time PCR analysis suggested that ABCG2, CD133, Oct4, Sox2 and NANOG were expressed in the SP cells at higher levels than the NSP cells by about 7. 132, 4. 985, 8. 642, 5.095 and 5. 164 folds, respectively ( P ＜0. 01 ). FCM analysis revealed that the expression of ABCG2, CD133, Oct4, Sox2 and NANOG proteins in SP cells was (92. 65 ±3.92)%, (12.75 ±1.62)%, (17.35 ±2.31)%, (9.57 ± 1.71)% and (28.39 ±5.28)% respectively,while in NSP cells that was (0. 26 ±0. 06)%, (2. 51 ±0. 17)%, ( 1.74 ±0. 38)%, ( 1.52 ±0. 41 )% and ( 3.37 ± 1.02) % respectively ( P ＜ 0. 01 ). Conclusion The SP cells sorted from SMMC-7721 cell line may enrich tumor stem cells. Purified liver cancer stem cells may be obtained by screening SP cells using a variety of stem cell markers.     

全反式维甲酸及三氧化二砷诱导肝癌细胞株HepG-2凋亡及与胞内钙相关性研究

目的 观察不同浓度全反式维甲酸(ATRA)与三氧化二砷(As2 O3)单独及联合作用于人肝癌细胞株HepG-2细胞,检测其体外生长增殖抑制率、细胞凋亡率及细胞内钙离子浓度([Ca2+]i)的变化,探讨其诱导肝癌细胞凋亡可能机制.方法 分别及联合应用不同浓度的ATRA(0.1、1.0、10.0 μmol/L)、As2O3(0.5、1.0、2.0μmol/L)处理HepG-2细胞,用噻唑蓝(MTr)法测定不同作用时间细胞增长抑制率;流式细胞仪(FCM)膜联蛋白V/碘化丙锭(Annexin V/PI)双染色法检测细胞凋亡率的变化;采用荧光探针技术(Fluo-3),应用激光共聚焦显微镜,测定细胞内[Ca2+]i的变化.结果 ATRA、As2O3对人肝癌HepG-2细胞增殖有抑制作用,抑制率与药物浓度、作用时间呈正相关,联合用药优于单独用药;ATRA、As2O3能够诱导人肝癌细胞HepG-2的凋亡,随药物作用时间的延长、药物浓度的增高,细胞凋亡率逐渐增高,联合组更明显;作用时间为24、48 h时,细胞内Ca2+荧光强度与药物浓度、作用时间呈正相关,联合组荧光增强更明显,与对照组比较差异有统计学意义(P＜0.01),而作用时间至72 h时,荧光强度又恢复至正常水平(P＞0.05).结论 ATRA、As2O3能够诱导人肝癌细胞HepG-2凋亡,其诱导凋亡分子机制可能与细胞内钙离子浓度变化有关;ATRA、As2O3体外联合应用有协同抗肝癌作用.     

Bee venom induces apoptosis through intracellular Ca2+ -modulated intrinsic death pathway in human bladder cancer cells

Objectives: To focus on bee venom‐induced apoptosis in human bladder cancer TSGH‐8301 cells and to investigate its signaling pathway to ascertain whether intracellular calcium iron (Ca²⁺) is involved in this effect. Methods: Bee venom‐induced cytotoxic effects, productions of reactive oxygen species and Ca²⁺ and the level of mitochondrial membrane potential (ΔΨm) were analyzed by flow cytometry. Apoptosis‐associated proteins were examined by Western blot analysis and confocal laser microscopy. Results: Bee venom‐induced cell morphological changes and decreased cell viability through the induction of apoptosis in TSGH‐8301 cell were found. Bee venom promoted the protein levels of Bax, caspase‐9, caspase‐3 and endonuclease G. The enhancements of endoplasmic reticulum stress‐related protein levels were shown in bee venom‐provoked apoptosis of TSGH‐8301 cells. Bee venom promoted the activities of caspase‐3, caspase‐8, and caspase‐9, increased Ca²⁺ release and decreased the level of ΔΨm. Co‐localization of immunofluorescence analysis showed the releases of endonuclease G and apoptosis‐inducing factor trafficking to nuclei for bee venom‐mediated apoptosis. The images revealed evidence of nuclear condensation and formation of apoptotic bodies by 4′,6‐diamidino‐2‐phenylindole staining and DNA gel electrophoresis showed the DNA fragmentation in TSGH‐8301 cells. Conclusions: Bee venom treatment induces both caspase‐dependent and caspase‐independent apoptotic death through intracellular Ca²⁺‐modulated intrinsic death pathway in TSGH‐8301 cells.     

HDAC抑制剂SAHA对人肝癌细胞分化诱导作用的研究

目的 探讨组蛋白去乙酰化酶(histone deacetylases,HDAC)抑制剂SAHA(suberoylanilide hydroxamic acid)对人肝癌SMMC-7721细胞的分化诱导作用.方法 倒置显微镜观察SAHA对SMMC-7721细胞形态的影响;MTT比色法测定SAHA对SMMC-7721细胞增殖的抑制情况;免疫细胞化学检测SAHA对SMMC-7721细胞中甲胎蛋白(AFP)和增殖细胞核抗原(PCNA)表达的影响;流式细胞术(FCM)分析细胞周期;RT-PCR方法榆测处理前后SMMC-7721细胞p21WAF1基因mRNA的表达变化.结果 实验组细胞增殖速度显著减慢,与正常细胞形念变化相似;MTT比色法测定结果显示不同浓度SAHA对SMMC-7721细胞的增殖均有抑制作用,并有明显的剂量依赖和时间依赖关系;免疫细胞化学检测显示SAHA能显著降低PCNA和AFP在SMMC-7721细胞中的表达;流式细胞仪检测结果显示,SMMC-7721细胞经SAHA处理后,G0/G1期细胞明显增加,S期细胞则明显减少,细胞被阻滞于G0/G1期;RT-PCR检测结果表明,实验组细胞中p21WAF1 mRNA的表达明显增加.结论 SAHA对人肝癌细胞具有显著的诱导分化作用,诱导肝癌细胞分化的机理可能与抑制HDAC的活性,上调p21 WAF1 mRNA表达,及阻滞肝癌细胞G0/G1期有关.