HMBA诱导人肝癌SMMC-7721细胞分化的观察

本文研究HMBA对人肝癌SMMC-7721细胞的诱导分化作用.细胞生长曲线测定和细胞分裂指数观察显示HMBA可明显抑制细胞增殖,细胞生长抑制率达64.14%,分裂指数抑制率为53.88%.光镜和透射电镜观察可见HMBA能诱导人肝癌SMMG-7721细胞形态和超微结构发生恢复性改变.生化检测或免疫细胞化学方法观察显示,HM-BA处理后细胞γ.谷氨酸转肽酶(γ-GT)活性和甲胎蛋白(AFP)、增殖细胞核抗原(PCNA)表达均降低,而酪氨酸.酮戊二酸转氨酶(TAT)活性增强.流式细胞仪分析表明HMBA引起细胞发生G0/G1期阻滞.以上结果表明HMBA能有效抑制人肝癌细胞恶性增殖活性,逆转肝癌细胞恶性形态与超微结构特征,改变肝癌细胞相关酶活性和抗原表达,引发G0/G1期阻滞,从而对肝癌细胞具有明显的诱导分化作用.     

HMBA对人肝癌SMMC-7721细胞周期相关基因表达的影响

本文研究HMBA对人肝癌SMMC-7721细胞周期G_0/G_1期阻滞相关基因表达的影响。免疫细胞化学和核酸原位杂交检测结果显示,HMBA可明显上调p21~(WAFl/CIPl)、p16蛋白表达并增强p21~(WAFl/CIPl)基因转录,同时对CDK4、Cyclin D1蛋白表达以及c-myc基因转录均具有明显的下调作用。结果表明,HMBA可通过增强p21~(WAFl/CIPl)、p16基因表达而抑制Cyclin D1-CDK4活性,最终导致细胞进入S期所需的c-myc等基因转录活性下降,从而将细胞周期阻滞于G_0/G_1期,诱导人肝癌细胞分化。     

HMBA诱导人成骨肉瘤MG-63细胞分化过程中prohibitin的表达与定位变化

本文以HMBA诱导处理前后的人成骨肉瘤MG-63细胞为对象,对prohibitin在核基质中存在、分布及其与相关基因产物在HMBA处理前后MG-63细胞中的共定位关系进行观察研究.蛋白印迹杂交结果确证prohibitin存在于人成骨肉瘤MG-63细胞核基质蛋白组分中,并在HMBA处理后细胞核基质中表达下调,免疫荧光显微镜观察显示prohibitin定位在核基质上,经HMBA处理后出现分布位置与表达水平的变化.激光共聚焦显微镜观察可见prohibitin与MG-63细胞中c-fos、c-myc、p53和rb基因产物均存在共定位关系,但在HMBA处理后细胞中其共定位分布区域出现变化.研究结果证实prohibitin是一种核基质蛋白,定位于核基质上,prohibitin在人成骨肉瘤MG-63细胞诱导分化过程中的表达分布及其与相关癌基因、抑癌基因产物的共定位现象值得进一步探索和研究.     

HMBA诱导人肝癌SMMC—7721细胞分化的观察

In this paper, the effects of HMBA on the differentiation of human hepatocarcinoma cell line SMMC-7721 were investigated. After treated with 5 mmol/L HMBA, the proliferation of SMMC-7721 cells was inhibited remarkably, the cell growth inhibitory rate amounted to 64.14%, the cell mitotic index was declined by 53.88%. Light microscopy and transmission electron microscopy showed that the morphology and ultrastructure of the cells treated with HMBA undergone restorational alteration. Cytochemistry and immunocytochemistry assay revealed that the activities of gamma-GT declined and the levels of AFP and PCNA downregulated while the activity of TAT increased significantly after HMBA treatment. In the meantime, flow cytometry analysis showed that HMBA could arrest the cells in G0/G1 phase. The results showed that HMBA could effectively inhibit the proliferation, reverse the malignant morphology and ultrastructure, alter the levels of enzymes and antigens, arrest the cells in G0/G1, and induce the differentiation of human hepatocarcinoma SMMC-7721 cells in vitro.     

自养小球藻多糖对人肝癌细胞SMMC-7721凋亡的诱导

应用超声破碎碱提法制备自养小球藻多糖。在体外培养条件下,用不同浓度的小球藻多糖处理肝癌细胞,并通过MTT,双苯并咪唑(Hoechst33258)染色,免疫细胞化学等方法检测。结果表明,1g/L的小球藻多糖作用肝癌细胞SMMC-77211 2h,对其增殖有显著抑制作用,且能通过下调抗凋亡蛋白Bcl-2及上调凋亡执行蛋白Capase-3的表达来诱导其发生凋亡。     

人参皂甙Rg1组合诱导人成骨肉瘤 MG-63细胞分化过程中Nucleophosmin的表达与定位变化

选择性抽提经中药有效成分人参皂甙Rg1组合(简称RCT)诱导处理前后的人成骨肉瘤MG-63细胞核基质,对nucleophosmin(NPM)在核基质中的存在、分布及其与相关基因产物在MG-63细胞中的共定位关系进行了观察研究.双向凝胶电泳和质谱鉴定结果显示NPM存在于MG- 63细胞核基质蛋白组分中,在RCT处理后细胞核基质蛋白中表达下调.蛋白质印迹杂交结果证实了NPM在RCT处理前后的MG-63细胞核基质蛋白中的存在及其表达下调变化.免疫荧光显微镜观察显示NPM定位于MG-63细胞核基质上,经RCT处理后出现分布位置与表达水平的变化.免疫荧光-激光扫描共聚焦显微镜的观察结果显示NPM在MG-63细胞中与c-fos、c-myc、RB、p53 等基因产物具有共定位关系,并在RCT处理后细胞核内其共定位区域发生了变化.研究结果证实了NPM存在于核基质上,是一种核基质蛋白,其在RCT诱导人成骨肉瘤MG-63分化过程中的表达与分布变化及其与相关癌基因、抑癌基因的关系显然对MG-63细胞分化具有重要影响,这为深入揭示中药有效成分诱导肿瘤细胞分化的机制提供了重要科学依据和深入探索的新方向.     

人参皂苷Rg1组合诱导人成骨肉瘤MG-63细胞分化过程中Prohibitin的表达与定位变化

选择性抽提经人参皂苷Rg1组合（RCT）诱导处理前后的人成骨肉瘤MG-63细胞核基质,对prohibitin在核基质中的存在、分布及其与相关基因产物在RCT处理前后MG-63细胞中的共定位关系进行观察研究.蛋白质组学分析结果显示,prohibitin存在于人成骨肉瘤MG-63细胞核基质蛋白组分中,并在RCT处理后细胞核基质中表达下调;蛋白质印迹杂交确证了prohibitin在MG-63细胞核基质中的存在及其在RCT处理后下调变化;免疫荧光显微镜观察进一步证实prohibitin定位在核基质上,经RCT处理后出现分布位置与表达水平变化;激光共聚焦显微镜观察可见prohibitin与c-Fos、c-Myc、p53和Rb基因产物均存在共定位关系,并在RCT处理后共定位分布区域出现变化.本研究证实了prohibitin是一种新发现的核基质蛋白,其在核基质上的定位与表达在RCT诱导分化前后发生显著变化,并与相关癌基因、抑癌基因产物存在共定位关系.实验表明RCT处理引起的prohibitin的变化与人成骨肉瘤MG-63细胞的诱导分化与调控具有密切关系,为深入揭示RCT等中药有效成分诱导肿瘤细胞分化的机理提供了重要科学依据和深入探索的新方向.     

姜黄素通过MAPK信号通路诱导人肝癌SMMC-7721细胞凋亡

本文阐述了姜黄素(Curcumin)对体外培养的人肝癌SMMC-7721细胞增殖和凋亡的影响,并探讨了其诱导凋亡的信号转导机制。采用MTT法和细胞计数法检测不同浓度姜黄素对人肝癌细胞株SMMC-7721增殖的影响,利用流式细胞术检测姜黄素对人肝癌SMMC-7721细胞凋亡的影响,通过RT-PCR及Western blot检测姜黄素对人肝癌SMMC-7721细胞中凋亡相关蛋白Caspase-3、Survivin、Bcl-2和Bax表达的影响,最后通过检测MAPK的磷酸化水平分析姜黄素诱导SMMC-7721细胞凋亡的信号转导机制,通过MAPK抑制剂实验进一步证实诱导凋亡的分子机制。研究结果显示,姜黄素呈时间和剂量依赖性抑制人肝癌SMMC-7721细胞的增殖,其中40μmol/L姜黄素可明显诱导SMMC-7721细胞的凋亡,并呈时间依赖性上调促凋亡蛋白Caspase-3和Bax的表达、下调抗凋亡蛋白Survivin和Bcl-2的表达,姜黄素对凋亡相关蛋白表达的调节及诱导凋亡可以通过激活JNK、抑制ERK和p38 MAPK信号通路实现。表明姜黄素可诱导人肝癌SMMC-7721细胞凋亡,其机制与姜黄素激活JNK、抑制ERK和p38 MAPK信号通路从而上调Caspase-3和Bax的表达,下调Survivin和Bcl-2的表达有关。     

URI基因稳定表达对肝癌SMMC-7721细胞增殖能力的影响

目的:瞬转以及筛选出能够稳定表达URI(RPB5-mediating protein)基因的SMMC-7721细胞株,以其为模型研究URI基因对人肝癌SMMC-7721细胞增殖的影响.方法:首先,抽提URI的重组质粒并转染到SMMC-7721细胞中,在G418药物的筛选下选出能够稳定表达URI基因的细胞株,RT-PCR法和酶切检测该稳定细胞中URI基因的表达效率以确认URI是否稳定表达,MTT法和克隆形成实验检测URI基因对SMMC-7721细胞增殖的影响.结果:成功建立URI基因过表达的稳定细胞株.与SMMC-7721细胞对照组比较,其URI mRNA表达水平显著上调,能稳定表达URI细胞株的增殖,克隆形成率明显升高.结论:pFLAG-CMV-4-URI重组质粒能使URI在肝癌SMMC-7721细胞内稳定表达,过表达URI基因有可能帮助细胞通过G2/M期检验点来提高肝癌细胞SMMC-7721的增殖能力.     

HMBA对人肝癌SMMC—7721细胞周期相关基因表达的影响

本文研究HMBA对人肝癌SMMC－7721细胞周期G0／G1期阻滞相关基因表达的影响。免疫细胞化学和核酸原位杂交检测结果显示,HMBA可明显上调p21^WAF1/CIP1、p16蛋白表达并增强p21^WAF1/CIP1基因转录,同时对CDK4、Cyclin D1蛋白表达以及c-myc基因转录均具有明显的下调作用。结果表明,HMBA可通过增强p21^WAF1/CIP1、p16基因表达而抑制Cyclin D1-CDK4活性,最终导致细胞进入S期所需的c-myc等基因转录活性下降,从而将细胞周期阻滞于G0／G1期,诱导人肝癌细胞分化。     

衣霉素对人肝癌细胞表面胰岛素受体的影响

衣霉素(Tunicamycin)是一种糖蛋白N-连接型糖链合成的抑制剂。它可抑制人肝癌细胞抹SMMC-7721的生长,其抑制率和剂量及处理时间有相关性。衣霉素处理细胞18h尚可显著抑制~3H-甘露糖和~3H-氨基葡萄糖参入细胞,但仅轻度抑制~3H-亮氨酸的参入。这些标记化合物的参入抑制都有量效关系。0.1gg/mL衣霉素处理18h后,细胞表面胰岛素受体和胰岛素的结合容量下降,而对照细胞和处理细胞的胰岛素竞争结合曲线基本平行。这主要由于衣霉素抑制新合成的胰岛素受体的糖基化所致。我们对糖基化障碍引起细胞膜胰岛素受体结合容量降低的机理作了讨论。     

Localization of nucleophosmin in nuclear matrix and changes in its expression during the differentiation of human neuroblastoma induced by retinoic acid

In this article, we selectively extracted the nuclear matrix and intermediate filament system of human neuroblastoma SK‐N‐SH cells pre‐ and post‐treated with retinoic acid (RA). The distribution of nucleophosmin (NPM) in the nuclear matrix and its colocalization with several products of related genes were investigated. Results from two‐dimensional gel electrophoresis and MALDI‐TOF showed that NPM was a component of the nuclear matrix and its expression in SK‐N‐SH cells post‐treated with RA was down‐regulated. Immunofluorescent microscopy observations further showed that NPM was localized in the nuclear matrix of SK‐N‐SH cells, and its expression level and distribution were altered after treatment with RA. The colocalization of NPM with c‐myc, c‐fos, p53, and Rb in SK‐N‐SH cells was observed under a laser scanning confocal microscope, but the colocalization region was changed by RA. Our results prove that NPM is a nuclear matrix protein, which is localized in nuclear matrix fibers. The colocalization of NPM with its related genes and oncogenes affect the differentiation of SK‐N‐SH cells. The expression of NPM and its distribution in the process of cell differentiation deserve more intensive investigation. J. Cell. Biochem. 111: 67–74, 2010. © 2010 Wiley‐Liss, Inc.     

双丁酰环磷酸腺苷耐人肝癌细胞株SMMC－7721表面N－糟链类型及天线数的影响

本文采用系列凝集素柱层析法,并配合外切精苷酶研究了在双丁酰环磷酸腺苷（ｄＢ－ｃＡＭＰ）作用１～５天过程中人肝癌细胞株ＳＭＭＣ－７７２１细胞表面Ｎ－糖链类型及复杂型糖链天线数的变化。结果表明,ｄＢ－ｃＡＭＰ促进３Ｈ—Ｍａｎ参入细胞表面Ｎ－糖链,使高甘露糖型Ｎ－糖链的百分比下降,并促进二天线Ｎ－糖链的生物合成。使多天线特别是四天线和Ｃ２Ｃ２Ｃ６三天线Ｎ－糖链的百分比减少．结果提示,Ｎ－糖链结构的这些变化可能是ｄＢ－ｃＡＭＰ诱导ＳＭＭＣ－７７２１细胞向正常方向分化的结果。     

视黄酸和亚硒酸钠对肝癌细胞某些表型逆转的加和作用

 人肝癌细胞株SMMC-7721经1μmol/L视黄酸和或2.5μmol/L亚硒酸钠处理后,膜上纤维连接蛋白沉着量逐日上升,且较相应天数的对照组细胞增加,而甲胎蛋白分泌量和~3H-TdR参入率被明显抑制。视黄酸和亚硒酸钠同时处理的联合组作用强度接近于两者单独使用时作用强度的加和。对以上结果和视黄酸及亚硒酸钠使肝癌细胞接触抑制恢复及表型逆转的关系作了讨论。     

Induction of Apoptosis in Human Hepatocarcinoma SMMC-7721 Cells In Vitro by Psoralen from Psoralea corylifolia

Psoralen is a major active component of Psoralea corylifolia. In the present study, we analyzed psoralen-induced changes in human hepatocarcinoma cell viability and apoptosis, and investigated the underlying mechanisms of the proapoptotic effect of the compound on SMMC-7721 cells. We measured human hepatocarcinoma cell viability by MTT assay and Annexin V-FITC/PI double staining, and evaluated the activity of caspase 3 and the expression of p53, Bax, and Bcl-2 proteins, involved in regulating cell apoptosis. Psoralen was able to inhibit the growth of SMMC-7721 cells in a dose- and time-dependent manner and had a strong proapoptotic effect on these cells. We show a dose-dependent increase in caspase-3 activity, and elevated levels of p53 and Bax proteins in psoralen-treated cells, that coincided with dose-dependent decrease in Bcl-2 expression. These results suggest that psoralen induces apoptosis in cancer cells via mechanisms that involve caspase-3, p53, Bax, and Bcl-2 pathway. Our results may provide a molecular basis for the further development of natural compounds as novel anticancer agents for human hepatomas.     

PTEN对SMMC—7721人肝癌细胞迁移增殖及粘着斑激酶磷酸化水平的影响

抑癌基因PTEN编码产物具有双专一性磷酸酶活性 ,并与细胞骨架张力蛋白同源。PTEN可参与粘着斑的形成和解聚而影响细胞迁移。现用PTEN表达质粒转染SMMC 772 1肝癌细胞 ,研究SMMC 772 1细胞运动能力的变化及PTEN与粘着斑激酶 (FAK)酪氨酸磷酸化水平之间的关系。PTEN过表达能够显著抑制细胞在Fn基质上的活动 :细胞在Fn基质上的迁移下降了 35 % ;在 30min和 6 0min两个时间点 ,Fn基质上细胞铺展分别降低了 2 9%和 2 6 % ;而在多聚赖氨酸基质上细胞铺展并没有变化。运用免疫沉淀和Western印迹方法 ,分析FAK及其酪氨酸磷酸化水平 ,发现PTEN过表达不影响FAK表达 ,但显著降低Fn诱导的FAK酪氨酸磷酸化水平 ,两者水平呈负相关。流式细胞仪分析细胞周期结果表明 ,PTEN抑制细胞 ,S期细胞下降了 16 %。上述结果提示 ,PTEN抑制肝癌细胞迁移铺展和增殖 ;PTEN对细胞运动的影响可能通过调节FAK酪氨酸磷酸化水平而实现。     

Ganglioside Expression During Differentiation of Chick Retinal Cells In Vitro

The neural retina has been widely used to study the developmental patterns of ganglioside metabolism. Recent findings about in vitro differentiating chick embryo retina cells showed that: a) GD3 and GD1a ganglioside patterns undergo the most dramatic changes; b) when the cells emit neurites, GD3 ganglioside and a group of complex gangliotetraosylgangliosides (GTOG) are transiently coexpressed; c) synchronized developmental phenomena are dissociated by anti-GM1 antibodies; d) GD3 remains as a major ganglioside in differentiated neurons, though it is almost not immunoexpressed; e) GTOG affect antibody binding to GD3; f) the content of gangliosides involved in neural differentiation modifies their immunostain localization on cell membrane; g) after exogenous GTOG uptake, immature neurons mimic GD3 immunoflourescent localization of mature cells; h) a subset of purified retinal ganglion cells express GTOG characteristic of mature neurons.     

Cytotoxic Terpenes from the Flowers of Inula japonica against Human Hepatocarcinoma Cells

Two new 1,10-seco-eudesmanolides ( 1 and 2 ) were isolated from the flowers of Inula japonica together with two eudesmanolide analogs ( 3 and 4 ) and two monoterpene derivatives ( 5 and 6 ). Their structures were established on the basis of detailed spectroscopic analyses and electronic circular dichroism data. All isolates were evaluated for their antiproliferative activities against human hepatocarcinoma HepG2 and SMMC-7721 cells. Japonipene B ( 3 ) exhibited the most potent effect with the IC₅₀ values of 14.60±1.62 and 22.06±1.34 μM against HepG2 and SMMC-7721 cells, respectively. Furthermore, japonipene B ( 3 ) showed significant efficacies of arresting the cell cycle at the S/G2-M stages, inducing mitochondria-mediated apoptosis, and inhibiting cell migration in HepG2 cells.     

视黄酸对人肝癌细胞蛋白激酶的影响

 视黄酸(RA)处理SMMC-7721人肝癌细胞株72小时后,胞浆、膜性组分的蛋白激酶C(PK-C)的活力及比活力均下降,活力分别下降44.9％和48.8％,比活力下降42.7％和35.0％。然而,胞浆与膜性组分的活力,比活力比值在RA处理前后并无十分明显的变化,这提示在RA作用过程中,未发生PK-C的膜-浆转位。蛋白激酶A(PK-A)的变化则相反,RA处理72小时,活力、比活力上升了295％,258％。PK-A/PK-C的活力比值则从0.342增加到1.849,比活力比值从0.210增加到0.897。因PKC和PKA分别和细胞的去分化性增殖和分化有关,故上述结果和我们已报道的RA可抑制SMMC-7721细胞株的增殖和促进其分化相一致。