转醛醇酶在胃癌组织中的特异性表达

目的:在胃癌组织、癌旁组织及正常胃组织之间比较转醛醇酶(TAL)的活性与表达的变化,探讨转醛醇酶活性或表达变化与肿瘤发生机制的可能联系.方法:转醛醇酶活性测定,Western免疫印迹测定TAL蛋白表达.结果:胃癌组织中TAL活性明显高于癌旁组织及正常皮肤组织(p＜0.05).癌旁组织中TAL活性低于正常组织(p＜0.05).TAL在胃癌组织和正常胃组织中的蛋白水平没有显著性差异.结论:转醛醇酶的活性升高参与胃组织癌变过程.     

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。     

D-核糖产生菌转酮醇酶活性分析

芽孢杆菌属的转酮醇酶(Transketolase,TKT)变异株可利用培养基中的D-葡萄糖,将其转化成D-核糖分泌于细菌培养基中。阐述了D-核糖产生菌转酮醇酶的活性测定方法,制备了测定所必需的2种酶。     

绿茶提取物对人肝癌细胞株SMMC-7721体外凋亡的影响

从茶多酚中分离提纯表没食子儿茶素没食子酸酯(Epigallocatechin gallate,EGCG)单体。体外培养人肝癌SMMC-7721细胞,采用不同浓度的EGCG对其进行干预,用流式细胞仪检测其对肝癌细胞凋亡的影响;免疫细胞化学法和RT-PCR检测EGCG作用于SMMC-7721细胞后对Survivin、突变型P53 mRNA和蛋白表达的影响。结果显示在EGCG的作用下,SMMC-7721凋亡明显增加(P0.05);Survivin和突变型P53蛋白和mRNA表达水平均下调(P0.05)。表明EGCG可能通过下调人肝癌细胞株Survivin和突变型p53的表达,进而促进人肝癌细胞株SMMC-7721凋亡,以达到抗癌作用。     

蚕蛹复合氨基酸对人肝癌细胞SMMC-7721的抑制作用

本实验探讨蚕蛹复合氨基酸对人肝癌细胞株SMMC-7721的抑制作用.实验经MTT法检测药物对人正常肝脏细胞株QSG-7701的毒性后,计算药物安全浓度.将不同浓度的蚕蛹复合氨基酸与人肝癌细胞SMMC-7721共培养,采用MTT法测定OD值,评定蚕蛹蛋白复合氨基酸对肝癌细胞株的增殖抑制作用;经Hoechst33258染色和倒置显微镜进行形态学观察以检测细胞凋亡率;流式细胞法测定细胞周期和Annexin V/PI双染色法检测细胞凋亡.细胞毒性实验表明:蚕蛹蛋白复合氨基酸的最大无毒浓度为10 mg/mL.不同浓度的蚕蛹蛋白复合氨基酸对SMMC-7721细胞均有抑制作用,各组与对照组相比差异有显著性(P＜0.05),并呈剂量和时间依赖性.Hoechst33258染色和流式细胞术结果亦证实,蚕蛹复合氨基酸能显著促进SMMC-7721细胞的凋亡.     

大肠杆菌K-12转醛醇酶基因talB的克隆及在运动发酵单胞菌CP4中的表达

研究了E.coliK-12转醛醇酶基因(talB)在自身启动子和在Z.mobilisCP4eno基因启动子的启动下在E.coliDH5α和Z.mobilisCP4中的表达情况。首先克隆了E.coliK-12talB基因,并连接到穿梭载体pZB1上构建成pZB1-talB;然后利用PCR重叠延伸技术将E.coliK-12talB自身的启动子换成Z.mobilisCP4eno的启动子,构建得到pZB1-Peno-talB。将这两个质粒分别转化E.coliDH5α和Z.mobilisCP4。对转化子粗酶液进行的转醛醇酶酶活力测定结果表明,E.coli talB自身启动子和Z.mobilis eno启动子能以基本相同的效率启动talB基因在E.coli和Z.mobilis中的表达。     

地塞米松对人肝癌细胞系SMMC-7721凋亡的影响

观察不同浓度的地塞米松(dexamethasone,Dex)对人肝癌细胞系SMMC-7721凋亡的影响。SMMC-7721细胞培养于DMEM(高糖,含10%胎牛血清)培养液中,待细胞融合度达70%时,分别加入地塞米松,使其终浓度分别达到0.5μg/m L(低浓度组)、1.0μg/m L(中浓度组)和2.0μg/m L(高浓度组),另设一生理盐水对照组。诱导24 h后,Hoechst 33258染色,荧光倒置显微镜观察凋亡细胞形态和比例;发现随着Dex浓度的变化,凋亡的肝癌细胞数量、形态变化明显,Dex浓度越高,凋亡的肝癌细胞越多,并呈现浓度依赖性,即凋亡数与Dex浓度呈正相关。提取细胞总RNA,逆转录成为c DNA,荧光定量PCR法测定凋亡相关基因Bcl-2、Caspase 3、Caspase 5、Caspase 7、Caspase 9、Fas及P53的表达。与对照组比较,3种浓度Dex处理的细胞其凋亡细胞形态和比例均发生显著差异,凋亡细胞数量显著增加,各相关基因表达均显著下降。Dex对SMMC-7721细胞具有诱导凋亡的作用。     

大田环境下转Bt基因玉米对土壤酶活性的影响

在大田自然条件下,比较研究了转Bt基因玉米和非转基因亲本玉米在种植和秸秆分解时对土壤酶活性影响的差异。结果表明,与亲本非转基因玉米相比,在各生育期内种植转Bt玉米对土壤蛋白酶和土壤脲酶活性均没有显著影响;在喇叭口期和抽雄期,土壤蔗糖酶和土壤酸性磷酸酶活性显著提高。在秸秆还田后,两种玉米秸秆对土壤酸性磷酸酶活性的影响没有显著差异,但使用转Bt玉米秸秆的土壤蔗糖酶、土壤脲酶和土壤蛋白酶的活性则有显著提高。与亲本玉米相比,在所有观测期内,种植Bt玉米及秸秆还田对土壤酶活性的影响,在影响的幅度及趋势上随玉米生育期和土壤酶种类的不同而产生差异,但没有观测到显著不利影响;商业化Bt玉米的环境释放仍有待长期定位观测和评价。     

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。     

不同生长期转Bt基因水稻秸杆还土对淹水土壤酶活性的影响

在实验室条件下通过秸杆还土试验比较了不同生长期转Bt基因克螟稻及其亲本稻秸杆对淹水土壤酶活性的影响。研究结果表明,与同一生长期的亲本稻秸杆相比,孕穗期和成熟期克螟稻秸杆对淹水土壤磷酸酶活性的影响较小;相反,对淹水土壤脱氢酶活性的影响非常显著,并且孕穗期秸杆与成熟期秸杆的添加对淹水土壤脱氢酶活性的影响趋势也存在较大差异。推测造成淹水土壤脱氢酶活性的显著性差异的主要原因可能是由于Bt插入基因表达的多效性所致。结果认为土壤脱氢酶活性可作为转Bt基因水稻生态安全风险性评价的潜在指标。     

Transforming growth factor-beta1 stimulated protein kinase B serine-473 and focal adhesion kinase tyrosine phosphorylation dependent on cell adhesion in human hepatocellular carcinoma SMMC-7721 cells

Transforming growth factor-beta1 (TGF-beta1) is a potent growth inhibitor and apoptosis inducer for most normal cells. However, tumor cells are commonly nevertheless sensitive to the tumor-suppressing effects of TGF-beta1. In this paper, we focus on the effects of TGF-beta1 on two important anti-apoptotic protein kinases, protein kinase B (PKB), and focal adhesion kinase (FAK), in SMMC-7721 cells. We found that PKB-Ser-473 phosphorylation was apparently up-regulated by TGF-beta1. In the meantime, PKB-Thr-308 phosphorylation was slightly up-regulated by TGF-beta1. TGF-beta1 could also enhance FAK-Tyr phosphorylation. We observed that integrin-linked kinase (ILK) was also up-regulated by TGF-beta1 in good accordance with PKB-Ser-473 phosphorylation. We first found that TGF-beta1 could stimulate PKB-Ser-473 phosphorylation possibly via up-regulating ILK expression. Furthermore, we also failed to detect PKB-Ser-473 and FAK-Tyr phosphorylation with various concentrations of TGF-beta1 treatment when cells were kept in suspension. The above results indicate that PKB-Ser-473 and FAK-Tyr phosphorylation stimulated by TGF-beta1 are both dependent on cell adhesion.     

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.     

EPA诱导人肝癌细胞SMMC-7721凋亡及端粒酶活性调控机制研究

目的:探究二十碳五烯酸(Eicosa Pentaenoic Acid.EPA)对SMMC-7721人肝癌细胞的凋亡、端粒逆转录酶h TERT的调控作用及端粒酶表达活性的影响。方法:体外培养SMMC-7721人肝癌细胞,用不同浓度的EPA(0μM、25μM、50μM、100μM、200μM)作用于SMMC-7721肝癌细胞(24 h、48 h、72 h)后,显微镜下观察其形态学变化;应用MTT法检测SMMC-7721肝癌细胞细胞增殖变化情况;Western-blot法检测h TERT、Bax、Bcl-2蛋白表达水平变化;Real Time-PCR检测h TERTm RNA的表达变化;ELISA法检测SMMC-7721肝癌细胞端粒酶活性的表达水平。结果:EPA可诱导肝癌细胞SMMC-7721发生细胞凋亡,具有明显的时间计量依赖关系。在此过程中Bcl-2蛋白表达的降低和Bax蛋白表达上调,同时端粒酶逆转录酶h TERT蛋白及其m RNA的表达水平和端粒酶活性均明显降低。结论:抑制端粒酶逆转录酶基因(h TERTm RNA)表达而抑制端粒酶的活性、诱导癌细胞凋亡,可能是EPA的抗癌作用机制之一。     

Redifferentiation of human hepatoma cells (SMMC-7721) induced by two new highly oxygenated bisabolane-type sesquiterpenes

Bisabolane-type sesquiterpenes are a class of biologically active compounds that has antitumour, antifungal, antibacterial, antioxidant and antivenom properties. We investigated the effect of two new highly oxygenated bisabolane-type sesquiterpenes (HOBS) isolated from Cremanthodium discoideum (C. discoideum) on tumour cells. Our results showed that HOBS induced morphological differentiation and reduced microvilli formation on the cell surface in SMMC-7721 cells. Flow cytometry analysis demonstrated that HOBS could induce cell-cycle arrest in the G1 phase. Moreover, HOBS was able to increase tyrosine-α-ketoglutarate transaminase activity, decrease α foetoprotein level and γ-glutamyl transferase activity. In addition, we found that HOBS inhibited the anchorageindependent growth of SMMC-7721 cells in a dose-dependent manner. Taken together, all the above observations indicate that HOBS might be able to normalize malignant SMMC-7721 cells by inhibiting cell proliferation and inducing redifferentiation.     

斑蝥素酸镁对人肝癌细胞SMMC-7721及其裸鼠皮下移植瘤的影响

【目的】探讨斑蝥素酸镁对人肝癌细胞SMMC-7721及其裸鼠皮下移植瘤的影响。【方法】1、采用磺酰罗丹明染色法(SRB法)检测不同浓度斑蝥素酸镁在体外对人肝癌细胞SMMC-7721增殖的抑制作用;2、流式细胞术检测斑蝥素酸镁对人肝癌细胞SMMC-7721细胞周期和细胞凋亡的影响;3、Hoechst33342染色观察斑蝥素酸镁对人肝癌细胞SMMC-7721细胞形态的影响;4、透射电镜观察斑蝥素酸镁作用后人肝癌细胞SMMC-7721超微结构的变化;5、建立人肝癌细胞SMMC-7721裸鼠皮下移植瘤模型,实验组每只裸鼠瘤周注射斑蝥素酸镁6.26×10?5 mmol,对照组给予相同容积的无菌生理盐水瘤周注射,计算抑瘤率;6、原位末端标记染色(TUNEL)法检测人肝癌裸鼠皮下移植瘤组织细胞凋亡情况。【结果】1、斑蝥素酸镁对人肝癌细胞SMMC-7721有比较明显的抑制作用,抑制率随药物浓度的增加而升高,呈剂量效应关系,其半数抑制浓度(IC50)为1.79?mol/L;2、流式细胞检测结果显示:人肝癌细胞SMMC-7721在斑蝥素酸镁的作用下,G0/G1期细胞减少,G2/M期细胞增加,细胞出现G2/M期阻滞;细胞凋亡率随斑蝥素酸镁浓度加大而逐渐增加;3、Hoechst33342染色镜下显示:斑蝥素酸镁作用后人肝癌细胞SMMC-7721出现凋亡细胞形态特征;4、透射电镜观察:斑蝥素酸镁作用后人肝癌细胞SMMC-7721出现细胞核异形、染色质聚集成团、边集,见凋亡小体;5、斑蝥素酸镁组肿瘤体积、重量显著小于生理盐水组(P﹤0.05),抑瘤率为49%;6、TUNEL法提示斑蝥素酸镁组移植瘤组织细胞凋亡率显著高于生理盐水组(χ2=92.609,P﹦0.000)。【结论】斑蝥素酸镁对人肝癌细胞SMMC-7721在体内外均有抑制增殖作用,并可以诱导肿瘤细胞凋亡,其凋亡的发生与细胞分裂期阻滞有关。     

5′-nucleotidase activity in permanent human lymphoid cell lines Implication for cell proliferation and aging in vitro

5′-Nucleotidase of 11 human lymphoid cell lines was measured. These cell ines were homogeneous B, T and Null cells, had an unlimited lifespan in vitro, and were subcultivated from leukemic cells of patients with Burkitt lymphoma, the blastic phase of chronic myelocytic leukemia, or acute lymphoblastic leukemia. 5′-Nucleotidase activities in normal human lymphocytes and in human fibroblasts (VA-13 and IMR-90) could be determined at a cellular protein concentration as low as 0.025 and 0.007 mg/ml, respectively. In all the eleven lymphoid cell lines, including 8 B-cell lines (RPMI 8422, B46M, RPMI 1788, DAUDI, HRIK, B411-4, B85 and DND-3-9A), 2 T-cell lines (MOLT 3 and RPMI 8402) and 1 Null cell line (NALM-1) 5′-nucleotidase was undetectable with the protein concentration range from 0.033 to 8.543 mg/ml. Previously 5′-nucleotidase activity was found to increase 10-, 6- and 20-fold in normal human embryonic lung (WI-38 and IMR-90 cells) and chick embryo fibroblasts, respectively, from a young rapidly proliferative to a senescent non-proliferative stage (Sun, A.S., Aggarwal, B.B. and Packer, L. (1975) Arch. Biochem. Biophys. 170, 1 and Sun, A.S., Alvarez, L.J. Reinach, P.S. and Rubin, E. (1979) Lab. Invest. 41, 1). These data demonstrate that the large increase in 5′-nucleotidase activity occurs concomitantly with the in vitro senescence of these normal cell lines. The present study suggests that this large increase in 5′-nucleotidase activity during cell aging is absent in these permanently lymphoid cell lines. The undetectable 5′-nucleotidase activity may be a biochemical characteristic of these homogeneous B, T and Null cells originating from the aforesaid leukemias. The implications of these results for cell proliferation and aging are discussed.     

两种藻胆蛋白对人肝癌SMMC-7721细胞的光动力体外杀伤效应

用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)法测定提取自条斑紫菜的R-藻红蛋白(R-PE)和C-藻蓝蛋白(C-PC)经激光介导对人肝癌SMMC-7721细胞的杀伤率,并检测细胞凋亡率。结果显示在120mg/L浓度下,R-PE在氩离子激光器100J/cm2辐照下对应的细胞存活率为27%,C-PC在He-Ne激光器35J/cm2辐照下对应的细胞存活率为47%;单用2种激光器辐照对应的细胞存活率分别为65%和70%;单用2种蛋白处理细胞在72h后出现对细胞生长的显著抑制,最大抑制率分别为31%(R-PE,120mg/L)和27%(C-PC,250mg/L)。120mg/L的2种藻胆蛋白在对应激光辐照下,于照后8h达到细胞凋亡率最大值,分别为31.54%(R-PE)和32.54%(C-PC)。本实验证明条斑紫菜R-PE和C-PC具有可开发成为光敏剂的应用前景。     

Investigation of xenotropic murine leukemia virus-related virus (XMRV) in human and other cell lines

Xenotropic murine leukemia virus-related virus (XMRV) was discovered in human prostate tumors and later in some chronic fatigue syndrome (CFS) patients. However, subsequent studies have identified various sources of potential contamination with XMRV and other murine leukemia virus (MLV)-related sequences in test samples. Biological and nucleotide sequence analysis indicates that XMRV is distinct from known xenotropic MLVs and has a broad host range and cell tropism including human cells. Therefore, it is prudent to minimize the risk of human exposure to infection by evaluating XMRV contamination in cell lines handled in laboratory research and particularly those used in the manufacture of biological products. Nested DNA PCR assays were optimized for investigating XMRV gag and env sequences in various cell lines, which included MRC-5, Vero, HEK-293, MDCK, HeLa, and A549, that may be used in the development of some vaccines and other cell lines broadly used in research. The sensitivity of the DNA PCR assays was <10 copies in approximately 1.8 x 10⁵ cells equivalent of human DNA. The results indicated the absence of XMRV in the cell lines tested; although in some cases DNA fragments identified as cellular sequences were seen following the first round of PCR amplification with the env primer pair.     

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。     

Circumventing the Crabtree Effect: A method to induce lactate consumption and increase oxidative phosphorylation in cell culture

Most cells grown in glucose-containing medium generate almost all their ATP via glycolysis despite abundant oxygen supply and functional mitochondria, a phenomenon known as the Crabtree effect. By contrast, most cells within the body rely on mitochondrial oxidative phosphorylation (OXPHOS) to generate the bulk of their energy supply. Thus, when utilising the accessibility of cell culture to elucidate fundamental elements of mitochondria in health and disease, it is advantageous to adopt culture conditions under which the cells have greater reliance upon OXPHOS for the supply of their energy needs. Substituting galactose for glucose in the culture medium can provide these conditions, but additional benefit can be gained from alternate in vitro models. Herein we describe culture conditions in which complete autonomous depletion of medium glucose induces a lactate-consuming phase marked by increased MitoTracker Deep Red staining intensity, increased expression of Kreb’s cycle proteins, increased expression of electron transport chain subunits, and increased sensitivity to the OXPHOS inhibitor rotenone. We propose these culture conditions represent an alternate accessible model for the in vitro study of cellular processes and diseases involving the mitochondrion without limitations incurred via the Crabtree effect.