微量量热法筛选抑菌特效药的研究

用微量最热计连续测定细菌代谢过程中的热显变化，可获得反映细菌代谢规律的完整的热谱图以按指数生长模型对指数生长期进行处理，可计算出细菌正常代谢的生长速率常数L’．若在培养基中加入合成药物，使细菌在药物抑制作用下生长，也可获得完整的热谱图，从而计算出在药物抑制作用下细菌的生长速率常数．本义对福氏志贺氏Zb菌和金黄色葡萄球莉在四种合成药物抑制作用下的热谱进行了测定，并计算了生长速率常数，找出了细菌生长速率常数与所用药物浓度之间的定量关系，可为筛选抑菌药物和确定用药显提供定量依据’1基本原理设细菌在代谢过…     

Schiff碱药物与产气杆菌作用的微量热研究

用生物活性监测系统(Bioactivity Monitor 2277)测定了五种Schiff碱药物与产气杆菌作用的热谱。从热谱图上可直接反映出不同的药物由于结构不同从而对产气杆菌的作用也不同。实验表明Zn-SG对产气杆菌的抑制作用居首,Zn-o-VG, SG次之, Co(Ⅲ)-SG较小, 其半抑制量也依次增大, 而Cu(Ⅱ)-o-VG对产气杆菌的生长代谢抑制几乎与浓度无关(SG: 水杨醛氨基葡萄糖Schiff碱,o-VG: 邻香草醛氨基葡萄糖Schiff碱)。     

微量量热法测定细菌生长的热谱

活体中细胞内的各种代谢过程都件随着一定的热效应,若使用具有足够灵敏的量热计对它进行探测,就提供了一种研究活细胞代谢过程及共有关特性的新方法。当用量热计连续跟踪监测细胞(如细菌)生长繁殖过程热效应的变化时,便获得该细胞生长的“热谱”。近期的研究实践证明,量热学方法用来进行细菌基本生长的研究是可行的。Boling等曾用Batch型量热计检测了某些细菌的生长热谱(见图1),但有些谱图不完整,因在它们的图     

硝酸镧引起Escherichia coli B代谢过程热爆发及其机理研究

采用微量热法研究了硝酸镧对Escherichia coli B生长代谢过程的影响, 发现高浓度硝酸镧引起E. coli B热谱图出现异常变化: 生长速率常数k值增大、产热峰显著升高和总发热量异常增加. 当硝酸镧浓度为300和500 mg/L时, 培养物在培养过程的总发热量分别是正常条件下的3.89和2.54倍. 用生物学方法对细胞存活率和生物量进行测定结果表明, 细胞在高浓度硝酸镧条件下增殖受到抑制、细胞生物量减少. 表明高浓度的硝酸镧存在时, E. coli B细胞生长受到抑制反而释放出比正常生长细胞多得多的热量, 将抑制状态细胞释放大量热量的现象称为热爆发. 分析热爆发的原因, 认为是La^3＋离子破坏细胞壁外膜而增加其透性, 导致细胞膜与外膜间的质子电化学势因质子外泄而降低或者不能形成, 氧化磷酸化过程中的能量不能有效地转化为ATP, 而以热能的方式释放出来. 细胞由于缺乏生物通用能量ATP, 因而其生长受到抑制.     

癌细胞的热化学研究

生物热化学是一门新兴的交叉学科,它从热化学的角度进行生物学方面的研究。组织细胞常态生长的热谱图反映了组织细胞新陈代谢的情况,热谱图峰高和组织细胞新陈代谢的活动强度成正比。但到目前为止,组织细胞的生长过程热谱图的测量国内还未见报导,因为其正常生长的条件要求较高,测量重复性差。本文在达方面进行了探索性研究。实验部分本工作使用的是MS80标准型Calvet微量量热计,工作温度为37℃,放大器用10μV档,     

重金属铅胁迫下镧对螺旋藻生长及生理特性的影响

以鄂尔多斯高原碱湖钝顶螺旋藻为实验材料,在Pb(NO3)2(70 mg·L-1)胁迫下,采用生理学方法研究了不同浓度La(NO3)3对螺旋藻生长速率、叶绿素a含量、硝酸还原酶及谷氨酸脱氢酶活性的影响。结果表明:各处理组在培养初期生长速率差异不大(P0.05),6 d后添加La(NO3)3的各处理组螺旋藻的生长速率显著高于对照(P0.05);当La(NO3)3处理浓度为8μg·L-1时可显著促进螺旋藻叶绿素a的合成,有效缓解Pb(NO3)2对硝酸还原酶及谷氨酸脱氢酶活性的影响(P0.05),高浓度La(NO3)3则会与Pb(NO3)2协同、抑制螺旋藻的生长。     

稀土Ce3+对盐胁迫下螺旋藻生理特性的影响

以非洲乍得湖钝顶螺旋藻为实验材料,在0.3 mol.L-1NaCl胁迫下,通过生理学方法研究了不同浓度Ce3+对螺旋藻生长的影响。结果表明:Ce3+浓度在8.104～16.208μmol.L-1之间时,促进了螺旋藻的生长以及叶绿素a的合成;Ce3+浓度在16.208～48.62μmol.L-1之间时,促进可溶性蛋白的合成;Ce3+浓度为8.104μmol.L-1时藻体MDA含量最高,之后MDA含量随着Ce3+处理浓度的增加逐步降低。这说明Ce3+在一定的浓度范围内可减轻NaCl对螺旋藻的胁迫作用,促进螺旋藻可溶性蛋白和叶绿素a的积累,降低藻细胞MDA的浓度,高浓度Ce3+则会与NaCl协同、抑制螺旋藻的生长。     

关于宫颈癌变细胞红外光谱测定方法的研究

提出了一种测定癌变细胞的新方法，利用高分辨傅里叶变换红外光谱（ＦＴＩＲ），对正常人的细胞和千余名宫颈癌患者的细胞进行了对比研究，初步得出该法能在分子水平上揭示出肿瘤细胞与正常细胞的明显差别，并且通过谱图解析，可直接阐明引起谱图变化的主要原因、细胞癌变的可能机理，从而对病程进行预测，为肿瘤疾病的早期诊断展现了良好的前景。     

乙醇/硫酸铵双水相体系萃取螺旋藻多糖的研究

考察了低分子有机溶剂与无机盐-乙醇/硫酸铵双水相体系萃取螺旋藻多糖的可行性及影响因素。研究结果表明:为除去并有效回收螺旋藻细胞中的蛋白质成分,在藻细胞破碎后进行盐析沉淀蛋白质,再采用传统的热水浸提法,可得到螺旋藻多糖的溶出率为38.44±1.12mg/g干燥粉;通过乙醇/硫酸铵双水相体系的萃取分配,在w(乙醇)=19%,w(硫酸铵)=27.5%(即双水相体系系线长度TLL=42.9),体系相比VR=1.05,pH=7.0时,螺旋藻多糖的收率可达84.5±1.45%,富集因子可达6.2。该研究结果表明廉价的乙醇/硫酸铵双水相萃取螺旋藻多糖将有望开发成为一条简洁、高效、低成本的螺旋藻多糖分离提取工艺。     

10-羟基喜树碱的光谱性质、抗肿瘤活性及应用于细胞标记研究

通过紫外-可见光谱、荧光光谱和红外光谱等方法研究了药物10-羟基喜树碱(HCPT)的光谱性质.采用溴化噻唑蓝四氮唑(MTT)法测定了HCPT对3种肿瘤细胞(HeLa,MCF-7和HT1080)的抗肿瘤活性,其IC50值分别为16.37,16.73和19.24μg/mL.测定了HCPT对正常细胞人胚肾细胞系HEK293T的生长抑制活性,最高抑制率达88.72%,表明正常细胞比3种肿瘤细胞对药物HCPT更敏感.以HeLa细胞为模型,利用Annexin V-FITC细胞凋亡检测试剂盒研究了HCPT的抗肿瘤作用机制,发现几乎所有细胞均同时被Annexin V-FITC和碘化丙啶(PI)染色,细胞膜为绿色,而细胞核为红色,表明HCPT诱导了HeLa细胞的晚期凋亡.通过荧光显微镜观察了HCPT在HeLa细胞中的分布,为其在细胞标记中的应用提供了科学依据.     

Expression of Biologically Active Human Recombinant Interferon Alpha 2b in Human Breast Cancer Cell Line Bcap-37

Human interferon alpha 2b (IFNα-2b) is a pleiotropic cytokine used to treat various viral diseases and cancers. Conventionally, recombinant human IFNα-2b used in clinics was produced by prokaryotic expression system, which always lack of enough biological activity due to limitations on proper folding and post-translational modifications, so the eukaryotic expression system are becoming prevailing method for the production of recombinant proteins. In this study, human breast cancer cell Bcap-37 was firstly used as host for the expression of human IFNα-2b, with the expression vector pIRES2-IFN-EGFP, in which IFNα-2b gene is under the control of CMV promoter. The expression of recombinant IFNα-2b was detected by Western blot and ELISA. Results showed that the concentration of the secreted recombinant IFNα-2b in culture medium was 435.7 pg/mL/24 h. Biological activity of the recombinant IFNα-2b was assayed by detecting the expression of IFN-inducible genes, including MxA, OAS, PKR, and Caspase1 through QRT-PCR. Results demonstrated that recombinant IFNα-2b possess the biological activities. Compared to non-transgenic cells, the expression levels of the aforementioned four IFN-inducible genes were increased by 18.098-, 1.843-, 2.21-, and 3.066-folds, respectively. We got to a conclusion that the human breast cancer cell Bcap-37 could express bioactive recombinant IFNα-2b.     

A parametric study of human fibroblasts culture in a microchannel bioreactor

The culture of cells in a microbioreactor can be highly beneficial for cell biology studies and tissue engineering applications. The present work provides new insights into the relationship between cell growth, cell morphology, perfusion rate, and design parameters in microchannel bioreactors. We demonstrate the long-term culture of mammalian (human foreskin fibroblasts, HFF) cells in a microbioreactor under constant perfusion in a straightforward simple manner. A perfusion system was used to culture human cells for more than two weeks in a plain microchannel (130 microm x 1 mm x 2 cm). At static conditions and at high flow rates (>0.3 ml h(-1)), the cells did not grow in the microchannel for more than a few days. For low flow rates (<0.2 ml h(-1)), the cells grew well and a confluent layer was obtained. We show that the culture of cells in microchannels under perfusion, even at low rates, affects cell growth kinetics as well as cell morphology. The oxygen level in the microchannel was evaluated using a mass transport model and the maximum cell density measured in the microchannel at steady state. The maximum shear stress, which corresponds to the maximum flow rate used for long term culture, was 20 mPa, which is significantly lower than the shear stress cells may endure under physiological conditions. The effect of channel size and cell type on long term cell culture were also examined and were found to be significant. The presented results demonstrate the importance of understanding the relationship between design parameters and cell behavior in microscale culture system, which vary from physiological and traditional culture conditions.     

In vitro antitumor activity of triterpenes from Ceriops tagal

The embryo of Ceriops tagal was extracted with 95% ethanol at room temperature, and four triterpenes (1-4) were separated from this extract. For the first time these triterpenes were the separated from this plant. Compounds (1-4) were tested in vitro for antitumor activity against three cell lines (human liver cancer cell (H-7402), human B-lymphoblastoid cell (Raji), and human cervical carcinoma cell (Hela)). Compounds 1 and 3 were effective to inhibit cell proliferation and growth of H-7402 and Hela, the IC(50) of them on H-7402 were 14.42 microg mL(-1) and 9.97 microg mL(-1), and the IC(50) of them on Hela were 11.84 microg mL(-1) and 11.32 microg mL(-1). All compounds 1-4 were not effective to inhibit cell proliferation and growth of Raji. The effects of compound 4 on inhibiting proliferation and growth of these three cancer cells was also not obvious.     

Resveratrol-induced cell inhibition of growth and apoptosis in MCF7 human breast cancer cells are associated with modulation of phosphorylated akt and caspase-9

Resveratrol (trans-3,4N,-5-trihydroxystilbene), a phytoalexin present in grapes and red wine, is emerging as a natural compound with potential anticancer properties. Here we show that resveratrol affects the growth of human breast cancer cell lines MCF7, MDA-MB-231, SK-BR-3, and Bcap-37 in a dose-dependent manner and that MCF7 is the most sensitive among the four cell lines. MCF7 cells treated with resveratrol showed typical characteristics of apoptosis including the poly (ADP-ribose) polymerase cleavage, TdT-mediated dUTP nick end labeling-positive staining, and morphologic changes. Phosphorylation of the oncogene product Akt was significantly reduced followed by decreased phosphorylation and increased processing of pro-caspase-9 on resveratrol treatment. These results indicate that resveratrol seems to exert its growth-inhibitory/apoptotic effect on the breast cancer cell line MCF7 via the Akt-caspase-9 pathway.     

A possible modulation of acetylcholine receptors of embryonic chick muscle cells by alpha-bungarotoxin

Acetylcholine receptors were assayed with alpha-bugarotoxin on embryonic chick skeletal muscle growing in primary cell culture. Toxin was bound specifically to muscle cells and could be competed with D-tubocurarine. Two dissociation constants were obtained by equilibrium binding: 7.2 x 10(-9)M and 2.7 x 10(-7)M at 25 degrees C. Two sets of rate constants were also obtained from dissociation kinetics. There are five times more low affinity sites on cells than high affinity sites. The average density of high-affinity receptors is about 200/micrometers2. A time course of toxin binding to receptors at 37 degrees C in growth medium revealed that under conditions permitting growth and metabolism, toxin bound to cells was lost. The possibility that the growth medium was inactivating toxin molecules was ruled out by showing that unbound toxin molecules in the medium were fully capable of binding to fresh cultures.     

Synthesis and antitumor activity of α-aminophosphonates containing thiazole[5,4-b]pyridine moiety

A new procedure is developed for the synthesis of α-aminophosphonates containing thiazole[5,4-b]pyridine moiety from conveniently available starting materials. The target compounds were characterized by infrared, (1)H NMR, (13)C NMR, (31)P NMR, mass spectrometry and elemental analysis. The newly synthesized compounds were evaluated for their anticancer activities against PC-3, Bcap-37, H460 cells in vitro by the MTT method. Compounds 3b and 3f are highly effective against PC-3, Bcap-37 cells and good to H460 cells. Further study is necessary to find out the potential antitumor activities.     

The role of the p53 tumor suppressor in the response of human cells to photofrin-mediated photodynamic therapy

Although there is evidence that the p53 tumor suppressor plays a role in the response of some human cells to chemotherapy and radiation therapy, its role in the response of human cells to photodynamic therapy (PDT) is less clear. In order to examine the role of p53 in cellular sensitivity to PDT, we have examined the clonogenic survival of normal human fibroblasts that express wild-type p53 and immortalized Li-Fraumeni syndrome (LFS) cells that express only mutant p53, following Photofrin-mediated PDT. The LFS cells were found to be more resistant to PDT compared to normal human fibroblasts. The D37 (LFS cells)/D37 (normal human fibroblasts) was 2.8 +/- 0.3 for seven independent experiments. Although the uptake of Photofrin per cell was 1.6 +/- 0.1-fold greater in normal human fibroblast cells compared to that in LFS cells over the range of Photofrin concentrations employed, PDT treatment at equivalent cellular Photofrin levels also demonstrated an increased resistance for LFS cells compared to normal human fibroblasts. Furthermore, adenovirus-mediated transfer and expression of wild-type p53 in LFS cells resulted in an increased sensitivity to PDT but no change in the uptake of Photofrin per cell. These results suggest a role for p53 in the response of human cells to PDT. Although normal human fibroblasts displayed increased levels of p53 following PDT, we did not detect apoptosis or any marked alteration in the cell cycle of GM38 cells, despite a marked loss of cell viability. In contrast, LFS cells exhibited a prolonged accumulation of cells in G2 phase and underwent apoptosis following PDT at equivalent Photofrin levels. The number of apoptotic LFS cells increased with time after PDT and correlated with the loss of cell viability. A p53-independent induction of apoptosis appears to be an important mechanism contributing to loss of clonogenic survival after PDT in LFS cells, whereas the induction of apoptosis does not appear to be an important mechanism leading to loss of cell survival in the more sensitive normal human fibroblasts following PDT at equivalent cellular Photofrin levels.     

Cellular Uptake of the ATSM−Cu(II) Complex under Hypoxic Conditions

The Cu(II)-diacetyl-bis (N4-methylthiosemicarbazone) complex (ATSM−Cu(II)) has been suggested as a promising positron emission tomography (PET) agent for hypoxia imaging. Several in-vivo studies have shown its potential to detect hypoxic tumors. However, its uptake mechanism and its specificity to various cancer cell lines have been less studied. Herein, we tested ATSM−Cu(II) toxicity, uptake, and reduction, using four different cell types: (1) mouse breast cancer cells (DA-3), (2) human embryonic kidney cells (HEK-293), (3) breast cancer cells (MCF-7), and (4) cervical cancer cells (Hela) under normoxic and hypoxic conditions. We showed that ATSM−Cu(II) is toxic to breast cancer cells under normoxic and hypoxic conditions; however, it is not toxic to normal HEK-293 non-cancer cells. We showed that the Cu(I) content in breast cancer cell after treatment with ATSM−Cu(II) under hypoxic conditions is higher than in normal cells, despite that the uptake of ATSM−Cu(II) is a bit higher in normal cells than in breast cancer cells. This study suggests that the redox potential of ATSM−Cu(II) is higher in breast cancer cells than in normal cells; thus, its toxicity to cancer cells is increased.