siRNA抑制精胺氧化酶的表达可降低人A549肺癌细胞对多胺类似物CPENSpm的敏感性

目的研究精胺氧化酶(spermine oxidase,SMO)基因表达抑制对多胺类似物CPENSpm抗瘤活性的影响。方法用siRNA技术获得SMO基因表达抑制的人A549肺癌细胞株,QT-RT-PCR法和酶活性分析法用于鉴定SMO基因的表达水平,MTT法检测细胞的存活率,DNA降解分析和流式细胞/亚凋亡峰分析检测细胞凋亡状况。结果成功获得SMO基因表达抑制的人A549肺癌细胞株。用SMO-siRNA质粒转染的细胞中,SMO mRNA和酶活性的基础水平分别降低53%和14%。用10μmol·L-1 CPENSpm处理对照细胞24h可使SMO mRNA和酶活性分别升高10倍和20倍,但在SMO-siRNA质粒转染的细胞中,这种药物对SMO的表达诱导被抑制。细胞增殖实验发现,SMO表达抑制的细胞对CPENSpm(0～20μmol·L-1)的药物敏感性低于对照细胞。细胞凋亡分析发现,与对照细胞相比,CPENSpm诱导SMO表达,抑制细胞发生细胞凋亡的能力明显减弱。结论CPENSpm诱导肿瘤细胞内SMO高表达可能是其抗癌药理活性的分子基础之一。     

Polyamine dendrimer-based MRI contrast agents for functional kidney imaging to diagnose acute renal failure

PURPOSE: To choose an efficacious renal functional MRI contrast agent to image early renal tubular damage. We synthesized and compared smaller polyamine dendrimer-based MRI contrast agents (<60 kD) that, unlike Gd-[DTPA], transiently accumulate in renal tubules and can be used to visualize renal structural and functional damage. MATERIALS AND METHODS: Six dendrimer-based MRI contrast agents smaller than 60 kD were studied by high resolution dynamic micro-MRI and compared to Gd-[DTPA]-dimeglumine and Gadomer-17. The best agent, DAB-G2, was further tested in a mouse ischemia/reperfusion model to validate its efficacy. RESULTS: Despite unequal renal clearance rates, all polyamine dendrimer agents visualized the renal functional anatomy of the mice better than Gd-[DTPA]-dimeglumine and Gadomer-17. DAB-G2 was excreted most rapidly, yet was able to visualize mild renal tubular injury very early after injury. CONCLUSION: DAB-G2 was found to be the best candidate for functional kidney imaging and enabled early diagnosis of acute renal injury.     

Enzymatic oxidation products of spermine induce greater cytotoxic effects on human multidrug-resistant colon carcinoma cells (LoVo) than on their wild-type counterparts

The occurrence of resistance to cytotoxic agents in tumor cells, associated with several phenotypic alterations, is one of the major obstacles to successful anticancer chemotherapy. A new strategy to overcome MDR of human cancer cells was studied, using BSAO, which generates cytotoxic products from spermine, H(2)O(2) and aldehyde(s). The involvement of these products in causing cytotoxicity was investigated in both drug-sensitive (LoVo WT) and drug-resistant (LoVo DX) colon adenocarcinoma cells. Evaluation of clonogenic cell survival showed that LoVo DX cells are more sensitive than LoVo WT cells. Fluorometric assay and treatments performed in the presence of catalase demonstrated that the cytotoxicity was due mainly to the presence of H(2)O(2). Cytotoxicity was eliminated in the presence of both catalase and ALDH. Transmission electron microscopic observations showed more pronounced mitochondrial modifications in drug-resistant than in drug-sensitive cells. Mitochondrial functionality studies performed by flow cytometry after JC-1 labeling revealed basal hyperpolarization of the mitochondrial membrane in LoVo DX cells. After treatment with BSAO and spermine, earlier and higher mitochondrial membrane depolarization was found in LoVo DX cells than in drug-sensitive cells. In addition, higher basal ROS production in LoVo DX cells than in drug-sensitive cells was detected by flow-cytometric analysis, suggesting increased mitochondrial activity in drug-resistant cells. Our results support the hypothesis that mitochondrial functionality affects the sensitivity of cells to the cytotoxic enzymatic oxidation products of spermine, which might be promising anticancer agents, mainly against drug-resistant tumor cells.     

妇科肿瘤患者尿中多胺值测定

目的：阐明多胺对妇科肿瘤的诊断及治疗效果判断的意义。方法：本文用固相萃取及高效波相色谱测定法检测并比较了34例妇科恶性肿瘤（恶性组）、35例良性肿瘤患者（良性组）及37例健康女性问4照组）的尿多胺值，且短期随访了16例卵巢恶性肿瘤患者治疗（根治性手术或细胞灭减术及一次化疗）前、后尿中多胺值的变化。结果：恶性组（含复发癌4例）多胺值显著高于良性组及对照组;同样，卵巢恶性肿瘤患者的测定值亦显著高于卵巢良性病变及健康对照。16例卵巢恶性肿瘤患者经有效治疗后尿多胺值明显降低。结论：提示多胺不能作为某一特定肿瘤的特异性诊断标准，但对判断肿瘤的良、恶性以及治疗疗效和预测恶性肿瘤复发有一定意义。     

红细胞多胺水平与小儿急性淋巴细胞白血病临床关系的初步探讨

采用丹酰氯－聚酰胺薄层层析法定量检测６０例急性淋巴细胞白血病患儿红细胞内多胺水平改变。结果显示：（１）急性淋巴细胞白血病患儿红细胞多胺水平高于正常组，并且以精脒和精脒／精胺比值的变化最为显著（Ｐ＜０．００１）。（２）急性淋巴细胞白血病经治疗缓解后，红细胞多胺水平均有不同程度下降，其中以精脒下降幅度较大（Ｐ＜０．００１），但仍高于正常组（Ｐ＜０．００１），而精脒／精胺比值也下降，低于疗前组（Ｐ＜０．０５），但高于正常组（Ｐ＜０．０５）。结果提示急性淋巴细胞白血病患儿红细胞多胺的检测有助于白血病诊断、观察疗效和评估预后。     

Polyamines stimulate the phosphorylation of phosphatidylinositol in rat mast cell granules

Rat mast cell granules were obtained by homogenization of highly purified rat mast cells and isolated in a Percoll gradient. Diphosphoinositide (DPI) synthesis in rat mast cell granules was assayed by measuring the incorporation of 32P from [gamma 32P] ATP into DPI in the absence of exogenous phosphatidylinositol (PI). Lipids were isolated with methanol/chloroform/HCl and were separated by thin-layer chromatography on oxalic acid impregnated silica gel plates. DPI areas were identified by staining with iodine, scraped and measured for 32P radioactivity. The addition of polyamines, spermine and spermidine, to the granules caused an increase of DPI synthesis, which can be catalyzed by PI kinase. This effect of polyamines in the DPI synthesis was in a dose-dependent manner and maximal effects were observed at 1 mM spermine and 10 mM spermidine, respectively.     

A novel macrocyclic polyamine cationic lipid containing an imidazolium salt group: synthesis, characterization and its transfection activity as a gene delivery vector

The synthesis and characterization of a novel macrocyclic polyamine cationic lipid containing an imidazolium salt group is reported. Its interaction with plasmid DNA was studied by gel electrophoresis and fluorescence quenching experiments. The transfection activity of target compound as a gene delivery vector was also investigated. The results showed that the synthesized macrocyclic polyamine cationic lipid has high binding and condensation ability of DNA under physiological conditions probably because of the cooperation effect of macrocyclic polyamine (Cyclen) and an imidazolium salt group. This novel lipid could transfer plasmid DNA into cell in in vitro experiment without the use of any extraneous agent.     

螯合金属多胺型棉纤维的制备及其对大肠杆菌抑菌活性的测定

目的研究稳定性螯合金属多胺型抗菌棉纤维。方法采用改进工艺路线和振荡烧瓶方法,制作螯合金属棉纤维并对其抑菌效果进行了测定。结果采用改进工艺路线碱化棉纤维与环氧氯丙烷反应,接枝环氧基团,接枝产物开环反应,进一步与多乙烯多胺以-C-N-键相连接,生成多胺型棉纤维。二乙烯三胺-Cu(Ⅱ)棉纤维的最低抑菌浓度为12mg/ml;三乙烯四胺-Cu(Ⅱ)棉纤维的最低抑菌浓度为14mg/ml;四乙烯五胺-Cu(Ⅱ)棉纤维的最低抑菌浓度为10mg/ml。三种抗菌棉纤维的抑菌作用速度都比较快,抑菌率与作用时间呈正比直线关系。作用20min,三种抗菌棉纤维抑菌率平均都达到90%以上;作用80min,抑菌率都达到100%。经蒸馏水清洗和75%酒精浸泡洗涤的三种抗菌棉纤维,对大肠杆菌均保持了良好的抑菌效果,有很好的耐热性和耐洗性。结论改进工艺合成制作的抗菌棉纤维达到新工艺要求,实现了抑菌效果好、具有良好的耐洗性的要求。     

A sensitive colorimetric assay for polyamines in erythrocytes using oat seedling polyamine oxidase

Background: Most of the polyamines circulating in blood are spermidine (Spd) and spermine (Spm) with only trace amounts of putrescine (Put), and they are mainly localized in erythrocytes. We developed a simple and sensitive colorimetric assay for polyamines in erythrocytes using oat seedling polyamine oxidase (OSPO). The method is based on the unique substrate specificity of OSPO, which is active toward Spd and Spm, but not toward diamines such as Put and cadaverine and monoamines such as histamine. Methods: The polyamines, which were purified from packed erythrocytes by weak cation-exchange chromatography, were incubated with OSPO at 37 °C for 15 min. In the presence of the H₂O₂ produced by this polyamine oxidase reaction and a new chromogen, N-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)-diphenylamine sodium salt (DA-64), peroxidase (POD) catalyzes the formation of N-[4-[[4-(dimethylamino)phenyl]imino]-2,5-cyclohexadien-1-ylidene]-N-methylmethanaminium chloride (Bindschedler's Green) having an absorption maximum at 727 nm. Results: The detection limit was 0.2 μmol/l for packed erythrocytes. The within-run and between-run precisions (coefficient of variation, CVs) were 5.6–15.2% and 6.5–16.4%, respectively. Analytical recoveries were 93.3–97.4%. Polyamine values obtained by this assay correlated well with those obtained by an HPLC (y=0.948x+1.912; r=0.944; n=46). Conclusions: This colorimetric assay is simple and highly sensitive and practical for clinical use.     

新多胺类似物四丁基丙二胺对人肝癌HepG2细胞增殖及侵袭迁移的影响

目的研究新多胺类似物四丁基丙二胺(tetrabutyl propanediamine,TBP)对人肝癌HepG2细胞增殖及侵袭迁移的影响。方法 MTT比色法分析细胞增殖影响,流式细胞术检测细胞周期改变,Transwell体外迁移/侵袭实验检测迁移侵袭能力的变化,Western Blot用于分析细胞多胺代谢相关蛋白精胺氧化酶(SMO)的表达水平,化学发光法测定SMO活性,反相高效液相色谱法分析TBP对细胞多胺含量的影响。结果 TBP有效抑制HepG2细胞增殖水平,对细胞的侵袭、迁移能力也有明显抑制作用。TBP抑制细胞DNA复制,导致S期细胞比例显著性下降,G0/G1期细胞比例升高。细胞浆中SMO含量明显升高,但其酶活性下降。TBP显著性增加了细胞的腐胺水平,但对精脒和精胺无影响。结论 TBP能够抑制人肝癌HepG2细胞增殖、降低HepG2细胞侵袭和迁移的能力,其机制可能与影响肿瘤细胞DNA合成,改变细胞周期时相分布有关。