鬼臼毒素衍生物QW-83对人宫颈癌HeLa细胞凋亡的影响及其机制研究

目的:研究鬼臼毒素衍生物QW-83对人宫颈癌He La细胞凋亡的影响及其机制。方法:以0(阴性对照)、0.01、0.1、1、10μmol/L QW-83和阳性药物依托泊苷(VP-16)分别培养人宫颈癌He La细胞48 h后,采用MTT法测定细胞的增殖抑制率和半数抑制浓度(IC50);以0(阴性对照)、2.5、5、10μmol/L QW-83培养细胞48 h后,采用Hochest 33342染色观察细胞形态,采用流式细胞术测定细胞凋亡率,采用半定量逆转录-聚合酶链反应(RT-PCR)法检测细胞中凋亡相关基因P53、Bax、Casepase-3、Casepase-8、Casepase-9和Bcl-2 m RNA的表达。结果:与阴性对照比较,1、10μmol/L VP-16和QW-83对He La细胞增殖有明显的抑制作用(P<0.05或P<0.01),IC50值分别为(5.11±0.43)、(4.96±0.54)μmol/L;染色结果显示,QW-83作用后细胞凋亡明显,细胞固缩;流式细胞检测结果显示,QW-83作用后细胞凋亡率呈浓度依赖性升高,为16.89%~62.56%;RT-PCR结果显示,QW-83作用后细胞中P53、Bax、Casepase-3、Casepase-8、Casepase-9 m RNA的表达增强,Bcl-2 m RNA的表达减弱,Bax/Bcl-2比例升高(P<0.05)。结论:鬼臼毒素衍生物QW-83能诱导人宫颈癌He La细胞的凋亡,其机制可能与调控凋亡相关基因m RNA的表达有关。     

新型鬼臼毒素衍生物的合成及其抗肿瘤活性

目的 设计并合成新型鬼臼毒素类衍生物。方法 5-甲氧基吲哚与氯代物或酰氯反应得到一位氮取代的5-甲氧基吲哚;其与草酰氯反应,再以4-氨基4-脱氧表鬼臼毒素为原料,经缩合反应得到目标化合物。体外活性采用Hela细胞筛选模型进行评价。结果与结论 合成了7个新化合物,其中化合物7a, 7b 的活性优于阳性对照依托泊苷（etoposide）。     

纳米二氧化硅搭载4'-去甲基表鬼臼毒素对人宫颈癌HeLa细胞的增殖抑制作用研究

目的 研究鬼臼毒素、4''-去甲基表鬼臼毒素及其与不同比例纳米二氧化硅（SiO₂）的联合用药对人宫颈癌HeLa细胞的体外增殖抑制作用,并探讨其机制。方法 采用正硅酸乙酯水解法制备25 nm SiO₂样品,将其进行表面改性后搭载4''-去甲基表鬼臼毒素;MTT法检测SiO₂、鬼臼毒素、4''-去甲基表鬼臼毒素和联合用药（12.500、1.250、0.125 μg/mL纳米SiO₂分别搭载6.25 μg/mL 4''-去甲基表鬼臼毒素）对HeLa细胞体外增殖的抑制作用;Hoechst 33342染色法检测25 nm SiO₂的细胞相容性、鬼臼毒素和4''-去甲基表鬼臼毒素对细胞凋亡影响;倒置显微镜观察联合用药对细胞形态的影响;Western blotting技术检测4''-去甲基表鬼臼毒素和联合用药对HeLa细胞凋亡相关蛋白表达的影响。结果 4''-去甲基表鬼臼毒素对HeLa细胞的增殖抑制作用优于鬼臼毒素,联合用药对HeLa细胞的抑制作用优于单一4''-去甲基表鬼臼毒素,且0.125 μg/mL纳米SiO₂搭载6.25 μg/mL 4''-去甲基表鬼臼毒素时,抑制作用最明显;鬼臼毒素、4''-去甲基表鬼臼毒素和联合用药均可诱导细胞凋亡;4''-去甲基表鬼臼毒素和联合用药可以上调Bax/Bcl-2比值及Caspase-3、p53、p38的表达水平。结论 SiO₂联合用药对HeLa细胞的增殖抑制作用优于单一4''-去甲基表鬼臼毒素,其作用机制可能为,通过影响Bcl-2、Bax、Caspase-3、p53、p38等凋亡相关蛋白表达诱导细胞凋亡。     

聚合物胶团在肿瘤治疗中的应用

聚合物胶团(polymeric micelles)是近几年来,在药剂学领域崭露头角的一类新型的纳米载体[1],聚合物胶团由于其增溶性、靶向性、低毒性和长循环性引起了人们的广泛重视.     

鬼臼毒素纳米脂质体对人白血病细胞的凋亡诱导作用

目的研究鬼臼毒素纳米脂质体对人白血病细胞K562的凋亡诱导效应。方法薄膜超声分散法制备鬼臼毒素纳米脂质体。不同浓度的鬼臼毒素和鬼臼毒素纳米脂质体分别处理K562细胞,MTT法检测细胞的增殖活性,细胞形态学改变和AnnexinⅤ/PI染色检测细胞凋亡。结果鬼臼毒素纳米脂质体对K562细胞增殖的抑制作用明显高于鬼臼毒素,24、48、72h的半数抑制浓度(IC50)分别为0.71、0.02、0.003μg·ml-1,而鬼臼毒素为1.50、0.12、0.05μg·ml-1;0.01μg·ml-1鬼臼毒素纳米脂质体处理24、72h,AnnexinⅤ/PI染色K562细胞凋亡率分别为91.1%和92.7%,但72h时以晚期凋亡为主;而0.01μg·ml-1鬼臼毒素处理的细胞凋亡率分别为63.4%和66.8%。鬼臼毒素纳米脂质体和鬼臼毒素处理后,K562细胞出现皱缩、胞膜起泡、凋亡小体等典型的细胞凋亡形态学改变。结论鬼臼毒素纳米脂质体较鬼臼毒素对白血病K562细胞具有更强的增殖抑制和凋亡诱导效应。     

4-酰胺基-4-脱氧-4′-去甲表鬼臼毒素衍生物的合成和抗肿瘤活性

4′-去甲表鬼臼毒素与叠氮酸在三氟化硼乙醚存在下缩合,并经还原得4-氨基-4-脱氧-4′-去甲表鬼臼毒素。该中间体与酸或酸酐反应得相应酰胺化合物24个。经体外筛选,多数化合物抑制L₁₂₁₀和KB细胞活性相当或超过依托泊甙。与相应的醚、酯、胺等类型相比,这一类化合物活性最强。     

4-酰胺基-4-脱氧-4′-去甲表鬼臼毒素衍生物的合成和抗肿瘤活性

4′-去甲表鬼臼毒素与叠氮酸在三氟化硼乙醚存在下缩合,并经还原得4-氨基-4-脱氧-4′-去甲表鬼臼毒素。该中间体与酸或酸酐反应得相应酰胺化合物24个。经体外筛选,多数化合物抑制L_(1210)和KB细胞活性相当或超过依托泊甙。与相应的醚、酯、胺等类型相比,这一类化合物活性最强。     

壳寡糖嫁接硬脂酸阳离子聚合物胶团的制备及其理化性质

目的考察阳离子型壳寡糖硬脂酸嫁接物胶团的理化性质及载药胶团的体外药物释放。方法以碳二亚胺为交联偶合剂制备壳寡糖硬脂酸嫁接物；芘荧光法测定该聚合物的临界聚集浓度，微粒粒度及电泳分析仪测定聚合物胶团的粒径和表面电位；以甲氨蝶呤为模型药物，考察胶团作为药物载体的可行性。结果嫁接物的临界聚集浓度为0.05 g·L^-1；去离子水中的胶团粒径为26.7 nm，表面电位为(55.9±0.1) mV；三聚磷酸钠修饰可使胶团粒径增加、表面电位降低、药物包封率增加；降低胶团溶液的pH值，可使胶团的粒径和表面电位上升，药物包封率下降，体外释药速度加快。 结论壳寡糖硬脂酸嫁接物胶团是一种良好的药物载体，其体外释药具有一定的pH依赖性。     

4-β-硫酯-4-脱氧-4′-去甲表鬼臼毒素衍生物的合成及其抗肿瘤活性

为考察 4′ 去甲鬼臼毒素C4位上联结碳原子对活性的影响 ,作者设计并合成C4位 β构型的硫酯取代的衍生物 18个 .这类化合物在体外的抑制L12 10细胞和KB细胞的药理实验中显示出一定的抗肿瘤活性 ,但均弱于etoposide     

4-β-酯取代-4-脱氧-4′-去甲表鬼臼毒素衍生物的合成及其抗肿瘤活性

目的　寻找高效低毒的抗肿瘤药物。方法和结果　本文设计并合成了新的C₄位β构型的酯取代的衍生物38个(II-1～II-38),并进行了体外对L₁₂₁₀细胞和KB细胞的抗肿瘤活性实验。 结论　脂肪酯的活性强于芳香酯,其中以II-10的活性最强,超过etoposide 2个数量级。脂肪酯中主链不超过3个碳原子的活性较好。在芳香酯中,苯环上连接吸电子基的化合物的活性稍强于连接有供电子基的化合物。     

叶酸修饰多柔比星聚合物胶束的制备及体外抗肿瘤活性评价

合成了叶酸-聚乙二醇-二硬脂酰磷脂酰乙醇胺(Folate-PEG-DSPE),并用其作为靶向肿瘤的功能性材料,以甲氧基聚乙二醇-二硬脂酰磷脂酰乙醇胺(MPEG-DSPE)作为骨架材料,采用成膜水化法制备载多柔比星(1)胶束。所得胶束呈球状结构,粒径为(20±5)nm,叶酸修饰和非叶酸修饰胶束的包封率和载药量分别为(78.8±1.52)%、(79.2±147)%和(1 3.6±1.26)%、(1 3.9±1.19)%。流式细胞结果显示,分别给予游离罗丹明B(Rh B)、叶酸修饰和非叶酸修饰的载RhB胶束,摄取荧光的巨噬细胞分别占细胞总数的36.6%、1.5%和4.4%,说明聚合物胶束可显著减弱巨噬细胞的吞噬作用。体外抗KB人口腔上皮癌细胞活性的试验结果显示,1、叶酸修饰和非叶酸修饰胶束的IC_(50)分别为29 7、0.61和4.12μmol/L,表明叶酸修饰的聚合物胶束能显著提高1的抗肿瘤活性。     

Enhanced Intercellular Retention Activity of Novel pH-sensitive Polymeric Micelles in Wild and Multidrug Resistant MCF-7 Cells

Purpose The purpose of this work was to demonstrate the advantage of using pH-sensitive polymeric mixed micelles (PHSM) composed of poly(l-histidine) (polyHis)/poly(ethylene glycol) (PEG) and poly(l-lactic acid) (pLLA)/PEG block copolymers with folate conjugation to increase drug retention in wild-type and MDR tumor cells. Materials and Methods Both wild-type and multidrug resistant (MDR) human breast adenocarcinoma (MCF-7) cell lines were used to investigate the accumulation and elimination of doxorubicin (DOX), PHSM with folate (PHSM/f), and pH-insensitive micelles composed of pLLA/PEG block copolymer with folate (PHIM/f). Results Cells treated with PHSM/f showed decelerated elimination kinetics compared to cells treated with PHIM/f. MDR cells treated with drug-containing PHSM/f for 30 min retained 80% of doxorubicin (DOX) even after incubation for 24 h in the absence of drug. On the other hand, cells treated with drug-containing PHIM/f retained only 40% of DOX within the same period of time. Flow cytometry and confocal microscopy confirmed these results. Conclusions Cellular entry of the micelles occurred via receptor-mediated endocytosis using folate receptors. The pH-induced destabilization of PHSM/f led to rapid distribution of drug and polymer throughout the cells, most likely due to polyHis-mediated endosomal disruption. This reduced the likelihood of drug efflux via exocytosis from resistant tumor cells.     

Magnetically Guided Self-Assembled Protein Micelles for Enhanced Delivery of Dasatinib to Human Triple-Negative Breast Cancer Cells

Magnetic nanocarriers are useful in targeted cancer therapy. Dasatinib (DAS)-loaded magnetic micelles were prepared for magnetically guided drug delivery. The magnetic nanoplatform is composed of hydrophobic oleic acid–coated magnetite (Fe₃O₄) core along with DAS encapsulated in amphiphilic zein-lactoferrin self-assembled polymeric micelles. Transmission electron microscope analysis manifested formation of these magnetic micelles with a mean diameter of about 100 nm. In addition, drug-loaded magnetic micelles displayed a saturation magnetization of about 10.01 emu.g^?1 with a superparamagnetic property. They also showed good in vitro serum stability and hemocompatibility accompanied with a sustained release of DAS in acidic pH. More importantly, they exhibited 1.35-fold increase in their in vitro cytotoxicity against triple-negative human breast cancer cell line (MDA-MB-231) using an external magnetic field compared to drug-loaded magnetic micelles in the absence of a magnetic field. Enhanced inhibition of p-c-Src protein expression level and in vitro cellular migration under the effect of magnetic field was noted owing to the dual-targeting strategy offered by the presence of a magnetic sensitive core, as well as the active targeting property of lactoferrin corona. Taken all together, these results suggest that DAS-loaded magnetic micelles possess a great potential for targeted therapy of breast cancer.     

Antiproliferative Activity of Purine Nucleoside Phosphorylase Multisubstrate Analogue Inhibitors Containing Difluoromethylene Phosphonic Acid against Leukaemia and Lymphoma Cells

Potent inhibitors of purine nucleoside phosphorylase (PNP) are expected to act as selective agents against T-cell tumours. Five compounds with guanine, three with hypoxanthine, and five with 9-deazaguanine, all connected by a linker with difluoromethylene phosphonic acid, were studied on their inhibitory potential against human and calf PNPs. Antiproliferative activity of these analogues against lymphocytes as well as lymphoma and leukaemia cells has been also investigated. All tested compounds act as multisubstrate analogue inhibitors of PNP with the apparent inhibition constants in the range 5–100 nm , and also show a slight antiproliferative activity. Analogues with 9-deazaguanine aglycone have better anti-leukaemic and anti-lymphoma activities compared to the guanine and hypoxanthine analogues, and applied in the concentration of 100 μm , caused a statistically significant decrease in the cell viability in all human leukaemia and lymphoma cells used. Despite the high PNP inhibitory potential of tested analogues, no differences were observed between the effects on the growth of tumour cells sensible to the inhibition of PNP, such as human adult T-cell leukaemia and lymphoma cells, and other investigated cells. Obtained poor effects on cell proliferation could be explained probably by a poor ability of tested compounds to penetrate cell membranes.     

三氧化二砷复合物抑制人脑胶质瘤U87细胞生长的机制研究

目的 研究三氧化二砷复合物对人脑胶质瘤U87细胞凋亡的影响,并进一步探讨其作用机制。方法 采用激光共聚焦技术研究三氧化二砷各复合物进入细胞的方式;采用细胞划痕愈合试验观察其对U87细胞迁移能力的影响;采用血管形成试验观察其对新生血管形成能力的影响;流式细胞仪检测各组细胞周期及细胞凋亡;免疫蛋白印迹法检测凋亡相关蛋白Bcl-2、Bax和caspase-3的表达情况。结果 三氧化二砷复合物主要通过内吞方式进入细胞,能显著抑制U87细胞的迁移以及新生血管的形成;其可诱导U87细胞周期阻滞于G2/M期,促进细胞发生凋亡;三氧化二砷复合物促进Bax和caspase-3的表达,抑制Bcl-2的表达。结论 三氧化二砷复合物对U87细胞的抑制作用机制可能是通过内吞方式进入细胞后,上调Bax的表达,下调Bcl-2的表达,最终诱导细胞发生凋亡。     

十二碳烯酸对人H4细胞毒性作用研究

目的探讨十二碳烯酸对人H4细胞的毒性作用机制。方法采用MTT法、流式细胞仪检测十二碳烯酸对人H4细胞的毒性作用。结果分别给予人H4细胞0．3,0．9,2．7mg·L^-1十二碳烯酸培养48h,十二碳烯酸对人H4细胞增殖抑制率分别为41．64％,70．97％,81．52％,半数抑制浓度（IC50）为0．40mg·L^-1,与对照组相比,差异有统计学意义（P〈0．01）;凋亡率分别为10．16％,18．22％,24．06％。结论十二碳烯酸对人H4细胞具有显著的毒性作用,其机制可能与损伤线粒体结构,抑制细胞分裂等有关。     

Anti-tumor Effect of All-<Emphasis Type="BoldItalic">Trans</Emphasis> Retinoic Acid Loaded Polymeric Micelles in Solid Tumor Bearing Mice

Purpose All-trans retinoic acid (ATRA) polymeric micelles were developed for parenteral administration. The distribution characteristics and antitumor activities of ATRA polymeric micelles were evaluated after intravenous administration to mice bearing CT26 solid tumors. Methods ATRA incorporated in poly(ethylene glycol)-poly(benzyl aspartate) block copolymer was prepared by the evaporation method. The levels of [³H]ATRA in blood and tissue including tumor were determined by measuring the radioactivity after injection into mice. The tumor volume and the survival of the mice were determined to assess the anticancer activity. Results The delivery of ATRA by polymeric micelles prolonged the blood circulation and enhanced the accumulation of ATRA in the tumor tissue compared with the administration of free ATRA. Tumor growth was significantly delayed and the survival time of mice was prolonged following the treatment by ATRA polymeric micelles demonstrating the improved anticancer activity of ATRA. Conclusion Polymeric micelles are a promising and effective carrier of ATRA in order to enhance tumor delivery and they have a promising potential application in the treatment of solid tumors.     

重组人血小板型磷脂酶A_2体外抗金黄色葡萄球菌的活性及其影响因素研究

目的:体外评价具有强正电荷性的重组人血小板型磷脂酶A2(RHP-PLA2)对金黄色葡萄球菌的杀菌活性,观察其在不同影响因素及水平下杀菌活性的变化。方法:用琼脂平板计数法测定杀菌活性,并对影响其杀菌活性的pH、Ca2+、Mg2+、肝素(带负电荷)等因素及水平进行测定。结果:RHP-PLA2杀灭99%金黄色葡萄球菌的最低浓度(MBC)为0.08ng.L-1。在MBC时,当pH为5.0、7.4、9.0时,RHP-PLA2杀菌率分别为44.4%、99.8%、99.5%;1～5mmol·L-1Ca2+适于RHP-PLA2发挥杀菌活性;5.12mmol·L-1乙二醇二乙醚二胺四乙酸、10mmol·L-1MgCl2及0.30×10-4IU.L-1肝素可完全抑制RHP-PLA2杀菌作用。结论:RHP-PLA2杀菌活性受pH值和Ca2+浓度的影响。PLA2分子带的正电荷性是其发挥杀菌活性的关键因素。     

Paradoxical Effect of 2,3-Dimercapto-1-propanesulfonic Acid (DMPS) on Enhancing Antitumor Activity of Cisplatin in Ascites Sarcoma 180 Cells

We investigated the enhancing effect of two metal-chelating compounds, 2,3-dimercapto-1-propanesulfonic acid (DMPS) and meso-2,3-dimercaptosuccinic acid (DMSA), on the antitumor activity of cisplatin (CDDP). In the in vivo experiments, DMPS showed a clear synergistic effect and significantly enhanced the antitumor activity of CDDP in terms of survival and life span in mice transplanted with ascites sarcoma 180 cells (S180 cells) at a dose of <100 μ mol/ kg, s.c., but not at a dose of >500 μ mol/kg. On the other hand, DMSA did not enhance the antitumor activity of CDDP. DMPS (50 μ mol/kg, s.c.) combined with CDDP also potently suppressed [³H]thymidine uptake in S180 cells implanted in mice, whereas DMSA did not. In the in vitro experiments, DMPS (10^?6 to 10^?5 M) produced a time- and dose-dependent decrease in intracellular Ca²⁺ concentrations ([Ca²⁺] _i ) in S180 cells and, in combination with CDDP, yielded a significant increase in intracellular platinum accumulation compared to that in cells treated with CDDP alone. These results indicate that DMPS used in combination with CDDP may be of considerable benefit in enhancing the cytotoxicity of CDDP in tumor cells, especially at a low dose. The results also suggest that the enhancing effect of DMPS is closely related to a decrease in [Ca²⁺] _i and that the suitable dose and adequate administrational time of DMPS are important for its effective action.