去甲基斑蝥素纳米粒大鼠体肠吸收动力学研究

目的:考察去甲基斑蝥素(NCTD)及其壳聚糖纳米粒(NCTD-CS-NP)制剂的大鼠在体吸收特征与相关动力学。方法:采用大鼠在体回流试验,并用紫外分光光度法和HPLC分别测定酚红和NCTD的质量浓度;并采用在体循环法比较不同质量浓度NCTD及NCTD-CS-NP在大鼠小肠区段内的吸收规律。结果:NCTD在十二指肠、空肠、回肠、结肠的吸收速率常数(ka)分别为:0.0306,0.0278,0.0151,0.0044h-1,NCTD-CS-NP在对应肠段的ka分别为:0.0689,0.0592,0.0353,0.0095h-1;质量浓度分别为140,160,180μg/mL的NCTD小肠ka分别为:0.0499,0.0525,0.0489h-1;对应质量浓度的NCTD-CS-NPka分别为:0.0814,0.0764,0.0734h-1。结论:NCTD的质量浓度对其大鼠全肠道的吸收无显著影响,吸收呈一级动力学过程,且以肠道中上部的吸收为主,NCTD-CS-NP可有效促进药物在肠粘膜的吸收。     

两种去甲斑蝥素注射剂型对小鼠移植性肝癌抑制作用比较

目的:比较去甲斑蝥素注射液普通剂型和缓释剂型的抗肿瘤效果,研发增强去甲斑蝥素抗肿瘤作用的新剂型。方法:选择药物缓释辅料泊洛沙姆407,将去甲斑蝥素制成注射用缓释剂,观察其对小鼠H 22皮下移植瘤和腹水瘤的抗肿瘤效果。结果:缓释剂型组对小鼠皮下肿瘤的抑瘤率为53.7%,优于普通剂型组的26.0%(P<0.05);缓释剂型组对小鼠腹水瘤的生命延长率为40.2%,优于普通剂型组的19.6%(P<0.01)。结论:去甲斑蝥素缓释剂型注射液的抗肿瘤效果优于普通剂型注射液。     

去甲斑蝥素缓释制剂毒性与疗效的实验研究

目的：初步探讨去甲斑蝥素缓释制剂的减毒增效作用。方法：选择泊洛沙姆407作为去甲斑蝥素局部用药的载体,将去甲斑蝥素制成缓释剂型,在初步证实该制剂兔肝内注射后确能缓慢释放的前提下,比较不同剂型的去甲斑蝥素的毒性及对荷W256肿瘤大鼠的抗肿瘤效果。结果：（1）去甲斑蝥素缓释制剂肝内注射后在局部至少能停留4 h;（2）去甲斑蝥素缓释制剂的毒性明显低于单纯去甲斑蝥素;（3）去甲斑蝥素缓释剂型治疗组大鼠肿瘤生长受到显著抑制,肿瘤组织坏死广泛,治疗后平均生存期延长（与单纯去甲斑蝥素组相比,P<0.05）,综合疗效优于单纯去甲斑蝥素组。结论：去甲斑蝥素缓释制剂肝内局部注射能够通过延缓去甲斑蝥素的释放速度,增加其与肿瘤细胞直接作用的时间等途径达到减毒增效的目的。     

去甲斑蝥素微球介入治疗大鼠肝癌疗效及其机制研究

目的探讨去甲斑蝥素-海藻酸/聚酸酐微球(norcantharidin-alginicacid/polyacidanhydridemicro-spheres,N-MS)介入治疗大鼠肝癌的作用及其机制。方法采用乳化-化学交联法制备N-MS。建立大鼠肝癌模型,将荷瘤大鼠随机分为空白对照组、去甲斑蝥素(norcantharidin,NCTD)组、空白微球(blankmicro-sphere,B-MS)组、NCTD-碘油组和N-MS组。各组荷瘤大鼠分别经肝动脉注入生理盐水、NCTD、B-MS、NCTD-碘油和N-MS。治疗后,观察各组大鼠生存时间、肝肿瘤体积和肝肿瘤坏死程度;采用TUNEL法检测各组大鼠肝肿瘤细胞凋亡指数;采用免疫组织化学链霉菌抗生物素蛋白-过氧化物酶连接法(streptavidin-biotinperoxidasemethod,SP)检测各组大鼠肝肿瘤细胞Ki-67的表达。结果治疗后,N-MS组大鼠的生存期较其他各组明显延长;肝肿瘤体积小于其他各组;肿瘤生长率和肝肿瘤细胞Ki-67的表达明显低于其他各组;肿瘤坏死程度和肝肿瘤细胞凋亡指数均高于其他各组,差异均有统计学意义。结论N-MS经肝动脉介入对大鼠肝癌具有较好的治疗作用,其作用机制与栓塞肿瘤微血管、缓慢释放NCTD、诱导肿瘤细胞凋亡和下调Ki-67的表达,从而抑制肿瘤细胞增殖有关。     

去甲斑蝥素抑制黑色素瘤M14细胞增殖和诱导细胞凋亡的研究

Objective To investigate the effects of norcantharidin on the proliferation and apoptosis related proteins Bax and Bcl-2 of human melanoma M14 cells. Methods Melanoma M14 cell line cultured in vitro was treated with concentrations of 50, 100 and 500 μg / L norcantharidin respectively, and untreated cells were used as control. Cell proliferation was detected by CCK-8 method after treatment for 24 h and 48 h. Morphology of cells was observed under inverted microscope. DAPI nuclear staining was used to observe the morphological changes of apoptosis under fluorescence microscope. The expressions of Bax and Bcl-2 were detected by Western blot assay. Results CCK-8 results showed that concentrations of 100 and 500 μg/L norcantharidin inhibited the proliferation of melanoma M14 cells after treatment for 24 h and 48 h (P＜0.05), and the higher the concentration, the more significant the inhibitory effect. The cells showed irregular contours, nucleus shrinkage, small volume, and poor cell adhesion of apoptosis changes under inverted microscope and fluorescence microscope. Western blot analysis showed that the relative expression of Bcl-2 decreased gradually with the increased concentration of norcantharidin (P＜0.05), and the relative expression of Bax increased gradually (P＜0.05).Conclusion Norcantharidin can inhibit the proliferation of melanoma M14 cells, and its mechanism may be related to the promotion of apoptosis.     

NCTD通过靶向Hedgehog/SOX2 axis抑制急性髓系白血病HL60细胞的增殖

目的:研究去甲斑蝥素(NCTD)通过抑制Hedgehog信号通路(SHH)关键调节因子及其下游转录因子SOX2对急性髓系白血病细胞增殖的影响.方法:HL60和NB4细胞系经NCTD、SMO和GLI1抑制剂培养24 h后,用CCK8检测细胞数目;HL60细胞系经NCTD处理后,利用免疫印迹法检测GLI1、SMO、SOX2...     

The targeting effect of Hm2E8b–NCTD–liposomes on B-lineage leukaemia stem cells is associated with the HLF–SLUG axis

To identify an agent with specific activity against B-lineage leukaemia stem cells (B-LSCs), we generated norcantharidin (NCTD)-encapsulated liposomes modified with a novel humanised anti-human CD19 monoclonal antibody, Hm2E8b (Hm2E8b–NCTD–liposomes). These liposomes were specially designed to recognise and kill B-LSCs in vitro, and to decrease non-specific cytotoxicity to untargeted cells. Hm2E8b–NCTD–liposomes selectively ablated B-LSCs through targeting hepatic leukaemia factor (HLF), which is implicated in haematopoietic stem cell regulation and is overexpressed in LSCs. Hm2E8b–NCTD–liposomes decreased HLF protein levels and induced apoptosis in the HAL-01 cell line harbouring the oncoprotein E2A–HLF. This resulted in modulation of the expression of several molecules that govern survival pathways, including HLF, SLUG, NFIL3 and C-Myc, thereby causing the induction of p53 and the mitochondrial caspase cascade. Therefore, the potent in vitro effect of Hm2E8b–NCTD–liposomes on B-LSC activity and survival pathways have the potential to be exploited clinically with appropriate drug combinations.     

去甲基斑蝥素纳米粒肠道吸收的体内外相关性研究

目的 考察去甲基斑蝥素壳聚糖纳米粒(NCTD-CS-NP)的体外释放以及其在体、离体肠吸收特征,并建立三者之间的相关性.方法 采用离子诱导交联法制备NCTD-CS-NP,动态透析法考察其体外释药动力学;以去甲基斑蝥素(NCTD)水溶液为对照,采用大鼠离体外翻肠法与在体同流法研究纳米粒制剂在大鼠小肠区段内的吸收规律;并对体外释放、离体及在体肠吸收进行相关性研究.结果 NCTD与NCTD-CS-NP在大鼠小肠部位的吸收速率常数(K_a)依次为:十二指肠>空肠>回肠>结肠;两种剂型在十二指肠的K_a分别为15.69、29.16 h~(-1),纳米粒制剂的吸收显著好于水溶液;同一剂型,不同质量浓度药物的大鼠小肠部位的吸收速率之间无显著影响(P>0.05),均符合一级吸收动力学过程;离体小肠外翻吸收的趋势与在体吸收一致.仅K_a稍有降低,二者呈现良好的相关性(r=0.999 4).结论 壳聚糖纳米粒有利于药物释放,可有效促进NCTD在肠黏膜的吸收;NCTD在肠道中上部吸收明显,药物质量浓度对吸收速率尤显著影响;NCTD体外释药、在体、离体肠吸收三者之间相关性良好.     

去甲斑蝥素对12类主要人类癌症细胞株生长状态的影响

目的:确定去甲斑蝥素(norcantharidin,NCTD)对12类,72种人类癌症细胞的半数抑制浓度(half maximal inhibitory concentration,IC_(50)),并以此作为生长抑制效应的指标。方法:采用长时间细胞观察及功能分析系统(Incu CyteTM ZOOM),用不同浓度NCTD分别与癌细胞株共同孵育72 h,实时监测和观察细胞增殖情况,分析NCTD对12种人类癌症细胞的细胞毒性。此外,长时间细胞观察及功能分析系统的成像系统在每个时点进行拍照存档,展示了各细胞生长及存活的状态,并且进行实时观察。结果:NCTD对72株癌细胞系的生长抑制效应存在广泛的差异[IC_(50)2~66μmol·L-1,平均(24.63±12.97)μmol·L~(-1)]。NTCD对不同细胞生长抑制的影响差异明显,体现在不同癌种之间以及同种癌种内不同个体之间。这充分反映了NTCD临床疗效个体化的特征,并提示了未来个体化治疗的可能性。肝细胞癌、胃癌、前列腺癌、白血病、中枢神经系统癌、肾癌、胆管癌细胞的细胞系IC_(50)平均值均低于总平均值,且前列腺癌、白血病、中枢神经系统癌、肾癌、胆管癌细胞NCTD IC_(50)平均值与肝癌细胞系和胃癌细胞系IC_(50)平均值相近。结论:NTCD可能对前列腺癌、白血病、中枢神经系统癌、肾癌、胆管癌这几类癌症可产生显著疗效。     

去甲斑蝥素诱导人肝癌细胞凋亡的研究

目的探讨去甲斑蝥素（NCTD）对人肝癌细胞SMMC-7721凋亡的影响。方法用MTT方法检测人肝癌细胞SMMC-7721的生长抑制率。应用流式细胞仪检测细胞周期和凋亡。结果 NCTD对人肝癌细胞SMMC-7721有生长抑制作用,随浓度升高、时间延长作用增强,呈剂量和时间效应关系。流式细胞仪示SMMC-7721细胞的S期细胞明显增多,G0/G1期细胞减少,凋亡率上升（P〈0.05或〈0.01）。结论 NCTD能显著抑制人肝癌SMMC-7721细胞的生长,其可能与抑制SMMC-7721细胞增殖,诱导细胞凋亡有关。