【作者】 陆鹏；

【导师】 曾甫清；

【作者基本信息】 华中科技大学 ， 外科学， 2006， 博士

【摘要】 第一部分姜黄素前体药物合成及其鉴定目的:制备姜黄素前体药物,使其在肿瘤细胞内高选择性活化,为进一步开展肿瘤靶向性化疗奠定基础。方法:以姜黄素分子结构为基础,化学合成N-马来酰-L-缬氨酸酯姜黄素、N马来酰-甘氨酸酯姜黄素;红外光谱、核磁共振法对合成的药物进行鉴定;MTT比色分析法比较两种姜黄素前体药物分别作用6-24h后,人膀胱癌EJ细胞及肾小管上皮HKC细胞生长抑制的差异。结果: 20μmol/L-40μmol/L的N-马来酰-L-缬氨酸酯姜黄素、N-马来酰-甘氨酸酯姜黄素作用6-24h后,EJ细胞生长抑制率分别为6.71%-65.13%(P<0.05)、10.96%-73.01%(P<0.05),呈浓度、时间依赖性。与同浓度姜黄素比较,两种前体药物对HKC细胞生长的抑制作用均显著降低(P<0.01)。结论:合成的两种姜黄素前体药物通过细胞内酯酶的水解作用,促使姜黄素前体药物活化,能在体外有效抑制肿瘤细胞的生长活性,降低对正常二倍体细胞的毒副作用,为深入研制肿瘤靶向性化疗药物提供了新的途径。第二部分姜黄素前体药物诱导膀胱癌细胞凋亡的研究目的观察姜黄素前体药物活化系统诱导膀胱癌细胞凋亡的生物学效应,并探讨其可能的机制。方法N-马来酰-L-缬氨酸酯姜黄素、N-马来酰-甘氨酸酯姜黄素分别作用于人膀胱癌EJ细胞、肾小管上皮HKC细胞6 24h后,采用台盼兰活细胞拒染法检测细胞生长活性,透射电镜观察细胞超微结构改变,DNA片段化原位荧光标记技术、流式细胞术-检测细胞凋亡及比率。结果10μmol/L-40μmol/L的N-马来酰L-缬氨酸酯姜黄素、N-马来酰-甘氨酸酯姜黄素作用6-24h后,EJ细胞生长抑制率分别为(6.11-66.23)%(P<0.05)、(8.96-68.21)%(P<0.05),呈浓度、时间依赖性;部分癌细胞出现凋亡形态学改变,DNA直方图上可见“亚G1峰”,12 h细胞凋亡率分别为(10.13-23.36)%(P<0.05)、(12.42-28.56)%(P<0.05)。两种前体药物对HKC细胞生长的抑制作用较同浓度姜黄素显著降低(P<0.05)。结论通过细胞内酯酶的水解作用,促使姜黄素前体药物活化,姜黄素前体药物能在体外有效诱导膀胱癌EJ细胞凋亡,降低对正常二倍体细胞生长的抑制作用,为深入研制肿瘤靶向性化疗药物提供了新的途径。第三部分姜黄素载药纳米微球的制备及表征目的:制备姜黄素载药纳米微球,为进一步开展肿瘤靶向性化疗研究奠定基础。方法:使用改进的自乳化-溶剂扩散法制备聚乳酸载姜黄素纳米微球,分别采用丙烯碳酸酯-乙醇混合溶剂、丙酮-乙醇混合溶剂制备载药微粒,并计算姜黄素包裹率和包封率。结果:经检测计算,聚乳酸载姜黄素纳米微球的载药率为3.15%,包封率18.9%。结论:经包裹后,肉眼比较可发现载姜黄素纳米微球可较好的分散于水中形成颜色均匀稳定的悬乳液,这表明药物不溶于水的性质得到了极大的改善。这一成果可使载姜黄素纳米微球更好的应用于肿瘤治疗研究中,具有广泛的应用前景。第四部分核酸特异性触发姜黄素前体药物活化系统的合成及其初步表征目的:在前面研究的基础上把已经合成的姜黄素前体药物、催化剂咪唑分别和寡链核苷酸结合。方法:采用化学合成,液相色谱、长链烷基胺载体等方法将药物和反义核酸铰链在一起,并通过高压液相等对合成的药物初步表征结果:经过合成的姜黄素前药-寡聚核苷酸复合物经过高压液相色谱初步表征,已经被初步证实合成成功。结论:核酸特异性触发姜黄素前体药物活化体系是一个切实可行的靶向化疗的治疗体系。第五部分核酸特异性触发姜黄素前体药物活化系统在体内的抑瘤作用的初步研究目的:了解核酸特异性触发姜黄素前体药物催化剂系统在体内的抗肿瘤活性及其毒副作用。方法:通过建立裸鼠皮下膀胱肿瘤模型,肿瘤体内注射法干预肿瘤生长,组织切片等方法了解姜黄素前体药物活化系统对动物体内肿瘤生长的抑制作用,并且检测对实验动物肝肾功能的毒副作用。结果:前期合成的核酸特异性触发姜黄素前体药物活化体系对肿瘤有靶向杀伤作用,其作用与原化疗药物相比,其对肿瘤的生长抑制率相当,但其毒副作用明显降低。结论:核酸特异性触发姜黄素前体药物活化系统是一个能有效杀伤肿瘤细胞且对正常组织细胞毒性极低的靶向化疗策略,具有广泛的应用前景。更多还原

【Abstract】 Part 1Preparation of curcumin prodrugs and their anti-tumor activities in vitroObjectives To prepare the prodrugs of curcumin, which could be selectively activated in tumor cells, in order to establish a basis for further development of targeted chemotherapy for cancer. Methods Based on the molecular structure of curcumin, the N-maleoyl-L-valine-curcumin (NVC), N-maleoyl-glycine-curcumin (NGC) were chemically synthesized and identified by IR spectroscopy. After treatment with these two prodrugs for 6～24h, the growth inhibition rates on human bladder cancer EJ cells and renal tubular epithelial (HKC) cells were detected by MTT colorimetry. Results After treatment with 20μmol/L～40μmol/L MVC and MGC for 6～24h, the growth inhibitory effects on EJ cells were 6.71%～65.13%% (P<0.05), 10.96%～73.01% (P<0.05), respectively, with dose- and time-dependent characters. When compared with the curcumin of same concentrations, the growth inhibitory effects of these two prodrugs on HKC cells were significantly decreased (P<0.01). Conclusion The two-curcumin prodrugs: N-maleoyl-L-valine-curcumin (NVC), N-maleoyl-glycine- curcumin (NGC) were chemically synthesized successfully and were identified that they both could inhibit the growth of tumor cell in vitro. Furthermore, the two prodrugs of curcumin were found less toxic in HKC cell than curcumin.Part 2Apoptosis of bladder cancer cells induced by curcumin prodrugs in vitroObjectives: To study the growth inhibition effects of N-maleoyl-L-valine-curcumin (NVC), N-maleoyl-glycine- curcumin (NGC) on human bladder cancer cell line EJ, renal tubular epithelial (HKC) cells, and the mechanisms for further developing researches on their anti-tumor activity and apoptosis effects. Methods After treatment with these two prodrugs prepared before for 6～24h, the growth inhibition rates on human bladder cancer EJ cells and renal tubular epithelial (HKC) cells were detected by MTT colorimetry, cell count by Typan blue. Cell cycle phases were inspected by flow cytometery (FCM). The apoptosis was detected by TUNEL and DNA ladder methods. The morphological changes of cancer cells were observed under electronic microscopy. Results After treatment with 20μmol/L～40μmol/L NVC and NGC for 6～24h, the growth inhibitory effects on EJ cells were 6.71%～65.13% (P<0.05), 10.96%～73.01% (P<0.05), respectively, with dose- and time-dependent manners. FCM, DNA ladder and TUNEL methods exhibited that apoptosis occurred in some cancer cell with trapeziform bands on electrophoresis. Conclusion Activation of curcumin prodrugs via hydrolysis functions of cellular esterase, could inhibit the growth activities of tumor cells, and reduce the side effects on normal diploid cells through blocking cellular proliferation and inducing apoptosis.Part 3The preparation of curcumin nanoparticles and their characterizationObjectives: To prepare the nanoparticles of curcumin and study their characteristic, in order to establish a basis for further development of targeted chemotherapy of nanotechnology for cancer. Methods With a modified spontaneous emulsification solvent diffusion method to make various types of PLGA and PLA polymers for preparation of nanoparticles of curcumin, through acetone/ethanol and PC/ethanol respectively and calculate the drug loading and drug trapping efficiency of curcumin Results: The drug loading of curcumin nanoparticles should be 3.15% and the drug trapping efficiency of curcumin nanoparticles should be 18.9%. Conclusion After been packed, the curcumin nanoparticles can be seen dissipated in the water and formed a stable suspensive emulsion; These phenomenons shows that the diffluent characteristic of curcumin has been changed through packed, which afford a new way to the chemotherapy therapy of cancer. Part 4Synthesis of specific nucleic acid-triggered prodrugs of curcumin and primary characterization of the prodrugsObjectives On the basis of our former study, We have linked the prodrugs of curcumin and catalyst imidazole with antisense oligodeoxynucleotide of survivin. Methods: With the usage of chemical synthesis; high performance liquid chromatography and controlled pore glass, the prodrugs of curcumin and catalyst imidazole were linked with the antisense oligodeoxynucleotide of survivin. The new material was identified by the high performance liquid chromatography. Results: The identification of the high performance liquid chromatography showed that the prodrugs of curcumin and catalyst imidazole were linked with the antisense oligodeoxynucleotide of survivin successfully. Conclusion: The specific nucleic acid-triggered curcumin prodrug activation system is an efficient targeted therapy of tumor.Part 5Specific nucleic acid-triggered prodrugs of curcumin in vivoObjectives To investigate the antitumor activity and side effect of the specific nucleic acid-triggered curcumin prodrug activation system in vivo. Methods: With the subcutaneous tumor model of nude mouse; drug injection inside tumor and histological section; the side effect and the tumor growth inhibition were detected. Results: The specific nucleic acid-triggered curcumin prodrug activation system is an efficient targeted therapy of tumor and has a less cytotoxin than curcumin. Conclusion: The specific nucleic acid-triggered curcumin prodrug activation system is an efficient targeted therapy of tumor, which established a basis for further development of targeted chemotherapy for cancer.更多还原