【作者】 姜子荣；

【导师】 兰青；

【作者基本信息】 苏州大学 ， 神经外科学， 2008， 博士

【摘要】 第一部分阿霉素聚氰基丙烯酸丁酯纳米粒的制备及载药研究目的:制备阿霉素(doxorubicin,DOX)聚氰基丙烯酸丁酯( polybutylcyanoacrylate, PBCA)纳米粒(DOX-PBCA-NP),并对其进行测定。方法:应用乳化聚合法制备DOX-PBCA-NP,用正交设计优选DOX-PBCA-NP的制备条件。纳米粒的粒径及分布用马尔文激光粒度分析仪测试,透射电镜(TEM)观察纳米粒形态、大小,紫外分光光度法在波长481nm处测定纳米粒中DOX的载药量和包封率。结果:激光粒度分析仪测定纳米粒的平均粒径为120nm(载药)、100(空白),多分散系数(Polydispersity Index)为0.12。TEM下,纳米粒呈圆形,无粘连,纳米粒的载药量与包封率分别为10.58%与87.43%。结论:应用乳化聚合的方法制备的纳米粒大小形态一致,性能稳定,可为进一步的体内和体外实验提供基础。第二部分阿霉素聚氰基丙烯酸丁酯纳米粒抑制脑胶质瘤SHG44细胞的研究目的:制备阿霉素(doxorubicin)聚氰基丙烯酸丁酯(polybutylcyanoacrylate)纳米粒(DOX-PBCA-NP),并研究其体外对脑胶质瘤SHG44的抑制作用。方法:乳化聚合法制备DOX-PBCA-NP;MTT法测定细胞抑制率;流式细胞仪(flow cytometry,FCM)测定细胞凋亡和细胞周期的改变;倒置显微镜观察细胞生长情况;免疫组织化学法检测SHG44细胞端粒酶蛋白的表达。结果:1、用药24h后,DOX的三个不同浓度组(按DOX浓度分别为2μg/ml,5μg/ml,10μg/ml)对SHG44细胞的抑制率分别为:(32.3±0.2)%、(48.7±0.7)%、(67.1±0.4)%;DOX-PBCA-NP组分别为:(45.7±0.2)%、(61.6±0.4)%、(76.2±0.8)%;PBCA-NP组为(1.6±0.7)%。2、在DOX,DOX-PBCA-NP三个不同浓度组(按DOX浓度分别为2μg/ml,5μg/ml,10μg/ml),DOX组细胞端粒酶蛋白阳性率分别为(86.4±3.7)%、75.6±2.1)%、(69.7±1.3)%,DOX-PBCA-NP组分别为(72.3±1.6)%、63.2±3.1)%、(55.2±0.8)%,各组端粒酶蛋白的表达阳性率均与对照组有显著性差异(P<0.01)。3、抑制效应表现为细胞周期改变明显,G1/Go期细胞增多,S期细胞明显减少,细胞凋亡增多,端粒酶蛋白表达明显下降。DOX组、DOX-PBCA-NP组与对照组比较均有显著性差异(P<0.01),DOX-PBCA-NP组较DOX组对SHG44细胞的抑制效应更明显,两者比较差异有统计学意义(P<0.05)。。结论:阿霉素聚氰基丙烯酸丁酯纳米粒与阿霉素比较,对脑胶质瘤SHG44细胞的抑制作用明显增强。第三部分端粒酶反义寡核苷酸聚氰基丙烯酸丁酯纳米粒的制备及载药研究目的:制备端粒酶反义寡核苷酸聚氰基丙烯酸丁酯纳米粒(hTERT ASODN- PBCA-NP),并测定其粒径大小、形态、Zeta电位等。方法:应用乳化聚合法制备hTERT ASODN-PBCA-NP,正交设计优选hTERT ASODN-PBCA-NP的制备条件。纳米粒的粒径及分布用马尔文激光粒度分析仪测试,透射电镜(TEM)观察纳米粒大小、形态,高效液相色谱法测定纳米粒中ASODN的载药量和包封率。结果:激光粒度分析仪测定纳米粒的平均粒径为120nm,多分散系数(Polydispersity Index)为0.11。TEM下,纳米粒呈圆形,颗粒间无粘连,纳米粒的载药量与包封率分别为71.17%与95.20 %,Zeta为+41.3mV。。结论:应用乳化聚合的方法制备的纳米粒大小形态一致,表面带正电荷,性能稳定,具有较高的载药量和包封率。第四部分端粒酶反义寡核苷酸聚氰基丙烯酸丁酯纳米粒抑制脑胶质瘤SHG44细胞的研究目的:以聚氰基丙烯酸丁酯纳米粒(polybutylcyanoacrylate nanoparticles, PBCA-NP)为载体,以端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)反义寡核苷酸(antisense oligodeoxynucletide,ASODN)为目的基因体外转染脑胶质瘤SHG44细胞,研究其体外对SHG44细胞生长的抑制作用。方法:应用乳化聚合法制备端粒酶反义寡核苷酸聚氰基丙烯酸丁酯纳米粒(hTERT ASODN-PBCA-NP),琼脂糖凝胶电泳法分析其对抗核酸酶的降解作用,MTT法检测细胞的生长抑制率;倒置显微镜观察细胞的生长状况;流式细胞仪(flow cytometry,FCM)测定细胞周期的改变以及5’-FITC标记的ASODN(FASODN-NP)转染后的细胞内荧光强度;免疫细胞化学法测定SHG44细胞端粒酶蛋白的表达,RT-PCR检测hTERT mRNA的表达。结果: 1、琼脂糖凝胶电泳结果表明ODN-PBCA-NP具有不被核酸酶降解的作用;转染后48h空白对照组、NP组、FASODN组和FASODN-NP组的胞内荧光道数分别为138.20,140.40,142.06和490.00,FASODN-NP组细胞内荧光强度增加,与其它各组相比有显著性差异(P<0.01);2、hTERT mRNA表达的相对值在ASODN1-3-NP各组分别为1.45±0.11、1.39±0.22、1.43±0.14,空白对照组为(2.23±0.12);3、端粒酶蛋白表达的阳性率在ASODN1-3各组分别为(53.1±1.8)%、(56.7±1.5)%和(59.6±4.3)%,空白对照组为(94.6±1.3)%;4、ASODN-NP组与空白对照组、SODN-NP组、ASODN组、NP组相比,SHG44细胞的抑制率、细胞周期的改变以及hTERT mRNA、端粒酶蛋白的表达均有显著性差异(P<0.05),ASODN-NP组细胞在G1/G0期增多,S期细胞减少,hTERT mRNA、端粒酶蛋白的表达下降。结论: PBCA-NP为载体介导的hTERT-ASOND可以有效地透过细胞膜到达SHG44细胞内部,阻断hTERT mRNA的表达,抑制肿瘤细胞的生长。第五部分端粒酶反义寡核苷酸聚氰基丙烯酸丁酯纳米粒体内治疗裸鼠脑胶质瘤动物模型的研究目的:以聚氰基丙烯酸丁酯纳米粒(polybutylcyanoacrylate nanoparticles, PBCA-NP)为载体,以端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)反义寡核苷酸(antisense oligodeoxynucletide,ASODN)为目的基因转染裸鼠脑胶质瘤动物模型,研究其体内对裸鼠脑胶质瘤动物模型的治疗作用。方法:制备裸鼠脑胶质瘤动物模型,尾静脉注射端粒酶反义寡核苷酸聚氰基丙烯酸丁酯纳米粒,测定裸鼠颅内肿瘤的大小;免疫组织化学法检测肿瘤组织端粒酶蛋白的表达、病理改变,RT-PCR检测hTERT mRNA的表达。结果:1、肿瘤组织端粒酶蛋白的阳性表达率在空白对照组为(92.6±2.7)%,NP组为(91.2±3.4)%,SODN-NP组为(94.7±1.3)%,ASODN组为(90.5±2.6)%,ASODN-NP组为(65.2±2.1)%;2、空白细胞对照组、NP组、SODN-NP组、ASODN组肿瘤组织hTERT mRNA表达的相对值分别为2.34±0.15、2.46±0.22、2.41±0.35、2.39±0.24,ASODN-NP组为1.56±0.39;3、ASODN-NP组与对照组、SODN-NP组、ASODN组、NP组相比,肿瘤体积、端粒酶蛋白的表达以及hTERT mRNA表达均有显著性差异(P<0.05),ASODN-NP组肿瘤体积小,端粒酶蛋白、hTERT mRNA的表达下降。结论: PBCA-NP为载体介导的hTERT-ASOND在体内具有抑制裸鼠脑胶质瘤生长的作用。更多还原

【Abstract】 PartⅠPreparation and drug loading of doxorubicin–polybutylcyano -acrylate nanoparticlesObjective To prepare the doxorubicin–polybutylcyanoacrylate nanoparticles. Methods The DOX-PBCA-NP was prepared using the emulsion polymerization method under the optimal conditions of orthogonal design.The size and size distribution of nanoparticles were measured using Malven laser mastersizer 3000HS, nanoparticle morphology observed by transmission electron microsope.The Drud loading and entrapment efficiency of doxorubicin in the nanoparticles were measured by means of UV spectra at weave length of 481 nm.Results The nanoparticles were discrete and uniform spheres with average diameter of 120nm and 100nm for bare and drug loaded nanoparticles respectively.Conclusion Nanoparticles obtained by emulsion polymerization method were steady and the same size and morphology, which can be provided for the further in vitro and in vivo tests.PartⅡInhibition of SHG44 cells by polybutylcyanoacrylate nanoparticles loaded with Doxorubicin Objective To investigate the effect of polybutylcyanoacrylate nanoparticles loaded with Doxorubicin on the SHG44 cells.Methods The polybutylcyanoacrylate nanoparticles loaded with Doxorubicin were prepared by emulsion polymerization process .The inhibitory rate of SHG44 cells were detected by MTT assay, cell modalities were observed using inverse microscope and the cell cycles determined by flow cytometry. The expression of telomerase protein was measured by immunocytochemical method.Results 1. At three concentration levels of DOX, DOX-PBCA-NP, the inhibitory rate of SHG44 cells were (32.3±0.2)%、(48.7±0.7)%、(67.1±0.4)%% for DOX groups and(45.7±0.2)%、(61.6±0.4)%、(76.2±0.8)% for DOX-PBCA-NP groups, when the concentration of DOX were 2μg/ml, 5μg/ml and 10μg/ml for each group,with PBCA-NP group for (1.6±0.7)%.2. The telomerase protein expression were (86.4±3.7)%,75.6±2.1)% and(69.7±1.3)% for DOX groups, (72.3±1.6)%、63.2±3.1)% and(55.2±0.8)% for DOX-PBCA-NP groups , with the concentration of DOX were 2μg/ml, 5μg/ml and 10μg/ml for each group. 3. Compared with other groups, DOX-PBCA-NP groups displayed higher inhibitory rate, obviously cell modality change and cell cycle variety, reduced telomerase protein expression. Their differences are evidently remarkable(P<0.05).Conclusion Polybutylcyanoacrylate nanoparticles loaded with doxorubicin were more effective on the SHG44 cells than doxorubicin as to cell inhibition .PartⅢPreparation and drug loading of polybutylcyanoacrylate nanoparticles loaded with antisense oligodeoxynucleotide of hTERT mRNA Objective To prepare the hTERT-ASODN-polybutylcyanoacrylate nanoparticles. Methods The hTERT ASODN-PBCA-NP was prepared using the emulsion polymerization method under the optimal conditions of orthogonal design.The size , size distribution and Zeta potential of nanoparticles were measured using Malven laser Mastersizer 3000HS, nanoparticle morphology observed by transmission electron microsope.The drud loading and entrapment efficiency of ASODN in the nanoparticles were measured by means of HPLC method at weave length of 260 nm.Results The nanoparticles were discrete and uniform spheres with average diameter of 120nm PBCA nanoparticles, with drud loading and entrapment efficiency for 71.17% and 95.20%, and +41.3mV for Zeta potential.Conclusion Nanoparticles obtained by emulsion polymerization method were steady and the same size and morphology,with higher drud loading and entrapment efficiency of oligodeoxynucletide in the nanoparticles, and a high positive Zeta potential.PartⅣInhibition of SHG44 cells by polybutylcyanoacrylate nanoparticles loaded with antisense oligodeoxynucleotide of hTERT mRNAObjective: To investigate the effect of polybutylcyanoacrylate nanoparticles loaded with antisense oligodeoxynucleotide of hTERT mRNA on SHG44 cells.Methods: The polybutylcyanoacrylate nanoparticles loaded with antisense oligodeoxynucleotide of hTERT mRNA were prepared using the emulsion polymerization process .The inhibition rate of SHG44 cells were detected by MTT assay, cell modalities were observed using inverse microscope and the cell cycles and intracellular fluorescence intensity determined by flow cytometry after transfection . The expression of hTERT mRNA was measured using RT-PCR method, and expression telomerase protein was measured by immunocytochemical method. Results: Nanoparticles obtained by emulsion polymerizations process were the same size and morphology. Compared with FASODN group ( NP-free ) , the intracellular fluorescence intensity of FASODN-NP group was obviously stronger after transfection of 48h(P<0.01). The telomerase protein expression were (53.1±1.8)%, (56.7±1.5)% and(59.6±4.3)% for ASODN1-NP group, ASODN2-NP group and ASODN3-NP group, with control group for(94.6±1.3)%. The hTERT mRNA expression were 2.23±0.12、2.31±0.14、2.26±0.19 for ASODN1-NP group, ASODN2-NP group and ASODN3-NP group, with control group for 1.45±0.11.The ASODN-NP groups displayed higher inhibitory rate, obviously cell modality change and cell cycle variety, reduced hTERT mRNA and telomerase protein expression than that of ASODN groups(NP-free), SODN group , NP group and control group, their differences are evidently remarkable.Conclusion: Polybutylcyanoacrylate nanoparticles loaded with antisense oligodeoxynucleotide of hTERT mRNA can enter the inside of SHG44 cells, preventing the expression of hTERT mRNA, and inhibit cell proliferation .PartⅤChemotheraphy of glioma nude mice using polybutylcyanoacrylate nanoparticles loaded with antisense oligodeoxynucleotide of hTERT mRNAObjective: To investigate the chemotheraphic effect of polybutylcyanoacrylate nanoparticles loaded with antisense oligodeoxynucleotide of hTERT mRNA on SHG44 cell glioma nude mice.Methods: The polybutylcyanoacrylate nanoparticles loaded with antisense oligodeoxynucleotide of hTERT mRNA were prepared by emulsion polymerization process . The glioma nude mice models were established by means of tumor tissue pieces of SHG44 cells inoculated into BALB/c-nu nude mice. The preparation of drugs were injected i.v. into the tail vein.The expression of hTERT mRNA was measured using RT-PCR method,the telomerase protein and the pathology of the tumor tissue were measured by immunohistochemical method.Results: The glioma models in nude mice were successfully established by means of tumor tissue pieces inoculation.The telomerase protein expression were (92.6±2.7)%,(91.2±3.4)%,(94.7±1.3)%,(90.5±2.6)%,(65.2±2.1)% for control group, NP group, SODN-NP group, ASODN group and ASODN-NP group, and the hTERT mRNA expression were 2.34±0.15, 2.46±0.22, 2.41±0.35, 2.39±0.24, 1.56±0.39 respectively . Compared with other groups , the ASODN-NP group displayed reduced hTERT mRNA and telomerase protein expression,smaller tumor size, their differences are evidently remarkable(P<0.05). The above differences between control group, NP group, SODN-NP group and ASODN group were no statistical meaning.Conclusion: Polybutylcyanoacrylate nanoparticles loaded with antisense oligodeoxynucleotide of hTERT mRNA were chemotheraphic effective to SHG44 glioma nude mice .更多还原