【作者】 罗国培；

【导师】 倪泉兴； 傅德良； 金忱； 虞先浚；

【作者基本信息】 复旦大学 ， 外科学， 2010， 博士

【摘要】 目的研究载MBD1-siRNA质粒的PLGA/poloxamer纳米微粒在体内、外治疗胰腺癌效果。方法(1)设计并合成针对MBD1基因的siRNA,通过乳剂溶解挥发法制备包裹MBD1 siRNA质粒的PLGA/poloxamer纳米微粒,研究不同制作条件对纳米微粒形成和物理表征的影响,并通过激光粒度仪和透射电子显微镜对其进行物理表征测定；通过荧光分光光度法测量纳米微粒对含MBD1siRNA质粒DNA的包裹效率和载药量,并进一步测定纳米微粒缓释性、结构稳定性、保护性测定以及生物毒性。(2)在合成包裹MBD1siRNA质粒的纳米微粒的基础上,进一步研究载MBD1siRNA质粒的纳米微粒在体外的转染效率及其对胰腺癌细胞生长和基因表达的干扰作用。通过流式细胞仪检测MBD1 siRNA质粒的PLGA/poloxamer纳米微粒体外的转染效率,通过荧光染色法进一步证实纳米微粒在细胞内的分布,MTT法检测MBD1 siRNA质粒的PLGA/poloxamer纳米微粒不同浓度和不同时间对胰腺癌细胞生长的抑制作用,RT-PCR法检测基因转染后胰腺癌细胞内MBD1mRNA表达的变化,Western blot法检测基因转染后胰腺癌细胞内MBD1蛋白表达的变化,流式细胞仪、TUNEL、Hoechst法检测基因转染后细胞凋亡的改变,流式细胞仪法检测基因转染后细胞周期的改变。(3)在合成包裹MBD1siRNA质粒的纳米微粒的基础上,进一步研究载MBD1siRNA质粒的纳米微粒在体内的转染、抗肿瘤效果及其对基因表达的干扰作用。建立胰腺癌裸鼠腋下荷瘤模型,腹腔注射给药,观察肿瘤体积的改变,通过荧光染色法和透射电镜证实纳米微粒在肿瘤组织内的分布,RT-PCR法检测基因转染后胰腺癌细胞内MBD1mRNA表达的变化,Western blot法检测基因转染后胰腺癌细胞内MBD1蛋白表达的变化,流式细胞仪、TUNEL法检测基因转染后细胞凋亡的改变,流式细胞仪法检测基因转染后细胞周期的改变,HE染色观察肿瘤超微结构的改变。结果(1)PLGA/poloxamer纳米微粒呈圆形,粒径约为200nm。不同的包裹内容物对纳米微粒的粒径无明显影响,载质粒DNA的纳米微粒较载水的纳米微粒的zeta电位略有增加。当PLGA/poloxamer比例为1：1、PLGA分子量为8000Da和涡旋时间为60秒时形成的纳米微粒粒径最小,Zeta电位负值较低,P.I.较小,粒径分布较为集中。PLGA/poloxamer纳米微粒对质粒DNA的包裹效率约为31%,载药量约为1.55μg/mg。从纳米微粒释放的质粒DNA的结构在制备过程中未受到损害。PLGA/poloxamer纳米微粒具有缓释性、保护性和低或无生物毒性等特点。(2)纳米微粒在体外的转染效率约为28%,并且在转染后第一至第七天都能保持相似的转染效率。溴化乙锭标记的MBD1siRNA纳米微粒被细胞吞噬,并且部分纳米微粒已进入细胞核。抗增殖实验中,随着载MBD1siRNA PLGA/poloxamer纳米微粒浓度的增高,细胞的活力逐渐下降；另外,浓度为2.0mg/ml的载MBD1siRNA PLGA/poloxamer纳米微粒在不同时间的抗增殖效应相似。MBD1 mRNA基因和蛋白的表达量都逐渐减少。TUNEL法、流式细胞仪和Hoechst染色法检测发现,MBD1siRNA纳米微粒给药组的BxPC-3细胞中存在明显的凋亡细胞,单纯质粒组凋亡比例为4.79%,而MBD1siRNA纳米微粒组凋亡比例为24.19%。流式细胞仪检测发现,MBD1siRNA表达后,胰腺癌BxPC-3细胞的细胞周期发生明显变化。(3)MBD1siRNA纳米微粒组动物肿瘤体积明显小于空白对照组和单纯质粒组。透射电镜下纳米微粒分别存在于肿瘤细胞间隙间和肿瘤细胞浆及细胞核内。荧光显微镜下观察到纳米微粒分布于动物肿瘤组织中。RT-PCR检测MBD1siRNA纳米微粒组MBD1mRNA基因的表达量逐渐减少,Western blot检测MBD1siRNA纳米微粒组MBD1蛋白的表达量逐渐减少。流式细胞仪检测空白对照组细胞凋亡比例为10.87%,S期占总细胞周期的8.01%,而载MBD1 siRNA纳米微粒给药组细胞凋亡比例为21.53%,S期占总细胞周期的15.74%。TUNEL法可见MBD1siRNA纳米微粒组存在更多的凋亡细胞。病理切片检查发现,载siRNA纳米微粒给药组动物肿瘤组织中存在明显的坏死细胞。结论(1)PLGA/poloxamer纳米微粒是胰腺癌基因治疗的理想载体,具有缓释性、无毒性等优点；(2)MBD1是胰腺癌的理想治疗靶点之一；(3)载MBD1siRNA的PLGA/poloxamer在体内外都能发挥抗胰腺癌BxPC-3细胞的效应,能促进肿瘤细胞凋亡和周期的改变。更多还原

【Abstract】 Objective To investigate the anti-tumor effect of MBD1-siRNA plasmid loading PLGA/poloxamer nanoparticles to pancreatic cancer in vitro and in vivo.Methods (1)The siRNA sequence of MBD1 gene was designed and synthesized and inserted it into plasmid. Nanoparticles were prepared by a modified solvent diffusion technique. Nanoparticle characterization was investigated at different synthesis condition through dynamic light scattering and transmission electron microscopes. Plasmid DNA encapsulating and loading efficiencies were determined by spectrofluorometry. Stability of release of MBD1 siRNA plasmid DNA, DNaseⅠdigestion and cytotoxicity were also examined.(2) The transfection efficiency, gene expression level and anti-tumor effect of MBD1-siRNA plasmid loading PLGA/poloxamer in vitro were tested. Flow cytometry was used to test the transfection efficiency of MBD1-siRNA plasmid loading PLGA/poloxamer. Distribution of nanoparticles in the pancreatic cancer cells was examined by fluorescence microscopy. MTT tests were used to test the anti-tumor effect of MBD1-siRNA plasmid loading PLGA/poloxamer nanoparticles at different concentrations and times. RT-PCR was used to examine the change of MBD1 mRNA level after transfection. Western blot was carried to test the change of MBD1 protein level after transfection. Flow cytometry, TUNEL, and Hoechst staining were carried to test the apoptosis of tumor cells. Flow cytometry was carried to examine the cell cycle changes of tumor cells.(3) The transfection efficiency, gene expression level and antitumor effect of MBD1-siRNA plasmid loading PLGA/poloxamer in vivo were tested. Tumor animal models were established by subcutaneous injection at subaxillary. MBD1-siRNA plasmid loading PLGA/poloxamer nanoparticles were delivered by intraperitoneal injection. Tumor volume was calculated through the whole process after injection of nanoparticles. Distribution of nanoparticles in the pancreatic cancer cells was examined by transmission electron micrography and fluorescence microscopy. RT-PCR was used to examine the change of MBD1 mRNA level after transfection. Western blot was carried to test the change of MBD1 protein level after transfection. Flow cytometry and TUNEL were carried to test the apoptosis of tumor cells. Flow cytometry was carried to examine the cell cycle changes of tumor cells. The change of tumor microstructure was tested through pathological examination.Results (1) No significant differences in particle size were observed between MBD1-siRNA plasmid loading PLGA/poloxamer (210.1±24.3 nm) and water loading PLGA/poloxamer (187.1±21.9 nm), suggesting that pDNA encapsulation did not affect particle size. Transmission electron micrography showed that the nanoparticles were spherical and were of almost equal size. No difference in the averageδpotential was found between MBD1-siRNA plasmid loading PLGA/poloxamer and water loading PLGA/poloxamer. MBD1-siRNA plasmid loading PLGA/poloxamer exhibited a similar biphasic plasmid DNA release pattern, characterized by a first initial rapid release (>30% of pDNA within the first day) followed by a slower, continuous release (90% released in 11 days). No DNA fragments were detected from the plasmid DNA released from MBD1-siRNA plasmid loading PLGA/poloxamer. MBD1-siRNA plasmid loading PLGA/poloxamer nanoparticles showed the protective effect against DNaseⅠ. PLGA/poloxamer nanoparticles did not exert toxic effects on the cells.(2)Though the intensity of transfection was low, the number of cells transfected was high and was maintained in a very similar manner throughout the duration of the experiment, which may be explained by the controlled release of MBD1-siRNA plasmid loading PLGA/poloxamer nanoparticles. Relative cell viability levels decreased as the concentration of MBD1-siRNA plasmid loading PLGA/poloxamer nanoparticles increased from 1 mg/ml to 5 mg/ml. From days 3 to 7, the relative viability of BxPC-3 cells was among 20% and 35% compared to that of controls at a MBD1-siRNA plasmid loading PLGA/poloxamer nanoparticles concentration of 2mg/ml, which also points to the controlled release of MBD1-siRNA plasmid loading PLGA/poloxamer. In MBD1-siRNA plasmid loading PLGA/poloxamer nanoparticles treated BxPC-3 cells, MBD1 gene expression decreased gradually from day 2 and MBD1 protein expression also decreased from day 2 and completely disappeared at day 5. Apoptotic cells were observed by immunostaining for the presence of DNA fragments and the apoptosis rate was 24.19% in the MBD1-siRNA plasmid loading PLGA/poloxamer nanoparticles transfected cells, which was higher than that of the blank control cells (4.79%).The percentage of S-phase in the MBD1-siRNA plasmid loading PLGA/poloxamer nanoparticles transfected cells (33.68%) was higher than that of the blank control cells (8.03%).(3) The distribution of PLGA/poloxamer nanoparticles in tumors was confirmed by transmission electron micrography and fluorescence microscopy. In MBD1-siRNA plasmid loading PLGA/poloxamer nanoparticles treated group, MBD1 gene expression decreased gradually from day 2 and MBD1 protein expression also decreased from day 2 and completely disappeared at day 5, which also points to the controlled release of MBD1-siRNA plasmid loading PLGA/poloxamer. Apoptotic cells were observed by immunostaining for the presence of DNA fragments and the apoptosis rate was 21.53% in the MBD1-siRNA plasmid loading PLGA/poloxamer nanoparticles treated group, which was higher than that of the blank control cells. The percentage of S-phase in the MBDl-siRNA plasmid loading PLGA/poloxamer nanoparticles transfected cells (15.74%) was higher than that of the blank control cells (8.01%). Death cells were found in the MBD1-siRNA plasmid loading PLGA/poloxamer nanoparticles treated group through pathological examination. The volume of tumors of the MBD1-siRNA plasmid loading PLGA/poloxamer nanoparticles treated group was smaller than naked plasmid treated group and water loading PLGA/poloxamer nanoparticles treated group.Conclusions MBD1 is an ideal target for treatment of pancreatic cancer. PLGA/poloxamer nanoparticles have controlled release quality. MBD1 siRNA plasmid can be successfully transfected into tumor cells and the MBD1 nanoparticle compound can inhibit cell growth and induce apoptosis. The MBD1 nanoparticle is a promising candidate for gene therapy of pancreatic cancer.更多还原