生物可降解微球PLGA疫苗运输载体研究

发布时间：2018-06-15 16:45

本文选题：微球 + HPV16-E7蛋白　；参考：《兰州大学》2011年硕士论文

【摘要】：目的用复乳溶剂挥发法制备聚乳酸-聚羟基乙酸(PGLA)微球和以牛血清白蛋白(BSA)为模型蛋白,聚乳酸-聚羟基乙酸为包裹材料,制BAS-PLGA微球,探索微球的制备方法,微球的形态及包封率。最后以包封率高的方法制备人乳头状瘤病毒(HPV)16-E7蛋白(PGLA)微球,考察粒径分布情况并通过皮下免疫途径免疫C57BL/6小鼠做免疫学评价。方法用复乳溶剂挥发法制备两种微球,用扫描电子显微镜观察微球的大小形态,用CBB法测量蛋白浓度,微球的体外释放,然后用PLGA通过表面吸附人乳头状瘤病毒(HPV)16-E7蛋白制备成聚乳酸聚羟基乙酸(PGLA)微球,考察粒径分布情况及体外释放水平,通过吸附有相同抗原的HPV16-E7蛋白的佐剂包括E7-DDA组,E7-PLGA组,E7+AL(OH)2组皮下免疫注射途径免疫C57BL/6小鼠,注射小鼠6周后(第2周加强免疫1次),用间接ELISA法检测免疫鼠血清中的抗体水平,由此评价PLGA微球疫苗运输载体的佐剂效应结果(1)用复乳溶剂挥发法制备的BSA-PLGA微球表面成球形颗粒,外观圆整,表面光滑,直径5-10umm。包封率用提取水解法测的是39.58%(2)制备的PLGA为微球表面成球形,大小均匀,直径1-2um,PLGA纳米粒吸附BSA蛋白蛋白测蛋白吸附率结合率56.3%(3) PLGA微球通过表面吸附人乳头状瘤病毒(HPV) E7蛋白制备成聚乳酸聚羟基乙酸(PGLA)微球,吸附E7蛋白时,电子显微镜下可见形状各异,有的黏附成团,或成条索状,没有吸附蛋白的仍成球形。微球的包封率均值(n=4)是21.4%,通过皮下免疫注射途径免疫C57BL/6小鼠,免疫小鼠6周后(第2周加强免疫1次),可见不同的佐剂免疫C57BL/6小鼠后产生的特异性抗体不同,微球疫苗组诱导产生的IgG1抗体水平较同剂量的铝佐剂组和溴化二甲基双十八胺(DDA)组明显升高,(平均滴度分别为3805、1270、2262)；微球疫苗诱导产生IgG2b的抗体水平明显高于铝佐剂组,略低于DDA组,(平均滴度分别为1131、475、2653)且微球组和DDA组跟对照组(E7蛋白)相比有统计学意义,而诱导产生的IgG2c的抗体水平明显高于铝佐剂组和DDA组(平均滴度分别为150、36、106)。结论(1)用复乳溶剂挥发法制备的两种微球表面光滑,成球形,且大小均匀,但是以微球吸附蛋白的吸附率较高,因为在微球的制备中,药物的包封率是一个非常重要的参考因素。 (2)人乳头状瘤病毒E7蛋白聚乳酸羟基乙酸微球作为疫苗输送体可以明显的提高抗原的免疫原性,为治疗性宫颈癌疫苗及宫颈癌术后联合免疫治疗提供理论依据,开发更好的疫苗进行探索。
[Abstract]:Objective to prepare PLGA microspheres using polylactic acid-poly-glycolate (PGLAA) microspheres and bovine serum albumin (BSA) as model proteins and polylactic acid-poly-glycolic acid (PAA) as encapsulating materials, and to explore the preparation method of microspheres. Morphology and encapsulation efficiency of microspheres. Human papillomavirus HPV-16-E7 protein PGLA-microspheres were prepared by the method of high encapsulation efficiency. The particle size distribution was investigated and immunological evaluation of C57BL / 6 mice was carried out by subcutaneous immunization. Methods two kinds of microspheres were prepared by double emulsion solvent volatilization method. The size and morphology of microspheres were observed by scanning electron microscope (SEM). The protein concentration was measured by CBB method and the microspheres were released in vitro. Then PLGA was used to prepare poly (lactic acid) polylactide polyglycolic acid (PGLA) microspheres by adsorbing human papillomavirus HPV16 E7 protein on the surface. The particle size distribution and release level in vitro were investigated. C57BL / 6 mice were immunized with the adjuvant of HPV16-E7 protein with the same antigen, including E7-DDA group, E7-PLGA group and E7-PLGA group. C57BL / 6 mice were immunized by subcutaneous injection route. After 6 weeks of injection (the second week, the mice were immunized once), the antibody level in the serum of immunized mice was detected by indirect Elisa. Therefore, the adjuvant effect of PLGA microsphere vaccine transport carrier was evaluated. 1) the BSA-PLGA microspheres prepared by solvent volatilization method were spherical in appearance, smooth in appearance and 5-10 umm in diameter. The encapsulation efficiency of PLGA prepared by the method of extraction and hydrolysis is 39.58 and 39.58. The PLGA is spherical on the surface of microspheres, and the size of PLGA is uniform. The binding rate of BSA protein adsorbed by PLGA nanoparticles was 56.3%. PLGA microspheres adsorbed human papillomavirus (HPVV) E7 protein onto the surface to prepare polylactic acid poly-glycolate (PGLA) microspheres, which adsorbed E7 protein. Under the electron microscope, various shapes can be seen, some of which are adherent or cord-like, and those without adsorbent proteins are still globular. The average entrapment efficiency of microspheres was 21.4. C57BL / 6 mice were immunized by subcutaneous immunization. After 6 weeks of immunization (the second week, the mice were immunized once), the specific antibodies produced by different adjuvants in C57BL / 6 mice were different. The level of IgG1 antibody induced by microsphere vaccine group was significantly higher than that of aluminum adjuvant group and DDAs group (mean titer was 3805 / 12702262C, respectively), and the level of IgG2b antibody induced by microsphere vaccine was significantly higher than that of aluminum adjuvant group. It was slightly lower than that of DDA group (average titer was 1131), and there was significant difference between the microspheres group and DDA group compared with the control group. The level of IgG2c antibody induced by DDA group was significantly higher than that of aluminum adjuvant group and DDA group (the average titer was 150 ~ 36106). Conclusion 1) the surface of the two kinds of microspheres prepared by solvent volatilization method is smooth, spherical and uniform in size, but the adsorption rate of protein adsorbed by microspheres is higher, because in the preparation of microspheres, Encapsulation efficiency of drugs is a very important reference factor. 2) Human papillomavirus E7 protein poly (lactic acid) acetic acid microspheres as vaccine carriers can significantly improve the immunogenicity of the antigen. To provide theoretical basis for the treatment of cervical cancer vaccine and postoperative combined immunotherapy of cervical cancer, and to develop better vaccine for exploration.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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