大豆淀粉微载体的制备及在细胞培养中的初步应用

发布时间：2018-01-27 12:22

  本文关键词： 大豆淀粉 淀粉微球 微载体 细胞培养　出处：《西北民族大学》2017年硕士论文　论文类型：学位论文

【摘要】：淀粉微球在药物缓释、包埋剂和吸附剂方面的应用已有很多报道,但在贴壁动物细胞培养方面的应用却鲜有研究报道。用淀粉制备细胞培养用微载体,具有其独特的优势。本研究以价廉易得的淀粉为原料,研究制备适合动物细胞贴壁培养用的微载体,旨在开发具有高附加值、可生物降解、可用淀粉酶消化以收获细胞、非动物来源材料制备的淀粉微载体,避免在生物制药领域使用动物源性材料和高成本微载体的弊端。本文探讨了微载体的研究进展、高分子微球的基本性质、淀粉微球的制备方法及其研究应用进展。论证了淀粉微球作为动物细胞培养用微载体的可行性。在前人制备淀粉微球的基础上,经过制备工艺改进和参数优化,探讨了制备淀粉微球的最佳工艺条件。结果表明:以机油和异辛烷为油相,6%糊化淀粉碱溶液为水相,聚乙二醇二缩水甘油醚为交联剂制备交联淀粉微球时,油水相体积比4:1,搅拌转速500 rpm,交联剂10%为制备交联淀粉微球的最佳工艺条件,制备的交联淀粉微球能够耐受121℃C 30min的高压灭菌。通过正交试验、配基筛选,在淀粉微球表面偶联了二乙氨基乙基(DEAE),使淀粉微球表面具有了和商品化Cytodex 1微载体近似的电荷和电荷量。在测定、分析了所制交联淀粉微球的基本理化性质后,本研究以BHK-21、Marc-145和MDCK三种疫苗生产常用贴壁细胞为实验细胞,以Cytodex 1微载体为对照,进行的微载体细胞培养,并对实验结果进行了总结分析。研究结果表明:所制备的淀粉微球,在偶联DEAE后,能够耐受高压灭菌。在细胞培养后,偶联了 DEAE、粒径与Cytodex 1微载体相近的淀粉微球,上述三种细胞均能够良好贴附、增殖。在相同悬浮培养条件下,淀粉微载体的细胞贴附、增殖量与Cytodex 1微载体近似,培养至144h,上述三种细胞的增殖量均达到最大值。其中,在自制淀粉微球上BHK-21细胞的最大增殖量为 2.5 ×106cells/mL,略大于 Cytodex 1 的 2.2 ×106cells/mL。Marc-145 细胞的最大增殖量为前者1.4×106cells/mL,后者1.6×106cells/mL,后者略大于前者;MDCK细胞的最大增殖量为前者2.2×106cells/mL,后者2.4×106cells/mL,后者略大于前者。用自制微载体培养细胞,三种贴壁细胞的生长与生化代谢曲线也和商品化的Cytodex 1相似。综上可知,本研究制备的淀粉微球,通过偶联DEAE后,能够用于BHK-21、Marc-145和MDCK贴壁细胞的微载体悬浮培养。其细胞培养效果与Cytodex 1相似,有良好的开发与应用前景。
[Abstract]:The application of starch microspheres in drug delivery, encapsulation and adsorbents has been reported, but few studies have been reported on the application of starch microspheres in cell culture of adherent animals. This study is based on the cheap and easily available starch as raw material to study the preparation of microcarriers suitable for animal cell adhesion culture in order to develop a high value-added biodegradable microcarriers. Starch microcarriers that can be digested by amylase to harvest cells and non-animal materials. To avoid the disadvantages of using animal-derived materials and high-cost microcarriers in the field of biopharmaceuticals, this paper discusses the research progress of microcarriers and the basic properties of polymer microspheres. The preparation and application of starch microspheres. The feasibility of starch microspheres as microcarriers for animal cell culture was demonstrated. On the basis of the previous preparation of starch microspheres, the preparation process was improved and the parameters were optimized. The optimum conditions for preparation of starch microspheres were discussed. The results showed that when oil and isooctane were used as oil phase and 6% gelatinized starch alkali solution as water phase, polyethylene glycol diglycidyl ether was used as crosslinking agent to prepare cross-linked starch microspheres. The optimum conditions for the preparation of cross-linked starch microspheres were oil / water phase volume ratio 4: 1, stirring speed 500rpm, crosslinking agent 10%. The cross-linked starch microspheres were able to withstand high pressure sterilization at 121 鈩，

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