【作者】 赵黛坚；

【导师】 金一；

【作者基本信息】 浙江大学 ， 药剂学， 2004， 硕士

【摘要】 80年代初，Lim和Sun发明的APA微囊膜(alignate-poly(1)lysine-alignate，APA)，是两层海藻酸钠夹一层聚左赖氨酸的“三明治”结构膜。将特定的细胞包裹于该微囊膜中，利用微囊膜的半透性，氧、营养物质等小分子可透过膜供给细胞，使之保持生理活性，而细胞分泌的生物活性产物也可从微囊中透出，起到特定作用；同时具有免疫活性的大分子物质、细胞等不能通过微囊膜攻击囊内细胞，从而起到免疫隔离的作用。目前，国内外对微囊化细胞在医学方面的研究主要集中在微囊化胰岛细胞、肝细胞、多巴胺分泌细胞、甲状旁腺细胞、甲状腺细胞及基因工程细胞治疗相关疾病的研究，并取得了不同程度的进展。1997年，日本Okada等人提出“细胞制剂”(cytomedicine)，利用微囊内细胞代谢产物调节机体生理功能，治疗相关疾病，微囊化细胞首次在药学方面作为细胞性体内长期药物传递系统(drug delivery system,DDS)提出。 多发性骨髓瘤(multiple myeloma,MM)是一种原发性恶性单克隆丙种球蛋白病(monoclonal gammapthy)，其新生浆细胞常分泌一种M蛋白成分，即单克隆免疫球蛋白。由于恶性浆细胞增生，抑制正常Ig的产生，因此蛋白所属Ig显著增高，而非M蛋白所属Ig含量显著下降。轻链型由于瘤细胞产生重链的能力被选择性地抑制、只能合成轻链，不能合成完整的Ig，导致疾病。如能抑制轻链的过度合成，将有利于MM的治疗。K(?)hler和Milstein于1975年创立了杂交瘤技术，制备的各种单克隆抗体已广泛应用于医学基础研究以及临床诊断治疗。但是mAb的应用中有血清中半衰期短的缺陷，在体内长期给药方面存在着一定的限制。浙江大学硕士学位论文·人PA徽囊化杂交瘤细胞的制剂学研究 我们通过建立能分泌抗人IgG‘轻链单克隆抗体的杂交瘤细胞，用高压静电成囊法将其包裹于A卫A微囊内，杂交瘤细胞在囊内能保持生理活性，分泌抗人IgG轻链的单抗，而其又受微囊保护，不受小鼠免疫系统排斥。ApA微囊化杂交瘤细胞作为细胞性体内长期药物传递系统，用于治疗MM，有一定的临床应用前景。 本课题第一部分研究探讨了制备A卫A微囊的最优化方法及参数条件。首先建立对APA微囊初步评价的质量评价体系，即微囊的形态、粒径、成囊率、微囊膜机械物理强度、微囊膜化学强度、长期稳定性等性能评价指标。通过比较，选择高压静电成囊制备APA微囊的方法，即用自动注入机将注射器中的海藻酸钠溶液通过针头推出，同时利用高压静电发生器产生的静电场将其迅速滴入CaCI:溶液中，反应形成海藻酸钙微胶珠，再利用微胶珠表面负电荷与带正电荷的聚赖氨酸咖ty士一lysine，PLL)溶液进行聚电解质络合，加入低浓度的海藻酸钠去除表面多余的.NH:基团，以提高生物相容性，最后用55 Inmol·L一‘柠檬酸钠溶液液化囊心，最终形成APA微囊。 为确定制备APA微囊的处方及仪器参数，我们首先进行单因素考察，发现浓度为1 .5%的海藻酸钠溶液及浓度为100~ol·L一，的CaC12溶液为最佳处方参数，并选择7“针头用于制备微囊。通过实验还发现最先生成的微胶珠粒径不同，与不同浓度PLL反应导致不同的成囊率。根据单因素试验筛选出的主要影响因素，如自动注入机的推进速度、高压发生器的成囊电压、针头与CaC12溶液的液面距等，进行多因素正交设计优化。结果发现推进速度因素对指标1海藻酸钙微胶珠粒径及指标2形态圆整度的影响最大，而液面距对指标3大小均匀度的影响最大。最后主要考察指标1，综合考虑指标2、3，选择与B3CZ为最佳工艺条件，制备粒径150阿左右的微胶珠，与浓度为0.02%的PLL反应，最终生成形态圆整、表面光滑、大小均匀的粒径250娜左右的APA微囊，成囊率为%%。 本课题的模型细胞株为能分泌具治疗作用单抗的杂交瘤细胞系，我们采用K6hler和Milstein发明的杂交瘤技术，制备既具有体外长期生存传代能力，又能分泌生物活性物质—抗人IgGK链单克隆抗体(monodonal剐皿lbo勿，mAb)的杂交瘤细胞株。首先用纯化的人I药1抗原多次重复免疫BLAB/C小鼠，取免疫后脾细胞与小鼠骨髓瘤细胞系SPZ/0细胞用PEG 3350融合，经培养筛选，最终浙江大学硕士学位论文·A卫A徽囊化杂交瘤细胞的制剂学研究获得分泌抗人lgGK链单克隆抗体(mAb)的杂交瘤细胞株，命名为JY-AI，单抗亚型检测为小鼠IgGI亚型。 根据上述得到的最优化制备参数，将J丫Al细胞包裹于ARA微囊中得到平均粒径为260卿的微囊，其形态圆整，大小均匀。体外培养考察发现包裹JY-AI细胞的APA微囊膜强度与空A卫A微囊相似。体外培养条件下，囊内JY-AI细胞至少能存活2一3个月，用ELISA法能检测到培养液中由囊内JY-AI细胞分泌的mAb的释放，而囊内细胞并没有漏出微囊，可见微囊化对杂交瘤细胞分泌单抗的活性没有影响，且通透性符合要求。 将APA微囊化JY-AI细胞注射入小鼠腹腔，考察其安全性、生物相容性及囊内细胞的生长情况。结果证明，注射后7、14、28天回收的微囊形态完整，表面无结缔组织包绕;注射56天后回收的大部分微囊形态完整，但少数微囊有变形甚至破裂的现象。对于回收的微囊，观察发现囊内细胞生长快于体外培养情况，且多数?更多还原

【Abstract】 AIginate-poly(L)lysine-alginate (APA) membrane has been developed for the microencapsulation of living cells which protects these cells from the immune system. It was preparaed for the first time by Lim and Sun in 1980’s. APA microcapsules have semipermeable membranes which permit passage of low molecular weight substance, such as nutrients and oxygen, but not of cells and high molecular weight substance. APA microencapsulated biological active molecules, tissue, and cells for allo- or xenograft transplanting have been studied by many researchers. Recently, someone has proposed an term "cytomedicine" for cell therapy. This novel medical treatments against diseases using living cells makes the best use of cell functions which naturally act as a drug delivery system (DDS) in vivo. APA microcapsules acting as the vehicle of the cytomedicine will be potential for clinical use.Multiple myeloma (MM) is a disease of immunoglobulin structural abnormality. B-cell malignancies produce myeloma protein (M protein). In patients of MM with the IgG type kappa light chain subclass, kappa is dominantly produced, resulting in a marked change of light chain/heavy chain ratio in the early phase of the disease. If the excessive light chain is suppressed, it will be useful for clinical therapy.In this study, we optimized the parameters during the process of APA microencapsulation. Firstly, the morphologic study, the mean diameter, the suface finish, the uniformity, the mechanical strength and the chemical intensity of APA microcapsules were established as standards. Then, we performed the APAmicrocapsules with a high-voltage electrostatic system. Briefly, with the syringe pump, alginate solution was extruded at a controlled rate through a needle. When the suspension was forced out of the end of the needle, spherical droplets were pulled off in the electrical field by the high-voltage electrostatic generator. The spherical droplets were collected and transformed to alginate microbeads by gelling in 100 mmol -L CaCl2 solution for 10 minutes. After washing three times with 0.9 % saline, the microbeads suspended with poly-L-lysine (PLL) solution. Before and after treatment with 0.15 % alginate, the microbeads were washed three times with 0.9 % saline. In order to liquefy their inner cores, the microcapsules were allowed to react with 55 mmol L sodium citrate, and then washed three times with 0.9 % saline to remove excess citrate and liquefied alginate.After optimized the parameters individially, we conformed the best concentration of first alginate solution 1.5 %, the CaCh solution 100 mmol L, and the suitable needle 7. Also we found that different concentration of PLL solution reacted with different diameter of alginate microbeads led to varied surface finish. After orthogonal experiment design, the other parameters were optimized, such as push speed of the syringe pump, the voltage of high-voltage electrostatic generator, the distance between needle and the CaCl2 solution. It was confirmed that qualityl (the alginate microbeads diameter) and quality 2 (roundness) were affected by the push speed mostly, and quality 3 (homogeneity) was decided by the distance. Thus, the microbeads of 150m can be prepared with the parameters of the push speed 4.5 ml min, the voltage 12.5 kv, the distance 2.0 cm, followed with the reaction of 0.02 % PLL solution. With optimum parameters, the APA microcapsules of mean diameter 250 um can be made which were found round shape, homogeneous and had the best membrane strength.To get the cell line we need in this study, the hybridoma cell line secreting anti-human IgG1 type monoclonal antibodies was established. The spleen cells taken from BALB/C mice immunized repeatedly by purified human IgG1 type were fused with mouse myeloma cells SP2/0. After culture and screened by ELISA, thehybridoma cell lines secreting monoclonal antibodies (mAb) against human IgG1. type named JY-A1 was established. The subclass of mAb secreted by JY-A1 is belonged to IgGl.By the high-voltage electrostatic system with the op更多还原