APA微囊化杂交瘤细胞的制剂学研究
|
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?
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 APA
microcapsules 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, the
hybridoma 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
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?
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 APA
microcapsules 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, the
hybridoma 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
引文
[1] Lim F, Sun A.M. Microencapsulated islets as bioartifical endocrine pancreas [J]. Science, 1980, 210: 908-910.
[2] 梁治齐编著．微胶囊技术及其应用[M]．北京：中国轻工业出版社，1999．
[3] Gerbsch N, Buchholz R. New processes and actual trends in biotechnology [J]. FEMS Microbiology Reviews, 1995, 16: 259-269.
[4] Tai I.T, Sun A.M. Microencapsulation of recombinant cells [J]. Methodology, 1993, 7: 1061-1068.
[5] Okada N, Miyamoto H, Kaneda Y, et al. Medical application of microencapsulating hybridoma cells in agarose microbeads "cytomedicine": therapeutic effect on IgG1 plasmacytosis and mesangio-proliferative glomerulonephritis in the interleukin 6 transgenic mouse [J]. J of Controlled Release, 1997, 44: 195-200.
[6] Okada N, Miyamoto H, Yoshioka T, et al. Therapeutic effect of cytomedicine on mesangio-proliferative glomerulonephritis in human interleuldn-6 transgenic mice [J]. Bio Pharm Bull, 1997, 20 (3): 255-258.
[7] De Vos P, De Haan B.J, Wolters G.H.J, et al. Improved biocompatibility but limited graft survival after purification of alginate for microeneapsulation of pancreatic islets [J]. Diabetologia, 1997, 40: 262-270.
[8] 李保国，华泽钊，田泽正等．医用微胶囊成型装置的研制[J]．医疗卫生装备，2001，(3)：1-4．
[9] Goosen M.F.A, O'shea G.M, Gharapetian H.M, et al. Optimization of microencapsulation parameters: semipermeable microcapsules as a bioartificial pancreas [J]. Biotechnol Bioeng, 1985, 27: 146-150.
[10] Thu B, Bruheim P, Espevik T, et al. Alginate polycation microcapsules Ⅰ. Interaction between alginate and polycation [J]. Biomaterials, 1996, 17: 1031-1040.
[11] Thu B, Bruheim P, Espevik T, et al. Alginate polycation microcapsules Ⅱ. Some functional properties [J]. Biomaterial, 1996, 63: 425-432.
[12] Yoshioka Y, Suzuki R, Oka H, et al. A novel cytomedical vehicle capable of protecting cells against complement [J]. Biochem Biophys Res Comm, 2003, 305: 353-358.
[13] Goosen M.F.A, King G.A, Mcknight C.A, et al. Animal cell culture engineering using alginate polycation microcapsules controlled membrane molecular weight cut-off [J]. J of Membrane Science, 1989, 41: 323-343.
[14] 李保国，刘占杰．高压静电场制备微胶囊的研究[J]．上海理工大学学报，2000，22(3)：189-193．
[15] 陈爱政，王士斌，刘源岗等．高压微胶囊成型装置制备用于成囊的海藻酸钙胶珠[J]．山东生物医学工程，2003，21(4)：21-23．
[16] 李新建，薛毅珑，罗芸等．海藻酸盐-多聚赖氨酸-海藻酸盐微胶囊膜的强度和生物相容性测定[J]．军医进修学院学报，2001，22(2)：94-96．
[17] Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefmed specificity [J]. Nature, 1975, 256: 495-497.
[18] Galfre G, Howe S.C, Milstein C, et al. Antibodies to major histocompatibility antigens produced by hybrid cell lines [J]. Nature, 1977, 266: 550-552.
[19] 辛颜彬，薛小平，朱关福等．杂交瘤细胞体外大量培养研究的进展[J]．单克隆抗体通讯，1990，6(2)：63-67．
[20] Ohe Y, Podack E.R, Olsen K.J, et al. Interleukin-6cDNA transfected Lewis lung carcinoma cells show unaltered net tumour growth rate but cause weight loss and shorten survival in syngenic mice [J]. Br. J. Cancer, 1993, 67: 939-944.
[21] Jeong Y.H, Wang S.S. Role of glutarnine in hybridoma cell culture: Effects on cell growth, antibody production, and cell metabolism [J]. Enzyme Microb. Technol, 1995, 17: 47-55.
[22] 袁清安，俞炜源，黄翠芳．鼠单克隆抗体人源化[J]．细胞与分子免疫学杂志．1997,13(1)：68-72．
[23] 刘尚勤，陈燕．恶性淋巴瘤分类研究进展[J]．临床血液学杂志，1999，12(3)
[24] Nakano T, Nagata A. ELISAs for free light chains of human immunoglobulins using monoclonal antibodies: comparison of their specificity with available polyclonal antibodies [J]. J of Immunological Methods, 2003, 275: 9-17.
[25] McKirmey K.L, Dilwith R, Belfort G. Optimizing antibodies production in batch hybridoma cell culture [J]. Biotechnology, 1995, 40:31-48.
[26] Surujballi O, Elmgren C. Monoclonal antibodies suitable for incorporation into a competitive enzyme-linked immunosorbent assay (ELISA) for detection of specific antibodies to Leptospira interrogans serovar pomana. Veterinary Microbiology, 2000, 71: 149-159.
[27] 何洋，解玉冰，王勇等．APA微囊扩散数学模型的改进[J]．高等学校化学学报，2000，21(2)：278-282．
[28] Martinsen A, Storre I, Skjak-Braek G, et al. Alginate as immobilization material Ⅲ. Diffusional properties [J]. Biotechnol Bioeng, 1992, 39: 186-194.
[29] Constantinidis I, Rask I, Jr R.C.L, et al. Effects of alginate composition on the metabolic, secretory and growth characteristics of entrapped β TC3 mouse insulinoma cells [J]. Biomaterials, 1999, 20:2019-2027.
[30] Okada N, Miyamoto H, Yoshioka T, et al. Immunological studies of SK2 hybridoma cells microencapsulated with alginate-poly(L)lysine-alginate (APA) membrane following allogeneic transplantation [J]. Biochem Biophys Res Comm, 1997, 230: 524-527.
[31] Orive G; Ponce S, Hemandez R.M, et al. Biocompatibility of microcapsules for cell immobilization elaborated with different type of alginate [J]. Biomaterials, 2003, 23: 3825-3831.
[32] Reach G. Bioartificial pancreas [J]. Diabetes Medicine, 1993, 10: 105-109.
[33] Lanza R.P, Jackson R, Sullivan A, et al. Xenotransplantation of cells using biodegradable microcapsules [J]. Transplantation, 1999, 67 (8): 1105-1111.
[34] Okada N, Miyamoto H, Yoshioka T, et al. Cytomedical therapy for IgG1 plasmacytosis in human interleukin-6 transgenic mice using hybridoma cells microencapsulated in alginate-poly(L)lysine-alginate membrane [J]. Biochim et BiophysActa, 1997, 1360: 53-63.
[2] 梁治齐编著．微胶囊技术及其应用[M]．北京：中国轻工业出版社，1999．
[3] Gerbsch N, Buchholz R. New processes and actual trends in biotechnology [J]. FEMS Microbiology Reviews, 1995, 16: 259-269.
[4] Tai I.T, Sun A.M. Microencapsulation of recombinant cells [J]. Methodology, 1993, 7: 1061-1068.
[5] Okada N, Miyamoto H, Kaneda Y, et al. Medical application of microencapsulating hybridoma cells in agarose microbeads "cytomedicine": therapeutic effect on IgG1 plasmacytosis and mesangio-proliferative glomerulonephritis in the interleukin 6 transgenic mouse [J]. J of Controlled Release, 1997, 44: 195-200.
[6] Okada N, Miyamoto H, Yoshioka T, et al. Therapeutic effect of cytomedicine on mesangio-proliferative glomerulonephritis in human interleuldn-6 transgenic mice [J]. Bio Pharm Bull, 1997, 20 (3): 255-258.
[7] De Vos P, De Haan B.J, Wolters G.H.J, et al. Improved biocompatibility but limited graft survival after purification of alginate for microeneapsulation of pancreatic islets [J]. Diabetologia, 1997, 40: 262-270.
[8] 李保国，华泽钊，田泽正等．医用微胶囊成型装置的研制[J]．医疗卫生装备，2001，(3)：1-4．
[9] Goosen M.F.A, O'shea G.M, Gharapetian H.M, et al. Optimization of microencapsulation parameters: semipermeable microcapsules as a bioartificial pancreas [J]. Biotechnol Bioeng, 1985, 27: 146-150.
[10] Thu B, Bruheim P, Espevik T, et al. Alginate polycation microcapsules Ⅰ. Interaction between alginate and polycation [J]. Biomaterials, 1996, 17: 1031-1040.
[11] Thu B, Bruheim P, Espevik T, et al. Alginate polycation microcapsules Ⅱ. Some functional properties [J]. Biomaterial, 1996, 63: 425-432.
[12] Yoshioka Y, Suzuki R, Oka H, et al. A novel cytomedical vehicle capable of protecting cells against complement [J]. Biochem Biophys Res Comm, 2003, 305: 353-358.
[13] Goosen M.F.A, King G.A, Mcknight C.A, et al. Animal cell culture engineering using alginate polycation microcapsules controlled membrane molecular weight cut-off [J]. J of Membrane Science, 1989, 41: 323-343.
[14] 李保国，刘占杰．高压静电场制备微胶囊的研究[J]．上海理工大学学报，2000，22(3)：189-193．
[15] 陈爱政，王士斌，刘源岗等．高压微胶囊成型装置制备用于成囊的海藻酸钙胶珠[J]．山东生物医学工程，2003，21(4)：21-23．
[16] 李新建，薛毅珑，罗芸等．海藻酸盐-多聚赖氨酸-海藻酸盐微胶囊膜的强度和生物相容性测定[J]．军医进修学院学报，2001，22(2)：94-96．
[17] Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefmed specificity [J]. Nature, 1975, 256: 495-497.
[18] Galfre G, Howe S.C, Milstein C, et al. Antibodies to major histocompatibility antigens produced by hybrid cell lines [J]. Nature, 1977, 266: 550-552.
[19] 辛颜彬，薛小平，朱关福等．杂交瘤细胞体外大量培养研究的进展[J]．单克隆抗体通讯，1990，6(2)：63-67．
[20] Ohe Y, Podack E.R, Olsen K.J, et al. Interleukin-6cDNA transfected Lewis lung carcinoma cells show unaltered net tumour growth rate but cause weight loss and shorten survival in syngenic mice [J]. Br. J. Cancer, 1993, 67: 939-944.
[21] Jeong Y.H, Wang S.S. Role of glutarnine in hybridoma cell culture: Effects on cell growth, antibody production, and cell metabolism [J]. Enzyme Microb. Technol, 1995, 17: 47-55.
[22] 袁清安，俞炜源，黄翠芳．鼠单克隆抗体人源化[J]．细胞与分子免疫学杂志．1997,13(1)：68-72．
[23] 刘尚勤，陈燕．恶性淋巴瘤分类研究进展[J]．临床血液学杂志，1999，12(3)
[24] Nakano T, Nagata A. ELISAs for free light chains of human immunoglobulins using monoclonal antibodies: comparison of their specificity with available polyclonal antibodies [J]. J of Immunological Methods, 2003, 275: 9-17.
[25] McKirmey K.L, Dilwith R, Belfort G. Optimizing antibodies production in batch hybridoma cell culture [J]. Biotechnology, 1995, 40:31-48.
[26] Surujballi O, Elmgren C. Monoclonal antibodies suitable for incorporation into a competitive enzyme-linked immunosorbent assay (ELISA) for detection of specific antibodies to Leptospira interrogans serovar pomana. Veterinary Microbiology, 2000, 71: 149-159.
[27] 何洋，解玉冰，王勇等．APA微囊扩散数学模型的改进[J]．高等学校化学学报，2000，21(2)：278-282．
[28] Martinsen A, Storre I, Skjak-Braek G, et al. Alginate as immobilization material Ⅲ. Diffusional properties [J]. Biotechnol Bioeng, 1992, 39: 186-194.
[29] Constantinidis I, Rask I, Jr R.C.L, et al. Effects of alginate composition on the metabolic, secretory and growth characteristics of entrapped β TC3 mouse insulinoma cells [J]. Biomaterials, 1999, 20:2019-2027.
[30] Okada N, Miyamoto H, Yoshioka T, et al. Immunological studies of SK2 hybridoma cells microencapsulated with alginate-poly(L)lysine-alginate (APA) membrane following allogeneic transplantation [J]. Biochem Biophys Res Comm, 1997, 230: 524-527.
[31] Orive G; Ponce S, Hemandez R.M, et al. Biocompatibility of microcapsules for cell immobilization elaborated with different type of alginate [J]. Biomaterials, 2003, 23: 3825-3831.
[32] Reach G. Bioartificial pancreas [J]. Diabetes Medicine, 1993, 10: 105-109.
[33] Lanza R.P, Jackson R, Sullivan A, et al. Xenotransplantation of cells using biodegradable microcapsules [J]. Transplantation, 1999, 67 (8): 1105-1111.
[34] Okada N, Miyamoto H, Yoshioka T, et al. Cytomedical therapy for IgG1 plasmacytosis in human interleukin-6 transgenic mice using hybridoma cells microencapsulated in alginate-poly(L)lysine-alginate membrane [J]. Biochim et BiophysActa, 1997, 1360: 53-63.