转基因充质干细胞微胶囊化试验研究

【作者】 张武杰；

【导师】 李保国；

【作者基本信息】 上海理工大学 ， 食品科学， 2008， 硕士

【摘要】 基于干细胞(stem cells)的组织再生技术是近年来研究的热点。间充质干细胞(mesenchymal stem cells,MSCs)属于未分化的成体干细胞,是良好的基因载体,可作为基因治疗的载体细胞。如将转BMP-2基因的MSCs细胞移植入宿主体内,可用于治疗骨折和骨缺损等。上海交通大学医学院将转染人BMP-2基因的自体骨髓间充质干细胞移植修复大鼠、兔、羊等动物负重长骨的节段性缺损,均获得初步成功。但病毒转染的细胞移植后对宿主有一定免疫原性,取自身细胞有风险,而进行异体、异种细胞移植需使用免疫抑制剂。若将具有免疫隔离平台作用的微囊化技术与基因重组间充质干细胞技术结合,可望为克服移植排斥反应和细胞来源缺乏等问题提供途径。即利用重组细胞的代谢产物调节机体生理功能,治疗相关疾病。本文对海藻酸-聚赖氨酸-海藻酸(APA)微囊进行了改进,用新微囊体系包裹转BMP-2基因的MSCs细胞研究其成骨效应。主要进行了如下研究:1.传统APA微囊改进。利用纯化海藻酸钠制备的细胞微囊,细胞存活率高,微囊强度和生物相容性改善。海藻酸-壳聚糖微囊内MSCs存活率高于80%。载间充质干细胞海藻酸-壳聚糖-海藻酸(ACA)微囊制备工艺优化为:选用1.0～2.5×105分子量的壳聚糖、壳聚糖溶液的浓度为0.1%和壳聚糖覆膜时间7min。此外,利用聚乙烯乙二醇(PEG)对海藻酸钠体系微囊进行化学修饰。结果表明,化学修饰的微胶囊生物相容性改善。采用静电喷雾法制备了膜强度高的海藻酸钙-羧甲基纤维素钠液芯微胶囊。2.建立微囊化细胞动态检测新方法。Alamar Blue是一种无细胞毒性的活体染料,可用于连续检测细胞的增殖或代谢试验。Alamar Blue Assay的优点为:不用破坏微囊结构,无细胞毒性可以实现动态连续检测。Alamar Blue Assay是微囊内细胞增殖检测的有效方法。并利用alamar Blue Assay研究了化学修饰对微囊化细胞活力的影响。3.转BMP-2基因MSCs微囊化及其成骨效应的研究。转BMP-2基因MSCs微囊化后的蛋白渗透试验、MSCs成骨诱导以及ALP定量结果说明,微囊化MSCs具有活性能合成、分泌BMP-2且BMP-2能透过微囊且具有生理活性。微囊化转BMP-2基因MSCs不同种类鼠体内注射移植一月后,有新生骨组织形成。是骨组织工程化新方法。同时,微囊化基因重组干细胞技术开辟了基因治疗的新领域。更多还原

【Abstract】 Stem cells based tissue-restoration has been widely used in many medical fields. Mesenchymal stem cells (MSCs) are known as undifferentiated adult stem cells, and can be used as good gene vector for gene therapy. For example, MSCs can be transplanted in vivo after BMP-2 gene transfection for fracture and bone defect treatments. BMP-2 is a kind of growth factor that could stimulate osteogenic cell activity and differentiation. However, the high doses and repetitive medication of BMP always increase risk and cost. Researchers from Shanghai Jiaotong University succeesfully transplanted the autologous MSCs after BMP-2 gene transfection to repair segmental tibial bone defects of animals such as sheep etc. But the gene vectors of cells may arouse immune reaction of the host and allo- or xeno- cell grafts tends to be lack of undesired immunosuppression and availale cells. The combination of microencapsulation, immunoisolation and controlled-release tool, and gene engineered stem cells provide a possible way for overcoming immune rejection and limited cell sources. Recombinant gene products could regulate body physiological function to treat diseases.In this study, common used Alginate-PLL-Alginate (APA) microcapsule was improved and microcapsulation of transgenic MSCs using improved alginate-base system and its bone generation effects were also investigated. This research focued on the following aspects:Firstly, typical APA microcapsule was improved. Microcapsules prepared using purified sodium alginate had obvious improved biocompatibility and membrane strength. Microcapsules of uniform size and good morphology could be obtained using purified sodium alginate and chitosans of medium-molecular weight (1.0~2.5×105). The optimal alginate-chitosan microcapsule preparation technological parameters were chitosan solution concentration of 0.1% (w/v) and 7-min-long reaction time between alginate and chitosan. Moreover, Chemical modification of alginate-chitosan microcapsule using PEG could not change the microcapsule-formation capacity and the results of viabilities of microencapsulated cells and implantation in KM mice showed the improved biocompatibility of alginate-chitosan microcapsules after PEG modification (ACP). Calcium alginate-Sodium carboxymethyl cellulose liquid core microcapsules of high membrane strength could be prepared using electrostatic spray.Secondly, a dynamic method on viability determination of microencapsulated cells was proposed. Alamar Blue has the features of non-toxic, allowed monitorings cell growth continuously and safely, and less likely to interfere with normal metabolism.The advatages of alamar Blue Assay were: convenient, quick and precise, dynamic method for monitoring microencapsulated cells. Cell viability of chemical modified MSCs microcapsule was deterimined using alamar Blue Assay and the results showed this method was feasible.Lastly, microencapsulated Trans-BMP2 gene MSCs’s osteogenesis effects were also studied. Protein permeation, osteoblastic differentiation of MSCs and determination of ALP activity indicated that microencapsulated Trans-BMP2 gene MSCs could synthetize and secrete BMP-2 with physiological activity, while the BMP-2 could be released from the microcapsule membrane. Moreover, microencapsulated trans-BMP2 gene MSCs’s bone-generation effect was testified in vivo which provides a promising method for bone tissue engineering. Microencapsulation of transgenic MSCs provides a novel solution for gene therapy.更多还原