转基因充质干细胞微胶囊化试验研究

发布时间：2018-01-26 05:00

本文关键词： 微囊化细胞间充质干细胞海藻酸钠壳聚糖细胞治疗活力检测骨形态发生蛋白　出处：《上海理工大学》2008年硕士论文　论文类型：学位论文

【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。
[Abstract]:Tissue regeneration based on stem cells is a hot topic in recent years. Mesenchymal stem cells (MSCs). MSCs) is an undifferentiated adult stem cell, which is a good gene vector, and can be used as a vector cell for gene therapy. For example, the MSCs cells transformed with BMP-2 gene are transplanted into the host. Bone marrow mesenchymal stem cells transfected with human BMP-2 gene were transplanted to repair segmental defects of long bone bearing weight in rats, rabbits and sheep. However, the virus transfected cells had certain immunogenicity to the host after transplantation, and there was a risk to take their own cells and carry out allograft. Immunosuppressive agents are required for xenotransplantation. Microencapsulation techniques with immune isolation platform are combined with recombinant mesenchymal stem cells. It is expected to provide a way to overcome the problems of transplant rejection and lack of cell sources, that is, to regulate the physiological function of the body by using the metabolites of recombinant cells, and to treat related diseases. In this paper, the microcapsules of alginic acid-poly (lysine-alginate) APA were improved. The osteogenic effect of MSCs cells transfected with BMP-2 gene was studied with a new microcapsule system. 1. The improvement of traditional APA microcapsules. The cell microcapsules prepared by purified sodium alginate have high cell survival rate. Improvement of strength and biocompatibility of microcapsules. Survival rate of MSCs in alginic acid-chitosan microcapsules is higher than 80%. The optimum preparation process of microcapsules was as follows: 1. 0. 0. 5 脳 10. 5 molecular weight chitosan. The concentration of chitosan solution was 0.1% and chitosan film covering time was 7 min. In addition, the microcapsules of sodium alginate system were chemically modified by polyethylene glycol (PEG). The biocompatibility of chemically modified microcapsules was improved. Calcium alginate and sodium carboxymethylcellulose core microcapsules with high membrane strength were prepared by electrostatic spray method. 2. To establish a new method for the dynamic detection of microencapsulated cells. Alamar Blue is a non-cytotoxic living dye. Alamar Blue Assay can be used to continuously detect cell proliferation or metabolism. The advantage of this method is that it does not destroy the structure of microcapsules. Non-cytotoxicity can realize dynamic continuous detection. Alamar Blue Assay is an effective method for the detection of cell proliferation in microcapsules. Alamar Blue is also used to detect cell proliferation. Assay was used to study the effect of chemical modification on the viability of microencapsulated cells. 3. Study on the microencapsulation and osteogenic effect of BMP-2 gene MSCs. The protein permeation test of BMP-2 gene MSCs microencapsulated. The results of MSCs osteogenesis induction and ALP quantitative analysis showed that microencapsulated MSCs could be synthesized with activity. BMP-2 was secreted and BMP-2 passed through microcapsules and had physiological activity. The microencapsulated BMP-2 gene MSCs was injected into mice of different species on January. New bone tissue formation is a new method of bone tissue engineering. At the same time, microencapsulated gene recombinant stem cell technology has opened up a new field of gene therapy.
【学位授予单位】：上海理工大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R346

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