本文關(guān)鍵詞： 微囊化細(xì)胞 間充質(zhì)干細(xì)胞 海藻酸鈉 殼聚糖 細(xì)胞治療 活力檢測 骨形態(tài)發(fā)生蛋白　出處：《上海理工大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

【摘要】： 基于干細(xì)胞(stem cells)的組織再生技術(shù)是近年來研究的熱點。間充質(zhì)干細(xì)胞(mesenchymal stem cells,MSCs)屬于未分化的成體干細(xì)胞,是良好的基因載體,可作為基因治療的載體細(xì)胞。如將轉(zhuǎn)BMP-2基因的MSCs細(xì)胞移植入宿主體內(nèi),可用于治療骨折和骨缺損等。上海交通大學(xué)醫(yī)學(xué)院將轉(zhuǎn)染人BMP-2基因的自體骨髓間充質(zhì)干細(xì)胞移植修復(fù)大鼠、兔、羊等動物負(fù)重長骨的節(jié)段性缺損,均獲得初步成功。但病毒轉(zhuǎn)染的細(xì)胞移植后對宿主有一定免疫原性,取自身細(xì)胞有風(fēng)險,而進(jìn)行異體、異種細(xì)胞移植需使用免疫抑制劑。若將具有免疫隔離平臺作用的微囊化技術(shù)與基因重組間充質(zhì)干細(xì)胞技術(shù)結(jié)合,可望為克服移植排斥反應(yīng)和細(xì)胞來源缺乏等問題提供途徑。即利用重組細(xì)胞的代謝產(chǎn)物調(diào)節(jié)機(jī)體生理功能,治療相關(guān)疾病。 本文對海藻酸-聚賴氨酸-海藻酸(APA)微囊進(jìn)行了改進(jìn),用新微囊體系包裹轉(zhuǎn)BMP-2基因的MSCs細(xì)胞研究其成骨效應(yīng)。主要進(jìn)行了如下研究: 1.傳統(tǒng)APA微囊改進(jìn)。利用純化海藻酸鈉制備的細(xì)胞微囊,細(xì)胞存活率高,微囊強(qiáng)度和生物相容性改善。海藻酸-殼聚糖微囊內(nèi)MSCs存活率高于80%。載間充質(zhì)干細(xì)胞海藻酸-殼聚糖-海藻酸(ACA)微囊制備工藝優(yōu)化為:選用1.0～2.5×105分子量的殼聚糖、殼聚糖溶液的濃度為0.1%和殼聚糖覆膜時間7min。此外,利用聚乙烯乙二醇(PEG)對海藻酸鈉體系微囊進(jìn)行化學(xué)修飾。結(jié)果表明,化學(xué)修飾的微膠囊生物相容性改善。采用靜電噴霧法制備了膜強(qiáng)度高的海藻酸鈣-羧甲基纖維素鈉液芯微膠囊。 2.建立微囊化細(xì)胞動態(tài)檢測新方法。Alamar Blue是一種無細(xì)胞毒性的活體染料,可用于連續(xù)檢測細(xì)胞的增殖或代謝試驗。Alamar Blue Assay的優(yōu)點為:不用破壞微囊結(jié)構(gòu),無細(xì)胞毒性可以實現(xiàn)動態(tài)連續(xù)檢測。Alamar Blue Assay是微囊內(nèi)細(xì)胞增殖檢測的有效方法。并利用alamar Blue Assay研究了化學(xué)修飾對微囊化細(xì)胞活力的影響。 3.轉(zhuǎn)BMP-2基因MSCs微囊化及其成骨效應(yīng)的研究。轉(zhuǎn)BMP-2基因MSCs微囊化后的蛋白滲透試驗、MSCs成骨誘導(dǎo)以及ALP定量結(jié)果說明,微囊化MSCs具有活性能合成、分泌BMP-2且BMP-2能透過微囊且具有生理活性。微囊化轉(zhuǎn)BMP-2基因MSCs不同種類鼠體內(nèi)注射移植一月后,有新生骨組織形成。是骨組織工程化新方法。同時,微囊化基因重組干細(xì)胞技術(shù)開辟了基因治療的新領(lǐng)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.
【學(xué)位授予單位】：上海理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R346

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