腺病毒介导hIL-1Ra基因转染大鼠骨髓MSC及对其免疫调节因子表达调控的研究

发布时间：2018-05-12 12:51

本文选题：间充质干细胞 + 白介素1受体拮抗剂　；参考：《吉林大学》2008年博士论文

【摘要】： 白细胞介素1受体拮抗剂(Interleukin 1 receptor antagonist, IL-1Ra)是天然的IL-1拮抗物质,可作为基因治疗的目的基因,通过合适的基因转移途径,在体内发挥抗炎作用。间充质干细胞(Mesenchymal stem cell, MSC)作为成体干细胞,不仅是目前组织工程和基因治疗领域理想的种子细胞和基因载体,而且免疫原性低、并具有免疫调节作用。如将二者优势综合,通过适当途径将外源性IL-1Ra基因转入MSC,使其以旁分泌方式在体内局部组织发挥抗炎和组织修复功能,将在类风湿关节炎等免疫病理性炎症损伤疾病的治疗领域具有诱人的应用前景,目前尚未见诸国内外研究报道。按照这一设想,本研究通过克隆人IL-1Ra基因,成功构建AdhIL-1Ra重组腺病毒;在成功分离、培养和鉴定大鼠骨髓MSC后,首次将hIL-1Ra基因在腺病毒载体介导下转入大鼠骨髓MSC,并证实hIL-1Ra基因在大鼠骨髓MSC中成功表达;采用RT-PCR法,观察外源性hIL-1Ra基因转染对大鼠骨髓MSC免疫调节因子的表达调控。结果表明,正常培养的MSC可表达多种免疫调节因子,这些因子间的表达变化具有较高的相关性。LPS模拟的炎性刺激可使大鼠骨髓MSC的炎性因子表达水平升高。外源性hIL-1Ra基因修饰对大鼠骨髓MSC免疫调节因子基因表达具有明显的调控作用,表现为增强抑炎性免疫调节因子表达及抑制LPS刺激下炎性因子表达水平的升高。另外,本研究发现hIL-1Ra基因修饰和LPS刺激均可影响MSC向软骨细胞方向的分化。以上结果初步证明hIL-1Ra基因修饰MSC,发挥免疫调节作用的可行性,为今后进一步开展相关机理研究和体内研究打下实验基础。
[Abstract]:Interleukin-1 receptor antagonist (IL-1Ra) is a natural IL-1 antagonist, which can be used as the target gene of gene therapy and play an anti-inflammatory effect in vivo through the appropriate gene transfer pathway. Mesenchymal stem cell, MSC), as an adult stem cell, is not only the ideal seed cell and gene vector in tissue engineering and gene therapy, but also has low immunogenicity and immunomodulatory effect. If the two advantages are combined, the exogenous IL-1Ra gene can be transferred into MSCs by appropriate way, so that the exogenous IL-1Ra gene can play an anti-inflammatory and tissue repair function in local tissues in vivo by paracrine way. It will have an attractive application prospect in the treatment of immune pathological inflammatory injury diseases such as rheumatoid arthritis, which has not been reported at home and abroad. According to this assumption, the recombinant adenovirus of AdhIL-1Ra was successfully constructed by cloning human IL-1Ra gene, and the rat bone marrow MSC was successfully isolated, cultured and identified. HIL-1Ra gene was transferred into rat bone marrow by adenovirus vector for the first time, and the expression of hIL-1Ra gene in rat bone marrow MSC was confirmed successfully. The expression of MSC immunomodulatory factor in rat bone marrow was observed by RT-PCR method. The results showed that MSC in normal culture could express a variety of immunomodulatory factors, and the expression of these factors had a high correlation. LPS-mimic inflammatory stimulation could increase the expression of inflammatory factors in rat bone marrow MSC. Exogenous hIL-1Ra gene modification can obviously regulate the expression of MSC immunomodulator gene in bone marrow of rats, which is shown by enhancing the expression of anti-inflammatory immunomodulatory factor and inhibiting the increase of the expression level of inflammatory factor stimulated by LPS. In addition, it was found that both hIL-1Ra gene modification and LPS stimulation could affect the differentiation of MSC into chondrocytes. The above results preliminarily prove the feasibility of hIL-1Ra gene modification to play the role of immune regulation, and lay the experimental foundation for further research on related mechanism and in vivo study.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392

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