【作者】 聂君；

【导师】 王建军；

【作者基本信息】 华中科技大学 ， 外科学， 2010， 博士

【摘要】 目的：探讨缺血再灌注诱导大鼠肺组织损伤不同时期基因表达谱的变化,阐明肺缺血再灌注的动态损伤机制,通过筛选损伤和修复的相关基因,为肺缺血再灌注损伤的干预治疗提供新的线索和思路。方法：雄性SD大鼠36只,实验随机分为对照组(假手术组,6只)和缺血再灌注组(I/R 0、1、3、6、24小时组,各组6只)。通过阻断肺门建立大鼠肺缺血再灌注损伤模型,于再灌注后不同时间点取大鼠肺组织并提取标本总RNA,运用包含22 226个大鼠基因点的Illumina RatRef-12全基因组表达谱微珠芯片检测对照组和模型组肺缺血再灌处理后不同时间点的基因表达情况,并采用SOM算法将具有相似表达模式的基因进行聚类分析。结果：缺血后再灌0,1,3,6,24h分别有648,340,711,1279,641个基因表达发生改变。根据基因的功能不同将部分发生显著改变的基因分为七类,包括炎性相关因子、凋亡调控相关蛋白、氧化与抗氧化分子、代谢相关酶类、离子通道及水通道、信号传导分子以及补体系统等七类。同时通过聚类分析将具有相同表达模式的基因被聚为12类。结论：缺血再灌注可以诱导肺的基因表达谱发生改变,基因表达谱芯片能够快速、高效地筛选差异表达基因,多种不同功能的基因相互影响共同造成肺缺血后再灌损伤,具有相似表达模式的基因可能具有相似的功能或相似的表达调控机制。目的：建立大鼠骨髓间充质干细胞(Mesenchymal Stem Cells, MSCs)的分离和培养方法,探讨MSCs的生物学特性和不同情况下的多向分化潜能,为将MSCs作为基因载体应用于体内细胞治疗和构建新型气管组织工程软骨种子细胞提供理论基础。方法：采用原代贴壁法获得间充质干细胞,观察细胞形态和生长变化,筛选细胞培养的最佳条件。流式细胞仪鉴定细胞表面标志,体外诱导间充质干细胞向脂肪细胞、成骨细胞和软骨细胞分化。采用EGFP慢病毒载体感染培养的间充质干细胞,比较细胞感染前后生物学特性。观察绿色荧光蛋白(Green Fluorescent Protein, GFP)基因通过慢病毒载体感染MSCs的表达情况。结果：原代贴壁法获可获得纯度较高的MSCs,稳定培养液的pH值能使MSCs稳定传代。流式细胞仪检测示大鼠MSCs的表型为CD11b-CD34-CD44+CD45-CD90+CD-106+。体外MSCs能诱导分化为脂肪细胞、成骨细胞和软骨细胞。慢病毒载体可有效感染大鼠MSCs, EGFP在MSCs中能长期稳定表达,聚凝胺可增强感染效率。结论：稳定pH值的培养液能使大鼠间充质干细胞保持旺盛的生长能力,间充质干细胞具有良好的多向分化能力。慢病毒能高效感染间充质干细胞,GFP基因能在间充质干细胞中长期稳定表达。本研究建立了一种有效、稳定、持久的大鼠骨髓MSCs分离培养及荧光基因标记技术,为研究MSCs的转归、可塑性及基因治疗奠定了基础。目的：探讨骨髓间充质干细胞(MSCs)植入大鼠肺缺血再灌注损伤(I/R)模型中的效应、分化及其相关的作用机制。方法：分离纯化培养MSCs,进行GFP标记,将MSCs注射大鼠肺I/R损伤模型及对照组,观察再灌注后6h、1d、3d、7d等时间点肺的病理变化及功能改变,以及不同时间点MSCs向肺内迁移和在肺内的存留情况,通过免疫组化方法观察在肺内MSCs的分化。实时荧光定量PCR检测肺组织TNF-a mRNA和C5 mRNA表达变化,ELISA检测IL-1β、IL-10、IL-17、KGF, MIP-1α等炎性因子含量的改变。结果：MSCs植入I/R模型可以降低肺湿重比和MPO活力,MSCs可以向肺内迁移,以24h至3d数量较多,再灌后7d仍可见存留于肺内,数量减少,但7d时观察到带有EGFP和SP-C双标阳性的MSCs。相对于单纯I/R损伤组,MSCs可以下调肺组织内致炎因子TNF-α、C5、IL-17、MIP-1α的表达和上调抗炎因子IL-10、KGF的表达。结论：MSCs可以减轻肺I/R损伤,通过向肺Ⅱ型上皮细胞分化以及免疫调节和旁分泌功能发挥保护效应。更多还原

【Abstract】 Objective In order to provide us new clues to induce some endogenous protective molecular mechanisms, the changes in gene expression profile induced by ischemia-reperfusion in pulmonary tissues of rats were investigated and the dynamic mechanism of pulmonary ischemia-reperfusion injury was elucidated.Methods Thirty male SD rats were randomly divided into 6 groups:5 ischemia-reperfusion (I/R) groups (I/R 0-h, I/R 1-h, I/R 3-h, I/R 6-h, I/R 24-h) and control group (n=6 in each). An in situ ischemia-reperfusion lung injury rat model was established by occluded hilus of lung. The RatRef-12 Expression Beadchip (22 226 gene probes per array) was used to analyze the pattern of gene expression in all groups.Results The results showed that 648,340,711,1279 and 641 genes were differentially expressed in I/R 0-,1-,3-,6- and 24-h groups respectively. The differentially expressed genes were classified as following 7 functional categories:cytokine, growth factor and apoptosis-related factor, oxidation and antioxidation molecule, metabolic enzyme, ion channel and aquaporin, signal transduction molecule and complement. Clusters analysis identified 12 clusters of genes in which each cluster had similar expression pattern.Conclusions It was suggested that gene chip technology was an effective and quick method for screening differentially expressed genes. Many differentially expressed genes with different functions interacted each other to result in pulmonary ischemia-reperfusion injury. Genes with similar expression pattern could have the similar regulated mechanism. Objective The multilineage capability of adult bone marrow mesenchymal stem cells (MSCs) supports the feasibility of tissue engineering multiphasic constructs using a single cell source. Therefore, it is important to establish a method of isolation and culture of the rat mesenchymal stem cells, make a study on the differentiation character and pluripotency of MSCs under different conditions and to investigate the expression of green fluorescent protein (GFP) gene carried by lentiviral vectors into MSCs.Methods MSCs, which were initially isolated from the bone marrow of rats, were cultured in vitro, isolated by trypsin digestion method, purified by adherence method and tested by flow cytomertry. After MSCs were transferred to osteogenic, adipogenic or chondrogenic differentiation medium respectively, the morphological characterization of induced cells was observed. The expression of marker genes was measured by RT-PCR analysis. Then MSCs were infected with lentiviral vectors. The results of the expression of GFP and infection efficiency were observed by fluorescence microscope.Results MSCs of high purity were obtained from bone marrow using adherence screening method. The stable pH of culture medium was important for cell growth and subculture. MSCs typically expressed the antigens CD44 (94.81%), CD90 (99.53%), CD106 (76.34%). They were negative for typical lymphocytic markers like CD45 (1.94%) and CD11b (1.42%) and for the early hematopoietic markers CD34 (0.04%). MSCs can differentiate to adipocyte, osteocyte and chondrocyte in vitro. The results of fluorescence microscopic imaging proved that the high level and stability of GFP expression in MSCs infected with lentiviral vector. So the exogenous GFP and multilineage potential of MSCs had no severe influences on each other.Conclusions Since the MSCs can be easily obtained and abundant, it is proposed that they may be promising candidate cells for further studies on tissue engineering. Imaging with expression of GFP facilitates the research on MSCs physiological behavior and application in tissue engineering during differentiation both in vitro and in vivo. Objective To elucidate effect of adult bone marrow mesenchymal stem cells (MSCs) on pulmonary ischemia-reperfusion (I/R) injury and make a research on differentiaon and regulatory mechanism of MSCs in I/R injury.Methods MSCs were initially isolated from the bone marrow of rats and cultured in vitro. Then MSCs were infected with lentiviral vectors to obtain EGFP mark. MSCs were injected into rats of I/R injury model and control groups. Pathological changes and function alterations of lung were observed at 6 h,1 d,3 d and 7 d after I/R injury. Migration to lung and persistence in lung of MSCs were also observed. Immunohistochemisty method was used to investigate the differentiation of MSCs. Real-time RT-PCR was used to monitor gene expression changes of TNF-αand complement 5 (C5) in all groups. ELISA was used to detect concentration alterations of IL-1β, IL-10, IL-17, KGF and MIP-1αin pulmonary tissue.Results MSCs could decrease pulmonary ratio of wet to dry and MPO activity. MSCs could migrate to lung and the number of cells at 24 h was larger than those at 7 d. However, the double positive of EGFP and SP-C in MSCs was found at 7 d after I/R injury. Concertaion of pro-inflammatory cytokines auch as TNF-α, C5, IL-17 and MIP-1αwere decreased and expression of antiinflammatory cytokines such as IL-10 and KGF were upregulated.Conclusions MSCs have beneficial effects in experimental pulmonary I/R injury model. The beneficial effect of MSCs derives from their differentiation to typeⅡalveolar cells and more from their capacity to secrete paracrine soluble factors that modulate immune responses.更多还原