bFGF转染MSCs移植对COPD大鼠模型IL-10、IL-4的影响及分化研究

发布时间：2018-01-19 23:01

  本文关键词： 碱性成纤维细胞生长因子 骨髓间充质干细胞 慢性阻塞性肺疾病 白介素-10 白介素-4 分化　出处：《南昌大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的:研究碱性成纤维生长因子(bFGF)转染骨髓间充质干细胞(MSCs)对COPD大鼠中IL-10、IL-4表达的影响及其分化方法:采用全骨髓贴壁细胞法提取、培养BMSCs。采用脂质体转染法导入bFGFpcDNA3.1质粒。脂多糖(LPS)联合烟熏制备COPD大鼠。实验分5个组,健康对照组(A组):经尾静脉注射入1ml PBS液至正常大鼠体内。COPD组(B组):经尾静脉注射入1ml PBS液至COPD大鼠体内。MSCs组(C组):经尾静脉注射入被CM-Dil标记的MSCs COPD大鼠体内,量为1×106/1mlPBS;pcDNA3.1-MSCs组(D组):经尾静脉注射入被CM-Dil标记的pcDNA3.1-MSCs至COPD模型大鼠体内,量为106/1mlPBS;bFGF-pcDNA3.1-MSCs组(E组):经尾静脉注射入被CM-Dil标记的bFGF-pcDNA3.1-MSCs至COPD大鼠体内,量为1×106/1mlPBS。各组大鼠分别在D7、D14、D28处死,HE染色观察COPD病理改变,ELISA检测外周血IL-10、IL-4炎症因子的水平,qRT-PCR法检测肺组织中IL-10、IL-4mRNA的表达;CM-Dil标记联合冰冻切片免疫荧光染色法检测MSCs在肺组织中的分化情况。结果:1.提取、培养的MSCs,流式细胞术表型鉴定结果:CD29(99.1%)、CD44(43.6%)高表达,CD34(1.8%)、CD45(1.5%)低表达;2.bFGF-pcDNA3.1质粒测序结果与数据库发表的一致,bFGF-pcDNA3.1质粒采用脂质体转染法成功转染MSCs,bFGF在体内高表达;3.HE染色病理学检测,C组、D组、E组均较B组,病理改变明显改善,E组较C组、D组病理改变相对明显;提示bFGF转染MSCs对大鼠COPD病理改变有改善作用。4.在相同时间节点,C、D、E三组外周血及肺组织中的IL-10、IL-4表达高于B组(均P0.05),C、D、E三组相比,C、D组外周血及肺组织中的IL-4、IL10水平差别不大,但E组较之二者相对较高,有统计学差异(P0.05)。随着时间推移,C、D、E各组中外周血及肺组织中的IL-10、IL-4表达水平均升高,提示抗炎作用增强。5.CM-Dil染色联合免疫荧光染色提示:第28天,在C、D、E组的肺组织切片中,部分CM-Dil阳性细胞同时SPC或CC16表达阳性,提示外源性MSCs分化为II型肺泡上皮细胞或支气管上皮细胞,并且E组分化最多。结论:bFGF转染MSCs移植入COPD大鼠体内,可改善COPD病理改变,提高炎症因子IL-10、IL-4的表达,增强抗炎作用,并可促进MSCs分化为肺泡上皮细胞或支气管上皮细胞。
[Abstract]:Objective: To study the effect of basic fibroblast growth factor (bFGF) transfection of bone marrow mesenchymal stem cells (MSCs) in IL-10 COPD rats, IL-4 expression and differentiation method by whole bone marrow adherent cells was extracted, cultured BMSCs. by liposome transfection into bFGFpcDNA3.1 plasmid. Lipopolysaccharide (LPS) combined with fumigation preparation COPD rats. The experiment is divided into 5 groups, healthy control group (A group): after intravenous injection of 1ml PBS solution to normal rats.COPD group (group B): after intravenous injection of 1ml PBS solution to COPD rats.MSCs group (group C) were injected via the caudal vein. CM-Dil labeled MSCs in COPD rats, the amount is 1 * 106/1mlPBS; pcDNA3.1-MSCs group (D group): after intravenous injection of pcDNA3.1-MSCs to COPD model rats were labeled with CM-Dil, volume 106/1mlPBS; bFGF-pcDNA3.1-MSCs group (E group): after intravenous injection of CM-Dil labeled bFGF-pcDNA3.1-MSCs to COPD In vivo, amount to 1 * 106/1mlPBS. rats were killed at D7, D14, D28, COPD, HE staining to observe the pathological changes of peripheral blood IL-10, ELISA test, IL-4 cytokine levels, IL-10 detection of lung tissue qRT-PCR, IL-4mRNA expression; CM-Dil markers with frozen sections immunofluorescence staining was used to detect MSCs in the lung tissue differentiation. Results: 1. extraction, cultured MSCs, flow cytometry phenotypic identification results: CD29 (99.1%), CD44 (43.6%) high expression of CD34 (1.8%), CD45 (1.5%) low expression; plasmid 2.bFGF-pcDNA3.1 sequencing results were consistent with the published database, bFGF-pcDNA3.1 plasmid by liposome transfection was successfully transfected into MSCs, the high expression of bFGF in vivo; 3.HE staining of pathological examination, C group, D group, E group compared with B group, the pathological changes were obviously improved, E group compared with C group, the pathological changes of the D group is relatively obvious; it indicated bFGF gene MSCs can improve the effect of.4. on pathological changes in COPD rats 鍦ㄧ浉鍚屾椂闂磋妭鐐，

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