發(fā)布時間：2018-01-19 23:01

  本文關(guān)鍵詞： 堿性成纖維細胞生長因子 骨髓間充質(zhì)干細胞 慢性阻塞性肺疾病 白介素-10 白介素-4 分化　出處：《南昌大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:研究堿性成纖維生長因子(bFGF)轉(zhuǎn)染骨髓間充質(zhì)干細胞(MSCs)對COPD大鼠中IL-10、IL-4表達的影響及其分化方法:采用全骨髓貼壁細胞法提取、培養(yǎng)BMSCs。采用脂質(zhì)體轉(zhuǎn)染法導(dǎo)入bFGFpcDNA3.1質(zhì)粒。脂多糖(LPS)聯(lián)合煙熏制備COPD大鼠。實驗分5個組,健康對照組(A組):經(jīng)尾靜脈注射入1ml PBS液至正常大鼠體內(nèi)。COPD組(B組):經(jīng)尾靜脈注射入1ml PBS液至COPD大鼠體內(nèi)。MSCs組(C組):經(jīng)尾靜脈注射入被CM-Dil標(biāo)記的MSCs COPD大鼠體內(nèi),量為1×106/1mlPBS;pcDNA3.1-MSCs組(D組):經(jīng)尾靜脈注射入被CM-Dil標(biāo)記的pcDNA3.1-MSCs至COPD模型大鼠體內(nèi),量為106/1mlPBS;bFGF-pcDNA3.1-MSCs組(E組):經(jīng)尾靜脈注射入被CM-Dil標(biāo)記的bFGF-pcDNA3.1-MSCs至COPD大鼠體內(nèi),量為1×106/1mlPBS。各組大鼠分別在D7、D14、D28處死,HE染色觀察COPD病理改變,ELISA檢測外周血IL-10、IL-4炎癥因子的水平,qRT-PCR法檢測肺組織中IL-10、IL-4mRNA的表達;CM-Dil標(biāo)記聯(lián)合冰凍切片免疫熒光染色法檢測MSCs在肺組織中的分化情況。結(jié)果:1.提取、培養(yǎng)的MSCs,流式細胞術(shù)表型鑒定結(jié)果:CD29(99.1%)、CD44(43.6%)高表達,CD34(1.8%)、CD45(1.5%)低表達;2.bFGF-pcDNA3.1質(zhì)粒測序結(jié)果與數(shù)據(jù)庫發(fā)表的一致,bFGF-pcDNA3.1質(zhì)粒采用脂質(zhì)體轉(zhuǎn)染法成功轉(zhuǎn)染MSCs,bFGF在體內(nèi)高表達;3.HE染色病理學(xué)檢測,C組、D組、E組均較B組,病理改變明顯改善,E組較C組、D組病理改變相對明顯;提示bFGF轉(zhuǎn)染MSCs對大鼠COPD病理改變有改善作用。4.在相同時間節(jié)點,C、D、E三組外周血及肺組織中的IL-10、IL-4表達高于B組(均P0.05),C、D、E三組相比,C、D組外周血及肺組織中的IL-4、IL10水平差別不大,但E組較之二者相對較高,有統(tǒng)計學(xué)差異(P0.05)。隨著時間推移,C、D、E各組中外周血及肺組織中的IL-10、IL-4表達水平均升高,提示抗炎作用增強。5.CM-Dil染色聯(lián)合免疫熒光染色提示:第28天,在C、D、E組的肺組織切片中,部分CM-Dil陽性細胞同時SPC或CC16表達陽性,提示外源性MSCs分化為II型肺泡上皮細胞或支氣管上皮細胞,并且E組分化最多。結(jié)論:bFGF轉(zhuǎn)染MSCs移植入COPD大鼠體內(nèi),可改善COPD病理改變,提高炎癥因子IL-10、IL-4的表達,增強抗炎作用,并可促進MSCs分化為肺泡上皮細胞或支氣管上皮細胞。
[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，

本文編號：1445847