高表达CXCR7的MSC向ARDS肺组织归巢及其肺保护作用的实验研究

发布时间：2019-06-26 17:25

【摘要】：目的：探讨高表达趋化因子受体7(CXCR7)是否有利于增加间充质干细胞(Mesenchymal Stem Cells, MSCs)向急性呼吸窘迫综合征(ARDS)小鼠损伤肺组织的归巢,从而有利于肺组织炎症反应的控制,促进MSC的肺保护作用。方法：将90只C57BI/6小鼠随机分为Control组(NS+PBS), ARDS组(LPS+PBS), MSC组(LPS+MSC), MSC-GFP组(LPS+MSC-GFP)及MSC-CXCR7 组(LPS+MSC-CXCR7, CXCR7基因通过慢病毒载体介导转染至MSC)。气道内滴入5mg/kg的脂多糖(LPS)复制ARDS小鼠模型,造模4h后按分组给予尾静脉注射等量的生理盐水或含MSC的细胞悬液,30min、 24h及72h后观察以下指标：1)MSC向肺组织的归巢比较：采用近红外离体肺组织成像、肺组织荧光镜检直接观察,应用酶联免疫吸附试验(ELISA)检测肺组织相关粘附因子如血管细胞粘附分子-1(VCAM-1)及重组人胶原蛋白-1(COL-1)的含量,从器官、组织及分子水平进行综合评价；2)小鼠肺损伤严重程度的比较：各时间点处死小鼠留取肺组织,观察大体病理损伤情况,HE染色行组织病理学检查并进行肺损伤评分,计算肺湿重/体重比评价肺水肿程度;3)肺组织局部炎症反应的比较：通过ELISA法检测肺组织中抑炎因子IL-10及促炎因子TNF-α的浓度反映。结果：1、本实验共分5组3个实验时间点(n=6),处死小鼠留取肺组织行病理学检查,观察结果表明气道滴入LPS后肺间质和肺泡明显出血水肿、大量炎性细胞浸润,肺泡结构破坏萎陷,提示均成功建立ARDS模型。2、各组MSC向ARDS小鼠损伤肺组织归巢的比较：1)近红外离体器官成像结果表明,与Control组相比,经小鼠尾静脉给予 MSC-GFP治疗后30min即可在肺组织内观察到明显的荧光信号,24h荧光信号达峰值,72h有所降低；在给予高表达CXCR7的MSC治疗后定性观察及定量分析肺组织中荧光信号强度,结果表明MSC-CXCR7组在24h及72h的信号均比MSC-GFP组显著增强(24h：301.62±187.12 vs. 71.75±32.37 scaled counts/mm2, 7?0.05; 72h: 217.02±126.38 vs. 67.08±26.44 scaled counts/mm2, #p0.05);2)肺组织荧光镜检定性观察结果表明,在MSC-GFP移植入小鼠体内后的24h即可见表达绿色荧光蛋白的MSC,72h后荧光信号有所减弱；然而MSC-CXCR7组小鼠在24h及72h肺组织内观察到的荧光信号均比MSC-GFP组更强；3)ELISA法检测小鼠肺组织中与MSC归巢相关的粘附因子水平结果表明,Control组小鼠肺内VCAM-1浓度较低,在ARDS模型成功构建后的24h及72h,其浓度有所升高,给与MSC-GFP治疗后肺组织内VCAM-1水平进一步升高,与之相比,给与MSC-CXCR7治疗的小鼠肺内粘附因子水平升高程度更为显著(24h: 0.873±0.021 vs. 0.463±0.021 ng/ml, *p0.05; 72h: 1.340±0.141 vs. 0.512±0.038 ng/ml,p0.05)；各实验组小鼠肺组织内COL-1水平变化趋势与VCAM-1相同,在给予MSC-CXCR7治疗后的24h及72h小鼠肺内COL-1浓度较MSC-GFP组显著升高(24h：1.738±0.247 vs. 0.977±0.133ng/ml, p0.05; 72h: 4.137±0.386 vs. 3.597±0.197ng/ml,p0.05)。3、各组小鼠肺损伤程度的比较：肺组织大体及组织病理损伤结果显示,高表达CXCR7的MSC治疗较MSC-GFP更有利于减轻肺组织出血、炎细胞浸润及透明膜形成,降低肺湿重/体重(24h: 5.98±0.63 vs. 7.33±0.53mg/g, p0.05; 72h: 7.37±0.85 vs. 8.97±1.25mg/g, */K0.05)及病理损伤评分(30min: 10.20±0.40 vs. 11.80±0.78, *p0.05; 24h: 8.33±0.67vs. 12.87±0.38, ^O.OOl; 72h: 10.00±0.26vs. 14.00±0.72, *p0.001). 4,各组小鼠肺部炎症反应程度的比较：ARDS组小鼠肺内促炎因子TNF-α水平较Control组升高,而抑炎因子IL-10水平降低,当给予MSC-GFP治疗24h及72h后检测到小鼠肺组织内TNF-α浓度显著降低,反而IL-10水平有所升高,而在给予MSC-CXCR7治疗后促炎因子TNF-α浓度较MSC-GFP组进一步下降(24h: 6.665±0.349 vs. 9.963±0.382 ng/ml, §p0.001; 72h: 7.592±0.434 vs. 10.718±0.769ng/ml, §p0.001), IL-10浓度则较MSC-GFP组显著升高(24h: 176.432±4.431 vs. 148.082±4.469ng/ml, p0.001; 72h: 176.300±2.508vs. 143.947±8.179ng/ml, */X0.05);并且在MSC-CXCR7MSC-CXCR7治疗72h后在小鼠肺组织内检测到的TNF-α浓度比治疗24h更高(sp0.05)。结论：MSC可向ARDS小鼠损伤的肺组织靶向归巢,发挥明显的调控炎症及组织修复作用,改善内皮功能,减轻肺损伤程度；高表达CXCR7则进一步促进了MSC向损伤肺组织的归巢,较普通的MSC更有利于抑制局部炎症反应,从而充分发挥MSC对肺组织的保护作用。
[Abstract]:Objective: To study whether the high-expression chemokine receptor 7 (CXCR7) is in favor of increasing the homing of the mesenchymal stem cells (MSCs) to the lung tissue of the acute respiratory distress syndrome (ARDS), thereby facilitating the control of the inflammatory response of the lung tissue and promoting the lung protection of the MSC. Methods:90 BBI/6 mice were randomly divided into control group (NS + PBS), ARDS group (LPS + PBS), MSC group (LPS + MSC), MSC-GFP group (LPS + MSC-GFP) and MSC-CXCR7 (LPS + MSC-CXCR7, and CXCR7 gene was transfected into MSC via lentiviral vector). 5 mg/ kg of lipopolysaccharide (LPS) was added to the airway to replicate the model of ARDS mice. After the model was made for 4 h, the following indexes were observed after the injection of the same amount of normal saline or the cell suspension containing the MSC (30 min,24 h and 72 h):1) Comparison of the homing of the MSC to the lung tissue: using the near-infrared in-vitro lung tissue imaging, The content of adhesion factor, such as vascular cell adhesion molecule-1 (VCAM-1) and recombinant human collagen-1 (COL-1), was measured by enzyme-linked immunosorbent assay (ELISA). 2) Comparison of the severity of lung injury in mice: the mice were sacrificed at all time points to take the lung tissue, the general pathological injury was observed, the pathological examination was performed by HE staining, and the lung injury score was carried out, and the lung wet weight/ body weight ratio was calculated to evaluate the degree of pulmonary edema; 3) Comparison of the local inflammatory response of the lung tissue: the concentration of the anti-inflammatory factor IL-10 and the pro-inflammatory factor TNF-1 in the lung tissue was detected by ELISA. Results:1. The experiment was divided into 5 groups and 3 experimental time points (n = 6), and the pathological examination of the lung tissue was performed in the mice. The results showed that the pulmonary interstitial and alveolar obvious hemorrhage and edema, the infiltration of a large amount of inflammatory cells and the collapse of the alveolar structure were observed in the airway. The results showed that the model of ARDS was successfully established.2. The comparison of each group of MSC to the homing of lung tissue in ARDS mice:1) The results of the near-infrared in-vitro organ imaging showed that compared with the control group, The obvious fluorescence signal can be observed in the lung tissue after 30 minutes after the mouse tail vein is administered to the MSC-GFP treatment, the peak value of the 24-hour fluorescence signal is reduced, and the fluorescence signal intensity in the lung tissue is qualitatively observed and quantitatively analyzed after the MSC of the high-expression CXCR7 is administered, The results showed that the signals of MSC-CXCR7 group were significantly enhanced in 24 h and 72 h than in the MSC-GFP group (24 h: 301.62-187.12 vs. 71.75-32.37 scalded counterts/ mm2,7-0.05;72 h: 217.02-126.38 vs. 67.08-26.44 scalded counterts/ mm2, # p0.05);2) the qualitative observation of lung tissue fluorescence microscopy showed that, The results showed that the fluorescence signal of the MSC-CXCR7 group was stronger than that of the MSC-GFP group at 24 h after the MSC-GFP was transplanted into the mouse, and the fluorescence signal was decreased after 72 h; however, the fluorescence signal observed in the lung tissues of the MSC-CXCR7 group in the 24 h and 72 h lung tissues was stronger than that of the MSC-GFP group; 3) The level of adhesion factor related to the homing of MSC in lung tissue of mice was detected by ELISA. The results showed that the concentration of VCAM-1 in the lung of the control group was lower, and the concentration of VCAM-1 in the lung tissue after the successful construction of the ARDS was increased, and the level of VCAM-1 in the lung tissue after the treatment with MSC-GFP was further increased. The level of adhesion factor in the lung of mice treated with MSC-CXCR7 was more significant (24 h: 0.873, 0.021 vs. 0.463, 0.021 ng/ ml, * p0.05;72 h: 1.340, 0.141 vs. 0.512, 0.038 ng/ ml, p0.05); the level of COL-1 in the lung tissue of each experimental group was the same as that of VCAM-1, In the 24 h and 72 h mice treated with MSC-CXCR7, the intra-lung COL-1 concentration was significantly higher than that in the MSC-GFP group (24 h: 1.738, 0.247 vs. 0.977, 0.133 ng/ ml, p0.05;72 h: 4.137, 0.386 vs. 3.597, 0.197 ng/ ml, p0.05). The MSC-GFP of high-expression CXCR7 was more beneficial to the reduction of lung tissue hemorrhage, inflammatory cell infiltration and the formation of hyaline membrane, and decreased the wet weight/ weight of the lung (24 h: 5.98, 0.63 vs. 7.33, 0.53 mg/ g, p0.05;72 h: 7.37, 0.85 vs. 8.97, 1.25 mg/ g, */ K0.05) and the pathological injury score (30 min: 10.20, 0.40 vs. 11.80) 0.78, * p0.05; 24h: 8.33卤0.67vs. 12.87卤0.38, ^O.OOl; 72h: 10.00卤0.26vs. 14.00卤0.72, *p0.001). 4. Compared with the control group, the level of TNF-1 in the lung of the ARDS group was higher than that of the control group, while the IL-10 level of the anti-inflammatory factor decreased, and the levels of TNF-1 in the lung tissue of the mice were significantly reduced after 24 h and 72 h after the treatment with MSC-GFP, but the level of IL-10 increased. However, in the treatment of MSC-CXCR7, the pro-inflammatory factor TNF-1 concentration decreased further (24 h: 6.665, 0.349 vs. 9.963, 0.382 ng/ ml, p.001;72 h: 7.592, 0.434 vs. 10.718, 0.769 ng/ ml, p0.001), and the IL-10 concentration was significantly higher than that of the MSC-GFP group (24 h: 176.432, 4.431 vs. 148.082, 4.469 ng/ ml, p0.001;72 h: 176.300) 2.508 vs. 143.947 ng/ ml, */ X0.05); and the TNF-concentration detected in the mouse lung tissue after 72 h at the MSC-CXCR7MSC-CXCR7 was higher than the treatment for 24 h (sp0.05). Conclusion: The MSC can target the lung tissue injured by ARDS to target the nest, play a significant role in regulating the inflammation and tissue repair, improve the endothelial function and reduce the degree of lung injury. The high expression of the CXCR7 further promotes the homing of the MSC to the injured lung tissue. The more common MSC is more beneficial to the inhibition of local inflammatory response, thus giving full play to the protective effect of the MSC on the lung tissue.
【学位授予单位】：东南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R563.8

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