發(fā)布時(shí)間：2019-06-26 17:25

【摘要】：目的：探討高表達(dá)趨化因子受體7(CXCR7)是否有利于增加間充質(zhì)干細(xì)胞(Mesenchymal Stem Cells, MSCs)向急性呼吸窘迫綜合征(ARDS)小鼠損傷肺組織的歸巢,從而有利于肺組織炎癥反應(yīng)的控制,促進(jìn)MSC的肺保護(hù)作用。方法：將90只C57BI/6小鼠隨機(jī)分為Control組(NS+PBS), ARDS組(LPS+PBS), MSC組(LPS+MSC), MSC-GFP組(LPS+MSC-GFP)及MSC-CXCR7 組(LPS+MSC-CXCR7, CXCR7基因通過慢病毒載體介導(dǎo)轉(zhuǎn)染至MSC)。氣道內(nèi)滴入5mg/kg的脂多糖(LPS)復(fù)制ARDS小鼠模型,造模4h后按分組給予尾靜脈注射等量的生理鹽水或含MSC的細(xì)胞懸液,30min、 24h及72h后觀察以下指標(biāo)：1)MSC向肺組織的歸巢比較：采用近紅外離體肺組織成像、肺組織熒光鏡檢直接觀察,應(yīng)用酶聯(lián)免疫吸附試驗(yàn)(ELISA)檢測(cè)肺組織相關(guān)粘附因子如血管細(xì)胞粘附分子-1(VCAM-1)及重組人膠原蛋白-1(COL-1)的含量,從器官、組織及分子水平進(jìn)行綜合評(píng)價(jià)；2)小鼠肺損傷嚴(yán)重程度的比較：各時(shí)間點(diǎn)處死小鼠留取肺組織,觀察大體病理?yè)p傷情況,HE染色行組織病理學(xué)檢查并進(jìn)行肺損傷評(píng)分,計(jì)算肺濕重/體重比評(píng)價(jià)肺水腫程度;3)肺組織局部炎癥反應(yīng)的比較：通過ELISA法檢測(cè)肺組織中抑炎因子IL-10及促炎因子TNF-α的濃度反映。結(jié)果：1、本實(shí)驗(yàn)共分5組3個(gè)實(shí)驗(yàn)時(shí)間點(diǎn)(n=6),處死小鼠留取肺組織行病理學(xué)檢查,觀察結(jié)果表明氣道滴入LPS后肺間質(zhì)和肺泡明顯出血水腫、大量炎性細(xì)胞浸潤(rùn),肺泡結(jié)構(gòu)破壞萎陷,提示均成功建立ARDS模型。2、各組MSC向ARDS小鼠損傷肺組織歸巢的比較：1)近紅外離體器官成像結(jié)果表明,與Control組相比,經(jīng)小鼠尾靜脈給予 MSC-GFP治療后30min即可在肺組織內(nèi)觀察到明顯的熒光信號(hào),24h熒光信號(hào)達(dá)峰值,72h有所降低；在給予高表達(dá)CXCR7的MSC治療后定性觀察及定量分析肺組織中熒光信號(hào)強(qiáng)度,結(jié)果表明MSC-CXCR7組在24h及72h的信號(hào)均比MSC-GFP組顯著增強(qiáng)(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)肺組織熒光鏡檢定性觀察結(jié)果表明,在MSC-GFP移植入小鼠體內(nèi)后的24h即可見表達(dá)綠色熒光蛋白的MSC,72h后熒光信號(hào)有所減弱；然而MSC-CXCR7組小鼠在24h及72h肺組織內(nèi)觀察到的熒光信號(hào)均比MSC-GFP組更強(qiáng)；3)ELISA法檢測(cè)小鼠肺組織中與MSC歸巢相關(guān)的粘附因子水平結(jié)果表明,Control組小鼠肺內(nèi)VCAM-1濃度較低,在ARDS模型成功構(gòu)建后的24h及72h,其濃度有所升高,給與MSC-GFP治療后肺組織內(nèi)VCAM-1水平進(jìn)一步升高,與之相比,給與MSC-CXCR7治療的小鼠肺內(nèi)粘附因子水平升高程度更為顯著(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)；各實(shí)驗(yàn)組小鼠肺組織內(nèi)COL-1水平變化趨勢(shì)與VCAM-1相同,在給予MSC-CXCR7治療后的24h及72h小鼠肺內(nèi)COL-1濃度較MSC-GFP組顯著升高(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、各組小鼠肺損傷程度的比較：肺組織大體及組織病理?yè)p傷結(jié)果顯示,高表達(dá)CXCR7的MSC治療較MSC-GFP更有利于減輕肺組織出血、炎細(xì)胞浸潤(rùn)及透明膜形成,降低肺濕重/體重(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?yè)p傷評(píng)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,各組小鼠肺部炎癥反應(yīng)程度的比較：ARDS組小鼠肺內(nèi)促炎因子TNF-α水平較Control組升高,而抑炎因子IL-10水平降低,當(dāng)給予MSC-GFP治療24h及72h后檢測(cè)到小鼠肺組織內(nèi)TNF-α濃度顯著降低,反而IL-10水平有所升高,而在給予MSC-CXCR7治療后促炎因子TNF-α濃度較MSC-GFP組進(jìn)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濃度則較MSC-GFP組顯著升高(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治療72h后在小鼠肺組織內(nèi)檢測(cè)到的TNF-α濃度比治療24h更高(sp0.05)。結(jié)論：MSC可向ARDS小鼠損傷的肺組織靶向歸巢,發(fā)揮明顯的調(diào)控炎癥及組織修復(fù)作用,改善內(nèi)皮功能,減輕肺損傷程度；高表達(dá)CXCR7則進(jìn)一步促進(jìn)了MSC向損傷肺組織的歸巢,較普通的MSC更有利于抑制局部炎癥反應(yīng),從而充分發(fā)揮MSC對(duì)肺組織的保護(hù)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鹵0.67vs. 12.87鹵0.38, ^O.OOl; 72h: 10.00鹵0.26vs. 14.00鹵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.
【學(xué)位授予單位】：東南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R563.8

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