骨髓間充質(zhì)干細(xì)胞不同時相干預(yù)對大鼠急性肺損傷炎性因子表達(dá)的影響及其治療作用
發(fā)布時間：2018-09-10 09:00
【摘要】：研究背景和研究目的:急性肺損傷(acute lung injury,ALI)是各種病因,包括肺內(nèi)因素和(或)肺外因素所致的肺泡上皮細(xì)胞和肺內(nèi)毛細(xì)血管內(nèi)皮損傷,并由此引起肺泡膜通透性改變、肺泡表面活性物質(zhì)被破壞、彌漫性肺間質(zhì)及肺泡水腫、透明膜形成、肺泡萎陷。急性呼吸窘迫綜合征(acute respiratory distress syndrome,ARDS)則是急性肺損傷發(fā)生、發(fā)展的結(jié)果。盡管機(jī)械通氣策略的進(jìn)步、體外膜肺氧合(extracorporeal membrane oxygenation,ECMO)的出現(xiàn),其病死率仍然居高不下。為了尋求更好的治療手段,學(xué)者們開始將目光聚焦在具有組織修復(fù)及再生能力的骨髓間充質(zhì)干細(xì)胞上。骨髓間充質(zhì)干細(xì)胞(bone marrow mesenchymal stem cells,BM-MSCs)應(yīng)用于ARDS的治療是一種非常有應(yīng)用前景的手段,研究表明,BM-MSCs能夠歸巢于組織受損部位,并通過細(xì)胞免疫調(diào)節(jié)在受損部位的炎癥微環(huán)境中發(fā)揮抗炎、抗水腫、減輕內(nèi)皮細(xì)胞通透性等作用,并且BM-MSCs能夠通過自身分泌抗菌肽、增強(qiáng)巨噬細(xì)胞吞噬能力、調(diào)節(jié)T細(xì)胞等方式,從而發(fā)揮抗菌作用。此外,近年研究表明,BM-MSCs可能通過線粒體跨細(xì)胞轉(zhuǎn)移到ALI/ARDS的受損肺泡上皮細(xì)胞中,挽救損傷的肺泡上皮,從而減少肺水腫的可能。盡管BM-MSCs具備眾多抗炎作用以及治療急性炎癥所致肺部疾病效果較理想,然而臨床上應(yīng)用其治療慢性炎癥所致肺部疾病仍有爭議。因此,探究BM-MSCs不同時相干預(yù)對急性肺損傷的炎癥因子變化及修復(fù)作用能在臨床應(yīng)用提供一定的理論依據(jù)。研究方法:1、通過從SD大鼠長骨中提取骨髓細(xì)胞,通過細(xì)胞貼壁法分離及純化骨髓間充質(zhì)干細(xì)胞;2、BM-MSCs傳代至第3代后,通過定向誘導(dǎo),誘導(dǎo)其向成骨、成脂肪、成軟骨方向分化;3、利用免疫熒光抗體對該細(xì)胞表面CD分子標(biāo)記,通過流式細(xì)胞術(shù)對細(xì)胞表面CD分子陽性表達(dá)率進(jìn)行檢測,對所培養(yǎng)的BM-MSCs進(jìn)行鑒定;4、急性肺損傷動物模型主要通過尾靜脈注射脂多糖(lipopolysaccharide,LPS)構(gòu)建SD大鼠急性肺損傷動物模型,并通過病理評分及動物動脈血?dú)夥治鰴z測建模情況,動物病理評分參照2011年《美國胸科協(xié)會動物急性肺損傷病理評分》進(jìn)行評分;5、120只SD大鼠隨機(jī)分成N組(對照組)、L組(LPS組)及L+M組(LPS+MSCs組),每組40只,并根據(jù)MSCs干預(yù)的不同時間將各組分成2h、8h、24h、48h、96h五個亞組,每亞組8只;N組動物給予等劑量PBS注射,L組均在起始時間尾靜脈注射LPS,劑量為5mg/kg,分別在上述時相予1x106/ml X1ml的劑量MSCs處理后的24h檢測支氣管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)中炎性細(xì)胞、炎性標(biāo)記物(TNF-α、IL-1α、IL-10)、動脈血血?dú)夥治�、肺組織病理評分等。研究結(jié)果:1、成功分離、培養(yǎng)并純化大鼠骨髓間充質(zhì)干細(xì)胞成功分離出骨髓貼壁細(xì)胞,將其純化后,誘導(dǎo)其向成脂、成骨、成軟骨分化,具備多向分化潛能;流式細(xì)胞術(shù)檢測細(xì)胞表面CD分子結(jié)果為,CD90,CD105陽性率分別為98.89%,97.37%(強(qiáng)表達(dá)間充質(zhì)抗原標(biāo)記物);CD45,CD34陽性率分別為3.17%,1.41%(弱表達(dá)血管內(nèi)皮表面抗原標(biāo)記物及造血系細(xì)胞表面抗原標(biāo)記物)。結(jié)果表明所分離純化的細(xì)胞即為大鼠骨髓間充質(zhì)干細(xì)胞。2、脂多糖明顯降低大鼠Pa O2,BM-MSCs干預(yù)使Pa O2升高在2h顯著與同時相N組相比,2h L組、8h L組、24h L組、48h L組Pa O2均有明顯下降(P0.01);與同時相L組比較,2h L+M組Pa O2明顯升高(P0.05),而其余L+M組無明顯差異(P0.05)。3、脂多糖明顯升高BALF中TNF-α、IL-1α、IL-10濃度,BM-MSCs干預(yù)可明顯降低BALF中TNF-α、IL-1α濃度,并明顯升高IL-10濃度TNF-α濃度比較:與同時相N組相比,各時相的L組TNF-α濃度均明顯增加(P0.05);與同時相L組比較,2h L+M組、8h L+M組、24h L+M組、48h L+M組TNF-α濃度均明顯降低(P0.05),而其余組無明顯差異(P0.05)。IL-1α濃度比較:與同時相N相比,各時相的L組IL-1α濃度均明顯增加(P0.05);與同時相L組比較,2h L+M組、8h L+M組、24h L+M組IL-1α濃度降低(P0.01),而其余L+M組無顯著性差異(P0.05)。IL-10濃度比較:與同時相N組相比,2h L組、8h L組IL-10的濃度明顯增加(P0.01),而其余L組無顯著性差異;與同時相L組比較,各時相L+M組IL-10的濃度均增加(P0.05)。4、脂多糖使BALF中PMN明顯增多,BM-MSCs干預(yù)使PMN降低在2h、8h顯著與同時相N組相比,各時相L組PMN總數(shù)均有明顯增加(P0.01);與同時相L組比較,2h L+M組、8h L+M組PMN總數(shù)明顯下降(P0.05),其余L+M組無顯著性差異(P0.05)。5、脂多糖使肺濕/干(W/D)明顯增加,BM-MSCs干預(yù)使PMN降低在2h、8h、24h顯著與同時相N組相比,各時相L組W/D均有增加(P0.01);與同時相L組比較,2h L+M組、8h L+M組、24h L+M組W/D均有降低(P0.05),而其余L+M組雖W/D無顯著性差異(P0.05)。6、脂多糖導(dǎo)致急性肺損傷,BM-MSCs干預(yù)對脂多糖誘導(dǎo)急性肺損傷病理評分降低在2h、8h、24h顯著與同時相N組比較,各時相L組ALI病理評分均有增加(P0.05);與同時相L組比較,2h L+M組、8h L+M組、24h L+M組ALI病理評分均有下降(P0.05),而其余L+M組無顯著性差異。結(jié)論:1、從骨髓中體外分離出貼壁細(xì)胞,通過貼壁法純化細(xì)胞,將其進(jìn)行定向誘導(dǎo)分化為骨細(xì)胞、脂肪細(xì)胞及軟骨細(xì)胞,并通過流式細(xì)胞術(shù)檢測細(xì)胞表面簇分化抗原(CD分子),結(jié)果提示所純化的貼壁細(xì)胞即為骨髓間充質(zhì)干細(xì)胞。2、在早期應(yīng)用BM-MSCs干預(yù),能夠更明顯地減輕脂多糖誘導(dǎo)的大鼠急性肺損傷BALF中的炎性因子,減輕炎性因子表達(dá)及炎性細(xì)胞浸潤,改善肺組織損傷程度,改善動脈血氧分壓。
[Abstract]:BACKGROUND AND RESEARCH OBJECTIVES: Acute lung injury (ALI) is caused by various etiologies, including alveolar epithelial cells and pulmonary capillary endothelial cells damaged by endopulmonary and/or extrapulmonary factors, resulting in changes in alveolar membrane permeability, destruction of alveolar surfactant, diffuse interstitial and alveolar edema, hyaline membrane. Acute respiratory distress syndrome (ARDS) is the result of acute lung injury. Despite advances in mechanical ventilation strategies and the emergence of extracorporeal membrane oxygenation (ECMO), mortality remains high. Bone marrow mesenchymal stem cells (BM-MSCs) can be used in the treatment of ARDS. It has been shown that BM-MSCs can homing in the damaged tissue and through fine-grained tissue. Cellular immune regulation plays an anti-inflammatory, anti-edema and anti-endothelial cell permeability role in the inflammation microenvironment of the damaged site. BM-MSCs can play an anti-bacterial role by secreting antimicrobial peptides, enhancing macrophage phagocytosis, regulating T cells and so on. In addition, recent studies have shown that BM-MSCs may be through mitochondrial transgranulation. Although BM-MSCs have many anti-inflammatory effects and are ideal for the treatment of acute inflammation-induced lung diseases, the clinical application of BM-MSCs in the treatment of chronic inflammation-induced lung diseases is still controversial. Methods: 1. Bone marrow cells were extracted from the long bones of SD rats, and bone marrow mesenchymal stem cells were isolated and purified by cell adherence method; 2. BM-MSCs were subcultured to the 3rd generation, and then directed to induce their orientation. Osteogenesis, adipogenesis and cartilage differentiation; 3. Immunofluorescent antibody was used to detect the expression of CD molecule on the cell surface, and the BM-MSCs were identified by flow cytometry; 4. Acute lung injury animal model was mainly constructed by tail vein injection of lipopolysaccharide (LPS). Animal models of acute lung injury were established in SD rats, and the pathological scores and arterial blood gas analysis were used to detect the model. The pathological scores of SD rats were scored according to 2011 < American Thoracic Association Animal Acute Lung Injury Pathological Score >; 5,120 SD rats were randomly divided into N group (control group), L group (LPS group) and L + M group (LPS + MSCs group), 40 rats in each group and 40 rats in each group. Each group was divided into five subgroups at different intervals of MSCs: 2h, 8h, 24h, 48h and 96h, with 8 rats in each subgroup; animals in group N were injected with the same dose of PBS, and rats in group L were injected with LPS at the tail vein at the beginning of the intervention at a dose of 5mg/kg. The bronchoalveolar lavage fluid (BALF) was detected 24 hours after MSCs treatment at the same time of 1x106/ml X1ml. Inflammatory cells, inflammatory markers (TNF-a, IL-1a, IL-10), arterial blood gas analysis, pathological score of lung tissue in BALF were isolated, cultured and purified successfully. Bone marrow adherent cells were successfully isolated and purified from rat bone marrow mesenchymal stem cells. The positive rates of CD90 and CD105 were 98.89% and 97.37% respectively (strongly expressed mesenchymal antigen markers), 3.17% and 1.41% respectively (weakly expressed vascular endothelial surface antigen markers and hematopoietic cell surface antigen markers). Bone marrow mesenchymal stem cells. 2, lipopolysaccharide significantly decreased PaO 2 in rats, BM-MSCs intervention significantly increased PaO 2 in 2 hours compared with the same phase N group, 2H L group, 8h L group, 24h L group, 48h L group PaO 2 significantly decreased (P 0.01); compared with the same phase L group, 2H L+M group PaO 2 significantly increased (P 0.05), while the other L+M group had no significant difference (P 0.05). The concentrations of TNF-a, IL-1a and IL-10 in BALF were significantly increased by BM-MSCs intervention. The concentrations of TNF-a and IL-1a in BALF were significantly decreased by BM-MSCs intervention, and the concentrations of TNF-a in BALF were significantly increased by BM-MSCs intervention compared with N group (P 0.05). Compared with the same phase N, the concentration of IL-1a in L group increased significantly (P 0.05); compared with the same phase L group, the concentration of IL-1a in 2H L+M group, 8h L+M group, 24h L+M group decreased significantly (P 0.01), but there was no significant difference in the other L+M group (P 0.05). Compared with 2H L group, the concentration of IL-10 in 8h L group increased significantly (P 0.01), but there was no significant difference in other L groups. Compared with the same phase L group, the concentration of IL-10 in L+M group increased significantly (P 0.05). Compared with the same phase L group, the total number of PMN in 2H L+M group, 8h L+M group decreased significantly (P 0.05), the other L+M group had no significant difference (P 0.05). The W/D of L+M group decreased significantly (P 0.05), while the W/D of other L+M group had no significant difference (P 0.05). LPS induced acute lung injury, BM-MSCs intervention on LPS-induced acute lung injury pathological score decreased significantly in 2 hours, 8 hours, 24 hours compared with the same N group, the ALI pathological score of L group increased significantly (P 0.05). Conclusion: 1. Adhesive cells were isolated from bone marrow in vitro and purified by adherent method. The cells were directionally induced to differentiate into osteocytes, adipocytes and chondrocytes, and the surface cluster differentiation antigens (CD scores) were detected by flow cytometry. The results suggest that the purified adherent cells are bone marrow mesenchymal stem cells.
【學(xué)位授予單位】：廣東藥科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R563.8
本文編號：2233985
[Abstract]:BACKGROUND AND RESEARCH OBJECTIVES: Acute lung injury (ALI) is caused by various etiologies, including alveolar epithelial cells and pulmonary capillary endothelial cells damaged by endopulmonary and/or extrapulmonary factors, resulting in changes in alveolar membrane permeability, destruction of alveolar surfactant, diffuse interstitial and alveolar edema, hyaline membrane. Acute respiratory distress syndrome (ARDS) is the result of acute lung injury. Despite advances in mechanical ventilation strategies and the emergence of extracorporeal membrane oxygenation (ECMO), mortality remains high. Bone marrow mesenchymal stem cells (BM-MSCs) can be used in the treatment of ARDS. It has been shown that BM-MSCs can homing in the damaged tissue and through fine-grained tissue. Cellular immune regulation plays an anti-inflammatory, anti-edema and anti-endothelial cell permeability role in the inflammation microenvironment of the damaged site. BM-MSCs can play an anti-bacterial role by secreting antimicrobial peptides, enhancing macrophage phagocytosis, regulating T cells and so on. In addition, recent studies have shown that BM-MSCs may be through mitochondrial transgranulation. Although BM-MSCs have many anti-inflammatory effects and are ideal for the treatment of acute inflammation-induced lung diseases, the clinical application of BM-MSCs in the treatment of chronic inflammation-induced lung diseases is still controversial. Methods: 1. Bone marrow cells were extracted from the long bones of SD rats, and bone marrow mesenchymal stem cells were isolated and purified by cell adherence method; 2. BM-MSCs were subcultured to the 3rd generation, and then directed to induce their orientation. Osteogenesis, adipogenesis and cartilage differentiation; 3. Immunofluorescent antibody was used to detect the expression of CD molecule on the cell surface, and the BM-MSCs were identified by flow cytometry; 4. Acute lung injury animal model was mainly constructed by tail vein injection of lipopolysaccharide (LPS). Animal models of acute lung injury were established in SD rats, and the pathological scores and arterial blood gas analysis were used to detect the model. The pathological scores of SD rats were scored according to 2011 < American Thoracic Association Animal Acute Lung Injury Pathological Score >; 5,120 SD rats were randomly divided into N group (control group), L group (LPS group) and L + M group (LPS + MSCs group), 40 rats in each group and 40 rats in each group. Each group was divided into five subgroups at different intervals of MSCs: 2h, 8h, 24h, 48h and 96h, with 8 rats in each subgroup; animals in group N were injected with the same dose of PBS, and rats in group L were injected with LPS at the tail vein at the beginning of the intervention at a dose of 5mg/kg. The bronchoalveolar lavage fluid (BALF) was detected 24 hours after MSCs treatment at the same time of 1x106/ml X1ml. Inflammatory cells, inflammatory markers (TNF-a, IL-1a, IL-10), arterial blood gas analysis, pathological score of lung tissue in BALF were isolated, cultured and purified successfully. Bone marrow adherent cells were successfully isolated and purified from rat bone marrow mesenchymal stem cells. The positive rates of CD90 and CD105 were 98.89% and 97.37% respectively (strongly expressed mesenchymal antigen markers), 3.17% and 1.41% respectively (weakly expressed vascular endothelial surface antigen markers and hematopoietic cell surface antigen markers). Bone marrow mesenchymal stem cells. 2, lipopolysaccharide significantly decreased PaO 2 in rats, BM-MSCs intervention significantly increased PaO 2 in 2 hours compared with the same phase N group, 2H L group, 8h L group, 24h L group, 48h L group PaO 2 significantly decreased (P 0.01); compared with the same phase L group, 2H L+M group PaO 2 significantly increased (P 0.05), while the other L+M group had no significant difference (P 0.05). The concentrations of TNF-a, IL-1a and IL-10 in BALF were significantly increased by BM-MSCs intervention. The concentrations of TNF-a and IL-1a in BALF were significantly decreased by BM-MSCs intervention, and the concentrations of TNF-a in BALF were significantly increased by BM-MSCs intervention compared with N group (P 0.05). Compared with the same phase N, the concentration of IL-1a in L group increased significantly (P 0.05); compared with the same phase L group, the concentration of IL-1a in 2H L+M group, 8h L+M group, 24h L+M group decreased significantly (P 0.01), but there was no significant difference in the other L+M group (P 0.05). Compared with 2H L group, the concentration of IL-10 in 8h L group increased significantly (P 0.01), but there was no significant difference in other L groups. Compared with the same phase L group, the concentration of IL-10 in L+M group increased significantly (P 0.05). Compared with the same phase L group, the total number of PMN in 2H L+M group, 8h L+M group decreased significantly (P 0.05), the other L+M group had no significant difference (P 0.05). The W/D of L+M group decreased significantly (P 0.05), while the W/D of other L+M group had no significant difference (P 0.05). LPS induced acute lung injury, BM-MSCs intervention on LPS-induced acute lung injury pathological score decreased significantly in 2 hours, 8 hours, 24 hours compared with the same N group, the ALI pathological score of L group increased significantly (P 0.05). Conclusion: 1. Adhesive cells were isolated from bone marrow in vitro and purified by adherent method. The cells were directionally induced to differentiate into osteocytes, adipocytes and chondrocytes, and the surface cluster differentiation antigens (CD scores) were detected by flow cytometry. The results suggest that the purified adherent cells are bone marrow mesenchymal stem cells.
【學(xué)位授予單位】：廣東藥科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R563.8
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