骨髓间充质干细胞不同时相干预对大鼠急性肺损伤炎性因子表达的影响及其治疗作用
发布时间：2018-09-10 09:00
【摘要】：研究背景和研究目的:急性肺损伤(acute lung injury,ALI)是各种病因,包括肺内因素和(或)肺外因素所致的肺泡上皮细胞和肺内毛细血管内皮损伤,并由此引起肺泡膜通透性改变、肺泡表面活性物质被破坏、弥漫性肺间质及肺泡水肿、透明膜形成、肺泡萎陷。急性呼吸窘迫综合征(acute respiratory distress syndrome,ARDS)则是急性肺损伤发生、发展的结果。尽管机械通气策略的进步、体外膜肺氧合(extracorporeal membrane oxygenation,ECMO)的出现,其病死率仍然居高不下。为了寻求更好的治疗手段,学者们开始将目光聚焦在具有组织修复及再生能力的骨髓间充质干细胞上。骨髓间充质干细胞(bone marrow mesenchymal stem cells,BM-MSCs)应用于ARDS的治疗是一种非常有应用前景的手段,研究表明,BM-MSCs能够归巢于组织受损部位,并通过细胞免疫调节在受损部位的炎症微环境中发挥抗炎、抗水肿、减轻内皮细胞通透性等作用,并且BM-MSCs能够通过自身分泌抗菌肽、增强巨噬细胞吞噬能力、调节T细胞等方式,从而发挥抗菌作用。此外,近年研究表明,BM-MSCs可能通过线粒体跨细胞转移到ALI/ARDS的受损肺泡上皮细胞中,挽救损伤的肺泡上皮,从而减少肺水肿的可能。尽管BM-MSCs具备众多抗炎作用以及治疗急性炎症所致肺部疾病效果较理想,然而临床上应用其治疗慢性炎症所致肺部疾病仍有争议。因此,探究BM-MSCs不同时相干预对急性肺损伤的炎症因子变化及修复作用能在临床应用提供一定的理论依据。研究方法:1、通过从SD大鼠长骨中提取骨髓细胞,通过细胞贴壁法分离及纯化骨髓间充质干细胞;2、BM-MSCs传代至第3代后,通过定向诱导,诱导其向成骨、成脂肪、成软骨方向分化;3、利用免疫荧光抗体对该细胞表面CD分子标记,通过流式细胞术对细胞表面CD分子阳性表达率进行检测,对所培养的BM-MSCs进行鉴定;4、急性肺损伤动物模型主要通过尾静脉注射脂多糖(lipopolysaccharide,LPS)构建SD大鼠急性肺损伤动物模型,并通过病理评分及动物动脉血气分析检测建模情况,动物病理评分参照2011年《美国胸科协会动物急性肺损伤病理评分》进行评分;5、120只SD大鼠随机分成N组(对照组)、L组(LPS组)及L+M组(LPS+MSCs组),每组40只,并根据MSCs干预的不同时间将各组分成2h、8h、24h、48h、96h五个亚组,每亚组8只;N组动物给予等剂量PBS注射,L组均在起始时间尾静脉注射LPS,剂量为5mg/kg,分别在上述时相予1x106/ml X1ml的剂量MSCs处理后的24h检测支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)中炎性细胞、炎性标记物(TNF-α、IL-1α、IL-10)、动脉血血气分析、肺组织病理评分等。研究结果:1、成功分离、培养并纯化大鼠骨髓间充质干细胞成功分离出骨髓贴壁细胞,将其纯化后,诱导其向成脂、成骨、成软骨分化,具备多向分化潜能;流式细胞术检测细胞表面CD分子结果为,CD90,CD105阳性率分别为98.89%,97.37%(强表达间充质抗原标记物);CD45,CD34阳性率分别为3.17%,1.41%(弱表达血管内皮表面抗原标记物及造血系细胞表面抗原标记物)。结果表明所分离纯化的细胞即为大鼠骨髓间充质干细胞。2、脂多糖明显降低大鼠Pa O2,BM-MSCs干预使Pa O2升高在2h显著与同时相N组相比,2h L组、8h L组、24h L组、48h L组Pa O2均有明显下降(P0.01);与同时相L组比较,2h L+M组Pa O2明显升高(P0.05),而其余L+M组无明显差异(P0.05)。3、脂多糖明显升高BALF中TNF-α、IL-1α、IL-10浓度,BM-MSCs干预可明显降低BALF中TNF-α、IL-1α浓度,并明显升高IL-10浓度TNF-α浓度比较:与同时相N组相比,各时相的L组TNF-α浓度均明显增加(P0.05);与同时相L组比较,2h L+M组、8h L+M组、24h L+M组、48h L+M组TNF-α浓度均明显降低(P0.05),而其余组无明显差异(P0.05)。IL-1α浓度比较:与同时相N相比,各时相的L组IL-1α浓度均明显增加(P0.05);与同时相L组比较,2h L+M组、8h L+M组、24h L+M组IL-1α浓度降低(P0.01),而其余L+M组无显著性差异(P0.05)。IL-10浓度比较:与同时相N组相比,2h L组、8h L组IL-10的浓度明显增加(P0.01),而其余L组无显著性差异;与同时相L组比较,各时相L+M组IL-10的浓度均增加(P0.05)。4、脂多糖使BALF中PMN明显增多,BM-MSCs干预使PMN降低在2h、8h显著与同时相N组相比,各时相L组PMN总数均有明显增加(P0.01);与同时相L组比较,2h L+M组、8h L+M组PMN总数明显下降(P0.05),其余L+M组无显著性差异(P0.05)。5、脂多糖使肺湿/干(W/D)明显增加,BM-MSCs干预使PMN降低在2h、8h、24h显著与同时相N组相比,各时相L组W/D均有增加(P0.01);与同时相L组比较,2h L+M组、8h L+M组、24h L+M组W/D均有降低(P0.05),而其余L+M组虽W/D无显著性差异(P0.05)。6、脂多糖导致急性肺损伤,BM-MSCs干预对脂多糖诱导急性肺损伤病理评分降低在2h、8h、24h显著与同时相N组比较,各时相L组ALI病理评分均有增加(P0.05);与同时相L组比较,2h L+M组、8h L+M组、24h L+M组ALI病理评分均有下降(P0.05),而其余L+M组无显著性差异。结论:1、从骨髓中体外分离出贴壁细胞,通过贴壁法纯化细胞,将其进行定向诱导分化为骨细胞、脂肪细胞及软骨细胞,并通过流式细胞术检测细胞表面簇分化抗原(CD分子),结果提示所纯化的贴壁细胞即为骨髓间充质干细胞。2、在早期应用BM-MSCs干预,能够更明显地减轻脂多糖诱导的大鼠急性肺损伤BALF中的炎性因子,减轻炎性因子表达及炎性细胞浸润,改善肺组织损伤程度,改善动脉血氧分压。
[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.
【学位授予单位】：广东药科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R563.8
本文编号：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.
【学位授予单位】：广东药科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R563.8
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