骨髓间充质干细胞增殖及对肺损伤修复的研究

发布时间：2018-06-07 11:38

本文选题：骨髓间充质干细胞 + 增殖　；参考：《大连医科大学》2012年硕士论文

【摘要】：目的：本研究以SD大鼠为动物模型，研究大鼠BMSCs体外分离培养、鉴定和增殖，评价不同浓度的LPS对BMSCs增殖能力的影响；并探讨通过增殖的BMSCs对ALI修复的可行性。方法： 1.原代BMSCs的分离培养、诱导分化鉴定、流式细胞鉴定，并观察不同代的BMSCs的生长状况。给予不同浓度的LPS与BMSCs共培养，利用MTT法判断BMSCs增殖时LPS的最适浓度。 2.随机从28只雌性大鼠中取24只，经腹腔注入0.5ml浓度为5mg/kg的LPS建立ALI，造模0.5h后，将其随机分成3组：治疗组A（8只，经尾静脉注入浓度为1×104/ml的BMSCs，体积为0.5ml），治疗组B（8只，，经尾静脉注入预先用浓度为0.01μg/mlLPS共培养的BMSCs1×104/ml，体积为0.5ml），损伤组（8只，经尾静脉注入0.5mlPBS），对照组（4只，经尾静脉注入等量的PBS1ml）。24h后检测各组大鼠的血气指标、肺湿干比、病理变化、及支气管肺泡灌洗液中TNF-α和IL-10的变化。结果： 1.分离培养的BMSCs呈现长梭形，紧密排列似漩涡状。经诱导液诱导后，能分化成脂肪细胞和成骨细胞。流式鉴定表面抗原CD29和CD90表达阳性，CD45和CD11表达阴性，符合骨髓间充质干细胞特性。MTT法显示第3代和第5代相比第7代的BMSCs增殖较旺盛。浓度为0.01μg/ml的LPS对BMSCs增殖作用最显著。 2.成功造模24h后，损伤组明显出现低氧、二氧化碳潴留和酸中毒，肺湿干比值较正常高，病理切片示肺泡间隔断裂增宽，组织疏松，并有炎性细胞侵润。BALF中的TNF-α较对照组明显增高、IL-10较对照组明显降低。治疗组血气分析指标趋向正常，肺湿干比趋向正常，病理切片肺泡结构相对均匀完整，肺泡间隔增宽不明显，炎性细胞侵润明显减少，BALF中的TNF-α较损伤组明显降低、IL-10较损伤组明显增高，而且治疗组B比治疗组A效果佳。结论： 1.BMSCs经诱导可分化成脂肪细胞、成骨细胞。 2.本实验用浓度为0.01μg/ml的LPS刺激BMSCs后其增殖能力增强。 3.本实验经腹腔注入浓度5mg/kg的LPS可建立ALI模型。 4.注入增殖的BMSCs后可改善病理学变化，有助于促炎因子TNF-α的降低和抗炎因子IL-10的增高。
[Abstract]:Aim: to study the effects of different concentrations of LPS on the proliferation of BMSCs and to explore the feasibility of ALI repair by proliferating BMSCs in this study, using SD rats as an animal model to study the isolation, culture, identification and proliferation of rat BMSCs in vitro, and to evaluate the effect of different concentrations of LPS on the proliferation of BMSCs. Methods: 1. Isolation and culture of primary BMSCs, identification of induced differentiation, flow cytometry, and observation of the growth status of BMSCs in different generations. Different concentrations of LPS and BMSCs were co-cultured and the optimum concentration of LPS was determined by MTT method. 2. Twenty-four female rats were randomly divided into three groups after intraperitoneal injection of LPS with 0.5ml concentration of 5mg/kg for 0.5 h. The rats in the treatment group were randomly divided into three groups: 8 rats in the treatment group were injected with 1 脳 104/ml BMSCs through the caudal vein, 8 rats in the treatment group were injected with BMS at the concentration of 1 脳 104/ml, and 8 rats in the treatment group were injected with LPS at the concentration of 1 脳 104/ml. The rats in the injury group were injected with 0. 01 渭 g/mlLPS co-cultured BMSCs1 脳 104 / ml (0.5 ml / ml), 8 rats in the injury group and 4 rats in the control group were injected through the tail vein. The blood gas index, lung wet / dry ratio and pathological changes were measured after the same amount of PBS1ml).24h was injected into the tail vein. The changes of TNF- 伪 and IL-10 in bronchoalveolar lavage fluid. Results: 1. The isolated BMSCs showed a long fusiform shape, closely arranged like a whirlpool. After induction, adipocytes and osteoblasts could be differentiated into adipocytes and osteoblasts. Flow cytometry showed that the positive expression of CD29 and CD90 was negative, which was consistent with the characteristics of bone marrow mesenchymal stem cells. The proliferation of BMSCs in the third and fifth generation was stronger than that in the seventh generation. LPS at the concentration of 0. 01 渭 g/ml had the most significant effect on the proliferation of BMSCs. 2. 24 hours after successful modeling, hypoxia, carbon dioxide retention, acidosis, wet / dry ratio of lung were higher than normal in the injury group. Pathological sections showed that the alveolar septum was broken and widened, and the tissue was loose. TNF- 伪 in BALF with inflammatory cell infiltration was significantly higher than that in control group and IL-10 was significantly lower than that in control group. In the treatment group, the indexes of blood gas analysis tended to be normal, the wet / dry lung ratio tended to be normal, the alveolar structure was relatively uniform and intact, and the alveolar septum was not widened obviously in the treatment group. TNF- 伪 in BALF was significantly reduced by inflammatory cells, and IL-10 was significantly increased in BALF than in injury group, and the effect of treatment group B was better than that of treatment group A. Conclusion: 1.BMSCs can be induced to differentiate into adipocytes and osteoblasts. 2. In this experiment, the proliferation ability of BMSCs was enhanced by LPS at a concentration of 0. 01 渭 g/ml. 3. In this experiment, ALI model could be established by intraperitoneal injection of LPS with concentration of 5mg/kg. 4. Injection of proliferative BMSCs can improve pathological changes and contribute to the decrease of pro-inflammatory factor TNF- 伪 and the increase of anti-inflammatory factor IL-10.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R563

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