KGF基因修饰的骨髓间充质干细胞移植治疗内毒素致小鼠急性肺损伤及相关机制的实验研究

发布时间：2018-07-31 21:01

【摘要】：背景和目的：急性肺损伤（acute lung injury，ALI）和急性呼吸窘迫综合征（acute respiratorydistress syndrome，ARDS）是各种致病因素导致的急性进行性呼吸衰竭，其最常见的致病因素为肺部感染。国内外学者对ARDS的治疗进行了大量的研究并取得了一定的进展，但并不能从根本上改善不良预后，其死亡率仍高达40%。骨髓间充质干细胞（mesenchymal stem cells，MSCs）作为一种多潜能干细胞，取材方便，易于分离扩增，且能免疫耐受并能转染和长期表达外源性基因，是理想的组织修复来源和基因转染的稳定载体，因而其在ARDS/ALI治疗中具有重要的应用价值。角质细胞生长因子（keratinocyte growth factor，KGF）是成纤维细胞生长因子(fibroblast growth factor, FGF)家族的第7位成员，具有促进上皮细胞有丝分裂、调控细胞分化、抗凋亡及促进表面活性物质结合蛋白（surfactant-associatedprotein，SP）的合成等作用，在ALI的治疗研究中具有广阔的应用前景。本研究旨在观察KGF基因修饰的间充质干细胞回输体内对内毒素（lipopolysaccharide, LPS）所致的肺损伤模型的生物学作用并探讨其相关的机制，，为ALI的治疗提供一种新的具有突破性的实验线索。方法和结果采用全骨髓贴壁法分离MSCs，培养和扩增至第四代，经流式细胞仪和分化鉴定，得到纯度较高的MSCs。同时构建KGF重组质粒感染293T细胞，浓缩纯化后收集病毒颗粒，再分别感染小鼠MSCs，荧光显微镜观察两组感染效率均达90%，经real-time定量PCR法和Western-blot法分析KGF基因表达良好。经小鼠气管内滴注LPS10mg/kg成功构建ALI模型后，160只ALI模型小鼠随机分为生理盐水对照组(NS组)、单纯MSCs对照组（MSCs组）、MSCs空载对照组（MSCs-vec组）和携带KGF的MSCs实验组（MSCs-KGF组）。结果显示：与对照组相比，MSCs可定植于肺组织内；在24h和3d时间点，MSCs-KGF实验组KGF的RNA和蛋白质在体内表达明显增高（P0.05），同时对降低肺湿干比、BALF中总蛋白含量、BALF中中性粒细胞数量及肺组织髓过氧化物酶（MPO）活性有统计学意义（P0.05），而且显著降低了BALF中促炎因子水平而升高了抗炎因子水平。MSCs-KGF实验组肺组织病理切片的病变程度轻于对照组，病理评分有统计学意义（P0.05）。另取40只小鼠随机分为上述4组单独测生存率，结果提示其对改善生存率有一定优势（P0.05）。同时MSCs-KGF实验组SPB和SPC的mRNA水平与对照组比较明显增加（P0.05）,免疫组化示MSCs-KGF实验组PCNA阳性的细胞较对照组增多。结论： 1.运用全骨髓贴壁法能成功从小鼠骨髓中分离MSCs，并可在体外培养扩增。 2. KGF慢病毒载体能感染MSCs，并能高效率、长时间在MSCs内稳定表达KGF，MSCs是良好的基因治疗运载工具。 3. KGF基因修饰的MSCs移植后可归巢到受损肺组织，并能在肺内高表达KGF； 4. KGF与MSCs发挥协同作用，可改善肺血管渗透性，抑制肺组织的炎症反应，减轻内毒素诱导的急性肺损伤病变严重程度，并且具有提高ALI动物生存率的趋势，从而为ALI的基因治疗提供了实验依据。 5. KGF发挥作用的潜在机制可能与KGF刺激SP分泌及促进有丝分裂有关。
[Abstract]:Background and purpose:
Acute lung injury (acute lung injury, ALI) and acute respiratory distress syndrome (acute respiratorydistress syndrome, ARDS) are acute progressive respiratory failure caused by various pathogenic factors. The most common pathogenic factor is pulmonary infection. The domestic and foreign scholars have made a great deal of research on the treatment of ARDS and have made certain progress, but they have made some progress. It can not fundamentally improve the poor prognosis, the mortality rate is still as high as 40%.
Mesenchymal stem cells (MSCs), as a kind of pluripotent stem cells, is a kind of pluripotent stem cells, which is convenient, easy to be separated and amplified, and can be immune tolerance and can be transfected and long expressed exogenous genes. It is an ideal source of tissue repair and a stable carrier of gene transfection. Therefore, it has important application value in the treatment of ARDS/ALI.
Keratinocyte growth factor (KGF) is the seventh member of the fibroblast growth factor (fibroblast growth factor, FGF) family, which has the role of promoting mitosis in epithelial cells, regulating cell differentiation, anti apoptosis and promoting the synthesis of surfactant junction protein (surfactant-associatedprotein, SP). The therapeutic research of ALI has a broad application prospect.
The purpose of this study is to observe the biological effects of KGF gene modified mesenchymal stem cells on the model of lung injury induced by lipopolysaccharide (LPS) and explore its related mechanisms, providing a new and breakthrough experimental clue for the treatment of ALI.
Methods and results
The total bone marrow adherent method was used to isolate MSCs and be cultured and amplified to fourth generations. By flow cytometry and differentiation, the high purity MSCs. was obtained and KGF recombinant plasmid was constructed to infect 293T cells. After concentration and purification, the virus particles were collected and then infected with MSCs in mice respectively. The infection efficiency of the two groups was 90%, and PCR was quantified by real-time PCR. The expression of KGF gene was well expressed by method and Western-blot method. After the successful construction of ALI model by intratracheal infusion of LPS10mg/kg in mice, 160 ALI mice were randomly divided into normal saline control group (NS group), pure MSCs control group (MSCs group), MSCs empty control group (MSCs-vec group) and KGF MSCs experimental group (Group). The results showed that the control group was compared with the control group. Compared with the group, MSCs could be colonized in the lung tissue. In the time point of 24h and 3D, the expression of RNA and protein in KGF was significantly increased in the MSCs-KGF experimental group (P0.05), and the total protein content in BALF, the amount of neutrophils in BALF and the activity of myeloperoxidase (MPO) in lung tissue were statistically significant (P0.05), and significantly decreased. The degree of pathological section of lung tissue in the.MSCs-KGF experimental group of BALF was higher than that of the control group, and the pathological score of the lung tissue was less than that of the control group, and the pathological score was statistically significant (P0.05). The other 40 mice were randomly divided into 4 groups, and the results showed that they had some advantages (P0.05) for the improvement of survival rate (P0.05). At the same time, MSCs-KGF was true. The levels of SPB and SPC mRNA in the test group were significantly higher than those in the control group (P 0.05). The number of PCNA positive cells in the MSCs-KGF test group was higher than that in the control group by immunohistochemistry.
Conclusion:
1. MSCs can be successfully isolated from mouse bone marrow by whole bone marrow adherent method and can be amplified in vitro.
2. KGF lentiviral vector can infect MSCs with high efficiency. It can stably express KGF in MSCs for a long time. MSCs is a good carrier for gene therapy.
3. KGF gene modified MSCs can homing to damaged lung tissue and express KGF in lung.
4. KGF and MSCs play a synergistic effect, which can improve pulmonary vascular permeability, inhibit the inflammatory response of lung tissue, reduce the severity of acute lung injury induced by endotoxin, and have a tendency to increase the survival rate of ALI animals, thus providing experimental basis for the gene therapy of ALI.
5. the underlying mechanism of KGF may be related to KGF stimulating SP secretion and promoting mitosis.
【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R563.8

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