骨髓间充质干细胞性别差异对急性肺栓塞治疗效果的影响及机制

发布时间：2018-02-24 19:14

本文关键词： 急性肺栓塞骨髓间充质干细胞细胞治疗连续切片性别差异急性肺栓塞甘油醛-3-磷酸脱氢酶骨髓间充质干细胞　出处：《华中科技大学》2013年博士论文　论文类型：学位论文

【摘要】：急性肺栓塞是临床常见疾病,具有高发病率、高死亡率、高致残率,以及临床表现的多样化,成为治疗的难点。目前,针对肺栓塞的治疗措施以抗凝治疗及溶栓治疗为主。但是药物治疗存在着特异性差、半衰期短、副作用大等缺点。干细胞治疗疾病的前景令人鼓舞,但目前的研究就其是否能够促进血栓溶解还存在争议,并且骨髓间充质干细胞对急性肺栓塞的治疗是否有效还未得到证实。本研究利用颈外静脉注射血栓复制急性肺栓塞动物模型,然后用骨髓间充质干细胞进行治疗,发现治疗有效。为评估治疗效果,在本研究中我们首创应用全肺连续切片的方法统计实际存在的每个栓子的体积,并且与传统的非连续切片统计血栓面积的方法进行比较,证实了对肺栓塞这种多血栓的情况进行定量评估时利用全肺连续切片的必要性。多项研究报道细胞存在性别差异,且发现干细胞在治疗多种疾病都表现为雌性干细胞比雄性干细胞更有优势。利用干细胞治疗血栓的报道很多,但都未探讨性别差异的问题。本研究首次探讨不同性别的骨髓间充质干细胞对急性肺栓塞的治疗效果,发现雌性骨髓间充质干细胞具有更优于雄性干细胞的治疗效果。GAPDH作为一种管家蛋白除了参与糖酵解外,还参与包括DNA修复、tRNA转运、膜融合转运、细胞骨架构建甚至细胞死亡等多种细胞生命活动的调节。有学者对大鼠脑部的研究发现GAPDH的表达存在性别差异。我们的实验也证明正是雌、雄骨髓间充质干细胞的GAPDH表达差异造成了其对急性肺栓塞的治疗差异,具有较高GAPDH基础水平的雌性干细胞对急性肺栓塞的治疗效果更好,而GAPDH的这一作用,可能是通过调控尿激酶型纤溶酶原激活物来实现的。目的观察不同性别骨髓间充质干细胞对急性肺栓塞的治疗作用。方法利用小鼠颈外静脉注射血栓复制急性肺栓塞的动物模型。造模后4h、8h及16h分组经尾静脉注射不同性别的骨髓间充质干细胞2×106个,造模24h后处死小鼠,取肺固定包埋。全肺连续切片,HE染色,精确统计出现的所有栓子,并通过体积计算方法分析治疗差异。结果 1.成功复制急性肺栓塞的动物模型模型组经过血栓注射后24小时,通过切片染色可以看到非常明显的血栓。利用连续切片体积分析方法对模型组和治疗组所有的栓子进行分析,栓子体积主要集中于0.1-5×106μm3,利用非连续切片面积分析方法分析栓子面积主要位于1-10x103μm2间,两种分析方法都显示栓子在肺内的分布右肺多于左肺,且主要位于0-30μm直径的小血管内,这与临床血栓肺内分布基本一致。 2.用总体积比用总面积分析多发血栓更加科学、合理；雌性和雄性骨髓间充质干细胞均对急性肺栓塞有治疗效果,且雌性骨髓间充质干细胞更有优势。用文献常用的面积计算方法来分析模型动物肺血栓情况发现,较连续切片体积计算法血栓总数漏掉了54%,但栓子的分布特点和临床基本一致。用连续切片体积来分析,统计所有出现的栓子,更加精确和客观。栓塞4h后治疗,在雌性小鼠和雄性小鼠中,雌性和雄性骨髓间充质干细胞均有效果,且雌性骨髓间充质干细胞治疗效果更好；栓塞8h后治疗,对于雌性小鼠,雌、雄骨髓间充质干细胞均有效果,但未发现两者的治疗差异,而在雄性小鼠,雌、雄骨髓间充质干细胞均有效果,且雌性骨髓间充质干细胞治疗效果更好；栓塞16h后治疗,雌、雄骨髓间充质干细胞对雌性小鼠均有效果,但两者未发现治疗差异,而在雄性小鼠,仅仅雌性骨髓间充质干细胞治疗有效。结论利用颈外静脉注入血栓法可以成功复制急性肺栓塞动物模型,复制的模型与临床资料肺血栓的分布基本一致,用这种复制血栓的方法来研究肺栓塞是可行可靠的。相对于文献常用面积计算分析血栓的方法,我们独创全肺连续切片计算血栓体积来分析的方法更加准确和客观。雌、雄骨髓间充质干细胞在肺血栓发生的早期治疗均有效,且雌性骨髓间充质干细胞治疗效果更好；而在血栓发生的晚期,仅仅雌性骨髓间充质干细胞治疗有效。目的确定GAPDH在调控雌、雄骨髓间充质干细胞治疗急性肺栓塞效果差异中的作用,进一步探讨骨髓间充质干细胞治疗急性肺栓塞的机制。方法 1.雌性骨髓间充质干细胞稳转GAPDH干扰的质粒或空质粒,雄性骨髓间充质干细胞稳转GAPDH过表达的质粒或空质粒,western blotting检测各细胞中GAPDH的表达情况。 2.复制急性肺栓塞的动物模型。栓塞后4h分组注射调控了GAPDH表达的骨髓间充质干细胞(F-BMSCs-siRNA-GAPDH、F-BMSCs-siRNA vector、 M-BMSCs OE-GAPDH、M-BMSCs OE vector)2×106个,20h后处死小鼠,取肺固定包埋。全肺连续切片,HE染色,精确统计出现的所有栓子,分析治疗差异。 3.全肺连续切片中所有奇数片HE染色,发现有连续的片子出现血栓,其对应的偶数片子做免疫荧光,对干细胞进行追踪；western blotting检测各细胞中uPA的表达情况。结果 1. GAPDH稳定转染的情况未处理的雌性骨髓间充质干细胞中GAPDH的基础表达量要高于雄性骨髓间充质干细胞；雌性骨髓间充质干细胞转染干扰GAPDH的质粒后,其GAPDH表达量明显减少；雄性骨髓间充质干细胞转染GAPDH过表达的质粒后,其GAPDH表达量明显增高。 2. GAPDH转染后的骨髓间充质干细胞对急性肺栓塞的治疗情况急性肺栓塞4h后开始干细胞治疗,干扰GAPDH表达的雌性骨髓间充质干细胞治疗组的血栓体积明显大于单纯雌性骨髓间充质干细胞治疗组,而GAPDH过表达的雄性骨髓间充质干细胞治疗组比单纯雄性骨髓间充质干细胞治疗组血栓体积明显减少,治疗效果明显增强。 3.干细胞追踪及uPA表达情况免疫荧光实验发现绿色荧光标记的骨髓间充质干细胞主要在血管内栓子的外缘附着。雌性骨髓间充质干细胞中uPA表达量高于雄性骨髓间充质干细胞,GAPDH过表达以后的骨髓间充质干细胞uPA表达量明显增高。结论雌性骨髓间充质干细胞具有比雄性骨髓间充质干细胞更好地对肺血栓的治疗效果,其原因可能是雌性骨髓间充质干细胞中具有显著的高于雄性骨髓间充质干细胞的GAPDH表达量。通过干预GAPDH在雌雄骨髓间充质干细胞的表达更验证了雌、雄骨髓间充质干细胞中GAPDH表达水平的不同是造成这两种干细胞对急性肺栓塞治疗差异的原因。干细胞在血栓外缘的定位以及骨髓间充质干细胞中GAPDH可以调控uPA的表达,说明急性肺栓塞的早期骨髓间充质干细胞可能主要通过分泌一些溶栓因子来促进血栓的溶解,从而起到治疗作用。
[Abstract]:Acute pulmonary embolism is a common clinical disease with high morbidity, high mortality, high morbidity, and varied clinical manifestations, become the treatment difficulty. At present, the pulmonary embolism treatment measures to anticoagulant therapy and thrombolytic therapy. But the drugs have poor specificity, short half-life, side effects and other shortcomings stem cells in the treatment of disease. The prospects are encouraging, but the current study is whether it can promote thrombolysis is still controversial, and bone marrow mesenchymal stem cells in treatment of acute pulmonary embolism is effective. This study has not been confirmed by external jugular vein injection of thrombus in animal model of acute pulmonary embolism, and bone marrow mesenchymal stem cells for treatment, that treatment is effective. In order to evaluate the therapeutic effect, in this study we initiated each sub application of statistical methods of whole lung thrombus in serial sections of the actual volume, Compared with the traditional method of non continuous slice statistics for thrombus area, it is confirmed that the necessity of whole lung continuous slice is used for quantitative evaluation of pulmonary thromboembolism.
There are gender differences reported in many studies and found that cells, stem cells in the treatment of various diseases are expressed as female cells than male stem cells have more advantages. The use of stem cells in the treatment of thrombosis reported, but did not investigate the gender differences. This is the first study to explore the gender therapeutic effect of bone marrow mesenchymal stem cells on acute pulmonary embolism, found that female bone marrow mesenchymal stem cells is more than the male dry treatment effect of.GAPDH cells as a housekeeping protein in addition to glycolysis, also involved in DNA repair, tRNA transporter, membrane fusion transporter, regulate cytoskeletal structure and even cell death and other cellular activities. Some scholars the rat brain found gender differences in the expression of GAPDH. Our experiments have shown that it is female, male bone marrow mesenchymal stem cells which are caused by the differences in the expression of GAPDH on acute The treatment of pulmonary embolism is different from that of female stem cells with a higher level of GAPDH, which is better for the treatment of acute pulmonary embolism. The role of GAPDH may be achieved by regulating urokinase type plasminogen activator.
objective
To observe the therapeutic effect of different sex bone marrow mesenchymal stem cells (MSCs) on acute pulmonary embolism.
Method
Using a mouse external jugular vein injection of thrombus replication of acute pulmonary embolism animal model. After modeling, 4h, 8h and 16h in bone marrow and divided into different groups by intravenous injection of sex mesenchymal stem cells 2 * 106 24h after modeling, the mice were killed, the lung paraffin embedded lung. Serial sections, HE staining, all an accurate statistics appear, and the volume calculation method analysis of treatment differences.
Result
1. animal model of successfully replicating acute pulmonary embolism
The model group after 24 hours after injection of thrombus, can see very obvious thrombosis by slicing staining. Analysis method of model group and treatment group all emboli were analyzed by serial sections of volume, volume of emboli mainly focused on 0.1-5 * 106 m3, using the analysis method of non continuous slice area analysis of embolus area mainly located in 1-10x103 m2. Two, analysis methods are shown in the distribution of pulmonary emboli than the right lung left lung, small blood vessels and mainly located in 0-30 m in diameter, and the clinical thrombosis pulmonary distribution is basically the same.
2., it is more scientific and reasonable to analyze multiple thrombus with total volume ratio and total area. Both female and male bone marrow mesenchymal stem cells have therapeutic effect on acute pulmonary embolism, and female bone marrow mesenchymal stem cells have more advantages.
To analyze the animal model of pulmonary embolism was found in the literature area commonly used calculation method, a continuous slice volume calculation method of thrombus total missing 54%, but the distribution and clinical characteristics of emboli are basically the same. The serial sections were used to analyze the volume, statistics of all the emboli, more accurate and objective. 4h after embolization treatment in female mice male and female and male mice, bone marrow mesenchymal stem cells have the effect, and the female bone marrow mesenchymal stem cells to better treatment effect; embolism after 8h treatment for the female mice, female and male bone marrow mesenchymal stem cells have the effect, but did not find the difference between the two treatment, while in male mice, female male, bone marrow mesenchymal stem cells have the effect, and the female bone marrow mesenchymal stem cells to better treatment effect; estrogen therapy, embolism after 16h male bone marrow mesenchymal stem cells of female mice were effective, but they were not found The treatment was different, but in male mice, only female bone marrow mesenchymal stem cells were treated effectively.
conclusion
Successfully established an animal model of acute pulmonary embolism by external jugular vein injection of thrombus, the distribution model of reproduction and clinical data of pulmonary embolism are basically the same, the methods used in this research to copy thrombosis pulmonary embolism is feasible and reliable. Compared to the commonly used literature area calculation method analysis of blood thrombus, we original whole lung sections calculation to analyze the thrombus volume accurately and objectively. The female and male bone marrow mesenchymal stem cells were effective in the early treatment of pulmonary thromboembolism, and female bone marrow mesenchymal stem cells and better treatment effect; in late thrombosis, only female bone marrow mesenchymal stem cells treatment is effective.
objective
Objective to determine the role of GAPDH in regulating the difference between male and female bone marrow mesenchymal stem cells in the treatment of acute pulmonary embolism, and further explore the mechanism of bone marrow mesenchymal stem cells in the treatment of acute pulmonary embolism.
Method
1., the bone marrow mesenchymal stem cells were transferred to GAPDH interference plasmid or empty plasmid. Male bone marrow mesenchymal stem cells stably transferred to GAPDH over expressed plasmid or empty plasmid. Western blotting was used to detect GAPDH expression in all cells.
2. animal models of replication of acute pulmonary embolism after embolization. The regulation of the expression of GAPDH 4H packet injection of bone marrow mesenchymal stem cells (F-BMSCs-siRNA-GAPDH, F-BMSCs-siRNA vector, M-BMSCs OE-GAPDH, M-BMSCs OE vector) 2 * 106 20h, mice were sacrificed and lung paraffin embedded lung. Serial sections, HE staining, all. The precise statistics, analysis of treatment differences.
3. in all lung continuous sections, all the odd HE stains were found. There was continuous thrombus in the slices. The corresponding even slices were immunofluorescent, and the stem cells were tracked. Western blotting was used to detect the expression of uPA in all the cells.
Result
1. GAPDH stable transfection
The untreated female bone marrow mesenchymal stem cells based on the expression of GAPDH is higher than that of male bone marrow mesenchymal stem cells; female bone marrow mesenchymal stem cells transfected with plasmid GAPDH, the expression of GAPDH was significantly reduced; male bone marrow mesenchymal stem cells transfected with GAPDH expression plasmid, GAPDH the expression is significantly increased.
Treatment of acute pulmonary embolism by 2. GAPDH transfected bone marrow mesenchymal stem cells
Stem cell therapy to acute pulmonary embolism after 4H interference GAPDH expression of female bone marrow mesenchymal stem cells in the treatment group was significantly higher than that of pure thrombus volume female bone marrow mesenchymal stem cells for the treatment group, while the expression of GAPDH male bone marrow mesenchymal stem cells in the treatment group were significantly reduced compared with male bone marrow mesenchymal stem cell therapy group thrombosis volume, treatment effect is significantly enhanced.
3. stem cell tracking and uPA expression
Immunofluorescence experiments showed that outer green fluorescent labeled bone marrow mesenchymal stem cells in intravascular emboli attachment. Female bone marrow mesenchymal stem was higher than that of male bone marrow mesenchymal stem cells uPA cells, overexpression of GAPDH after bone marrow mesenchymal stem cells uPA expression was significantly increased.
conclusion
Female bone marrow mesenchymal stem cells compared with male bone marrow mesenchymal stem cells on pulmonary thrombosis and better treatment effect, which may be due to female bone marrow mesenchymal stem cells is significantly higher than that of male bone marrow mesenchymal stem cells GAPDH expression of mesenchymal stem cells. The expression was verified by more female intervention the GAPDH charge in male and female bone marrow, male bone marrow mesenchymal stem cells in the expression level of GAPDH is different from the two kinds of stem cells in the treatment of acute pulmonary embolism and the reasons for the differences.
The location of stem cells in the outer edge of thrombus and the expression of uPA in bone marrow mesenchymal stem cells can regulate the expression of uPA, indicating that early bone marrow mesenchymal stem cells in acute pulmonary embolism may play a therapeutic role by promoting the dissolution of thrombus by secreting some thrombolytic factors.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R563.5

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