肠道缺血再灌注损伤对骨髓间充质干细胞旁分泌作用的影响

发布时间：2018-12-11 19:18

【摘要】：肠道的缺血再灌注损伤在临床当中日益得到重视。目前认为,在氧自由基、中性粒细胞、炎症因子、补体等多种因素的的共同作用下,肠道成为在缺血再灌注损伤中极易受到伤害的器官。严重的肠道的损伤可导致导致全身炎症反应综合征(MIRS)和多器官功能不全综合征(MODS)。而对于肠道缺血再灌注损伤的治疗,目前仍然缺乏有效的治疗手段。骨髓来源的间充质干细胞(bone marrow-derived mesenchymal stem cells,BM-MSCs)是一种存在于骨髓内的具有多向分化潜能的成体干细胞。由于BM-MSCs具有易于获得、多向分化、高度增殖的特点,成为近年来器官损伤修复的重要手段。在动物试验已经证实同种异体BM-MSCs移植可定植于胃肠道,并参与肠道损伤的修复。对于BM-MSCs参与受损组织修复的具体的机制,目前认为有直接分化和旁分泌两种方式,但仍缺乏进一步的研究证实。本研究试图模拟肠道缺血再灌注损伤的病理生理环境,观察肠道的缺血再灌注刺激对BM-MSCs旁分泌的影响,以证实BM-MSCs在肠道修复中的旁分泌作用。研究目的 1、完成Wistar雄性大鼠的骨髓间充质干细胞分离、培养、鉴定、染色工作。 2、探讨并制备肠道缺血再灌注大鼠模型,证实其肠道损伤及功能障碍,为后续的治疗和检测奠定基础。 3、观察肠道的缺血再灌注损伤刺激对BM-MSCs旁分泌作用的影响,以证实BM-MSCs在肠道修复中的旁分泌作用。研究方法 1、分离、培养、鉴定、染色Wistar雄性大鼠的骨髓间充质干细胞。 2、用肠系膜上动脉夹闭的方式制备大鼠肠道缺血再灌注模型,并根据夹闭时间的不同分为20分钟组,30分钟组,40分钟组和50分钟组,并检测损伤后肠道的LPS和肠道损伤指数,以寻找最合适的夹闭时间。 3、分别提取肠道缺血再灌注模型大鼠、正常大鼠的肠上皮粘膜提取物,与BM-MSCs共同培养; 4、分别在培养的第0天、第1天、第3天和第5天留取条件培养基,ELISA检测不同细胞条件培养基以及普通培养基中TGF-α、VEGF、FGF、IGF-1的含量。结果 1、全骨髓培养法得到的贴壁细胞完全符合骨髓间充质干细胞的特性,经鉴定为BM-MSCs; 2、在制备大鼠肠道缺血再灌注模型的过程中,夹闭40分钟组和夹闭50分钟组死亡率过高,而夹闭20分钟肠道损伤程度不能达标,只有夹闭30分钟既可以控制死亡率又能够保证损伤达标; 3、四种生长因子在含有肠上皮提取物的条件培养基中的含量均明显高于普通培养基。其中含有缺血再灌注肠上皮提取物的培养基TGF-α、VEGF、FGF的含量明显高于含有正常肠上皮提取物的培养基。VEGF含量的高峰时间出现在第三天,而FGF、IGF-1含量的高峰时间出现在第五天,TGF-α含量在第1-5天未见到明显差异。结论 1、全骨髓培养法是简便、可靠的BM-MSCs培养方法,其获得BM-MSCs稳定可靠; 2、在制备大鼠肠道缺血再灌注模型的过程中,肠系膜上动脉夹闭的时间控制在30分钟左右是满足实验需要的方法。 3、BM-MSCs可以旁分泌TGF-α、VEGF、FGF、IGF-1等生长因子;而在缺血再灌注损伤的微环境下,BM-MSCs的分泌功能得到增强,TGF-α、VEGF两种生长因子的峰值出现在第3天,而FGF、IGF-1两种生长因子的水平呈时间依赖性。
[Abstract]:The intestinal ischemia-reperfusion injury is becoming more and more important in the clinic. It is believed that the intestinal tract is the most vulnerable organ of the ischemia-reperfusion injury under the co-action of various factors such as oxygen free radical, neutrophil, inflammation factor, complement and the like. Severe intestinal injury can lead to systemic inflammatory response syndrome (MIRS) and multiple organ dysfunction syndrome (MODS). For the treatment of intestinal ischemia-reperfusion injury, there is still a lack of effective means of treatment. Bone marrow-derived mesenchymal stem cells (BM-MSCs) are a kind of body with multi-directional differentiation potential in the bone marrow. Because BM-MSCs have the characteristics of easy to obtain, multi-directional differentiation and high proliferation, it becomes an important hand in organ damage repair in recent years. Section. Allogeneic BM-MSCs transplantation has been demonstrated in animal testing to be colonized in the gastrointestinal tract and participate in the repair of intestinal injury Complex. For BM-MSCs involved in the repair of damaged tissue, there are two ways of direct differentiation and paracrine, but there is still a lack of further study. Objective: To study the pathological and physiological environment of intestinal ischemia-reperfusion injury, and to observe the effect of ischemia-reperfusion on the secretion of BM-MSCs on the side of BM-MSCs in order to confirm the paracrine secretion of BM-MSCs in the intestinal repair. for use. Objective: To study the isolation, culture and identification of bone marrow mesenchymal stem cells in Wistar male rats. and to study and prepare the rat model of intestinal ischemia-reperfusion, to confirm the intestinal injury and dysfunction and to be a follow-up treatment. and the effect of the injury of the intestinal ischemia-reperfusion injury on the paracrine function of BM-MSCs was observed to confirm that BM-MSCs were in the intestinal tract. repair paracrine effect. Study method 1, isolation, culture, identification, staining of Wistar rats Bone marrow mesenchymal stem cells were prepared in male rats. The rat intestinal ischemia-reperfusion model was prepared by means of the superior mesenteric artery occlusion, and the rats were divided into 20 minutes, 30 minutes, 40 minutes and 50 minutes according to the time of the occlusion, and the LPS and the intestinal loss in the intestinal tract after injury were detected. the injury index was used to find the most suitable clipping time. 3, the intestinal epithelium of the normal rats was adhered to the rat of the intestinal ischemia-reperfusion model, respectively. membrane extract, co-cultured with BM-MSCs; 4, respectively at day 0 of culture on day 1, day 3 and day 5, conditioned medium was taken, and the culture medium of different cells and TGF-in the normal medium were detected by ELISA. in that case of a man, The content of VEGF, FGF, IGF-1 was 1. The adherent cells obtained by the method of full bone marrow culture were in full compliance. The characteristics of bone marrow mesenchymal stem cells were identified as BM-MSCs; 2. In the preparation of rat intestinal ischemia-reperfusion model in that course of the procedure, the mortality rate of the 40-minute group and the clamp-close 50-minute group was too high, while the degree of intestinal injury of the clip was not up to the standard for 20 minutes, only 30 minutes of the clamp can control the death rate and ensure the damage to the standard; and 3, the four growth factors are The content of the condition culture medium with the intestinal epithelial extract is obviously higher than that of the normal culture medium, wherein the culture medium TGF-1, VE containing the ischemia-reperfusion intestinal epithelial extract The content of GF and FGF was significantly higher than that of the medium containing the normal intestinal epithelium extract. The peak time of the content of VEGF appeared on the third day, while the peak time of the content of FGF and IGF-1 appear in that fifth day, the TGF-1 content did not see a significant difference in day 1-5. conclusion 1, the full bone marrow culture method was simple and the method for culturing the BM-MSCs obtained by the method has the advantages that the BM-MSCs are stable and reliable, and 2, after the rat intestinal ischemia-reperfusion model is prepared, In the course, the time control of the superior mesenteric artery occlusion is about 30 minutes to meet the experimental requirements. 3, BM-MSCs can secrete the growth factors such as TGF-1, VEGF, FGF, IGF-1, and the secretion function of BM-MSCs under the microenvironment of ischemia-reperfusion injury. the growth factors of the two growth factors of the enhanced, TGF-1 and VEGF are enhanced,
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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