CX3CR1在BM MSCs修复受损肠上皮细胞间紧密连接中的作用

发布时间：2018-07-05 18:43

本文选题：骨髓间充质干细胞 + CX3CR1　；参考：《天津医科大学》2016年硕士论文

【摘要】：目的:在体外成功分离、培养及鉴定大鼠骨髓间充质干细胞(Bone marrow me senchymal stem cells,BM MSCs)及成功利用肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)刺激结肠腺癌细胞Caco-2建立体外肠黏膜上皮损伤模型的基础上,探讨趋化因子受体CX3CR1在BM MSCs修复受损肠上皮细胞间紧密连接中所发挥的作用。方法:1.体外贴壁筛选法分离培养大鼠BM MSCs至3代,观察BM MSCs的细胞形态、流式检测细胞表型、成脂成骨诱导分化方法检测BM MSCs的分化能力。2.使用Caco-2模拟肠黏膜上皮细胞,免疫荧光、RT-PCR和Western blot技术检测紧密连接蛋白ZO-1和Occludin来评价Caco-2的肠道屏障功能。将TNF-α以0 ng/m L、50 ng/m L、100 ng/m L和200 ng/m L四种不同浓度处理Caco-2,探索TNF-α作用Caco-2的最佳损伤浓度;选择造模最佳浓度点作用Caco-2细胞,作用时间分别为0 h、12 h、24 h和48 h,筛选TNF-α作用Caco-2的最佳损伤时间,从而摸索出建立稳定肠黏膜上皮损伤模型的最佳条件。3.将BM MSCs与受损Caco-2通过transwell小室间接共培养,采用RT-PCR和Western blot技术检测Caco-2细胞上ZO-1和Occludin以及BM MSCs上CX3CR1的含量变化。4.用CX3CR1抗体孵育封闭BM MSCs上CX3CR1受体,将anti-CX3CR1-BM MSCs与受损Caco-2通过Transw ell小室共培养,RT-PCR和Western blot技术检测Caco-2细胞上ZO-1和Occludin的含量变化,进一步明确CX3CR1在BM MSCs修复受损肠上皮细胞间紧密连接中所发挥的作用。结果:1.体外成功提取培养BM MSCs。镜下BM MSCs贴壁成长,呈长梭形,似漩涡或菊花状排列,具备典型MSCs的形态特征。流式检测三代BM MSCs,93.0%的细胞表达CD90而不表达CD45,97.5%的细胞表达CD29而不表达CD34,97.0%的细胞表达RT1A而不表达RT1B,这说明我们提取的原代细胞培养至三代可以获得极纯的BM MSCs,几乎没有造血干细胞等混杂。将三代BM MSCs使用成脂诱导培养液培养后,油红O染色细胞质内出现橘红色脂滴,BM MSCs使用成骨诱导培养液培养后,Von Kossa染色细胞内出现黑色钙盐沉积。这些提示我们:采用贴壁筛选法提取的BM MSCs,正是我们需要的目的细胞。2.应用不同浓度及不同作用时间的TNF-a处理Caco-2,免疫荧光检测可见正常Caco-2中ZO-1连接呈蜘蛛网状,随着TNF-a浓度的增加、作用时间的延长,ZO-1网状结构遭到破坏、荧光强度减弱,RT-PCR和Western blot检测结果显示随着TNF-a作用浓度的增大、作用时间的延长,ZO-1、Occludin的表达水平降低越来越明显,呈浓度依赖和时间依赖,而100 ng/m L和200 ng/m L TNF-a对Caco-2中ZO-1、Occlud in的表达影响差异无统计学意义,最终证实本实验条件下TNF-α的最佳造模浓度为100 ng/m L,造模时间为48 h。3.BM MSCs与受损Caco-2共培养后,RT-PCR结果显示:ZO-1、Occludin m RNA的表达水平较受损Caco-2组增加,同时BM MSCs上CX3CR1 m RNA的表达量较正常BM MSCs组增加,具有统计学意义;Western blot结果显示:ZO-1、Occludin蛋白的表达水平较受损Caco-2组增加,同时BM MSCs上CX3CR1蛋白的表达量较正常BM MSCs组增加,具有统计学意义。m RNA水平与蛋白水平结果一致。提示:BM MSCs具有修复受损肠上皮细胞间紧密连接的作用。4.Anti-CX3CR1-BM MSCs与受损Caco-2共培养后,RTPCR结果显示:ZO-1、Occludin m RNA的表达量较阻断CX3CR1前减少,但高于受损Caco-2组的表达量。Western blot结果显示:ZO-1、Occludin蛋白的表达量较阻断CX3CR1前减少,但高于受损Caco-2组的表达量。揭示了BM MSCs上趋化因子受体CX3CR1参与其修复受损肠上皮细胞间紧密连接。结论:1.采用贴壁筛选法可以成功提取、培养获得纯度高、数量多的大鼠BM MSCs,该方法简单易行。2.在体外环境下,使用100 ng/ml TNF-α作用48h刺激Caco-2细胞,可成功建立稳定的肠黏膜上皮损伤模型。3.BM MSCs表面CX3C R1高表达促进受损肠黏膜上皮间紧密连接蛋白及m RNA的表达,发挥保护肠黏膜上皮细胞的作用。封闭BM MSCs上CX3CR1受体后,BM MSCs修复肠上皮紧密连接蛋白及m RNA表达的作用减弱,证实CX3CR1在BM MSCs修复受损肠黏膜上皮间紧密连接中发挥作用。
[Abstract]:Objective: to successfully isolate and cultivate rat bone marrow mesenchymal stem cells (Bone marrow me senchymal stem cells, BM MSCs) and to successfully use tumor necrosis factor alpha (tumor necrosis factor- alpha, TNF- a) to stimulate colon adenocarcinoma cells in vitro. 1 the role of BM MSCs in the repair of intercellular close connections between damaged intestinal epithelial cells. Methods: 1. the BM MSCs to 3 generations of rats were isolated and cultured in vitro, and the cell morphology of BM MSCs was observed, the phenotype of the cells was detected by flow cytometry, the differentiation method of lipid forming osteogenesis was used to detect the differentiation energy of BM MSCs by using Caco-2 to simulate intestinal mucosal epithelial cells and immunization. Fluorescence, RT-PCR and Western blot techniques were used to detect close connexin ZO-1 and Occludin to evaluate the intestinal barrier function of Caco-2. TNF- alpha was treated with 0 ng/m L, 50 ng/m L, 100 ng/m and 200 different concentrations. 0 h, 12 h, 24 h and 48 h respectively, screening the optimum damage time of TNF- alpha action Caco-2, thus finding out the best condition for establishing the model of the intestinal mucosal epithelium injury, which is the indirect co culture of BM MSCs and the damaged Caco-2 through the Transwell chamber. The content change of R1.4. was incubated with CX3CR1 antibody to seal the CX3CR1 receptor on BM MSCs. The anti-CX3CR1-BM MSCs and the damaged Caco-2 were co cultured in Transw ell chamber. Results: 1. the BM MSCs adherent growth under the BM MSCs. microscope was successfully extracted and cultured in vitro, with a long shuttle shape, like a whirlpool or Chrysanthemum like arrangement, with the morphological characteristics of a typical MSCs. Flow cytometry was used to detect three generation of BM MSCs, 93% of the cells expressed CD90 but did not express CD45,97.5% in the expression of CD29 but did not express CD34,97.0% cell expression RT1A but not Up to RT1B, which indicates that the primary cells extracted from the three generation can obtain extremely pure BM MSCs, almost no hybrid hematopoietic stem cells. After the use of the three generation BM MSCs in the culture medium of lipid induced culture, the orange red lipid droplets appear in the cytoplasm of the oil red O staining, and the BM MSCs is cultured in the bone induced culture medium, and the Von Kossa staining cells appear in the cells. Black calcium salt deposition. These suggest that the BM MSCs extracted by the adherent screening method is exactly what we need for the target cell.2. to treat Caco-2 with different concentrations and different time of action of TNF-a. The immunofluorescence detection shows that the ZO-1 connection in the normal Caco-2 is spider network, with the increase of TNF-a concentration, the prolongation of the action time, the ZO-1 reticular formation. The structure was destroyed and the fluorescence intensity was weakened. The results of RT-PCR and Western blot detection showed that as the concentration of TNF-a increased, the expression level of ZO-1 and Occludin decreased more and more obviously with the concentration dependence and time dependence, while the difference of the expression of 100 ng/m L and 200 ng/m L TNF-a was no statistical difference. The optimal model concentration of TNF- alpha was 100 ng/m L and the molding time was 48 h.3.BM MSCs and the damaged Caco-2 co culture. The RT-PCR results showed that the expression level of ZO-1 and Occludin m RNA was higher than that of the damaged Caco-2 group. The results of Western blot show that the expression level of ZO-1, Occludin protein is higher than that of the damaged Caco-2 group, and the expression of CX3CR1 protein on BM MSCs is higher than that of the normal BM MSCs group. After co culture of 3CR1-BM MSCs and damaged Caco-2, the results of RTPCR showed that the expression of ZO-1, Occludin m RNA decreased compared with that before blocking CX3CR1, but higher than that of the damaged Caco-2 group. The sub receptor CX3CR1 participates in the repair of the close intercellular connection between the damaged intestinal epithelial cells. Conclusion: 1. the adherent screening method can be successfully extracted and cultured to obtain BM MSCs with high purity and a large number of rats. This method is simple and easy to use.2. to stimulate Caco-2 cells in 48h by using the action of 100 ng/ml TNF- alpha in 48h, and a stable upper lesion of the intestinal mucosa can be successfully established. The high expression of CX3C R1 on the surface of the injury model.3.BM MSCs promotes the expression of interepithelial tight connexin and m RNA in the damaged intestinal mucosa, and plays a role in protecting intestinal epithelial cells. After blocking the CX3CR1 receptor on BM MSCs, BM MSCs repair the intestinal epithelial tight connexin and the role of M Expression. It plays a role in the interskin close connection.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R574

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