丙烯酰胺对猪骨髓间充质干细胞NFκB通路的影响

发布时间：2018-08-03 19:48

【摘要】：骨髓间充质干细胞(BMMSCs)具有免疫调节功能,参与多种免疫反应,在体内具有优先迁移到受损组织器官发挥其修复功能的能力。现阶段的BMMSCs体外分离方法是建立在Friedenstein分离方法的基础上形成的。目前,由于BMMSCs的鉴定没有一种明确统一的体系,只能从几个方面综合分析来作为其鉴定的依据。丙烯酰胺(ACR)具有致癌性、遗传毒性以及神经毒性。本试验通过BMMSCs的分离鉴定,建立BMMSCs的体外培养体系,并探讨ACR对BMMSCs上ROS、Hsp27以及NFκB通路的影响,以期为ACR的作用机制研究提供一条新的线索。本文采用全骨髓贴壁法体外分离0-6月龄五指山小型猪BMMSCs,并利用CCK-8检测其增殖能力。BMMSCs的鉴定是通过细胞形态学分析、成脂成骨诱导分化潜能鉴定以及CD34/CD45/CD90/CD29/CD44细胞表面抗原的检测等几种指标综合分析。使用0.5mmM的ACR刺激BMMSCs 72 h,分别用ROS检测试剂盒检测ROS分泌量、ELISA试剂盒在蛋白水平检测Hsp27和IL-8表达量、实时定量PCR在mRNA水平上检测IL-8的表达量、双荧光素酶系统检测NFκB通路的激活状况,并用试剂盒检测ACR处理前后BMMSCs的细胞周期和凋亡状况。结果表明分离得到的五指山小型猪BMMSCs形态呈梭形或多角形,类似成纤维样细胞,细胞群体呈旋涡状生长。成脂、成骨诱导培养21d分别用油红O及茜素红染色均呈阳性。流式细胞仪检测显示,BMMSCs表达CD90、CD29及CD44,不表达造血干细胞标志性抗原CD34和白细胞标志性抗原CD45。0.5 mM的ACR刺激BMMSCs 72 h后检测其ROS、Hsp27、IL-8表达量均明显升高,双荧光素酶系统检测到NFκB通路被激活(P0.05)。然而,ACR刺激后BMMSCs的细胞周期和凋亡未见明显变化。总之,本文摸索出五指山小型猪BMMSCs一套分离培养体系,通过形态学分析、分化潜能以及表面抗原三个方面检测,基本确定其为骨髓间充质干细胞。当0.5 mMACR刺激时,ROS、Hsp27及IL-8表达量均升高,细胞质中NFκB含量显著升高,说明NFκB通路可能被激活,而细胞凋亡和周期变化不明显。
[Abstract]:Bone marrow mesenchymal stem cells (BMMSCs) have immunomodulatory function, participate in various immune responses, and have the ability to migrate to damaged tissues and organs in vivo to play their repair function. At present, the method of BMMSCs isolation in vitro is based on the method of Friedenstein separation. At present, the identification of BMMSCs is not a clear and unified system, so it can only be analyzed from several aspects as the basis of its identification. Acrylamide (ACR) is carcinogenic, genotoxic and neurotoxic. Through the isolation and identification of BMMSCs, the in vitro culture system of BMMSCs was established, and the effects of ACR on the rosiform Hsp27 and NF 魏 B pathway on BMMSCs were investigated, in order to provide a new clue for the study of the mechanism of ACR. BMMSCs of 0-6 months old Wuzhishan miniature pig were isolated by whole bone marrow adherent method in vitro. The proliferative ability of BMMSCs was determined by CCK-8. The identification of adipogenic osteogenic differentiation potential and the detection of surface antigen of CD34/CD45/CD90/CD29/CD44 cells were comprehensively analyzed. 0.5mmM ACR was used to stimulate BMMSCs for 72 h. ROS secretion was detected by ROS assay kit and Hsp27 and IL-8 expression was detected by Elisa kit at protein level. IL-8 expression was detected by PCR at mRNA level in real time. The activation of NF 魏 B pathway was detected by double luciferase system, and the cell cycle and apoptosis of BMMSCs before and after treatment with ACR were detected by Kit. The results showed that the BMMSCs of Wuzhishan miniature pig was spindle-shaped or polygonal, similar to fibroblast, and the cell population was swirling. Oil red O and alizarin red were positive for 21 days of osteogenic induction. Flow cytometry analysis showed that BMMSCs expressed CD90, CD29 and CD44, and the expression of ROSS-Hsp27 IL-8 was significantly increased after 72 h stimulation of BMMSCs with ACR that did not express the iconic antigen of hematopoietic stem cell (CD34) and leukocyte iconic antigen (CD45.0.5 mm). NF 魏 B pathway was activated by double luciferase system (P0.05). However, the cell cycle and apoptosis of BMMSCs did not change after ACR stimulation. In a word, a set of isolation and culture system of Wuzhishan mini-pig BMMSCs was found out. It was identified as bone marrow mesenchymal stem cells by morphological analysis, differentiation potential and surface antigen detection. When stimulated with 0.5 mMACR, the expression of Hsp27 and IL-8 increased, and the content of NF 魏 B in cytoplasm increased significantly, indicating that NF 魏 B pathway might be activated, but apoptosis and cell cycle changes were not obvious.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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