间充质干细胞条件培养基通过NLRP3炎症小体增加胰岛素敏感性的机制研究
发布时间：2018-12-15 21:43
【摘要】：目的：①探讨分离大鼠骨髓间充质干细胞的方法并鉴定其相关表型,收集培养的上清液并将其制备成浓缩条件培养基保存备用。②证实NLRP3炎症小体及其下游分子在游离脂肪酸(FFA)和脂多糖(LPS)诱导HepG2细胞胰岛素抵抗过程中的表达情况。③初步明确PA和LPS能够通过慢性炎症过程诱导胰岛素抵抗,进一步探讨BMSCs-CM通过作用于NLRP3、IL-1β、IL-18及TNF-α所致炎症反应而提高HepG2细胞胰岛素敏感性的可能机制。方法：首先,常规无菌条件下分离、培养并鉴定BMSCs,通过贴壁培养法对其进行纯化,传代并收集第2-4代细胞的条件培养基,进一步将其进行浓缩处理。进一步将实验分为5组：以正常HepG2细胞作为空白对照,分别以BSA、PA、LPS及PA+LPS作为造模组,根据各组内葡萄糖利用情况筛选出最佳造模剂使用浓度及作用时间,同时测定造模组内炎症因子的表达量。最后,在慢性炎症诱导胰岛素抵抗成功的基础上加入BMSCs-CM,同时以加入同样经半透膜浓缩后的无血清L-DMEM作为阴性对照,分别采用葡萄糖及糖原检测试剂盒来分析各组葡萄糖利用情况;ELISA观察各组炎症因子的表达量;采用q RT-PCR方法来分析细胞内相关基因的表达;Western blot检测BMSCs-CM对胰岛素发挥作用的靶蛋白基因的表达的影响。结果：成功培养BMSCs并对收集的上清液进行了浓缩条件培养基的配制;构建了慢性炎症诱导的HepG2细胞胰岛素抵抗模型,造模组胞内糖原含量明显减低而培养基中葡糖糖的含量则较空白组显著增多；而在加入BMSCs-CM后上述葡萄糖代谢水平均得到了提高,ELISA及q RT-PCR去分别从蛋白质的分泌和基因表达两个水平说明间充质干细胞条件培养基能够发挥抗炎作用,Western blot结果提示,与胰岛素抵抗组相比,BMSCs-CM组HepG2细胞内的胰岛素作用蛋白表达得到了恢复。结论：本研究表明BMSCs-CM可以通过调控NLRP3炎症小体及其下游相关炎症因子的表达来发挥增加葡萄糖摄取及利用的作用,进而改善胰岛素抵抗。
[Abstract]:Objective: 1 to investigate the method of isolating rat bone marrow mesenchymal stem cells (BMSCs) and identify their phenotypes. The supernatant of culture was collected and prepared into a concentrated conditioned medium for preservation. 2 it was confirmed that NLRP3 inflammatory bodies and their downstream molecules were involved in the induction of insulin resistance in HepG2 cells by free fatty acid (FFA) and lipopolysaccharide (LPS). (3) it is preliminarily clear that PA and LPS can induce insulin resistance through chronic inflammation. To further explore the possible mechanism of increasing insulin sensitivity of HepG2 cells by BMSCs-CM acting on inflammatory responses induced by NLRP3,IL-1 尾, IL-18 and TNF- 伪. Methods: firstly, BMSCs, was isolated under normal aseptic condition, purified by adherent culture method, subcultured and collected the conditioned medium of 2-4 passage cells, and further concentrated. The experiment was further divided into five groups: normal HepG2 cells were used as blank control and BSA,PA,LPS and PA LPS were used as model groups respectively. According to glucose utilization in each group, the optimal concentration and time of action of the model maker were selected. At the same time, the expression of inflammatory factors in the model group was measured. Finally, on the basis of successful insulin resistance induced by chronic inflammation, BMSCs-CM, was added and serum-free L-DMEM, which was also thickened by semi-permeable membrane, was added as negative control. Glucose and glycogen detection kits were used to analyze glucose utilization in each group. ELISA was used to observe the expression of inflammatory factors in each group, and Q RT-PCR method was used to analyze the expression of related genes in cells.; Western blot was used to detect the effect of BMSCs-CM on the expression of target protein genes acting on insulin. Results: BMSCs was cultured successfully and the supernatant was prepared with conditional culture medium. The insulin resistance model of HepG2 cells induced by chronic inflammation was constructed. The intracellular glycogen content in the model group was significantly decreased, while the glucosamine content in the culture medium was significantly increased compared with the blank group. The level of glucose metabolism was increased after adding BMSCs-CM, and ELISA and Q RT-PCR were removed from protein secretion and gene expression, respectively, indicating that the conditioned medium of mesenchymal stem cells could play an anti-inflammatory effect. The results of Western blot showed that the expression of insulin acting protein in HepG2 cells in BMSCs-CM group recovered compared with insulin resistance group. Conclusion: this study suggests that BMSCs-CM can increase glucose uptake and utilization by regulating the expression of inflammatory corpuscles of NLRP3 and its downstream inflammatory factors, thereby improving insulin resistance.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R587.1
本文编号：2381340
[Abstract]:Objective: 1 to investigate the method of isolating rat bone marrow mesenchymal stem cells (BMSCs) and identify their phenotypes. The supernatant of culture was collected and prepared into a concentrated conditioned medium for preservation. 2 it was confirmed that NLRP3 inflammatory bodies and their downstream molecules were involved in the induction of insulin resistance in HepG2 cells by free fatty acid (FFA) and lipopolysaccharide (LPS). (3) it is preliminarily clear that PA and LPS can induce insulin resistance through chronic inflammation. To further explore the possible mechanism of increasing insulin sensitivity of HepG2 cells by BMSCs-CM acting on inflammatory responses induced by NLRP3,IL-1 尾, IL-18 and TNF- 伪. Methods: firstly, BMSCs, was isolated under normal aseptic condition, purified by adherent culture method, subcultured and collected the conditioned medium of 2-4 passage cells, and further concentrated. The experiment was further divided into five groups: normal HepG2 cells were used as blank control and BSA,PA,LPS and PA LPS were used as model groups respectively. According to glucose utilization in each group, the optimal concentration and time of action of the model maker were selected. At the same time, the expression of inflammatory factors in the model group was measured. Finally, on the basis of successful insulin resistance induced by chronic inflammation, BMSCs-CM, was added and serum-free L-DMEM, which was also thickened by semi-permeable membrane, was added as negative control. Glucose and glycogen detection kits were used to analyze glucose utilization in each group. ELISA was used to observe the expression of inflammatory factors in each group, and Q RT-PCR method was used to analyze the expression of related genes in cells.; Western blot was used to detect the effect of BMSCs-CM on the expression of target protein genes acting on insulin. Results: BMSCs was cultured successfully and the supernatant was prepared with conditional culture medium. The insulin resistance model of HepG2 cells induced by chronic inflammation was constructed. The intracellular glycogen content in the model group was significantly decreased, while the glucosamine content in the culture medium was significantly increased compared with the blank group. The level of glucose metabolism was increased after adding BMSCs-CM, and ELISA and Q RT-PCR were removed from protein secretion and gene expression, respectively, indicating that the conditioned medium of mesenchymal stem cells could play an anti-inflammatory effect. The results of Western blot showed that the expression of insulin acting protein in HepG2 cells in BMSCs-CM group recovered compared with insulin resistance group. Conclusion: this study suggests that BMSCs-CM can increase glucose uptake and utilization by regulating the expression of inflammatory corpuscles of NLRP3 and its downstream inflammatory factors, thereby improving insulin resistance.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R587.1
【参考文献】
相关期刊论文 前1条
1 Zhenghui Gordon Jiang;Simon C. Robson;Zemin Yao;;Lipoprotein metabolism in nonalcoholic fatty liver disease[J];Journal of Biomedical Research;2013年01期
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