間充質(zhì)干細胞條件培養(yǎng)基通過NLRP3炎癥小體增加胰島素敏感性的機制研究
發(fā)布時間：2018-12-15 21:43
【摘要】：目的：①探討分離大鼠骨髓間充質(zhì)干細胞的方法并鑒定其相關表型,收集培養(yǎng)的上清液并將其制備成濃縮條件培養(yǎng)基保存?zhèn)溆谩＂谧C實NLRP3炎癥小體及其下游分子在游離脂肪酸(FFA)和脂多糖(LPS)誘導HepG2細胞胰島素抵抗過程中的表達情況。③初步明確PA和LPS能夠通過慢性炎癥過程誘導胰島素抵抗,進一步探討B(tài)MSCs-CM通過作用于NLRP3、IL-1β、IL-18及TNF-α所致炎癥反應而提高HepG2細胞胰島素敏感性的可能機制。方法：首先,常規(guī)無菌條件下分離、培養(yǎng)并鑒定BMSCs,通過貼壁培養(yǎng)法對其進行純化,傳代并收集第2-4代細胞的條件培養(yǎng)基,進一步將其進行濃縮處理。進一步將實驗分為5組：以正常HepG2細胞作為空白對照,分別以BSA、PA、LPS及PA+LPS作為造模組,根據(jù)各組內(nèi)葡萄糖利用情況篩選出最佳造模劑使用濃度及作用時間,同時測定造模組內(nèi)炎癥因子的表達量。最后,在慢性炎癥誘導胰島素抵抗成功的基礎上加入BMSCs-CM,同時以加入同樣經(jīng)半透膜濃縮后的無血清L-DMEM作為陰性對照,分別采用葡萄糖及糖原檢測試劑盒來分析各組葡萄糖利用情況;ELISA觀察各組炎癥因子的表達量;采用q RT-PCR方法來分析細胞內(nèi)相關基因的表達;Western blot檢測BMSCs-CM對胰島素發(fā)揮作用的靶蛋白基因的表達的影響。結(jié)果：成功培養(yǎng)BMSCs并對收集的上清液進行了濃縮條件培養(yǎng)基的配制;構建了慢性炎癥誘導的HepG2細胞胰島素抵抗模型,造模組胞內(nèi)糖原含量明顯減低而培養(yǎng)基中葡糖糖的含量則較空白組顯著增多；而在加入BMSCs-CM后上述葡萄糖代謝水平均得到了提高,ELISA及q RT-PCR去分別從蛋白質(zhì)的分泌和基因表達兩個水平說明間充質(zhì)干細胞條件培養(yǎng)基能夠發(fā)揮抗炎作用,Western blot結(jié)果提示,與胰島素抵抗組相比,BMSCs-CM組HepG2細胞內(nèi)的胰島素作用蛋白表達得到了恢復。結(jié)論：本研究表明BMSCs-CM可以通過調(diào)控NLRP3炎癥小體及其下游相關炎癥因子的表達來發(fā)揮增加葡萄糖攝取及利用的作用,進而改善胰島素抵抗。
[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- 偽. 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.
【學位授予單位】：中國人民解放軍醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R587.1
本文編號：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- 偽. 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.
【學位授予單位】：中國人民解放軍醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R587.1
【參考文獻】
相關期刊論文 前1條
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
，本文編號：2381340
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