IL-17A对前成骨细胞MC3T3-E1功能的影响及其依赖AKT2的初步机制研究

发布时间：2018-07-02 08:35

本文选题：IL-17A + 前成骨细胞　；参考：《浙江大学》2014年硕士论文

【摘要】：目的：白介素-17A(IL-17A)在牙周炎牙槽骨改建过程中扮演重要角色,前期实验发现磷脂酰肌醇-3-激酶/丝氨酸苏氨酸蛋白激酶(PI3K/AKT)信号通路参与骨改建过程。本研究旨在探索IL-17A对前成骨细胞MC3T3-E1增殖、分化、钙化功能的影响及其依赖AKT2的初步机制研究。方法：前期实验中采用基因干扰技术成功将MC3T3-E1细胞AKT2基因沉默,并与正常的MC3T3-E1细胞分别于有或无IL-17A的培养基中培养。通过甲基噻唑基四唑法(methyl thiazolyl tetrazolium, MTT)法检测细胞增殖,流式细胞术检测细胞周期。在增加诱导培养条件后,蛋白免疫印迹实验(western-blot)检测PI3K及p-PI3K,实时荧光定量聚合酶链反应(real-time PCR)检测细胞成骨相关基因的表达,包括Runt相关转录因子2(Runx2)、碱性磷酸酶(ALP)、骨钙素(OCN)及IL-17A受体(IL-17RA).同时,采用碱性磷酸酶活性测定法、茜素红染色法、羟脯氨酸测定法检测分化、钙化功能。结果：在IL-17A作用于AKT2基因沉默及正常的MC3T3-E1细胞后,显示p-PI3K表达上调,IL-17A受体表达增加(P0.05)。MTT表明AKT2基因沉默后MC3T3-E1细胞增殖减慢(P0.05),而IL-17A的作用并无明显影响。细胞周期结果表明AKT2基因沉默后较正常组细胞更多出于G0/G1期,细胞所占比例分别为(81.793±1.227,62.043±1.317)(P0.05),但S期与G2/M期细胞比例下降(P0.05),而IL-17A作用后未发现明显改变。而且,在诱导条件下正常MC3T3-E1细胞与AKT2基因沉默细胞中Runx2、ALP及OCN基因表达均上升,碱性磷酸酶与羟脯氨酸活性,钙化结节染色增加均明显(P0.05),IL-17A作用后,对正常组与AKT2基因沉默细胞组进行比较：第7日Runx2mRNA表达(2.238±0.091,1.537±0.096),ALP mRNA表达(4.258±0.276,1.260±0.070)；第14日OCN mRNA表达(2.521±0.279,1.104±0.150)(P0.05).ALP浓度百分比(67.137±5.105,37.557±2.820)(P0.05),茜素红-S染色的积分光密度值(IOD)/105(19.638±0.960,5.533±0.251)(P0.05),培养基中羟脯氨酸含量(3.527±0.672,1.861±0.400)(P0.05),可知IL-17A对正常MC3T3-E1细胞的上述促进作用较AKT2基因沉默细胞更加明显。结论：AKT2与前成骨细胞MC3T3-E1增殖、分化、钙化相关；IL-17A依赖AKT2对MC3T3-E1细胞分化、钙化活动起促进作用。
[Abstract]:Aim: interleukin-17A (IL-17A) plays an important role in alveolar bone remodeling in periodontitis. Previous studies have found that phosphatidylinositol-3-kinase / serine threonine protein kinase (PI3K / AKT) signaling pathway is involved in bone remodeling. The aim of this study was to investigate the effects of IL-17A on the proliferation, differentiation and calcification of preosteoblast MC3T3-E1 and its mechanism of dependence on AKT2. Methods: gene interference technique was used to silencing AKT2 gene of MC3T3-E1 cells and cultured with or without IL-17A in MC3T3-E1 cells. Cell proliferation was detected by (methyl thiazolyl tetrazolium, assay and cell cycle was detected by flow cytometry. After culture conditions were increased, PI3K and p-PI3K were detected by Western blot assay (western-blot), and the expression of osteoblast-associated genes was detected by real-time fluorescence quantitative polymerase chain reaction (real-time). These include Runt associated transcription factor 2 (Runx2), alkaline phosphatase (ALP), osteocalcin (OCN) and IL-17A receptor (IL-17RA). At the same time, alkaline phosphatase activity assay, alizarin red staining and hydroxyproline assay were used to detect the function of differentiation and calcification. Results: after treated with IL-17A, the expression of p-PI3K was up-regulated in MC3T3-E1 cells (P0.05). MTT showed that the proliferation of MC3T3-E1 cells was inhibited by the silencing of AKT2 gene (P0.05), but the effect of IL-17A had no obvious effect on the proliferation of MC3T3-E1 cells. The results of cell cycle showed that the ratio of cells in G _ 0 / G _ 1 phase after AKT2 gene silencing was (81.793 卤1.227) 62.043 卤1.317 (P0.05), but the ratio of S phase to G _ 2 / M phase was decreased (P0.05), but IL-17A did not change significantly. In addition, the expression of Runx2, ALP and OCN genes in normal MC3T3-E1 cells and AKT2 gene silencing cells increased under induction condition. The activities of alkaline phosphatase and hydroxyproline, and calcified nodule staining increased significantly (P0.05) after treatment with IL-17A. The expression of Runx2 mRNA on the 7th day (2.238 卤0.091 卤1.537 卤0.096) was compared between the normal group and AKT2 gene silencing cell group. The expression of ALP mRNA was (4.258 卤0.276) 1.260 卤0.070; On the 14th day, the expression of OCN mRNA was (2.521 卤0.279) 1.104 卤0.150 (P0.05). The percentage of ALP concentration was (67.137 卤5.105) 卤(37.557 卤2.820) (P0.05). The integral optical density (IOD) of alizarin red S staining (IOD) was (19.638 卤0.9609) 5.533 卤0.251 (P0.05). The hydroxyproline content in culture medium was (3.527 卤0.672) 1.861 卤0.400 (P0.05). The above effects of IL-17A on normal MC3T3-E1 cells were more obvious than those of AKT2 silencing gene cells. Conclusion the proliferation and differentiation of MC3T3-E1 and calcification-related IL-17A may promote the differentiation and calcification of MC3T3-E1 cells.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R781.4

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