本文選題：IL-17A + 前成骨細胞　； 參考：《浙江大學》2014年碩士論文

【摘要】：目的：白介素-17A(IL-17A)在牙周炎牙槽骨改建過程中扮演重要角色,前期實驗發(fā)現(xiàn)磷脂酰肌醇-3-激酶/絲氨酸蘇氨酸蛋白激酶(PI3K/AKT)信號通路參與骨改建過程。本研究旨在探索IL-17A對前成骨細胞MC3T3-E1增殖、分化、鈣化功能的影響及其依賴AKT2的初步機制研究。 方法：前期實驗中采用基因干擾技術(shù)成功將MC3T3-E1細胞AKT2基因沉默,并與正常的MC3T3-E1細胞分別于有或無IL-17A的培養(yǎng)基中培養(yǎng)。通過甲基噻唑基四唑法(methyl thiazolyl tetrazolium, MTT)法檢測細胞增殖,流式細胞術(shù)檢測細胞周期。在增加誘導培養(yǎng)條件后,蛋白免疫印跡實驗(western-blot)檢測PI3K及p-PI3K,實時熒光定量聚合酶鏈反應(yīng)(real-time PCR)檢測細胞成骨相關(guān)基因的表達,包括Runt相關(guān)轉(zhuǎn)錄因子2(Runx2)、堿性磷酸酶(ALP)、骨鈣素(OCN)及IL-17A受體(IL-17RA).同時,采用堿性磷酸酶活性測定法、茜素紅染色法、羥脯氨酸測定法檢測分化、鈣化功能。 結(jié)果：在IL-17A作用于AKT2基因沉默及正常的MC3T3-E1細胞后,顯示p-PI3K表達上調(diào),IL-17A受體表達增加(P0.05)。MTT表明AKT2基因沉默后MC3T3-E1細胞增殖減慢(P0.05),而IL-17A的作用并無明顯影響。細胞周期結(jié)果表明AKT2基因沉默后較正常組細胞更多出于G0/G1期,細胞所占比例分別為(81.793±1.227,62.043±1.317)(P0.05),但S期與G2/M期細胞比例下降(P0.05),而IL-17A作用后未發(fā)現(xiàn)明顯改變。而且,在誘導條件下正常MC3T3-E1細胞與AKT2基因沉默細胞中Runx2、ALP及OCN基因表達均上升,堿性磷酸酶與羥脯氨酸活性,鈣化結(jié)節(jié)染色增加均明顯(P0.05),IL-17A作用后,對正常組與AKT2基因沉默細胞組進行比較：第7日Runx2mRNA表達(2.238±0.091,1.537±0.096),ALP mRNA表達(4.258±0.276,1.260±0.070)；第14日OCN mRNA表達(2.521±0.279,1.104±0.150)(P0.05).ALP濃度百分比(67.137±5.105,37.557±2.820)(P0.05),茜素紅-S染色的積分光密度值(IOD)/105(19.638±0.960,5.533±0.251)(P0.05),培養(yǎng)基中羥脯氨酸含量(3.527±0.672,1.861±0.400)(P0.05),可知IL-17A對正常MC3T3-E1細胞的上述促進作用較AKT2基因沉默細胞更加明顯。 結(jié)論：AKT2與前成骨細胞MC3T3-E1增殖、分化、鈣化相關(guān)；IL-17A依賴AKT2對MC3T3-E1細胞分化、鈣化活動起促進作用。
[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 鹵1.227) 62.043 鹵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 鹵0.091 鹵1.537 鹵0.096) was compared between the normal group and AKT2 gene silencing cell group. The expression of ALP mRNA was (4.258 鹵0.276) 1.260 鹵0.070; On the 14th day, the expression of OCN mRNA was (2.521 鹵0.279) 1.104 鹵0.150 (P0.05). The percentage of ALP concentration was (67.137 鹵5.105) 鹵(37.557 鹵2.820) (P0.05). The integral optical density (IOD) of alizarin red S staining (IOD) was (19.638 鹵0.9609) 5.533 鹵0.251 (P0.05). The hydroxyproline content in culture medium was (3.527 鹵0.672) 1.861 鹵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.
【學位授予單位】：浙江大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R781.4

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