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  本文選題：晚期糖基化終末產(chǎn)物 + 軟骨細(xì)胞��； 參考：《中南大學(xué)》2010年博士論文

【摘要】：第一部分AGEs對兔軟骨細(xì)胞TNF-a和MMP-13表達(dá)的影響與意義 目的：以外源性晚期糖基化終末產(chǎn)物(advanced glycation end products, AGEs)為損傷因子,在體外培養(yǎng)的家兔軟骨細(xì)胞模型上,觀察AGEs對腫瘤壞死因子-a (tumor necrosisfactor-a,TNF-α)和基質(zhì)金屬蛋白酶-13(Matrix metalloproteinase 13, MMP-13)的表達(dá)和影響,探討AGEs與骨關(guān)節(jié)炎(Osteoarthritis, OA)的關(guān)系及可能的信號通路及機制。 方法：在原代培養(yǎng)的家兔軟骨細(xì)胞模型上,(1)不同濃度的AGEs與軟骨細(xì)胞共孵育48h后檢測軟骨細(xì)胞TNF-a和MMP-13 mRNA的表達(dá)情況；(2)AGEs受體(Receptor for advanced glycation end products, RAGE)的抗體(anti-RAGE)及核因子，，B (nuclear factor-KB, NF-κB)的特異性阻斷劑吡咯烷二硫代氨基甲酸酯(pyrrolidine dithiocarbamate, PDTC)與軟骨細(xì)胞預(yù)孵12h后,再加入100μg/mlAGEs共同孵育48h,檢測軟骨細(xì)胞TNF-a及MMP-13mRNA的表達(dá)；(3)不同濃度的AGEs與軟骨細(xì)胞共孵育48h后檢測軟骨細(xì)胞過氧化氫酶(Catalase,CAT)、超氧化物歧化酶(superoxide dismutase, SOD)活性及丙二醛(Malondialdehyde, MDA)、活性氧(reactive oxygen species, ROS)水平。采用RT-PCR方法檢測TNF-α和MMP-13的mRNA表達(dá)量,試劑盒方法檢測CAT、SOD活性及MDA水平,熒光探針法檢測ROS水平。 結(jié)果：(1)不同濃度的AGEs (1,10,25,50,100μg/ml)與軟骨細(xì)胞共孵育48h后,TNF-a及MMP-13mRNA的表達(dá)較正常對照組明顯升高(P0.05或P0.01),且均以AGEs濃度為100μg/ml時作用最明顯；(2)Anti-RAGE (5μg/ml)與PDTC(0.1mmol/L)能顯著抑制由AGEs (100μg/ml)誘導(dǎo)的軟骨細(xì)胞TNF-α及MMP-13表達(dá)增多(P0.01),而anti-RAGE (5μg/ml)和PDTC(0.1mmol/L)單獨處理組與正常對照組相比差異均無統(tǒng)計學(xué)意義(P0.05)；(3)不同濃度的AGEs (1,10,25,50,100μg/ml)與軟骨細(xì)胞共孵育48h后,濃度依賴性地使軟骨細(xì)飽CAT、SOD活性降低, MDA、ROS含量增多,與正常對照組相比差異有統(tǒng)計學(xué)意義(P0.05) 結(jié)論：AGEs能顯著刺激軟骨細(xì)胞TNF-α和MMP-13表達(dá)增多,誘導(dǎo)軟骨細(xì)胞損傷,其機制與激活RAGE,誘導(dǎo)活性氧(ROS)生成增多,激活NF-κB信號通路有關(guān)。 第二部分AGEs對兔軟骨細(xì)胞PPARγ表達(dá)的影響與機制 目的：在體外培養(yǎng)的家兔軟骨細(xì)胞模型上,觀察AGEs對軟骨細(xì)胞過氧化物酶體增生物激活受體γ(Peroxisome proliferator-activated receptor-γ, PPARγ)表達(dá)的影響,探討AGEs與PPARγ的關(guān)系與機制。 方法：在原代培養(yǎng)的家兔軟骨細(xì)胞模型上,(1)不同濃度的AGEs與軟骨細(xì)胞共孵育48h后檢測軟骨細(xì)胞PPARγ的表達(dá)情況；(2)軟骨細(xì)胞與AGEs共孵育不同時間后檢測軟骨細(xì)胞PPARγ的表達(dá)情況；(3) RAGE的抗體(anti-PAGE)與軟骨細(xì)胞預(yù)孵1h后,再加入100μg/mlAGEs共同孵育48h,檢測軟骨細(xì)胞PPARγ的表達(dá)情況；(4)不同濃度的絲分裂原活化蛋白激酶(MAPK)信號路徑的阻斷劑(P38-MAPK阻斷劑SB203580,JNK-MAPK阻斷劑SP600125,ERK-MAPK阻斷劑PD98059)與軟骨細(xì)胞預(yù)孵30min,再加入100μg/mlAGEs共同孵育48h,檢測軟骨細(xì)胞PPARγ的表達(dá)情況。用RT-PCR方法檢測PPARγ的mRNA水平,用Western blot方法檢測PPARγ的蛋白含量。 結(jié)果：(1)不同濃度的AGEs (1,10,25,50,100μg/ml)與軟骨細(xì)胞共孵育48h后,軟骨細(xì)胞PPARγ的mRNA水平及蛋白含量較正常對照組明顯降低(P0.05),AGEs濃度越高PPARγ的mRNA水平及蛋白含量越低；(2)軟骨細(xì)胞與100μg/ml AGE共孵育不同時間(0,3,6,12,24,48h)后,PPARγ的mRNA表達(dá)和蛋白含量均隨時間的延長而降低,0h處理組與其他各組比較PPARγ的mRNA表達(dá)和蛋白含量差異均有統(tǒng)計學(xué)意義(P0.05)；(3)Anti-RAGE (5μg/ml) +AGEs處理組軟骨細(xì)胞PPARγ的mRNA表達(dá)顯著高于AGEs (100μg/ml)處理組(P0.05)；(4) P38-MAPK阻斷劑SB203580+AGEs和JNK-MAPK阻斷劑SP600125+AGEs處理組的軟骨細(xì)胞PPARγ的mRNA水平及蛋白含量明顯高于AGEs單獨處理組(P0.05),阻斷劑濃度越高PPARγ的mRNA水平及蛋白含量越高；ERK-MAPK阻斷劑PD98059+AGEs處理組與AGEs單獨處理組比較,軟骨細(xì)胞PPARγ的mRNA水平及蛋白含量均無明顯差異(P0.05) 結(jié)論：1.AGEs可誘導(dǎo)軟骨細(xì)胞PPARγ表達(dá)下調(diào),并具有濃度和時間依賴性；2.AGEs通過AGEs-RAGE-MAPK途徑實現(xiàn)對軟骨細(xì)胞PPARγ表達(dá)下調(diào)；3.MAPKs家族中P38-MAPK和JNK-MAPK信號通路參與了AGEs誘導(dǎo)軟骨細(xì)胞PPARγ表達(dá)下調(diào),ERK-MAPK信號通路與該效果無關(guān)。 第三部分PPARγ激動劑對AGEs誘導(dǎo)兔軟骨細(xì)胞TNF-α和MMP-13表達(dá)的影響與機制 目的：在體外培養(yǎng)的家兔軟骨細(xì)胞模型上,觀察PPARγ激動劑吡格列酮(pioglitazone)對AGEs誘導(dǎo)兔軟骨細(xì)胞TNF-a和MMP-13表達(dá)的影響,進一步探討PPARγ表達(dá)下調(diào)在AGEs致骨關(guān)節(jié)炎的作用、機制與意義。 方法：在原代培養(yǎng)的家兔軟骨細(xì)胞模型上,不同劑量的吡格列酮(1,10,50μM)與軟骨細(xì)胞預(yù)孵2h后,再加入100μg/mlAGEs共同孵育48h, (1)RT-PCR方法檢測軟骨細(xì)胞TNF-α和MMP-13的mRNA水平,用Western blot方法檢測TNF-α和MMP-13的蛋白含量；(2)試劑盒方法檢測軟骨細(xì)胞CAT、SOD活性及MDA水平；(3)熒光探針法檢測軟骨細(xì)胞ROS水平；(4)免疫熒光染色法檢測軟骨細(xì)胞的NF-κB-p65亞基轉(zhuǎn)運情況。 結(jié)果：(1)不同劑量的吡格列酮(1,10,50μM)+AGEs處理組軟骨細(xì)胞TNF-α和MMP-13mRNA水平及蛋白含量明顯低于AGEs處理組(P0.05),吡格列酮劑量越大TNF-α和MMP-13mRNA水平及蛋白含量越低,50μM匹格列酮+AGEs處理組及50μM匹格列酮單獨處理組與正常對照組相比差異無統(tǒng)計學(xué)意義(P0.05)；(2)1、10、50 u M口比格列酮與軟骨細(xì)胞預(yù)孵育1h后,濃度依賴性的拮抗由AGEs所致軟骨細(xì)胞CAT、SOD活性降低及MDA、ROS水平增高(P0.05),且均50μM毗格列酮作用最明顯；(3)1、10、50μM吡格列酮與軟骨細(xì)胞預(yù)孵育1h后,再加入100μg/mlAGEs共同孵育48h,軟骨細(xì)胞NF-κBP65的核轉(zhuǎn)位呈濃度依賴性明顯抑制,100μg/mlAGEs單純處理組則明顯高于正常對照組(P0.05)。 結(jié)論：1.存在一條AGEs誘導(dǎo)軟骨細(xì)胞TNF-a和MMP-13表達(dá)增多的信號通路,即：AGEs→RAGE→ROS↑→激活MAPK (P38-MAPK和JNK-MAPK)→下調(diào)PPARγ→F-κB活化→TNF-a和MMP-13↑; 2. PPARy下調(diào)在AGEs誘導(dǎo)軟骨細(xì)胞TNF-a和MMP-13表達(dá)增多的信號通路中起到了重要作用；3.PPARy激動劑吡格列酮能顯著抑制AGEs誘導(dǎo)軟骨細(xì)胞TNF-a和MMP-13表達(dá)增多；4.吡格列酮通過抑制RAGE/ROS/NF-κB信號通路,抵抗AGEs所致軟骨細(xì)胞損傷,從而起到防治骨關(guān)節(jié)炎病變的作用；5. AGEs致骨性關(guān)節(jié)炎病變可能存在以下細(xì)胞內(nèi)信號傳導(dǎo)途徑：AGEs與RAGE結(jié)合→氧自由基↑→激活MAPK→下調(diào)PPARy→細(xì)胞內(nèi)信號轉(zhuǎn)導(dǎo)使NF-κB活化→一系列炎癥和損傷因子表達(dá)→軟骨損傷。
[Abstract]:Part one the effect and significance of AGEs on the expression of TNF-a and MMP-13 in rabbit chondrocytes
Objective: To observe the expression and influence of AGEs on the tumor necrosis factor -a (tumor necrosisfactor-a, TNF- a) and matrix metalloproteinase -13 (advanced glycation end products, AGEs) as the damage factor, and to observe the expression and effect of AGEs on the tumor necrosis factor -a (tumor necrosisfactor-a, TNF- a) and matrix metalloproteinase -13 (13). Objective to investigate the relationship between AGEs and Osteoarthritis (OA) and possible signaling pathways and mechanisms.
Methods: on the original cultured rabbit chondrocyte model, (1) the expression of TNF-a and MMP-13 mRNA in cartilage cells was detected by different concentrations of AGEs and cartilage cells for 48h; (2) the specificity of the AGEs receptor (Receptor for advanced glycation end products) and the nuclear factor. The sex blocker, pyrrolidine two thiocarbamate (pyrrolidine dithiocarbamate, PDTC), was incubated with cartilage cells for 12h, and then incubated with 100 mu g/mlAGEs to incubate 48h, and to detect the expression of TNF-a and MMP-13mRNA in cartilage cells. (3) different concentrations of AGEs and chondrocytes were incubated for 48h after 48h and detected the chondrocyte catalase (Catalase, CAT). Superoxide dismutase (SOD) activity and malondialdehyde (Malondialdehyde, MDA), active oxygen (reactive oxygen species, ROS) level. The RT-PCR method was used to detect the expression of TNF- alpha and MMP-13.
Results: (1) after different concentrations of AGEs (1,10,25,50100 g/ml) and cartilage cells were incubated for 48h, the expression of TNF-a and MMP-13mRNA was significantly higher than that in the normal control group (P0.05 or P0.01), and the effect was the most obvious when the concentration of AGEs was 100 mu g/ml. (2) Anti-RAGE (5 mu g/ml) could significantly inhibit the cartilage induced by 100 micron. The expression of TNF- alpha and MMP-13 increased (P0.01), but there was no significant difference between the anti-RAGE (5 u g/ml) and PDTC (0.1mmol/L) alone group as compared with the normal control group (P0.05). (3) the concentration of AGEs (1,10,25,50100 u g/ml) and cartilage cells were reincubated with cartilage cells. The amount increased significantly compared with the normal control group (P0.05).
Conclusion: AGEs can significantly stimulate the expression of TNF- alpha and MMP-13 in cartilage cells and induce cartilage cell damage. The mechanism is related to the activation of RAGE, the induced activity of reactive oxygen species (ROS) and the activation of NF- kappa B signaling pathway.
The second part is the effect and mechanism of AGEs on PPAR gamma expression in rabbit chondrocytes.
Objective: To observe the effect of AGEs on the expression of peroxisome activation receptor gamma (Peroxisome proliferator-activated receptor- gamma (PPAR gamma), PPAR gamma, and the relationship and mechanism between AGEs and PPAR gamma in the chondrocyte model of rabbit in vitro.
Methods: on the original cultured rabbit chondrocyte model, (1) the expression of PPAR gamma in cartilage cells was detected after incubating with different concentrations of AGEs and cartilage cells for 48h; (2) the expression of PPAR gamma in cartilage cells was detected after CO incubation with AGEs at different time; (3) RAGE antibody (anti-PAGE) was incubated with cartilage cells and then added to 1H, and then added to the cartilage cells. The expression of PPAR gamma in cartilage cells was detected by 100 micron g/mlAGEs, and (4) the blockers of different concentrations of mitogen activated protein kinase (MAPK) signaling pathway (P38-MAPK blocker SB203580, JNK-MAPK blocker SP600125, ERK-MAPK blocker PD98059) were incubated with soft bone cells, and then 100 micron g/mlAGEs was incubated together. The expression of PPAR gamma in chondrocytes was measured. The mRNA level of PPAR gamma was detected by RT-PCR method and the protein content of PPAR gamma was detected by Western blot.
Results: (1) after different concentrations of AGEs (1,10,25,50100 g/ml) and chondrocytes were incubated with chondrocytes for 48h, the mRNA level and protein content of PPAR gamma in chondrocytes were significantly lower than that in the normal control group (P0.05), the higher the AGEs concentration was, the lower the mRNA level and the protein content of PPAR gamma, and (2) the time of incubation of the soft bone cells and 100 mu g/ml AGE was different. The expression of mRNA and protein content of PPAR gamma decreased with the prolongation of time. The mRNA expression and protein content of PPAR gamma in 0h treatment group were statistically significant (P0.05). (3) mRNA table of PPAR gamma in Anti-RAGE (5 u g/ml) +AGEs treatment group was significantly higher than that of AGEs (100 mu) treatment group; (4) The mRNA level and protein content of PPAR gamma in the chondrocyte of the blocker SB203580+AGEs and the JNK-MAPK blocker SP600125+AGEs treatment group was significantly higher than that of the AGEs alone group (P0.05). The higher the concentration of the blocker was, the higher the mRNA level and the protein content of PPAR gamma; the ERK-MAPK blocker was compared with the AGEs alone treatment group. The level of mRNA and protein content of gamma had no significant difference (P0.05).
Conclusion: 1.AGEs can induce down regulation of PPAR gamma expression in chondrocytes, and it has concentration and time dependence; 2.AGEs can reduce the expression of PPAR gamma in chondrocytes by AGEs-RAGE-MAPK pathway; P38-MAPK and JNK-MAPK signaling pathway in 3.MAPKs family is involved in AGEs induced down regulation of PPAR gamma in cartilage cells, ERK-MAPK signaling pathway and the effect of this effect are not. Close.
The third part is the effect and mechanism of PPAR gamma agonist on AGEs induced TNF- alpha and MMP-13 expression in rabbit chondrocytes.
Objective: To observe the effect of PPAR gamma agonist pioglitazone (pioglitazone) on the expression of TNF-a and MMP-13 in rabbit chondrocytes induced by AGEs in vitro, and further explore the effect, mechanism and significance of the down regulation of PPAR gamma expression in AGEs induced osteoarthritis.
Methods: on the original cultured rabbit chondrocyte model, after incubating 2H with different doses of pioglitazone (1,10,50 mu M) and cartilage cells, 48h was incubated with 100 mu g/mlAGEs, and (1) RT-PCR method was used to detect mRNA level of TNF- alpha and MMP-13 in cartilage cells. Western blot method was used to detect the content of TNF- alpha and protein. (2) reagent box The CAT, SOD activity and MDA level of cartilage cells were detected by the method. (3) the fluorescence probe method was used to detect the ROS level of cartilage cells, and (4) the transport of NF- kappa B-p65 subunits of cartilage cells was detected by immunofluorescence staining.
Results: (1) the level and protein content of TNF- alpha and MMP-13mRNA in the chondrocytes of different doses of pioglitazone (1,10,50 mu M) +AGEs treatment group were significantly lower than that in AGEs treatment group (P0.05). The higher the dose of pioglitazone, the lower the level of TNF- A and MMP-13mRNA and the protein content, the 50 micron pioglitazone + AGEs treatment group and 50 mu M pioglitazone alone treatment group and normal pairs. There was no significant difference in the contrast group (P0.05); (2) the concentration dependent antagonism of AGEs induced cartilage cells CAT, SOD activity decreased and ROS level increased (P0.05), and the action of 50 mu M vishaglitazone was the most obvious. (3) after incubating with chondrocytes, 1,10,50 u M mouth was preincubated with chondrocytes. (3) The nuclear transposition of NF- kappa BP65 in chondrocytes was significantly inhibited by the addition of 100 mu g/mlAGEs and co incubated for 48h. The 100 u g/mlAGEs treatment group was significantly higher than that of the normal control group (P0.05).
Conclusion: 1. there is a signal pathway of increased expression of TNF-a and MMP-13 in cartilage cells induced by AGEs, that is, AGEs, RAGE, ROS, MAPK (P38-MAPK and JNK-MAPK), down PPAR gamma, F- kappa B activation, etc. 3.PPARy agonist pioglitazone significantly inhibits the increased expression of TNF-a and MMP-13 in cartilage cells induced by AGEs; 4. pioglitazone can resist the injury of cartilage cells induced by AGEs by inhibiting the RAGE/ROS/NF- kappa B signaling pathway, and thus plays a role in the prevention and treatment of osteoarthritis, and 5. AGEs induced osteoarthritis may exist in the following cell The signal transduction pathway: the combination of AGEs and RAGE, oxygen free radical, activates MAPK, down PPARy and intracellular signal transduction makes NF- kappa B activation, a series of inflammation and damage factor expression, and cartilage damage.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R329

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 張林;陸軍;李永剛;;骨關(guān)節(jié)炎中介導(dǎo)軟骨細(xì)胞代謝失衡的相關(guān)信號通路研究進展[J];東南大學(xué)學(xué)報(醫(yī)學(xué)版);2013年04期

本文編號：2011374