本文選題：DYYG化合物 + 足細胞病　； 參考：《吉林大學》2017年博士論文

【摘要】：背景與目的:足細胞損傷與腎小球疾病的發(fā)生發(fā)展密切相關,本實驗采用的嘌呤霉素氨基核苷(PAN)誘導大鼠腎病模型(PAN腎病模型)與PAN體外誘導足細胞凋亡為兩個經(jīng)典的足細胞損傷模型,廣泛用于足細胞損傷研究當中。由我們的合作方美國劉宏宇教授課題組研發(fā)的DYYG化合物(C16H12N8O.HCL)是甲基并咪唑-VA二唑-吡啶并咪唑的衍生物的鹽酸鹽,是一種新型小分子化合物,其分子量為368。我們前期研究發(fā)現(xiàn)DYYG化合物對于足細胞損傷模型具有顯著修復作用,該論文主旨為探討DYYG化合物是否通過PI3K/Akt/m TOR信號通路誘導足細胞自噬,產(chǎn)生對PAN腎病足細胞損傷的修復,從而對腎臟起保護作用。方法:第一部分:體外培養(yǎng)足細胞實驗:分為CON組、DYYG組、PAN組、PAN+DYYG組,按分組條件培養(yǎng)24小時,Western blot、免疫熒光方法觀察PAN誘導足細胞損傷后,DYYG對其標志蛋白表達的影響。流式細胞儀檢測DYYG對PAN誘導足細胞凋亡的影響。第二部分:體外培養(yǎng)足細胞實驗:分為CON組、DYYG組、PAN組、PAN+DYYG組、PAN+DYYG+3-MA組、3-MA組,按分組條件培養(yǎng)24小時,應用共聚焦顯微鏡觀察DYYG對m RFP-GFP-LC3腺病毒轉染足細胞自噬的影響,透射電鏡下觀察足細胞內(nèi)自噬體數(shù)目的變化,Western blot檢測足細胞LC3-Ⅱ、Beclin-1、P62的表達水平。第三部分:60只雄性SD大鼠隨機分為CON組、PAN組,PAN+DYYG組,PAN+TAC組,每組15只,構建PAN腎病模型。于0、7、14、21、28天,分五批處死動物,12只/次,測每個時間點24小時尿量、尿蛋白定量、血Cr、血BUN。免疫組織化學方法檢測DYYG對PAN腎病模型腎小球足細胞標志蛋白Podcin表達的影響。免疫熒光方法共聚焦顯微鏡下觀察DYYG對PAN腎病模型腎小球足細胞特異性骨架蛋白Synaptopodin表達的影響。透射電鏡下觀察DYYG對PAN腎病模型腎小球足細胞病理結構的影響。第四部分:Western blot明確DYYG對PAN腎病模型足細胞LC3-Ⅱ、Beclin-1、P62以及PTEN、PI3K、Akt及其下游m TOR信號分子表達的影響。結果:第一部分:按分組條件培養(yǎng)足細胞24小時后,Western blot結果發(fā)現(xiàn),與CON組相比,PAN組足細胞標志蛋白Synaptopodin、Nephrin、Podcin、ZO-1蛋白表達下調(diào)(均P0.05)。與PAN組相比,PAN+DYYG組足細胞標志蛋白Synaptopodin、Nephrin、Podcin、ZO-1蛋白表達上調(diào)(均P0.05),而DYYG組與CON組相比則無明顯差異。免疫熒光方法結果顯示,PAN組足細胞特異性骨架蛋白Synaptopodin熒光強度顯著低于CON組,Synaptopodin絲狀結構破壞,皺縮于核周表達。PAN+DYYG組足細胞特異性骨架蛋白Synaptopodin熒光強度顯著高于PAN組。而DYYG組與CON組相比則無明顯差異。流式細胞儀檢測結果發(fā)現(xiàn),與CON組相比,PAN組足細胞凋亡比例明顯增加(均P0.05),與PAN組相比,PAN+DYYG組足細胞凋亡比例明顯降低(均P0.05),而DYYG組與CON組相比則無明顯差異。第二部分:應用共聚焦顯微鏡觀察DYYG對m RFP-GFP-LC3腺病毒轉染足細胞自噬的影響,結果顯示,CON組足細胞內(nèi)見紅色斑點及黃色斑點表達。與CON組相比,PAN組足細胞紅色斑點及黃色斑點聚集數(shù)量明顯減少(P0.05)。與PAN組相比,PAN+DYYG組足細胞紅色斑點及黃色斑點聚集數(shù)量明顯增加(P0.05)。經(jīng)3-MA預處理1小時后再給予PAN+DYYG,DYYG恢復足細胞自噬的作用減弱(P0.05)。DYYG組、3-MA組與CON組相比則無明顯差異。透射電鏡下觀察足細胞內(nèi)自噬體數(shù)量,結果顯示,與CON組相比,PAN組足細胞自噬體數(shù)量明顯減少(P0.05)。與PAN組相比,PAN+DYYG組足細胞自噬體數(shù)量明顯增加(P0.05)。DYYG組、3-MA組與CON組相比則無明顯差異。經(jīng)3-MA預處理1小時后再給予PAN+DYYG,足細胞內(nèi)自噬體數(shù)量無增加。Western blot檢測足細胞自噬相關蛋白的表達水平均驗證上述實驗結果,表明DYYG可增強PAN誘導足細胞損傷模型的自噬水平,從而達到足細胞保護作用。第三部分:成功建立SD大鼠PAN腎病模型,第7天PAN組、PAN+DYYG組、PAN+TAC組與CON組相比大鼠體重未見明顯變化,差異無統(tǒng)計學意義(均P0.05)。第14天、第21天及第28天CON組、PAN+DYYG組及PAN+TAC組與PAN組比較,大鼠體重逐漸增加,差異有統(tǒng)計學意義(均P0.05),PAN+DYYG組與PAN+TAC組比較差異無統(tǒng)計學意義(均P0.05)。第7天,PAN組、PAN+DYYG組、PAN+TAC組與CON組比較大鼠尿量明顯減少,出現(xiàn)蛋白尿,血Cr及血BUN值明顯升高(均P0.05)。第14天,PAN組、PAN+DYYG組、PAN+TAC組與CON組比較大鼠尿量明顯減少,24小時尿蛋白定量、血Cr及血BUN值較第7天降低,而PAN+DYYG組、PAN+TAC組與PAN組比較,24小時尿蛋白定量、血Cr及血BUN值降低更明顯(均p0.05)。第21天、第28天,PAN組、PAN+DYYG組、PAN+TAC組24小時尿蛋白定量、血Cr及血BUN濃度逐漸降低,與PAN組比較,PAN+DYYG組、PAN+TAC組降低更明顯,24小時尿量明顯增加,PAN+DYYG組與PAN+TAC組比較差異無統(tǒng)計學意義(均P0.05)。免疫組織化學方法檢測PAN組足細胞標志蛋白Podcin表達顯著低于CON組,PAN+DYYG組及PAN+TAC組足細胞標志蛋白Podcin表達顯著高于PAN組,兩者相比則無明顯差異(P0.05)。免疫熒光方法共聚焦顯微鏡結果顯示,PAN組足細胞特異性骨架蛋白Synaptopodin熒光強度顯著低于CON組,PAN+DYYG組足細胞特異性骨架蛋白Synaptopodin熒光強度顯著高于PAN組。而DYYG組與CON組相比則無明顯差異。透射電鏡結果顯示,PAN注射第7天,PAN腎病模型組,足突融合,溶酶體壞死,腎間質水腫,PAN腎病給予DYYG、TAC治療組,足突增寬,腫脹。PAN注射第14天,PAN腎病模型組,病變更加嚴重,足突廣泛融合,部分足突消失,而給予DYYG、TAC治療組,與第7天比較,足突形態(tài)好轉,但兩者未見明顯差異。PAN注射第21天,PAN腎病組及給予DYYG、TAC治療組,足突腫脹,融合好轉,與PAN腎病模型組相比,給予DYYG、TAC治療組好轉更明顯,與CON組足突結構相似,但兩者未見明顯差異。PAN注射第28天,給予DYYG、TAC治療組足突形態(tài)結構恢復正常,但PAN腎病模型組足突寬度仍高于CON組。通過對足突超微結構的觀察,與PAN腎病模型組相比,給予DYYG、TAC治療組足突融合程度明顯低于PAN腎病模型組,DYYG、TAC可以改善和逆轉足突形態(tài)改變。第四部分:Western Blot方法結果顯示:PAN組足細胞內(nèi)PTEN蛋白表達量下調(diào),PI3K、p Akt、m TOR表達量明顯增加,LC3Ⅱ、Beclin表達下調(diào);P62蛋白表達量增加,信號通路蛋白及自噬相關蛋白表達量與CON組相比,差異有統(tǒng)計學意義(均P0.05)。與PAN組比較,PAN+DYYG組足細胞PTEN表達上調(diào),PI3K、p Akt、m TOR表達下調(diào);LC3Ⅱ、Beclin表達上調(diào);P62蛋白表達量減少,信號通路及自噬相關蛋白表達量與PAN組相比,差異有統(tǒng)計學意義(均P0.05)。而PAN+LY294002組,PI3K抑制劑LY294002可抑制PI3K、p Akt、m TOR表達,上調(diào)自噬相關蛋白LC3Ⅱ、Beclin,下調(diào)P62表達(均P0.05),但對PTEN表達差異無統(tǒng)計學意義(P0.05)。結論:本研究證實PAN可抑制足細胞自噬,誘導足細胞損傷。DYYG干預可減輕PAN誘導的足細胞自噬抑制,增強足細胞自噬,改善足細胞損傷,其保護機制可能是通過上調(diào)PTEN表達,抑制PI3K/Akt/m TOR信號通路活化而實現(xiàn)的。
[Abstract]:Background and objective: podocyte injury is closely related to the development of glomerular disease. The rat model of nephrosis induced by PAN (PAN nephropathy model) and PAN induced foot cell apoptosis in vitro are two classic foot cell damage models, which are widely used in the study of foot cell injury. The DYYG compound (C16H12N8O.HCL), developed by Professor Liu Hongyu in the United States, is a salt of methyl and imidazole -VA two azol-pyridine and imidazole derivatives. It is a new small molecular compound. Its molecular weight is 368.. Our previous study found that DYYG compounds have a significant repair effect on the model of foot cell damage. The main purpose of this paper is to explore the main purpose of this paper. Whether DYYG compounds can induce autophagy through PI3K/Akt/m TOR signaling pathway to repair foot cell injury in PAN nephrosis and protect the kidneys. Method: Part 1: in vitro cultivation of podocyte experiment: CON group, DYYG group, PAN group, PAN+DYYG group, 24 hours, Western blot, immunofluorescence method according to the group conditions. The effect of DYYG on the expression of its marker protein after PAN induced podocyte injury. Flow cytometry was used to detect the effect of DYYG on PAN induced podocyte apoptosis. The second part: in vitro cultivation of podocyte experiment: CON group, DYYG group, PAN group, PAN+DYYG group, PAN+DYYG+3-MA group, 3-MA group, 24 hours by group conditions, and confocal microscopy The effect of DYYG on autophagy of M RFP-GFP-LC3 adenovirus transfected foot cells was observed. Under transmission electron microscope, the number of autophagosomes in podocytes was observed. Western blot was used to detect the expression level of LC3- II, Beclin-1 and P62 in podonote. The third part: 60 male SD rats were randomly divided into CON group, PAN group, PAN+DYYG group, and 15 rats each. On 0,7,14,21,28 days, five batches of animals were killed, 12 / time, 24 hours per hour urine volume, urine protein quantitative, blood Cr, and blood BUN. immunohistochemical method were used to detect the effect of DYYG on the expression of glomerular podocyte marker protein Podcin in PAN nephropathy model. DYYG against PAN nephropathy model glomerular foot thin under the immunofluorescence method. Effect of the expression of cellular specific skeleton protein Synaptopodin. The effect of DYYG on the pathological structure of glomerular podocytes in PAN nephropathy model was observed under transmission electron microscope. Fourth part: Western blot clearly the effect of DYYG on the expression of LC3- II, Beclin-1, P62 and PTEN, PI3K, and the lower reaches of the PAN nephropathy model. Results: the first part Western blot results showed that the expression of podocyte protein Synaptopodin, Nephrin, Podcin, ZO-1 protein was down regulated (all P0.05) in the PAN group compared with the CON group. The results of immunofluorescence showed that the fluorescence intensity of the specific skeleton protein Synaptopodin of the PAN group was significantly lower than that of the CON group, and the Synaptopodin filamentous structure was destroyed, and the expression of the specific skeleton protein Synaptopodin fluorescence intensity of the.PAN+DYYG group was significantly higher than that of the PAN group, while the DYYG group and the CON group phase were significantly higher than that of the group of Synaptopodin. Compared with the CON group, flow cytometry showed that the percentage of apoptotic podocytes in the PAN group was significantly increased (P0.05). Compared with the PAN group, the percentage of apoptotic podthe cells in the PAN+DYYG group was significantly lower (P0.05), while the DYYG group was not significantly different from that in the CON group. Second part: a confocal microscope was used to observe DYYG to m RFP-GFP-LC. The effect of 3 adenovirus transfection on autophagy of foot cells showed that red spots and yellow spots were expressed in the foot cells of CON group. Compared with group CON, the number of red spots and yellow spots in the foot cells of group PAN decreased significantly (P0.05). Compared with the PAN group, the number of red spots and yellow spots in the PAN+DYYG group increased significantly (P0.05). Through 3-, the number of the podocytes in the group of PAN was significantly increased. MA was given to PAN+DYYG after 1 hours of preconditioning, and the effect of DYYG on autophagy decreased (P0.05) in.DYYG group, and there was no significant difference between 3-MA and CON groups. The number of autophagic bodies in the podocytes was observed under transmission electron microscopy. The results showed that the number of autophagic bodies in the PAN group was significantly reduced (P0.05) compared with the CON group (P0.05). The PAN+DYYG group was more thin than the PAN group. There was a significant increase in the number of autophagic bodies (P0.05).DYYG, and there was no significant difference between the 3-MA group and the CON group. PAN+DYYG was given after 1 hours of 3-MA pretreatment. The number of autophagic in the podocytes was not increased by.Western blot to detect the expression level of autophagy related protein in the podocyte. The results showed that DYYG could enhance the injury of the foot cell induced by PAN. The third part: the model of PAN nephropathy in SD rats was successfully established. Seventh days PAN group, PAN+DYYG group, PAN+TAC group and CON group had no significant changes in weight (P0.05). Fourteenth days, twenty-first and 28 days CON, PAN+DYYG group and PAN+TAC group compared with PAN group, The weight of rats increased gradually, the difference was statistically significant (all P0.05). There was no significant difference between group PAN+DYYG and PAN+TAC group (P0.05). Seventh days, PAN group, PAN+DYYG group, PAN+TAC group and CON group were significantly reduced in urine volume, proteinuria, blood Cr and BUN value increased significantly (P0.05). Fourteenth days, PAN group, group, group and In group CON, urine volume decreased significantly, urine protein was 24 hours, blood Cr and blood BUN were lower than seventh days, while group PAN+DYYG, PAN+TAC group and PAN group, 24 hours urine protein quantitative, blood Cr and blood BUN value decreased more significantly (all P0.05). Twenty-first days, twenty-eighth days, PAN group, PAN+ DYYG group, 24 hours urine protein quantitative, blood and blood concentration by As compared with the PAN group, the decrease in the group PAN+DYYG and the PAN+TAC group was more obvious, the 24 hour urine volume increased significantly, and there was no significant difference between the PAN+DYYG group and the PAN+TAC group (P0.05). The expression of the foot cell marker protein Podcin in the PAN group was significantly lower than that in the CON group, and the PAN+DYYG and PAN+TAC group of the podpodic protein Podcin table was significantly lower than that in the PAN+TAC group. It was significantly higher than the PAN group, but there was no significant difference (P0.05). The immunofluorescence confocal microscopy showed that the fluorescence intensity of the foot cell specific skeleton protein Synaptopodin in the PAN group was significantly lower than that in the CON group, and the Synaptopodin fluorescence intensity of the foot cell specific skeleton protein of the PAN+DYYG group was significantly higher than that in the PAN group. The DYYG group was compared to the CON group. The transmission electron microscope showed that seventh days of PAN injection, PAN nephropathy model group, foot process fusion, lysosome necrosis, renal interstitial edema, PAN nephropathy given DYYG, TAC treatment group, widening of foot process, swelling.PAN injection, PAN nephropathy model group, more serious lesions, extensive fusion of foot process, partial foot process disappearing, and DYYG, TAC treatment group, Compared with the seventh days, the morphology of the foot process improved, but there was no significant difference between the two groups for twenty-first days. The PAN nephropathy group and the DYYG, TAC treatment group, the swelling of the foot process and the fusion improved. Compared with the PAN nephropathy model group, the improvement of the TAC treatment group was more obvious and similar to that of the CON group, but there was no significant difference between the two groups for twenty-eighth days, and DYYG, and there was no significant difference between the two groups. The morphological structure of foot process in the TAC treatment group returned to normal, but the width of the foot process in the PAN nephropathy model group was still higher than that of the CON group. Through the observation of the ultrastructure of the podocyte process, compared with the PAN nephropathy model group, the degree of foot process fusion in the TAC group was significantly lower than that of the PAN nephropathy model group, DYYG and TAC could improve and reverse the morphologic changes of the podocyte process. The fourth part: Western B. The results of lot showed that the expression of PTEN protein in the PAN group was down, the expression of PI3K, P Akt, m TOR increased obviously, the expression of LC3 II and Beclin decreased, the expression of P62 protein was increased, and the expression of signal pathway protein and autophagy related protein was significantly different from that of CON group. Up regulation, PI3K, P Akt, m TOR expression down-regulation; LC3 II, Beclin expression up-regulated, P62 protein expression reduced, signal pathway and autophagy related protein expression levels compared with the PAN group, the difference was statistically significant (P0.05). P62 expression (P0.05), but there is no significant difference in the expression of PTEN (P0.05). Conclusion: This study confirmed that PAN can inhibit autophagy of podgo, induced foot cell injury.DYYG intervention can reduce the inhibition of autophagy induced by PAN, increase the autophagy of podocyte and improve foot cell injury, and its protective mechanism may be by up regulation of PTEN expression and inhibiting PI3K/Akt/. The m TOR signal pathway is activated.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R692

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