本文關鍵詞： 常染色體顯性多囊腎病 二肽基肽酶 腺苷磷酸活化蛋白激酶 哺乳動物雷帕霉素靶蛋白　出處：《第二軍醫(yī)大學》2016年博士論文　論文類型：學位論文

【摘要】：研究背景及目的常染色體顯性多囊腎病(Autosomal dominant polycystic kidney disease, ADPKD)是人類發(fā)病率最高的單基因遺傳性腎臟病。接受替代治療的終末期腎病(ESRD)中7-10%是ADPKD患者。盡管在發(fā)現(xiàn)此病500年來進行了大量的研究,直至今日仍然無法治愈且對其機制尚不明了。所有人種皆不能幸免于ADPKD,7-10%的ESRD因此而起。全球范圍,接受腎臟替代治療(RRT)的第四大原因就是ADPKD。在不同的國家,ADPKD導致的ESRD發(fā)病率有所不同,在日本是每年百萬分之4.8,在美國是每年百萬分之7.9,歐洲各國每年百萬分之3.9到15.3。在丹麥哥本哈根[4]和美國奧姆斯特德進行的流行病學調(diào)查顯示,80歲以下人口中,ADPKD的患病率是400到1000分之一。此后在歐洲和日本進行的其他研究顯示不同地區(qū)ADPKD患病率波動于543到4000分之一。鑒于上述研究地理位置、觀測時間、采樣方法、診斷標準、家系調(diào)查、人口多寡都有不同,故而差異雖大尚可理解。然則,來自尸檢報告的數(shù)據(jù)要高于1/500,為1/339-1/492。這說明大量患者終其一生未能確診。ADPKD由多囊腎病基因1(Polycystic kidney disease 1 gene, PKD1)和多囊腎病基因2(Polycystic kidney disease 2 gene, PKD2)兩個基因突變造成,此二基因分別編碼多囊蛋白-1(Polycystin-1, PC1)及多囊蛋白.2(Polycystin-2, PC2)。其中PKD1突變的患者占80～85%,PKD2突變的患者占15-20%。所謂PKD3,經(jīng)過對相關病例的重新分析,已經(jīng)基本排除了其存在的可能性。PC1與PC2在腎小管上皮細胞初級纖毛上相互作用構(gòu)成復合體,后者屬于瞬時受體電位(transient receptor potential, T P)家族中的一種非選擇性鈣離子滲透通道。PC1的C末端結(jié)構(gòu)域可以易位至細胞核的結(jié)構(gòu),并充當基因轉(zhuǎn)錄的共激活劑。PC1和PC2在纖毛經(jīng)受機械刺激時引發(fā)Ca2+瞬間內(nèi)流。ADPKD的發(fā)病機制尚未清楚,有前途的治療方法主要集中于通過阻斷過度活化mTOR通路和cAMP通路來延緩疾病進展。動物實驗發(fā)現(xiàn)mTOR抑制劑(依維莫司和西羅莫司)改善嚙齒類動物模型囊性腎病的效果非常顯著,但依維莫司和西羅莫司都是強效的免疫抑制劑,可能帶來致命的副作用,且臨床試驗并未取得良好的療效。所以我們需要探索更為安全有效的手段。大量報道證實AMP活化蛋白激酶(AMP-activated protein kinase, AMPK)下調(diào)是：mTOR通路過度活化的原因之一,通過上調(diào)MPK水平可以抑制過度活化的mTOR通路,并在動物模型中取得了良好的效果。通過抑制二肽基肽酶4(dipeptidyl peptidase 4, DPP4)的活性可以上調(diào)AMPK水平是毫無疑問的,但能否抑制過度激活的mTOR通路改善ADPKD, ADPKD患者和動物模型中是否存在DPP4過表達從而過度激活了mTOR通路,這兩個問題尚無解答。本課題的目的是觀察ADPKD患者和實驗動物腎組織中DPP4是否存在表達上調(diào),以及使用DPP4抑制劑,是否能同過上調(diào)AMPK,抑制mTO R活性,達到治療ADPKD的目的。研究方法1、DPP4在ADPKD患者及動物模型中的表達水平檢測：搜集ADPKD患者腎組織與移植供腎組織標本,用western blot技術測定兩者的DPP4蛋白表達量,并進行比較；繁育多囊腎模型Han:SPRD大鼠,鑒定出野生型(WT)和發(fā)病模型(cy-／+),正常飼養(yǎng)至12周,取腎組織,采用western blot測定DPP4蛋白表達量,進行比較。2、動物實驗驗證抑制DPP4活性：干預藥物包括AMPK激活劑二甲雙胍(Metformin), DPP4抑制劑維格列汀(Vildagliptin)和利格列汀(Linagliptin)。繁育多囊腎模型Han:SPRD大鼠,出生后鑒定出野生型(WT)和發(fā)病模型(cy-/+)。將發(fā)病模型大鼠分為對照組(Ctrl)以及二甲雙胍(MET)組、維格列汀(VIG)組、利格列汀(LIG)組。后3組大鼠自出生后4周給藥,于第4/8/12周取血查腎功能,第12周代謝籠測24小時尿量,并處死動物,取雙腎測腎重體重比,提取蛋白,送病理行全片掃面。3、細胞實驗觀察抑制DPP4活性對細胞增殖的影響：采用人腎囊腫襯里上皮細胞株WT9.12進行培養(yǎng)。以MTT方法觀察在不同濃度的二甲雙胍、維格列汀、利格列汀作用下,24小時、48小時、72小時細胞增殖情況。4、抑制DPP4活性治療ADPKD的機制研究：取不同濃度二甲雙胍、維格列汀、利格列汀處理的WT9.12細胞,Western blot技術分析干預后AMPK的蛋白含量變化。取前述實驗提取WT、Ctrl、MET、VIG、LIG組大鼠腎組織蛋白,用western blot技術測定AMPK、Akt、p70S6K蛋白含量。研究結(jié)果1、ADPKD患者腎臟組織中DPP4的表達量顯著高于移植供腎(1.97 vs.0.60,P=0.002),升高了大約3倍。多囊腎模型大鼠(cy-/+)腎臟中情況類似,發(fā)病的模型大鼠腎臟中DPP4表達量升高了接近3倍(7.49 vs 1.14,P=0.001)。2、與對照組相比,二甲雙胍、維格列汀和利格列汀均可顯著降低Han:SPRD大鼠腎重體重比(KW/BW)、囊腫指數(shù),改善腎功能。3組給藥組之間上述指標沒有統(tǒng)計學差異。3、二甲雙胍、維格列汀、利格列汀都可以抑制WT9.12的增殖。三種藥物都存在濃度依賴性,在24小時、48小時、72小時三個時間節(jié)點50nM濃度的二甲雙胍對WT9.12細胞的增長抑制率分別為34%,60%,74%；20nM維格列汀對WT9.12細胞的增長抑制率分別為14%,20%,28%；1nM利格列汀對WT9.12細胞的增長抑制率分別為79%,86%,93%。4、經(jīng)給藥后,給予二甲雙胍、維格列汀、利格列汀的模型大鼠腎組織中pAMPK的表達均顯著高于Ctrl組(P0.001),分別升高了2.7倍、3.5倍和5倍;而且維格列汀組顯著高于二甲雙胍組(3.5 vs.2.7,P0.001),而利格列汀組又顯著高于維格列汀組(5.1 vs 2.7,P0.001)。和對照組相比,二甲雙胍(4.5 vs 5.1,P=0.029)、維格列汀(3.9 vs 5.1,P0.001)、利格列汀(3.0 vs 5.1,P0.001)都可以使模型大鼠腎組織中過度活化的1nTOR被抑制,下游產(chǎn)物p70S6K表達量顯著下降；維格列汀組p70S6K蛋白表達量低于二甲雙胍組(3.9 vs4.5,P=0.045),利格列汀組低于維格列汀組(3.0vs.3.9,P=0.001)。結(jié)論本課題發(fā)現(xiàn)/DPKD患者和多囊腎模型大鼠腎組織中DPP4過表達。使用DPP4抑制劑,可以上調(diào)AMPK抑制過度激活的mTOR通路,抑制囊腫細胞增殖,延緩病情進展。DPKD患者的DPP4過表達引起的mTOR過度激活可能是其發(fā)病機制之一,抑制DPP4活性,是治療ADPKD潛在的靶點。
[Abstract]:Background and objective: autosomal dominant polycystic kidney disease (Autosomal dominant polycystic kidney disease, ADPKD) is a human disease incidence of single gene hereditary kidney. The highest hormone replacement therapy of end-stage renal disease (ESRD) in 7-10% ADPKD patients. Although the disease has done a lot of research for 500 years, until today still can not the cure and the mechanism remains unknown. All races are not immune to ADPKD, 7-10% and ESRD. So the world, received renal replacement therapy (RRT) is the fourth leading cause of ADPKD. in different countries, the incidence of ESRD caused by ADPKD is different in Japan every year is 4.8 ppm, in the United States is the year 7.9 ppm, according to European countries every year 3.9 ppm to 15.3. in Denmark and the United States of Copenhagen [4] Olmsted epidemiological survey, the population under the age of 80, ADPKD The prevalence rate is 400 to 1000 points. One of the other studies since then in Europe and Japan show that different regions ADPKD prevalence rates from 543 to one in 4000. In view of the above research location, observation time, sampling methods, diagnostic criteria, family survey, population size are different, so the difference is big is understandable. However, from the autopsy report data for 1/339-1/492. is higher than 1/500, indicating a large number of patients in his life was not diagnosed.ADPKD from polycystic kidney disease 1 (Polycystic kidney disease 1 gene, PKD1) and polycystic kidney disease 2 (Polycystic kidney disease 2 gene, PKD2) two gene mutation caused the two gene encoding polycystins respectively -1 (Polycystin-1, PC1) and.2 (Polycystin-2, PC2, polycystins). The patients with PKD1 mutations accounted for 80 ~ 85%, PKD2 mutation patients accounted for 15-20%. called PKD3, after re analysis of related cases, Have basically ruled out the possibility of.PC1 and PC2 are interaction in renal tubular epithelial cells of primary cilia form complexes, which belong to the transient receptor potential (transient receptor potential, T P) is a non selective calcium channel family penetration in the.PC1 C terminal domain structure can be translocated to the nucleus, and act as the gene transcription coactivator.PC1 and PC2 subjected to mechanical stimulation in the cilia caused pathogenesis of.ADPKD Ca2+ within the moment is not yet clear, promising therapies focus on blocking the excessive activation of the mTOR pathway and cAMP pathway to delay the progression of the disease. Animal experiments showed that mTOR inhibitors (Bea Vimal S and sirolimus) improve the rodent animal model cystic kidney disease, the effect is very significant, but Bea Vimal S and are potent immunosuppressant sirolimus, may lead to fatal side-effects, and Clinical trials have not achieved good results. So we need to explore a more safe and effective means. A large number of reports confirmed that AMP activated protein kinase (AMP-activated protein kinase, AMPK) is one of the reasons: the down-regulation of mTOR pathway activation, by increasing the level of MPK can inhibit the excessive activation of the mTOR pathway system, and have achieved good results in in the animal model. By two inhibition of dipeptidyl peptidase 4 (dipeptidyl peptidase 4, DPP4) activity can increase the level of AMPK is no doubt, but can inhibit the excessive activation of the mTOR pathway to improve the ADPKD, whether the DPP4 expression of mTOR pathway leading to excessive activation of ADPKD patients and animal models, these two issues is no answer. The purpose of this study is to observe the renal tissue of ADPKD patients and animal in the presence of DPP4 expression, and the use of DPP4 inhibitors, whether can the same on AMPK inhibition The mTO activity of R, achieve the purpose of treatment of ADPKD. Methods: 1. To detect the expression of DPP4 in ADPKD patients and animal models: collect donor kidney tissue samples in patients with ADPKD and renal tissue transplantation, determination of expression of both DPP4 protein by Western blot technology, and compared the breeding of polycystic kidney; Han:SPRD rat model, identification the wild type (WT) and disease model (cy- / +), normal feeding to 12 weeks, the renal tissues, DPP4 protein expression was detected by using Western blot, compared to.2, the animal experiments to inhibit the activity of DPP4: drug intervention including AMPK activator (Metformin), metformin DPP4 inhibitor (Vildagliptin) and Leigh Glenn Dean Vee Glenn Dean (Linagliptin) breeding. Han:SPRD rat model of polycystic kidney, after the birth of identified wild type (WT) and model (cy-/+). The incidence of the disease model rats were randomly divided into control group (Ctrl) and metformin (MET) group, vildagliptin (VIG) group, linagliptin (LIG) group. After 3 groups of rats from birth 4 weeks after administration, in the 4/8/12 week blood check renal function, Twelfth Zhou Dai Xie cage was measured 24 h urine volume, and kill the animal, the two kidneys measured kidney weight / body weight ratio, protein extraction, pathology the line scanning.3 cells, experimental observation of effect of DPP4 on cell proliferation by human renal cyst lining epithelial cells WT9.12 were cultured. Vee Glenn Dean observed in different concentrations of metformin, using MTT method, under the action of linagliptin, 24 hours, 48 hours, 72 hours, the proliferation of.4 cells, inhibiting mechanism DPP4 activity in the treatment of ADPKD: different concentrations of metformin, Vee Glenn Dean, linagliptin treated WT9.12 cells, changes of protein content in Western intervention AMPK blot technology. The experiment from extraction of WT, Ctrl, MET, VIG, renal tissue in rats of LIG protein, AMPK was determined by Western blot technology, Akt, The content of p70S6K protein. Results: 1. Expression of DPP4 in renal tissue of ADPKD patients was significantly higher than that of donor kidney transplantation (1.97 vs.0.60, P=0.002), increased by about 3 times. The rat model of polycystic kidney (cy-/+) similar to the kidney, the expression of DPP4 increased by nearly 3 times the onset of kidney in model rats (7.49 vs 1.14, P=0.001.2), compared with the control group, metformin and vildagliptin and Leigh Glenn Dean could significantly decrease the Han:SPRD of rat kidney weight to body weight ratio (KW/BW), cyst index, improve renal function in.3 group administered group no significant difference between the index of.3, metformin, Vee Glenn Dean, linagliptin can inhibit the proliferation of WT9.12. Three kinds of drugs are concentration dependent, in 24 hours, 48 hours, three time node growth metformin concentration of 50nM for 72 hours on the WT9.12 cell inhibition rates were 34%, 60%, 74%; 20nM vildagliptin on WT9.12 cells increased The inhibition rates were 14%, 20%, 28%; Leigh Glenn Dean 1nM on WT9.12 cell growth inhibition rate were 79%, 86%, 93%.4 after treatment, metformin, Vee Glenn Dean, pAMPK expression of linagliptin in renal tissues in model rats were significantly higher than Ctrl group (P0.001), were increased by 2.7 times, 3.5 times and 5 times; and vildagliptin group was significantly higher than that of metformin group (3.5 vs.2.7, P0.001), while the Leigh Glenn Dean group was significantly higher than that of vildagliptin group (5.1 vs 2.7, P0.001). Compared with the control group, metformin (4.5 vs 5.1, P=0.029), Vee Glenn Dean (3.9 vs 5.1, P0.001, Leigh Glenn Dean) (3 vs 5.1, P0.001) can make the 1nTOR excessive activation of renal tissue in the rat model was inhibited, the expression of p70S6K downstream products decreased significantly; vildagliptin group p70S6K protein expression was lower than that of metformin group (3.9 vs4.5, P=0.045), linagliptin group was lower than that of group 3.0vs (vildagliptin .3.9, P=0.001). Conclusion: This study found that overexpression of DPP4 in renal tissue of rats with /DPKD and polycystic kidney model. Using the DPP4 inhibitor, can inhibit the mTOR pathway activated by upregulation of AMPK, inhibition of cyst cell proliferation, progression of.DPKD in patients with DPP4 overexpression caused by excessive activation of mTOR may be involved in its pathogenesis. Inhibition of DPP4 activity is potential therapeutic target for ADPKD.

【學位授予單位】：第二軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R692.1

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