本文選題：糖尿病腎病 + 血管緊張素Ⅱ　； 參考：《南方醫(yī)科大學(xué)》2014年碩士論文

【摘要】：研究背景 糖尿病(diabetes mellitus)的發(fā)病率逐年上升,我國已成為目前世界上DN患病人口最多的國家,由此導(dǎo)致的糖尿病腎病(diabetic nephropathy, DN)人數(shù)也越來越多,對(duì)DN的發(fā)病機(jī)制進(jìn)行研究勢(shì)在必行,但目前這一問題仍然懸而未決。足細(xì)胞(Podocyte)作為腎小球?yàn)V過屏障的重要組成部分,其在DN發(fā)生發(fā)展中占有的重要作用越來越受到關(guān)注,目前足細(xì)胞損傷已被公認(rèn)為是DN進(jìn)展的核心環(huán)節(jié),且DN早期時(shí)就可觀察到足細(xì)胞受損。雖然DN情況下造成足細(xì)胞損傷的機(jī)制尚不完全清楚,但有研究證明血管緊張素Ⅱ (angiotensin Ⅱ, Ang Ⅱ)是其可能的原因之一。自噬(autophagy)是細(xì)胞內(nèi)清除蛋白質(zhì)、受損及衰老細(xì)胞器的有效途徑,被認(rèn)為是維持細(xì)胞內(nèi)環(huán)境穩(wěn)定的細(xì)胞保護(hù)性機(jī)制。足細(xì)胞在正常情況下即具備高水平自噬,而病理情況下足細(xì)胞中的自噬水平紊亂可能將進(jìn)一步加重足細(xì)胞損傷,故自噬對(duì)足細(xì)胞的存亡具有重要意義。自噬有望成為治療早期DN的一個(gè)靶點(diǎn),然而目前對(duì)于自噬在DN腎損傷中的作用機(jī)制還不清楚。 目的 本研究首先通過建立DN大鼠模型,研究了Ang Ⅱ水平在早期DN中的變化情況及自噬在足細(xì)胞損傷中的作用；然后用血管緊張素轉(zhuǎn)換酶抑制劑(angiotensin converting enzyme inhibitor, ACEI)貝那普利和Ang Ⅱ受體拮抗劑(angiotensin II receptor blockers, ARBs)氯沙坦分別干預(yù)早期DN模型大鼠,觀察兩者對(duì)早期大鼠DN足細(xì)胞損傷的保護(hù)作用及對(duì)足細(xì)胞中自噬活性的影響,以探討自噬在早期DN發(fā)生中的作用機(jī)制。 方法 (1)將80只雄性Wistar大鼠隨機(jī)分為正常對(duì)照組(n=6)、單腎切除組(n=6)、單腎切除+貝那普利組(n=6)、單腎切除+氯沙坦組(n=6)、糖尿病腎病組(n=10)、糖尿病腎病+胰島素注射組(n=10)、糖尿病腎病+胰島素注射+貝那普利組(n=12)和糖尿病腎病+胰島素注射+氯沙坦組(n=12),共8組。 (2)大鼠適應(yīng)性飼養(yǎng)1周后行單側(cè)腎切除術(shù),4周后腹腔注射中低劑量(45mg/kg)鏈脲佐菌素(streptozotocin, STZ)建立DN模型。模型建立后連續(xù)3天監(jiān)測(cè)大鼠尾尖血糖,血糖持續(xù)超過16.7-33.3mmol/L則視為模型建立成功。此后每4周稱量大鼠體重,檢測(cè)鼠尾血糖,使模型組大鼠血糖始終高于16.7-33.3mmol/L,隨時(shí)剔除造模失敗及死亡實(shí)驗(yàn)對(duì)象。 (3)DN模型成功后第16周開始分別用貝那普利和氯沙坦對(duì)模型大鼠進(jìn)行干預(yù),單腎切除組實(shí)施相同干預(yù),空白對(duì)照組給予等量安慰劑。 (4)實(shí)驗(yàn)終點(diǎn)前測(cè)量各組大鼠體重；干預(yù)8周后處死大鼠,分別留取血尿標(biāo)本。 (5)測(cè)定血糖、血肌酐、血清白蛋白、放免法檢測(cè)血清中AngⅡ水平、24小時(shí)尿蛋白等指標(biāo)。 (6)留取腎臟組織,計(jì)算腎重／體重比值,一部分腎臟組織用于放免法測(cè)定腎臟組織中AngⅡ水平改變；一部分腎臟皮質(zhì)組織分別經(jīng)2.5%戊二醛和10%福爾馬林溶液固定后分別行光鏡及電鏡檢查腎臟組織病理改變；剩余腎臟組織用于提取腎小球,行Western Bloting檢測(cè)足細(xì)胞標(biāo)志蛋白Podocin和Nephrin表達(dá)情況的改變。 (7)電鏡下分別觀察各組大鼠腎小球足細(xì)胞中自噬體數(shù)目的變化情況；檢測(cè)各組大鼠腎小球組織中自噬體標(biāo)志物L(fēng)C-Ⅱ、自噬相關(guān)蛋白Atg5及P62蛋白表達(dá)情況的變化。 結(jié)果 (1)成功地建立DN大鼠模型。與正常對(duì)照組大鼠相比,模型組大鼠體重明顯減輕(P0.05),經(jīng)藥物干預(yù)8周后體重明顯增加(P0.05),單腎切除各組與正常對(duì)照組之間體重差異無統(tǒng)計(jì)學(xué)意義(P0.05)。 (2)與正常對(duì)照組大鼠相比,單腎切除各組和模型組大鼠腎質(zhì)量/體質(zhì)量比值均明顯升高(P0.01)且模型組大鼠較單腎切除組大鼠升高更為明顯(P0.01),經(jīng)藥物干預(yù)后模型大鼠的腎質(zhì)量/體質(zhì)量比值明顯降低(P0.01)。 (3)模型大鼠血糖與正常對(duì)照組相比明顯升高(P0.01),經(jīng)胰島素干預(yù)后血糖明顯降低(P0.01),且始終維持在16～17mmol/L這一水平,單腎切除各組血糖與正常對(duì)照組相比差異無統(tǒng)計(jì)學(xué)意義。 (4)模型大鼠肌酐值較正常對(duì)照組明顯升高(P0.01),經(jīng)藥物干預(yù)后肌酐值明顯降低(P0.01),單腎切除各組肌酐值與正常對(duì)照組相比差異無統(tǒng)計(jì)學(xué)意義。 (5)血清白蛋白水平模型組及單腎切除組與正常對(duì)照組相比均明顯降低(P0.01),其中模型組降低更為明顯(P0.01),經(jīng)藥物干預(yù)后模型大鼠血清白蛋白水平明顯升高(P0.01)。 (6)與正常對(duì)照組相比,模型大鼠24小時(shí)尿蛋白明顯增加(P0.01),經(jīng)貝那普利及氯沙坦治療后模型大鼠蛋白尿明顯減輕(P0.01),而單腎切除組大鼠24小時(shí)尿蛋白與正常對(duì)照組相比差異無統(tǒng)計(jì)學(xué)意義。 (7)接受氯沙坦治療的大鼠(單腎切除+氯沙坦組及DN+胰島素+氯沙坦組)血清中Ang Ⅱ水平與正常對(duì)照組相比明顯升高(P0.05),后者升高更為明顯(P0.05)其余各組與正常對(duì)照組相比差異無統(tǒng)計(jì)學(xué)意義。 (8)與正常對(duì)照組相比,單腎切除+氯沙坦組及DN各組大鼠腎組織中AngⅡ水平均明顯升高(P0.01),其中DN+胰島素+氯沙坦組大鼠腎組織Ang Ⅱ水平升高更為明顯(P0.01)。 (9)光鏡下,見模型組大鼠腎小球肥大,系膜膜細(xì)胞增生、基質(zhì)增多,可見蛋白管型；電鏡可見腎小球基底膜明顯增厚、足細(xì)胞肥大、足突廣泛融合。 (10)模型組及DN+胰島素+貝那普利組大鼠腎小球組織中的Podocin及Nephrin與正常對(duì)照組相比明顯升高(P0.05),其余各組與正常對(duì)照組相比差異無統(tǒng)計(jì)學(xué)意義。 (11)電鏡下觀察：模型組大鼠腎小球足細(xì)胞中自噬體數(shù)目較正常對(duì)照組明顯增加(P0.05),經(jīng)胰島素及氯沙坦治療后模型大鼠足細(xì)胞中自噬體數(shù)目明顯減少(P0.05)。 (12)模型組大鼠腎小球組織中的LC3-Ⅱ、Atg5及P62蛋白表達(dá)較正常對(duì)照組大鼠明顯增加(P0.01),氯沙坦干預(yù)后模型大鼠腎小球組織中的LC3-Ⅱ、Atg5及P62蛋白表達(dá)水平明顯降低(P0.01)。 討論 (1)本研究中采用的建模方法成功模擬了DN早期病理特點(diǎn)。產(chǎn)生的蛋白尿在貝那普利及氯沙坦的干預(yù)作用下得到明顯改善,但其對(duì)已產(chǎn)生的腎臟組織病理變化及足細(xì)胞損傷的改善并不十分明顯,可能由于藥物干預(yù)時(shí)間尚短。 (2)經(jīng)氯沙坦干預(yù)的大鼠(單腎切除+氯沙坦和DN+胰島素+氯沙坦)血清中及腎臟組織中Ang Ⅱ水平升高,是由于Ang Ⅱ與AT1受體的結(jié)合被氯沙坦阻滯后,血管阻力降低,醛固酮分泌減少,血漿AngⅡ水平增高。而作為血管緊張素轉(zhuǎn)換酶抑制劑的貝那普利治療的DN大鼠腎臟組織中的AngⅡ水平較正常對(duì)照組沒有明顯降低,則可能是由于其不能抑制由非血管緊張素轉(zhuǎn)換酶路徑合成的AngⅡ。 (3)本研究中DN大鼠腎小球組織中Podocin及Nephrin的表達(dá)明顯增加,這可能是DN早期足細(xì)胞代償性肥大、足突廣泛融合所導(dǎo)致的。 (4)DN大鼠腎小球足細(xì)胞中自噬體數(shù)目增加、腎小球組織中自噬活性增加,其可能機(jī)制是a.DN狀態(tài)下腎小球足細(xì)胞中的腎素-血管緊張素系統(tǒng)(renin-angiotensinsystem, RAS)活化,足細(xì)胞中的Ang Ⅱ水平升高激活了氧化應(yīng)激產(chǎn)生活性氧族(reactive oxygen species, ROS),活性氧族的產(chǎn)生可誘導(dǎo)自噬增強(qiáng)；b.高糖狀態(tài)直接激活氧化應(yīng)激,產(chǎn)生活性氧族誘導(dǎo)自噬增強(qiáng)。 (5)氯沙坦干預(yù)后可減輕氧化應(yīng)激誘導(dǎo)的自噬活性增強(qiáng),降低蛋白尿,延緩DN進(jìn)展。可能機(jī)制是通過選擇性阻斷血管緊張素受體1(AT1)阻斷了AngⅡ的生物學(xué)效應(yīng)。 (6)貝那普利干預(yù)后雖能減輕模型大鼠蛋白尿,但其腎小球組織中的自噬活性卻依然升高,表明在DN狀態(tài)下,除AngⅡ的作用外,還有其他機(jī)制影響足細(xì)胞中的自噬活性。本課題對(duì)于理解DN足細(xì)胞損傷的機(jī)制,及自噬在DN足細(xì)胞損傷中的作用,探索自噬活性在防治DN中的意義進(jìn)行了一定程度的研究。
[Abstract]:Research background
The incidence of diabetes (diabetes mellitus) is increasing year by year. China has become the country with the largest population of DN in the world, and the number of diabetic nephropathy (diabetic nephropathy, DN) is also increasing. It is imperative to study the pathogenesis of DN, but the problem is still in pending. Podocyte (Podocyte) is still in the air. As an important part of the glomerular filtration barrier, its important role in the development of DN has attracted more and more attention. Podocyte injury is now recognized as the core of the progress of DN, and the damage of the Poda can be observed at the early stage of DN. Although the mechanism of the foot cell injury in the case of DN is not completely clear, there is a study Angiotensin II (angiotensin II, Ang II) is one of the possible causes. Autophagy (autophagy) is an effective way to remove protein, damage and senescence organelles in cells. It is considered to be a cellular protective mechanism to maintain the stability of the cell environment. Autophagy in podocytes may further aggravate foot cell damage, so autophagy is of great significance to the survival of podocytes. Autophagy is expected to be a target for the treatment of early DN. However, the mechanism of autophagy in DN renal injury is still unclear.
objective
In this study, the changes of the level of Ang II in the early DN and the role of autophagy in the foot cell injury were studied by establishing the DN rat model, and then the angiotensin converting enzyme inhibitor (angiotensin converting enzyme inhibitor, ACEI) and Ang II receptor antagonists (angiotensin II receptor) were used. S) the effect of losartan on early DN model rats and the protective effect of both of them on DN podocyte injury in early rats and the effect on autophagy in podocytes were observed in order to explore the mechanism of autophagy in early DN.
Method
(1) 80 male Wistar rats were randomly divided into normal control group (n=6), single nephrectomy group (n=6), single nephrectomy + benazepril group (n=6), single nephrectomy + losartan group (n=6), diabetic nephropathy group (n=10), diabetic nephropathy + insulin injection group (n=10), diabetic nephropathy + insulin injection + benazepril group (n=12) and diabetic nephropathy + insulin injection. There were 8 groups in the group of shoot + losartan (n=12).
(2) the rats were fed with unilateral nephrectomy after 1 weeks of adaptive feeding, and the DN model was established by intraperitoneal injection of middle and low dose (45mg/kg) streptozotocin (streptozotocin, STZ) after 4 weeks. After the establishment of the model, the rat tail tip blood glucose was monitored continuously for 3 days, and the blood sugar continued to exceed 16.7-33.3mmol/L as a model. After the model, the rat weight was weighed every 4 weeks and the tail of the rat was detected. The blood glucose level of the model group was higher than that of 16.7-33.3mmol/L, and the model failure and death were excluded at any time.
(3) the model rats were intervened with Benner Pury and losartan respectively at sixteenth weeks after the DN model was successful. The same intervention was carried out in the single nephrectomy group, and the blank control group was given the same amount of placebo.
(4) the weight of rats in each group was measured before the end of the experiment. After 8 weeks of intervention, rats were sacrificed and blood and urine samples were kept.
(5) determination of blood glucose, creatinine, serum albumin, radioimmunoassay, serum Ang II level, 24 hour urine protein and other indicators.
(6) the renal tissue was retained and the ratio of kidney weight / weight was calculated. Some renal tissues were used for radioimmunoassay to determine the change of Ang II in renal tissue. A part of renal cortical tissue was fixed by 2.5% glutaraldehyde and 10% formalin solution respectively after fixation by light microscopy and electron microscopy to examine the pathological changes of kidney tissue, and the remaining renal tissue was used for extraction. Glomerular Western Bloting was used to detect the expression of podocyte marker protein Podocin and Nephrin.
(7) the changes in the number of autophagosomes in the glomerular podocytes were observed under the electron microscope, and the autophagosomes LC- II, the autophagy related protein Atg5 and the expression of P62 protein in the glomerular tissues of the rats were detected.
Result
(1) the DN rat model was successfully established. Compared with the normal control group, the body weight of the model rats was significantly reduced (P0.05), and the weight increased significantly after 8 weeks of drug intervention (P0.05). There was no significant difference in weight difference between the single nephrectomy group and the normal control group (P0.05).
(2) compared with the normal control group, the renal mass / body mass ratio of the rats in the single nephrectomy group and the model group were significantly increased (P0.01) and the rats in the model group were more significantly higher than those in the single nephrectomy group (P0.01), and the renal mass / body mass ratio of the rats after the drug dry prognosis was significantly decreased (P0.01).
(3) the blood glucose in the model rats was significantly higher than that in the normal control group (P0.01), and the blood sugar decreased significantly (P0.01) after the intervention of insulin, and remained at the level of 16 to 17mmol/L. There was no significant difference in blood sugar in the single nephrectomy group compared with the normal control group.
(4) the creatinine value of the model rats was significantly higher than that in the normal control group (P0.01), and the creatinine value decreased significantly after the drug intervention (P0.01). The creatinine value of the single nephrectomy group was not statistically significant compared with the normal control group.
(5) the serum albumin level model group and the single nephrectomy group were significantly lower than the normal control group (P0.01), in which the model group decreased more significantly (P0.01), and the serum albumin level of the rat model was significantly increased (P0.01) after the drug dry prognosis model.
(6) compared with the normal control group, the 24 hour urine protein in the model rats increased significantly (P0.01). The proteinuria of the model rats after the treatment of Benner Pury and losartan was significantly reduced (P0.01), but there was no significant difference between the 24 hours urine protein in the single nephrectomy group and the normal control group.
(7) the level of Ang II in the serum of rats treated with losartan (single nephrectomy plus losartan group and DN+ insulin + losartan group) was significantly higher than that of the normal control group (P0.05), the latter was more obvious (P0.05) and the difference between the other groups was not statistically significant compared with the normal control group.
(8) compared with the normal control group, the levels of Ang II in the renal tissue of the single nephrectomy + losartan group and the DN rats were all significantly increased (P0.01), and the level of Ang II in the renal tissue of the rats of DN+ insulin + losartan group was more obvious (P0.01).
(9) under the light microscope, the glomerular hypertrophy, the proliferation of mesangial cells, the increase of the matrix and the protein tube type were seen in the model group. The glomerular basement membrane was obviously thickened, the podocytes were hypertrophy, and the foot process was widely fused.
(10) the Podocin and Nephrin in the glomerular tissue of the model group and the DN+ insulin + benazepril group were significantly higher than that in the normal control group (P0.05), and the other groups had no statistical difference compared with the normal control group.
(11) observation under electron microscope: the number of autophagosomes in the glomerular podocytes of rat model group was significantly higher than that in the normal control group (P0.05). The number of autophagosomes in the rat foot cells after the treatment of insulin and losartan was significantly decreased (P0.05).
(12) the expression of LC3- II, Atg5 and P62 protein in the glomerular tissue of the model rats was significantly higher than that in the normal control group (P0.01). The expression level of LC3- II, Atg5 and P62 protein in the glomerular tissue of rats with losartan dry prognosis was significantly decreased (P0.01).
discuss
(1) the modeling method used in this study successfully simulated the early pathological characteristics of DN. The proteinuria produced by the intervention of Benner Pury and losartan was obviously improved, but the improvement of the pathological changes of kidney tissue and the improvement of podocyte injury was not very obvious, which may be due to the short time of drug intervention.
(2) the levels of Ang II in the serum and renal tissues of rats treated with losartan intervention (mononrennephrectomy + losartan and DN+ insulin + Losartan) were increased in the serum and renal tissues, due to the decrease of vascular resistance, the decrease of aldosterone secretion and the level of Ang II in plasma after the combination of the combination of Ang II and AT1 receptor by losartan, and the level of plasma Ang II. The level of Ang II in the renal tissue of the DN rats treated with nalpril was not significantly lower than that in the normal control group, perhaps because it could not inhibit the Ang II synthesized by the non angiotensin converting enzyme pathway.
(3) the expression of Podocin and Nephrin in the glomerular tissue of DN rats increased significantly in this study, which may be caused by the compensatory hypertrophy of the early podocytes in DN and the extensive fusion of the poddate.
(4) the number of autophagosomes in the glomerular podocytes of DN rats increased and the autophagic activity in the glomerular tissue increased. The possible mechanism was the activation of the renin angiotensin system (renin-angiotensinsystem, RAS) in the glomerulopodocyte (a.DN), and the increase of Ang II in the podocytes activated the oxidative stress to produce the living oxygen group (reactive oxyge). N species (ROS), the production of reactive oxygen species can induce autophagy enhancement; B. high glucose state directly activates oxidative stress and induces reactive oxygen species to induce autophagy enhancement.
(5) the intervention of losartan can reduce the enhancement of autophagy induced by oxidative stress, reduce proteinuria and delay the progress of DN. The possible mechanism is to block the biological effect of Ang II by selectively blocking angiotensin receptor 1 (AT1).
(6) although Benner Pury intervention can reduce the proteinuria of model rats, the autophagy activity in the glomerular tissue is still elevated, indicating that in the state of DN, there are other mechanisms that affect the autophagy in the podocytes except for the role of Ang II. This topic is the mechanism for understanding the damage of DN podocytes and the role of autophagy in DN podocyte injury. The significance of autophagy activity in the prevention and treatment of DN has been explored to some extent.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R587.2;R692

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相關(guān)期刊論文 前8條

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2 李惠秀;曹文富;;糖尿病腎病發(fā)病機(jī)制及治療進(jìn)展[J];重慶醫(yī)學(xué);2013年21期

3 趙秀文,蘇彤,高潔;血管緊張素Ⅱ與高血壓的研究進(jìn)展[J];華北煤炭醫(yī)學(xué)院學(xué)報(bào);2004年04期

4 陳世德;血管緊張素Ⅱ與纖溶酶原激活物抑制物-1的研究進(jìn)展[J];醫(yī)學(xué)綜述;2003年09期

5 李金紅;陶建瓴;李航;;足細(xì)胞損傷與糖尿病腎病的研究現(xiàn)狀[J];中國醫(yī)學(xué)科學(xué)院學(xué)報(bào);2010年05期

6 王小英;馮積容;周艷;林佳;潘競(jìng)鏘;王少紅;;2型糖尿病早期腎病大鼠模型制備及二甲雙胍干預(yù)作用[J];臨床醫(yī)學(xué)工程;2013年02期

7 張勇軍;劉冬;孫永蘋;莫鎮(zhèn)濤;;雷公藤多甙對(duì)糖尿病大鼠尿蛋白、腎小球硬化及足細(xì)胞的影響[J];新醫(yī)學(xué);2013年07期

8 戴厚永;范亞平;;足細(xì)胞損傷與糖尿病腎病關(guān)系的研究進(jìn)展[J];醫(yī)學(xué)綜述;2013年17期

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