标题: 探讨第二型血管收缩素转化酶与凝乳酶在糖尿病肾病变病程之扮演角色
Studying the Roles of Angiotensin Converting Enzyme II (ACE2) and Chymase on the Progression of Diabetic Nephropathy
作者: 王冠骅
林志生
Wang, Guan-Hua
Lin, Chih-Sheng
生物科技学系
关键字: 糖尿病肾病变;肾素-血管收缩素系统;第二型血管收缩素转化酶;凝乳酶;基因剔除小鼠;Diabetic nephropathy;Renin-angiotensin system;Angiotensin converting enzyme II;Chymase;Gene knockout mice
公开日期: 2017
摘要: 糖尿病肾病变(diabetic nephropathy, DN)是慢性肾脏疾病(chronic kidney disease, CKD)的主要病因之一,过去文献指出肾素-血管收缩素系统(renin angiotensin system, RAS)中 angiotensin II (Ang II)的调节失常,是加速 DN 病程发展的主因之一。据此,本研究目的为探讨在糖尿病(diabetes mellitus, DM)诱发之肾病变过程中第二型血管收缩素转化酶(angiotensin converting enzyme II,ACE2)与凝乳酶(chymase)扮演之角色。
本研究利用 BKS.Cg-Dock7 m +/+ Lepr db /JNarl 小鼠(db/db)与 ACE2 基因剔除小鼠(ACE2 KO)作为实验模型之动物,并成功地利用这两种小鼠配种出 db 与 ACE2 双基因剔除小鼠(db & ACE2 KO),应用于 DN 诱发模型之研究。我们使用Streptozotocin (STZ)腹腔注射以诱发 ACE2 KO 小鼠的胰岛 β 细胞死亡,使实验小鼠作为第一型糖尿病(Type 1 DM, T1DM);可自发性诱发高血糖的 db/db 小鼠则作为第二型糖尿病(Type 2 DM, T2DM)之研究。在上述两种实验动物模型中,小鼠于 8 周龄持续喂予高脂饲料(high-fat diet, HFD)4 周以加速糖尿病肾病变的病程发展。在 4 周肾病变诱发期后,牺牲 8 周龄及 12 周龄小鼠并收集其血液、尿液、肾脏及胰脏组织,分别进行生化、组织病理及分子检测。
经 HFD 喂予的 db/db 小鼠,其血清总胆固醇和尿液白蛋白较喂予正常饲料的db/db 小鼠显着提高,而肾脏组织切片也显示肾丝球间质扩散的病变情形较为严重。而以 STZ 与 HFD 处理诱发 ACE2 KO 小鼠高血糖后,小鼠的血清三酸甘油酯、总胆固醇、尿素氮及尿液白蛋白均显着上升,肾脏组织中则呈现大量白血球细胞浸润的炎症反应病征,而肾丝球间质扩散情形只有些微提升。db & ACE2 KO小鼠在 HFD 诱发 DN 的各项生化指标上,均显着较 db/db 小鼠严重。
在肾脏组织中 RAS 相关酵素之活性变化显示,喂予 HFD 会造成 db/db 小鼠肾脏组织的 ACE 和 chymase 活性显着上升,ACE2 活性则维持不变。由 STZ 诱发 T1DM 之 ACE2 KO 小鼠显示肾脏 ACE 活性提高,但不会影响 chymase 活性,而只喂予 HFD 的小鼠肾脏组织中 ACE 和 chymase 活性均会显着提高。经 HFD喂予后,db & ACE2 KO 小鼠肾脏组织中的 ACE 活性相较于 db/db 小鼠显着提高,但 chymase 活性不变。
实验结果显示 ACE2 基因剔除与糖尿病肾病变下的 chymase 活性变化有关且会加速 db/db 小鼠的 DN 病程发展,因此本研究所建立的 db & ACE2 KO 小鼠相较于 db/db 小鼠更易于作为 DN 之实验动物模型。上述的实验结果有助于医师与研究者们更了解 RAS 中各项重要酵素在 DN 疾病中扮演的角色,并据此研发新的肾脏疾病标的药物。
Diabetic nephropathy (DN) is a major cause of chronic kidney disease (CKD). Previous researches indicated that imbalanced angiotensin II (Ang II) in renin angiotensin system (RAS) can accelerate the progression of DN. Therefore, the aim of this study is to explore the roles of angiotensin converting enzyme II (ACE2) and chymase play in the progression of diabetes mellitus (DM)-induced nephropathy.
In this study, animal models of DN were performed by using BKS.Cg-Dock7m+/+Leprdb/JNarl (db/db) mice and ACE2 gene knockout (ACE2 KO) mice. Additionally, we
successfully generated db & ACE2 double gene KO (db & ACE2 KO) mice by a series of mating and breeding strategy using db/+ mice and ACE2 KO mice. The Type 1 DM (T1DM) of ACE2 KO mice was induced by intraperitoneal injection of Streptozotocin(STZ), a chemical to damage the pancreatic β-cells of mice. The Type 2 DM (T2DM) animal model was performed in db/db mice that high blood glucose level can be spontaneously induced. To accelerate the progression of DN, both of the animal models were subjected to high-fat diet (HFD) feeding at 8-week-old. The mice were sacrificed after 4 weeks chronic nephropathy development, and the blood, urine, kidney and pancreas of 8-week or 12-week-old mice were collected for further biochemical,
pathologic and molecular assays.
In the group of db/db mice fed with HFD, serum total cholesterol and urine albumin were significantly increased, and severe mesangial matrix expansion was also
observed compared with those of the db/db mice fed with normal diet. After high blood glucose in ACE2 KO mice was successfully induced by STZ and HFD treatment, serum
triglycerides, total cholesterol, blood urea nitrogen and urine albumin were significantly increased. In these mice, severe inflammation symptoms, inflammatory cells infiltrating in renal interstitial tissue, was observed, but there is slightly increasing in mesangial matrix expansion. Above biochemical and pathologic characters were more severe in db & ACE2 KO mice compared with those found in db/db mice.
Renal RAS-related enzyme activities in the db/db mice fed with HFD, show that ACE and chymase activities were significantly increased, but ACE2 activity remained
insignificant change. The ACE activity was increased in ACE2 KO mice by STZ treatment, but renal chymase activity was no significant change. After HFD feeding, both of renal ACE and chymase activities in ACE2 KO mice were significantly increased. The renal ACE activitiy was significantly increased but chymase activity was similar in db & ACE2 KO mice that compared with those found in db/db mice.
Our results show that ACE2 gene knockout related to the change of chymase activity in DN and accelerate the progression of DN in db/db mice. Therefore, db & ACE2 KO mice is easier to establish an animal model of DN compared with db/db mice. The discoveries of our study can help physicians and researchers to know more
information about the role of RAS in renal pathogenesis, and the information can be applied in the new drug development for nephropathy.
URI: http://etd.lib.nctu.edu.tw/cdrfb3/record/nctu/#GT070257037
http://hdl.handle.net/11536/141513
显示于类别:Thesis