【作者】 段玉蕊；

【导师】 陈宝平；

【作者基本信息】 河南大学 ， 内科学， 2012， 硕士

【摘要】 近年来，随着糖尿病患者族群的不断扩大，糖尿病目前已经成为了一个全球关注的健康问题。作为糖尿病比较常见的微血管并发症，糖尿病肾病（diabetic nephropathy, DN）的基本病理改变为细胞外基质（extra-cellular matrix, ECM）重构，引起肾小球硬化和肾间质纤维化。在肾间质纤维化的过程中，肾小管上皮细胞间充质转分化（Epithelial-mesenchymal-transition, EMT）起到关键的作用。α平滑肌肌动蛋白（α-smoothmuscle actin, α-SMA）的生成是EMT过程中关键的一步，提示着EMT发生的程度。转化生长因子-β（transforming growth factor-β, TGF-β）在体外是最有效的引起EMT的因子，骨成形蛋白-7(bone morphogenic protein-7, BMP-7)属于TGF-β超家族，一般来说主要表达于肾小管的上皮细胞已经以及部分足细胞。有研究显示，当肾小管的上皮细胞受到损伤时，肾组织中的BMP-7表达明显减少。而当对组织进行外源性的补充BMP-7后，肾功能明显得到改善，提示BMP-7可能为逆转肾间质纤维化的重要的肾脏保护因子。血管紧张素Ⅱ受体拮抗剂（Angiotensin Ⅱ receptor angtagonist, ARB）目前已应用于DN的临床治疗，主要是通过其血流动力学保护肾脏，在抑制肾间质纤维化方面的机制并不明了，冬虫夏草作为一种中药制剂对于肾脏的保护作用机制更是扑朔迷离。本课题通过研究糖尿病肾病大鼠模型肾组织中α-SMA、 BMP-7和TGF-β的表达情况，以及替米沙坦联合冬虫夏草干预下三者的表达情况，同时观察24小时尿蛋白定量和肾功能的变化，以此探讨其对于糖尿病肾病肾小管上皮间充质转分化的调节作用，并寻找替米沙坦联合冬虫夏草对于糖尿病肾病的保护机制，为糖尿病肾病的治疗提供更多的途径及理论依据。目的：通过研究糖尿病肾病大鼠模型肾组织中α-SMA、 BMP-7和TGF-β的表达情况，以及替米沙坦联合冬虫夏草干预下三者的表达情况，同时观察24小时尿蛋白定量和肾功能的变化，以此探讨其对于糖尿病肾病肾小管上皮间充质转分化的调节作用。方法：选用雄性Wistar大鼠作为实验对象，动物模型建立使用链脲佐菌素对大鼠一次性腹腔内注射，造模成功后，将大鼠随机分为4组：DN模型组（n＝9）、替米沙坦组（n＝9），冬虫夏草组（n＝9），替米沙坦联合冬虫夏草组（n＝10），另设正常对照组（n＝10）。替米沙坦组每日给予8mg/kg体重的替米沙坦灌胃一次；冬虫夏草组按药量2.5g/Kg虫草水提取液灌胃给药；联合治疗组两种药物均等量灌胃，对照组与模型组以等量饮用水灌胃，每日一次。所有大鼠不限食水。治疗8周后，对大鼠收集24小时尿量测定尿蛋白定量；心包取血测定血糖、血尿素氮、肌酐水平；处死大鼠后采集肾脏标本，测量大鼠肾重及肾重体重比；肾组织HE染色，测定各组大鼠肾小管-间质病理损伤评分，利用组织免疫化学法检测各组大鼠肾组织中BMP-7、α-SMA和TGF-β的表达情况。结果：（1）对血中BUN、SCr的影响：替米沙坦组、冬虫夏草组及联合用药组能明显降低BUN、SCr水平，与模型组比较有显著性差异（P<0.01），但仍高于正常对照组；联合用药组与替米沙坦组、冬虫夏草组比较有显著性差异（P<0.01）；替米沙坦组与冬虫夏草组比较差异无显著性意义（P>0.05）。（2）对24小时尿蛋白的影响：替米沙坦组、冬虫夏草组及联合用药组能明显降低24小时尿蛋白水平，与模型组比较有显著性差异（P<0.01），联合用药组与替米沙坦组、冬虫夏草组比较有显著性差异（P<0.01）；替米沙坦组与冬虫夏草组比较有显著性意义（P<0.01）。（3）肾组织病理：替米沙坦组、冬虫夏草组及联合用药组大鼠与正常对照组大鼠相比，出现了不同程度肾小球硬化、肾间质炎症细胞浸润、肾小管上皮细胞空泡变性及间质纤维化，肾小管-间质损伤指数明显升高（P<0.01），与DN组对比则明显减轻（P<0.01）。（4）肾组织免疫组化：替米沙坦组、冬虫夏草组及联合用药组大鼠肾间质α-SMA及TGF-β阳性表达与模型组相比均显著下调（P<0.01），BMP-7的阳性表达则显著上调（P<0.01），联合用药组与替米沙坦组、冬虫夏草组比较有显著性差异（P<0.01）；替米沙坦组与冬虫夏草组比较差异无显著性差异（P>0.01）。（5）α-SMA及TGF-β与24小时尿蛋白定量和肾功能成明显正相关（P＜0.01）；BMP-7与24小时尿蛋白定量和肾功能明显负相关（P＜0.01）；α-SMA与TGF-β呈明显正相关（P＜0.01）；BMP-7与α-SMA及TGF-β呈负相关，结果具有统计学意义（P＜0.01）。结论：1.糖尿病肾病模型组α-SMA表达明显升高，提示糖尿病肾病过程存在肾小管上皮细胞间充质转分化；2. α-SMA表达上调与TGF-β明显相关，提示TGF-β可诱导α-SMA的表达，促进EMT的发生；3. BMP-7与TGF-β、α-SMA表达明显负相关，提示BMP-7可能通过抑制TGF-β的表达，从而抑制EMT的进展；4.替米沙坦、冬虫夏草可以减少肾组织α-SMA，抑制EMT的发生发展，延缓和改善肾间质纤维化，且联合使用效果更佳。更多还原

【Abstract】 As the incidence of diabetes (DM) increased in recent years, it has become a global health burden.Diabetic nephropathy (DN) is one of the most common and severe microvascular complications of thediabetes, and its basic pathological change is extracellular matrix (ECM) reconstruction, which lead toglomerulosclerosis and renal interstitial fibrosis. EMT is now recognized as a key contributor to the loss ofrenal function throughout the nephron in diabetic nephropathy. The formation of α-smooth muscle actin(α-SMA) is a critical step in the EMT process, suggesting the extent of EMT. Studies have shown thattransforming growth factor-β (TGF-β) is the most effective factor in vitro caused the EMT. Bonemorphogenetic protein-7(BMP-7) belongs to the TGF-β superfamily, mainly expressed in tubular epithelialand the podocytes tubule cell, when the cells are injured, the expression of BMP-7is decreased. Throughexogenous BMP-7to protect kidney function now is considered as one of the most important factor toreversal renal fibrosis. Angiotensin II receptor antagonists (ARB) has been used clinically, primarilythrough its hemodynamic protection, but the mechanism of inhibition of renal interstitial fibrosis is notclear. Cordyceps sinensis as an important agents for renal protection mechanism is more complicated andconfusing. We will observe the expression of α-SMA、BMP-7and TGF-β in renal tissue of diabeticnephropathy rat model, as well as the change of the three indexes after the treatment of telmisartan andCordyceps, in order to find the effect to renal tubular epithelial to mesenchymal transdifferentiation and themechanism of telmisartan Cordyceps sinensis for protecting kidneys.Objective: To observe the expression of α-SMA、BMP-7and TGF-β in renal tissue of diabeticnephropathy rat model, as well as the change of the three indexes after the treatment of telmisartan andCordyceps, in order to find the effct of EMT in the development of DN and the mesenchymaltransdifferentiation and the mechanism of telmisartan Cordyceps sinensis for protecting kidneys.Methods: We established the diabetic nephropathy rat modle by single intraperitoneal injection ofstreptozotocin (STZ). Then randomly divided the DN model rats into four groups: non-treatment DN modelgroup (n=9), telmisartan-treated group (n=9), Cordyceps sinensis treated group (n=9),combination-treated group (n=10). In addition, we selected10normal rats served as control group. Thetelmisartan-treated group rats were gavaged once a day with telmisartan (8mg/kg of body weight). The Cordyceps sinensis-treated group rats were gavaged with Cordyceps sinensis(2.5g/kg of body weight) oncea day. The combination-treated group rats were gavaged with the two drugs once a day. The other twogroups were gavaged with drinking water everyday. The test time lasted for eight weeks, then determinatedthe urine of24hours, the volume of urinary protein and renal function markers. Then observed theexpreesion ofα-SMA、BMP-7and TGF-β by immunohistochemistry ABC method.Results:(1) The treatment groups rats, including one drug separately and two drugs combination,compare with DN model group rats, the kidney function (both BUN and Cr) is significantly decreased (P<0.01); but increased to normal group.(2) Compared with normal control group rats, the24hours urinaryprotein quality of the DN model group rats were significantly increased (P <0.01), after the treatment oftelmisartan and Cordyceps sinensis separately and both two drugs combination, the24hours urinaryprotein quality significantly decreased (P <0.01).(3) Inflammatory cells infiltration, tubular vacuolizationand tubulointerstitial fibrosis could be seen in DN group, and its index of renal tubular-interstitial injuryelevated significantly （P<0.01）when it was compared with normal group. Renal tubular-interstitial injuryin other three treatment groups significantly released（P<0.01）.(4) The expression of both α-SMA andTGF-β were significantly suppressed among subjects in DN group（P<0.01）, but the expression of BMP-7were contrast decreased (P <0.01). In other three treatment groups, the expression of both α-SMA andTGF-β were significantly reduced and the expression of BMP-7was increased compare with DN group (P<0.01).(5) α-SMA and TGF-β were positively correlated with urinary protein quality and renal functionmarkers significantly (P <0.01); but BMP-7was negatively correlated with them (P <0.01); α-SMA waspositively correlated with TGF-β(P <0.01), BMP-7was negatively correlated with both α-SMA and TGF-βsignificantly (P <0.01).Conclusions:1. The increased of expression of α-SMA in DN rats suggestted that there is EMT in DN rats.2. The expression of α-SMA is concerned with TGF-β significantly, suggesting that TGF-β caninduce the expression of α-SMA, and promote the occurrence of EMT3. The expression of BMP-7was significantly negatively correlated with α-SMA and TGF-β,suggesting that BMP-7maybe can through inhibition of TGF-β expression to inhibite the progressof the EMT 4. Telmisartan and Cordyceps sinensis can slow down the process of renal fibrosis decreasing ECMand impeding EMT, and the effect is better if they joint application.更多还原