摘要
目的观察整合素连接激酶(ILK)在单侧输尿管梗阻小鼠(UUO)肾脏中的表达,并探讨其在肾小管上皮细胞转化中的作用。方法将10周龄健康雄性 CD-1小鼠随机分为假手术组(C,n=20)和 UUO 组(n=40),分别于术后1、3、7、14 d 处死小鼠。采用 Masson 染色观察肾间质纤维化程度;用免疫组织化学(SP 法)的方法观察 ILK、E-钙黏蛋白(E-cadherin)和α-平滑肌肌动蛋白(α-SMA)的表达;Western blot 半定量分析梗阻侧肾组织 ILK 的蛋白水平;用即时荧光逆转录聚合酶链反应(RT-PCR)检测 ILK、E-cadherin 和α-SMA mRNA 的表达。结果免疫组织化学显示 ILK 在假手术组只有微量表达,主要位于肾小球脏层上皮细胞,UUO 术后1 d ILK 蛋白表达显著增多(t=16.5,P<0.01),7 d 达高峰。术后3 d 肾小管上皮细胞及肾间质α-SMA 阳性细胞数显著增多(t=21.0,P<0.01),肾小管上皮细胞表达 E-cadherin 明显减少(t=5.6,P<0.01)。根据免疫组织化学结果分析,术后1~7 d,ILK 蛋白表达与α-SMA 蛋白表达呈正相关(R=0.88,P<0.01),与 E-cadherin表达呈负相关(R=-0.87,P<0.01)。Western blot 结果显示 ILK 蛋白表达于术后1 d 明显增加,7 d达高峰,14 d 表达不再增加(P>0.05)。RT-PCR 结果显示术后1 d ILK mRNA 表达增多(t=141.6,P<0.01),E-cadherin mRNA 表达于术后3 d 显著减少(P<0.01),α-SMA mRNA 表达于术后3 d 开始增多(P<0.01)。结论 UUO 模型早期整合素连接激酶表达增高,可能通过介导肾小管上皮细胞-间充质转化而参与肾脏纤维化的发生发展。
Objective To investigate the expression of integrin-li0nked kinase (ILK) in kidneys of mice with unilateral ureteral obstruction and its relevance with the epithelial-mesenchymal transition. Methods Mice were randomly divided into two groups, sham operation ( C, n = 20) and unilateral ureteral obstruction ( UUO, n =40). The animals were sacrificed at day 1,3,7 and 14 respectively after the surgery. Tubttlointerstitial fibrosis (TIF) was graded according to Masson staining. The protein level of ILK was examined by Western blot. Tissue/cytological expression for ILK, α-SMA and E-cadherin were investigated by immunohistochemistry. The mRNA levels of ILK, α-SMA and E-cadherin were analyzed by quantitative real-time PCR. Results In the control animals (group C), weak staining for ILK was detected mainly in the pedocytes. Significant increase of staining for ILK in the experimental mice ( UUO group) was detected from day 1 onward ( t = 16. 5, P 〈0. 01 ), reaching the peak at day 7. The protein expression of E-cadherin was continuously down-regulated from day 3 onward after surgery ( t =21.0 ,P 〈0. 01 ), while expression for α-SMA was up-regulated. From day 1 to day 7, the protein expression of ILK was positively correlated with α-SMA(R = 0. 88, P 〈 0. 01 ), but negatively correlated with E-cadherin ( R = - 0. 87, P 〈 0. 01 ). The mRNA expression of ILK and α-SMA analyzed by real-time PCR increased from postoperative day 1 and 3 respectively, but the mRNA expression of E-cadherin decreased from day 3 onward. Conclusion Increasing expression of ILK occurs in the early phase of UUO mouse and may play an important role in the process of TIF through mediating the epithelial-mesenchymal transition.
出处
《中华病理学杂志》
CAS
CSCD
北大核心
2007年第1期43-47,共5页
Chinese Journal of Pathology
基金
国家自然科学基金(30471732)
江苏省医学重点人才基金(RC2002072)
关键词
肾硬化症
输尿管梗阻
黏蛋白类
肾小管
上皮细胞
Nephrosclerosis
Ureteral obstruction
Mucins
Kidney tubules
Epithelial cells