摘要
目的观察肾小管上皮细胞间充质转化(EMT)过程中,整合素连接激酶(ILK)的变化对基质金属蛋白酶-9/金属蛋白酶组织抑制剂-1(MMP-9/TIMP-1)比值的影响。方法培养人肾皮质近曲小管上皮细胞(HK-2),并随机分为正常组、转化生长因子-β1(TGF-β1)刺激组(以TGF-β1诱导HK-2细胞间充质转化)、TGF-β1+ILK-siRNA组[使用小干扰RNA (siRNA)技术抑制ILK基因表达]、TGF-β1+无序siRNA组(使用无序小干扰RNA转染)。后两组采用脂质体转染技术,将小干扰RNA转染入HK-2细胞,而后用10 ng/ml TGF-β1刺激细胞,分别提取不同组别细胞的mRNA和蛋白质,采用Real-time RT-PCR和Western blot检测细胞ILK、E-钙粘蛋白(E-cadherin)、α-平滑肌肌动蛋白(α-SMA)、MMP-9、TIMP-1的mRNA和蛋白质的表达,计算MMP-9、TIMP-1 mRNA和蛋白质表达的相对比值,比较其统计学差异。结果TGF-β1刺激的HK-2细胞E-cadherin表达减少,α-SMA表达增加,ILK表达显著增加,MMP-9/TIMP-1比值升高;转染ILK-siRNA可大幅度抑制HK-2细胞ILK基因的表达;且其E-cadherin表达增加,α-SMA表达减少,同时可减小MMP-9/TIMP-1比值升高的程度。结论ILK可通过影响MMP-9/TIMP-1比值,促进EMT进程。
Objective To investigate the possible regulating effect of integrin-linked kinase (ILK) towards matrix metalloproteinase-9/tissue inhibitor of metalloproteinase-1 ( MMP-9/TIMP-1 ) ratio in the process of transforming growth factor-β1 ( TGF-β1 ) -induced epithelial-mesenchymal transition (EMT) in human kidney proximal tubular epithelial (HK-2) ceils. Methods HK-2 ceils were cultured and stimulated with 10 ng/ml TGF-β1. Specific ILK-small interfering RNA (ILK-siRNA) was used to inhibit ILK expression. The characteristic epithelial marker (E-cadherin) and mesenchymal marker (α-SMA) of EMT were examined by Real-time reverse transeription polymerase chain reaction (real-time RT-PCR) and Western blot. The expressions of ILK, MMP-9, and TIMP-1 were also examined, to determine the regulating effect of ILK towards MMP-9/TIMP-1 ratio. Results In the HK-2 ceils cultured with TGF-β1, the expression of E-cadherin decreased, and α-SMA expression increased; overexpression of ILK and an abnormal changing of MMP-9/TIMP-1 ratio were observed. ILK inhibition by ILK-siRNA could adjust MMP-9/TIMP-1 ratio to near normal. Meanwhile, the overexpressed ILK and α-SMA were decreased. Conclusions Our data indicates that ILK-siRNA successfully inhibits ILK expression, which regulates the MMP-9/TIMP-1 ratio in HK-2 cells. The inhibition of ILK expression suppresses TGF-β1-induced EMT partially.
出处
《中国医师杂志》
CAS
2017年第3期357-361,366,共6页
Journal of Chinese Physician
基金
国家自然科学基金(30873331)
关键词
转化生长因子β
肾小管
上皮细胞
生物转化
整合素类
基质金属蛋白酶9
金属蛋白酶1组织抑制剂
Transforming growth factor betal
Kidney tubules
Epithelial cells
Biotransformation
Integrins
Matrix metalloproteinase 9
Tissue inhibitor of metalloproteinase-1