Tang Gong-yao, Chen Yi-fu, Zhang Mei, Rui Hong-liang, Cong Xiao, Lou Jin-ning
The Center of Nephrology, China-Japan Friendship Hospital, Beijing 100029, China.
Zhonghua Yi Xue Za Zhi. 2006 Jun 13;86(22):1540-4.
To investigate the expression and regulation of alpha 1 anti-trypsin (AAT) in tubular epithelial cells.
The expression of AAT in tubular epithelial cell line HKC was detected with indirect immunofluorescence assay and confirmed by reverse transcription polymerase chain reaction (PCR) in transcription level respectively, and PCR product was sequenced. In order to observe the response of HCK to LPS, the different concentrations of LPS (0, 0.5, 1.0, 2.0 microg/ml) were used in the research and the regulation of AAT in HKC was semi-quantitatively detected with Western Blot and Real-Time PCR respectively.
Indirect immunofluorescence staining showed that AAT was positive in HKC, but negative in renal interstitial fibroblast. By PCR, a significant messenger RNA band of AAT was found in HKC, but not in renal interstitial fibroblast. DNA sequencing indicated that the sequence of PCR product is consistent with AAT mRNA sequence in Gene Bank. Real-time PCR showed that the expression of AAT mRNA was up-regulated (fluorescence intensity ratio is 3.43 +/- 0.88 versus 1.22 +/- 0.20; P < 0.01) in HKC stimulated with 2.0 microg/ml LPS for 4 h, and Western Blot showed that the synthesis of AAT significantly increased (band density ratio is 0.88 +/- 0.12 versus 0.59 +/- 0.05; P < 0.01) in HKC stimulated with 2.0 microg/ml LPS for 8 h, as compared with unstimulated HKC. 0.5, 1.0 g/ml of LPS has no effect on the expression of AAT mRNA and AAT protein in HKC.
HKC express AAT and the expression can be up-regulated by LPS.
研究α1抗胰蛋白酶（AAT）在肾小管上皮细胞中的表达及调控。
分别采用间接免疫荧光法检测肾小管上皮细胞系HKC中AAT的表达，并通过逆转录聚合酶链反应（PCR）在转录水平进行验证，对PCR产物进行测序。为观察HKC对脂多糖（LPS）的反应，研究中使用不同浓度的LPS（0、0.5、1.0、2.0微克/毫升），分别采用蛋白质免疫印迹法和实时定量PCR半定量检测HKC中AAT的调控情况。
间接免疫荧光染色显示，AAT在HKC中呈阳性，而在肾间质成纤维细胞中呈阴性。通过PCR，在HKC中发现了一条明显的AAT信使核糖核酸条带，而在肾间质成纤维细胞中未发现。DNA测序表明，PCR产物的序列与基因库中AAT信使核糖核酸序列一致。实时定量PCR显示，用2.0微克/毫升LPS刺激HKC 4小时后，AAT信使核糖核酸的表达上调（荧光强度比值为3.43±0.88，而未刺激时为1.22±0.20；P<0.01），蛋白质免疫印迹法显示，用2.0微克/毫升LPS刺激HKC 8小时后，AAT的合成显著增加（条带密度比值为0.88±0.12，而未刺激时为0.59±0.05；P<0.01）。0.5、1.0微克/毫升的LPS对HKC中AAT信使核糖核酸和AAT蛋白的表达无影响。
HKC表达AAT，且其表达可被LPS上调。
