人肾脏近曲小管上皮细胞表达α_1抗胰蛋白酶的研究

Expression of alpha 1 anti-trypsin in proximal tubular epithelial cell line

目的探讨肾小管上皮细胞是否合成α1抗胰蛋白酶(AAT)以及脂多糖(LPS)对肾小管上皮细胞合成AAT的影响。方法分别用间接免疫荧光和逆转录多聚酶链反应(RT-PCR)检测人肾小管上皮细胞系(HKC)AATmRNA及蛋白表达,并对PCR产物进行DNA测序鉴定。用不同浓度LPS刺激HKC,根据LPS浓度实验分为0、0·5、1、2μg/ml4组,用实时荧光定量PCR和Western印迹法分别检测LPS刺激后HKCAATmRNA及蛋白质表达的变化。结果间接免疫荧光显示HKCAAT阳性,分布于胞质中;RT-PCR检测发现HKC表达AATmRNA;PCR产物经DNA测序与GeneBank中AATmRNA序列完全一致。与未刺激组比较,实时荧光定量PCR显示2·0μg/mlLPS刺激HKC4h,AATmRNA表达明显上调(荧光强度比值为3·43±0·88vs1·22±0·20;P<0·01),Western印迹法显示2·0μg/mlLPS刺激HKC8h,AAT蛋白质表达明显增高(条带密度比值为0·88±0·12vs0·59±0·05;P<0·01)。而0·5μg/ml和1·0μg/mlLPS对HKC的AATmRNA和蛋白质表达均无显著刺激作用。结论HKC能合成AAT,LPS可上调人肾近曲小管上皮细胞的AATmRNA和蛋白质表达。

Objective To investigate the expression and regulation of alpha 1 anti-trypsin （AAT） in tubular epithelial cells. Methods The expression of AAT in tubular epithelial cell line HKC was detected with indirect immunoflurescence assay and comfirmed 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 μg/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. Results Indirect immunoflurescence 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 （flurescence intensity ratio is 3.43 ±0. 88 versus 1.22 ±0. 20; P 〈0. 01 ） in HKC stimulated with 2. 0 μg/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 μg/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. Conclusions HKC express AAT and the expression can be up-regulated by LPS.