论文部分内容阅读
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 effects of α1 antitrypsin (AAT) and lipopolysaccharide (LPS) on the synthesis of AAT in renal tubular epithelial cells. Methods AAT mRNA and protein expression in human renal tubular epithelial cell line (HKC) were detected by indirect immunofluorescence and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. The PCR products were identified by DNA sequencing. HKCs were stimulated with different concentrations of LPS, and divided into groups of 0, 0.5, 1 and 2μg / ml according to LPS concentration test. HKCAAT mRNA and protein expression were detected by real-time fluorescence quantitative PCR and Western blotting respectively. Results The results of indirect immunofluorescence showed that HKCAAT was positive and located in the cytoplasm. The expression of AAT mRNA in HKC was detected by RT-PCR, and the sequence of AAT mRNA in GeneBank was identical with that of PCR. Compared with unstimulated group, real-time fluorescent quantitative PCR showed that HKC4h was stimulated by 2.0μg / ml LPS, the expression of AAT mRNA was significantly up-regulated (fluorescence intensity ratio was 3.43 ± 0.88vs1.22 ± 0.20; P <0.01) Western blotting showed that HKC8h was stimulated with 2.0μg / ml LPS, and the protein expression of AAT was significantly increased (the band density ratio was 0.88 ± 0.12 vs0.59 ± 0.05; P <0.01). While 0.5μg / ml and 1.0μg / ml LPS had no significant stimulation on AAT mRNA and protein expression in HKC. Conclusions HKC can synthesize AAT, and LPS up-regulates AAT mRNA and protein expression in human renal proximal tubule epithelial cells.