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探讨炎症状态下NF-κB通路、mTOR通路以及转录因子RUNX1对肾小管上皮细胞DC-SIGN表达调控。体外培养肾小管上皮细胞株(HK-2)并经TNF-α刺激,分别给予NF-κB抑制剂(BAY 11-7082)和mTOR抑制剂(Rapamycin)干预。采用免疫印迹试验和实时定量PCR法检测HK-2细胞DC-SIGN表达;免疫印迹试验检测mTOR的磷酸化水平。用上述经TNF-α刺激的HK-2以及建立的肾炎损伤小鼠模型,以实时定量PCR法检测HK-2细胞以及分离的模型鼠肾小管上皮细胞中RUNX1表达。进一步利用HK-2构建过表达RUNX1稳定转染细胞株并经TNF-α刺激,实时定量PCR法检测RUNX1和DC-SIGN表达。结果显示,HK-2经TNF-α刺激模拟炎症状态下,可促进mTOR磷酸化并诱导DC-SIGN表达;NF-κB抑制剂和mTOR抑制剂均能抑制HK-2细胞的DC-SIGN表达,NF-κB抑制剂亦可抑制mTOR的磷酸化。还发现炎症因素刺激下,人和小鼠肾小管上皮细胞体内体外均明显表达RUNX1,且过表达RUNX1的HK-2细胞稳转株显著表达DCSIGN,并在TNF-α刺激下可进一步上调DC-SIGN表达。本研究表明,肾脏微炎症状态下,NF-κB-mTOR通路以及转录因子RUNX1均参与了肾小管上皮细胞DC-SIGN的表达调控。提示此过程中,NF-κB通路作为mTOR上游通路,通过激活后者参与DC-SIGN表达,而转录因子RUNX1可能是启动DC-SIGN表达的关键调控因素。
To investigate the regulation of NF-κB pathway, mTOR pathway and transcription factor RUNX1 on DC-SIGN expression in renal tubular epithelial cells in inflammatory state. The renal tubular epithelial cell line (HK-2) was cultured in vitro and stimulated with TNF-α. The cells were treated with NF-κB inhibitor (BAY 11-7082) and mTOR inhibitor (Rapamycin) respectively. The expression of DC-SIGN in HK-2 cells was detected by Western blotting and real-time quantitative PCR. The phosphorylation of mTOR was detected by Western blotting. The expression of RUNX1 in HK-2 cells and isolated rat renal tubular epithelial cells was detected by real-time quantitative PCR using HK-2 stimulated with TNF-α as described above and established nephritis lesion mouse model. Furthermore, HK-2 cells were transfected with RUNX1 overexpressing cells and stimulated by TNF-α. Real-time quantitative PCR was used to detect RUNX1 and DC-SIGN expression. The results showed that HK-2 stimulated the mTOR phosphorylation and induced the expression of DC-SIGN under the stimulation of TNF-α, and inhibited the expression of DC-SIGN of HK-2 cells. NF-κB inhibitors also inhibit the phosphorylation of mTOR. Also found that inflammatory factors, human and mouse renal tubular epithelial cells were significantly in vitro and in vivo RUNX1, and overexpression of RUNX1 HK-2 cells stably transfected with significant DCSIGN expression and stimulation of TNF-α can be further increased DC- SIGN expression. This study shows that NF-κB-mTOR pathway and RUNX1, a transcription factor, are involved in the regulation of DC-SIGN expression in renal tubular epithelial cells under renal micro-inflammation. It is suggested that NF-κB pathway acts as an upstream pathway of mTOR and participates in DC-SIGN expression through activating the transcription factor RUNX1, which may be the key regulator of DC-SIGN expression.