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目的检验氯化铵是否能够特异性地刺激肝细胞,使其内部与氨离子转运有关的RHCG和AQP8基因表达发生变化。方法培养人正常肝脏细胞(LO2)、甲状腺癌细胞(TPC-1)、卵巢癌细胞(SKOVR-3)和食管癌细胞(9706),NH4Cl(2.5、5、10、20、40和50 mmol/L)对4种细胞作用24 h后,MTT法检测细胞的增殖;荧光定量PCR和蛋白免疫印迹技术检测LO2细胞经5、10和20 mmol/L的NH4Cl处理不同时间(6、12和24 h)后,AQP8和RHCG基因及蛋白的表达;10 mmol/L的NH4Cl处理TPC-1、SKOVR-3和9706细胞不同时间(6、12和24 h)后,AQP8和RHCG基因及蛋白的表达。结果随着NH4Cl浓度的增加,氨对肝细胞LO2的增殖抑制率逐渐增加且明显大于其他细胞系;10 mmol/L NH4Cl作用于LO2细胞6 h时,RHCG基因表达量明显下调(P<0.05),而AQP8基因的表达量在作用12 h后较对照组明显增加(P<0.05);10 mmol/L NH4Cl作用于LO2细胞24 h时,AQP8蛋白表达量较对照组显著增加(P<0.0.5),而RHCG无变化;相同浓度NH4Cl作用其他细胞系相同时间时,AQP8和RHCG的基因和蛋白水平均未见显著性变化。结论在氨升高的环境中,人肝细胞通过调节氨转运相关蛋白AQP8和RHCG的量来维持对氨摄取的平衡,减少氨对肝细胞的特异性损伤,在其他细胞系中无此现象。
Aim To investigate if ammonium chloride can specifically stimulate hepatocytes and change the expression of RHCG and AQP8 genes related to ammonia ion transport. Methods Human normal liver cells (LO2), thyroid cancer cells (TPC-1), ovarian cancer cells (SKOVR-3) and esophageal cancer cells (9706), NH4Cl (2.5, 5, 10, 20, 40 and 50 mmol / L) for 24 h, MTT assay was used to detect cell proliferation. Fluorescent quantitative PCR and Western blotting were used to detect the effects of 5, 10 and 20 mmol / L NH4Cl treatment on the proliferation of LO2 cells at different times (6, 12 and 24 h ), The expression of AQP8 and RHCG genes and proteins, and the expression of AQP8 and RHCG genes and proteins in TPC-1, SKOVR-3 and 9706 cells treated with 10 mmol / L NH4Cl for different time (6, 12 and 24 h) Results With the increase of NH4Cl concentration, the inhibitory rate of ammonia on LO2 proliferation increased gradually and significantly higher than that of other cell lines. The expression of RHCG gene was significantly down-regulated after treated with 10 mmol / L NH4Cl for 6 h (P <0.05) , While the expression of AQP8 increased significantly (P <0.05) at 12 h after treated with 10 mmol / L NH4Cl for 24 h. The expression of AQP8 protein in LO2 cells increased significantly (P <0.0) compared with the control group. 5), while there was no change in RHCG. No significant changes were found in the gene and protein levels of AQP8 and RHCG when the same concentration of NH4Cl was applied to other cell lines at the same time. Conclusion In the environment of elevated ammonia, human hepatocytes maintain the balance of ammonia uptake by regulating the amount of ammonia transport related proteins AQP8 and RHCG, and reduce the specific damage of ammonia to hepatocytes, but not in other cell lines.