NH_4Cl对人肝细胞系LO2氨转运相关蛋白RHCG和AQP8表达的影响

Effect of ammonium chloride on gene expression of RHCG and AQP8 in LO2 cells

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。

Objective To find whether ammonium RHCG and AQP8 gene expression. Methods The chloride can specifically stimulate liver cell to change the internal human liver cell line （LO2）, thyroid cancer cell line （TPC-1）, ovarian cancer cell line （ SKOV-3 ） and esophageal cancer cell line （9706） were cultured. MTT method was used to check the proliferation ability of those 4 cell lines after cells were treated with NH4Cl for 24 h. QT-PCR and West- ern blotting were used to detect the RHCG and AQP8 gene expression of LO2 cells after treated with NH4Cl for 6,12 and 24 h. The RHCG and AQP8 gene expression of TPC-1, SKOV-3 and 9706 cells was detected after treatment with NH4Cl （10 mmol/L）the same method above. Results The growth inhibitory rate in LO2 cells increase with ammonia concentration increasing, which is significantly greater than that in other 3 cell lines. RT-PCR finds thatRI-ICG mRNA in LO2 cells decreased when treated with 10 mmol/L ammonium chloride for 6 hours （ P 〈 0. 05 ）. while AQP8 mRNA in LO2 cells increased when treated with 10mmol/l ammonium chloride for 12 hours （P 〈 0. 05 ） ; 10 mmo/fL. AQP8 protein in LO2 cells increased when treated with 10 mmol/L ammonium chloride for 24 hours （P 〈 0. 05 ） , while RHCG protein expression has no change. No obvious change is found in the AQP8 and RHCG expression in other cells when treated with ammonia of the same concentration within the same time. Conclusions In the environment of high ammonia concentration, human liver cells can maintain the balance of absorbing ammo- nia by changing RHCG and AQP8 expression to reduce the harm of ammonia to liver cells, while other cells have no such function.