CAJ | 학술논문

目的探讨L-精氨酸(L-Arg)经一氧化氮途径对人结肠癌细胞株LS174的作用及其机制.方法 LS174细胞在不同浓度L-Arg干预后,采用MTT法检测细胞增殖活性;光镜和电子显微镜观察细胞形态和结构的变化;Annexin V/PI标记染色测定凋亡细胞:Western blotting和免疫细胞化学染色SP法检测VEGF、COX-2、p53、bcl-2和bax的表达.结果低浓度(0.125 mmol/L)L-Arg对LS174细胞的生长有促进作用,高浓度(0.5、2、8、32 mmol/L)L-Arg对LS174细胞的生长有抑制作用;在高浓度L-Arg组作用48h后引起了细胞核和细胞质一系列超微结构改变,电子显微镜可见LS174出现细胞缩小、染色质边集、固缩等细胞凋亡的形态改变;对照组凋亡细胞百分比为2.84%,低浓度L-Arg组凋亡细胞百分比为2.29%.随着L-Arg浓度增加,凋亡细胞百分比增加明显,分别为26.88%、28.95%、63.36%、84.51%;VEGF和COX-2在低浓度L-Arg组表达较对照组有增加趋势;p53和bcl-2在高浓度L-Arg组表达较对照组有明显降低趋势;bax在高浓度L-Arg组表达较对照组有明显增加趋势.结论低浓度L-Arg对LS174细胞的生长有促进作用,高浓度L-Arg对LS174细胞的生长有抑制作用;低浓度的L-Arg对LS174细胞的生长有促进作用的机制可能是在L-Arg代谢产物NO作用下,与上调VEGF与COX-2蛋白有关;高浓度的L-Arg对LS174细胞的生长有抑制作用的机制可能是在L-Arg代谢产物NO作用下,诱导肿瘤细胞凋亡;L-Arg诱导凋亡的机制与上调bax蛋白表达及下调p53和bcl-2蛋白表达有关.
목적 탐토L-정안산(L-Arg)경일양화담도경대인결장암세포주LS174적작용급기궤제.방법 LS174세포재불동농도L-Arg간예후,채용MTT법검측세포증식활성;광경화전자현미경관찰세포형태화결구적변화;Annexin V/PI표기염색측정조망세포:Western blotting화면역세포화학염색SP법검측VEGF、COX-2、p53、bcl-2화bax적표체.결과 저농도(0.125 mmol/L)L-Arg대LS174세포적생장유촉진작용,고농도(0.5、2、8、32 mmol/L)L-Arg대LS174세포적생장유억제작용;재고농도L-Arg조작용48h후인기료세포핵화세포질일계렬초미결구개변,전자현미경가견LS174출현세포축소、염색질변집、고축등세포조망적형태개변;대조조조망세포백분비위2.84%,저농도L-Arg조조망세포백분비위2.29%.수착L-Arg농도증가,조망세포백분비증가명현,분별위26.88%、28.95%、63.36%、84.51%;VEGF화COX-2재저농도L-Arg조표체교대조조유증가추세;p53화bcl-2재고농도L-Arg조표체교대조조유명현강저추세;bax재고농도L-Arg조표체교대조조유명현증가추세.결론 저농도L-Arg대LS174세포적생장유촉진작용,고농도L-Arg대LS174세포적생장유억제작용;저농도적L-Arg대LS174세포적생장유촉진작용적궤제가능시재L-Arg대사산물NO작용하,여상조VEGF여COX-2단백유관;고농도적L-Arg대LS174세포적생장유억제작용적궤제가능시재L-Arg대사산물NO작용하,유도종류세포조망;L-Arg유도조망적궤제여상조bax단백표체급하조p53화bcl-2단백표체유관.
Objective To study the effects of L-arginine (L-Arg) on human colon carcinoma cell line LS174 through NO pathway and the mechanism. Methods LS 174 cells were cultured in the presence of L-Arg at different concentrations for different time. MTT assay was employed to evaluate the cell proliferation, and the cell morphological changes were observed by optical and electron microscopy. The apoptosis of L-Arg-treated cells was observed by the Annexin V/PI staining. The expression levels of VEGF, COX-2, p53, bcl-2 and bax in the cells were determined using Western blotting and immunocytochemical staining. Results L-Arg promoted the growth of LS 174 cells at a low concentration (0.125 mmol/L) and inhibited the cell growth at higher concentrations (0.5, 2, 8, and 32 mmol/L). Under electron microscope, ultrastructual changes in the cytoplasm and nuclei of LS174 cells were observed 48 h after exposure to L-Arg. Some of the cells showed morphological changes characteristic of cell apoptosis such as cell size reduction, condensation and margioation of the nuclear chromatin. Low concentration of L-Arg resulted in a cell apoptosis rate of 2.29%, as compared with-the rate of 2.84% in the control group; at higher concentrations, L-Arg caused significantly increased cell apoptosis rates to 26.88%, 28.95%, 63.36%, and 84.51%, respectively. In cells treated with a low concentration of L-Arg, the expressions of VEGF and COX-2 were increased as compared with those in the control cells; higher concentrations of L-Arg obviously decreased the expressions of p53 and bcl-2 and increased the expression of bax. Conclusion Low-concentration L-Arg can promote the growth of LS174 cells probably by up-regulating VEGF and COX-2 protein under the influence of NO, the metabolite of L-Arg. The inhibitory effect of high concentrations of L-Arg is probably mediated by inducing cell apoptosis via NO. The mechanism of L-Arg-induced cell apuptosis may be related to the up-regulation ofbax protein and the down-regulation of p53 and bcl-2 proteins.