摘要
目的探讨L-精氨酸对人肝细胞LO2中内源凝血因子Ⅷ(Coagulation factorⅧ,FⅧ)表达的激活作用。方法将LO2细胞分为试验组和对照组,试验组加入L-精氨酸(终浓度为10 mmol/L)分别培养24、36、48和60 h,对照组用等体积的灭菌水取代L-精氨酸培养。采用RT-PCR法检测LO2细胞中人FⅧ基因mRNA的转录水平并测序鉴定,一期法检测细胞培养上清液中人FⅧ的促凝活性(FⅧ∶C),Western blot检测24、48 h时相点LO2细胞中人FⅧ蛋白的表达,免疫荧光染色结合激光共聚焦显微镜观察L-精氨酸作用48 h后LO2细胞中人FⅧ的表达。结果加入L-精氨酸培养36、48、60 h后,LO2细胞中有人FⅧ基因mRNA的转录,而对照组未出现人FⅧ基因mRNA的转录;各时相点两组细胞培养上清液中人FⅧ∶C的水平差异均无统计学意义(P>0.05);Western blot及激光共聚焦均观察到加L-精氨酸培养48 h后LO2细胞中出现人FⅧ的表达,而对照组细胞中均无人FⅧ的表达。结论 L-精氨酸可激活人正常肝细胞中内源FⅧ的表达。
Objective To explore the activating effect of L-arginine on expression of endogenous coagulation factor Ⅷ（FⅧ） in human normal liver LO2 cells. Methods LO2 cells were divided into test and control groups. The cells in test group were treated with L-arginine （at afinal concentration of 10 mmol/L） for 24, 36, 48 and 60 h respectively, while those in control group with phys- iological saline. The transcription level of FⅧ mRNA in LO2 cells was determined by RT-PCR, and the PCR product was sequenced. The cell culture supematant was collected and determined for clotting activity of human FⅧ （FⅧ ： C） by one-stage method. The ex- pressions of F~ in LO2 cells 24 and 48 h after treatment were determined by Western blot, and that 48 h after treatment by IFA combined with laser confocal microscopy. Results Transcription of F Ⅷ mRNA was observed in the cells 35, 48 and 50 h after treatment with L-arginine, while was not observed in those in control group. The expression levels of FⅧ in cell culture supernatant in test group at various time points after treatment showed no significant differences with those in control group （P 〉 0. 05）. Both Western blot and laser confocal microscopy showed expression of FⅧ in the cells 48 h after treatment with L-arginine but no expres- sion in those in control group. Conclusion L-arginine activated the expression of endogenous FV$ in human normal liver ceils.
出处
《中国生物制品学杂志》
CAS
CSCD
2012年第10期1303-1306,共4页
Chinese Journal of Biologicals
关键词
L-精氨酸
肝细胞
凝血因子Ⅷ
转录
表达
L-Arginine
Liver cells
Coagulation factor Ⅷ
Transcription
Expression
