【作者】 曹科；

【导师】 曹培国；

【作者基本信息】 中南大学 ， 内科学， 2007， 硕士

【摘要】 研究目的:以先前基因芯片结果为基础,确证维生素K2对人肝癌细胞株HuH-7增殖的抑制作用及其对成纤维细胞生长因子受体3(FGFR3)的下调作用,力图为维生素K2的临床应用及人类肝癌靶向治疗提供新的实验依据。研究方法:(1)、以MTT法检测维生素K2对HUH-7细胞增殖的抑制作用;(2)、以半定量RT-PCR法检测0、10、30μM维生素K2干预后HuH-7细胞中FGFR3和FGFR1的mRNA表达;(3)、以实时定量PCR法检测0、10、30μM维生素K2干预后HuH-7细胞中FGFR3和FGFR1的mRNA表达;(4)、以Western-blot法检测不同浓度维生素K2处理后的HuH-7细胞中FGFR3蛋白表达。研究结果:(1)、维生素K2对HuH-7细胞生长有抑制作用。10、30μM维生素K2干预组对HuH-7细胞的抑制率与无药对照组比较,72、96h有显著性差异(P＜0.01),0.01、0.1、1μM维生素K2干预组对HuH-7细胞的抑制率与无药对照组比较,48、72及96h均无显著性差异(P＞0.05);30μM维生素K2抑制HuH-7细胞增殖呈时间依赖性。(2)、RT-PCR示96h后10、30μM维生素K2干预组能呈剂量依赖性抑制HuH-7细胞中FGFR3 mRNA表达,而未明显抑制HuH-7细胞中FGFR1 mRNA表达;(3)、实时定量PCR示96h后10、30μM的维生素K2干预组中FGFR3 mRNA表达分别为无药对照组的0.692±0.087倍(p=0.0124,＜0.05)和0.385±0.053倍(p＜0.0001),均有显著性差异,10、30μM的维生素K2干预组之间亦存在显著性差异(p=0.0236,＜0.05);96h后10、30μM的维生素K2干预组中FGFR1 mRNA表达分别为无药对照组的0.938±0.051倍(p=0.2921,＞0.05)和0.933±0.096倍(p=0.5230,＞0.05),均无显著性差异;10、30μM的维生素K2干预组之间亦无显著性差异(p=0.9678,＞0.05);(4)、Western-blot法检测结果示96h后10、30μM的维生素K2干预组中FGFR3的蛋白表达较无药对照组及10、30μM维生素K2干预组之间(125 KDa,135 KDa)均存在显著性差异(p＜0.01)。研究结论:(1)、维生素K2能呈时间和剂量依赖性抑制HuH-7细胞增殖;(2)、维生素K2可能通过下调FGFR3的表达来抑制HuH-7细胞增殖;(3)、维生素K2抑制HUH-7细胞的增殖可能与FGFR1的表达量无关。更多还原

【Abstract】 Objective:To confirm the inhibitory effects of Vitamin K2 on the proliferation of human hepatocellular carcinoma cell HuH-7 and the expression of FGFR3,according to the previous results of DNA chip,and to provide convictive experimental bases for further clinical use of Vitamin K2 and the targeted therapy for liver cancer.Methods:(1)The inhibitory effect of Vitamin K2 on the proliferation of HuH-7 was measured by MTT assay.(2)Semi-quantitative RT-PCR(reverse-transcription-polymerase chain reaction)was used to confirm the mRNA levels of FGFR3 and FGFR1 after 96 hours exposure to 0,10 and 30μM of Vitamin K2.(3)Real-time PCR(quantitative polymerase chain reaction)was used to measure the mRNA levels of FGFR3 and FGFR1 after 96 hours exposure to 0,10 and 30μM of Vitamin K2.(4)Western-blot analysis was used to detect the protein level of FGFR3 after 96 hours exposure to 0,10 and 30μM of Vitamin K2.Results:(1)Vitamin K2 had the inhibitory effects on the proliferation of HuH-7 cells.The significant differences of cell numbers were observed after 72 hours and 96 hours in the 10 and 30μM Vitamin K2 groups (P＜0.01).No significant differences of the cell numbers were observed after 48 hours exposure of HuH-7 to 0.01,0.1,1,10 and 30μM Vitamin K2(P＞0.05)and after 72,96 hours in the 0.01,0.1 and 1μM Vitamin K2 groups(P＞0.05).The 30μM Vitamin K2 group showed a time-dependent inhibition on the proliferation of HuH-7 cells.(2)By RT-PCR,the inhibitory effect of Vitamin K2 on the FGFR3 mRNA was in a dose dependent manner.However,no remarkable inhibitory effect of Vitamin K2 on the FGFR1 mRNA was shown.(3)Real-time PCR revealed the significant reductions of the mRNA levels of FGFR3 in the 10 and 30μM Vitamin K2 groups as compared with the control group[0.692±0.087(p=0.0124,＜0.05)at 10μM and 0.385±0.053(p＜0.0001)at 30μM]after 96 hours treatments.The significant difference was demonstrated between 10 and 30μM Vitamin K2 groups(p=0.0236,＜0.05),too.No significant reductions of the mRNA level of FGFR1 was detected in the 10 or 30μM Vitamin K2 groups as compared with the control group[0.938±0.051(p=0.2921,＞0.05)at 10μM group and 0.933±0.096(p=0.5230,＞0.05)at 30μM group].No significant difference was shown between 10 and 30μM Vitamin K2 groups(p=0.9678,＞0.05),too.(4)Significant suppression of the FGFR3 protein expression was revealed in the 10 and 30μM Vitamin K2 groups as compared with the control group(p＜0.01).The significant difference between 10 and 30μM Vitamin K2 groups was shown(p＜0.01),too.Conclusions:(1)Vitamin K2 supresses the proliferation of HuH-7 in dosedependent and time-dependent manners.(2)Vitamin K2 probably inhibits the proliferation of HuH-7 through down-regulating FGFR3 expression.(3)The inhibitory effect of Vitamin K2 on the proliferation of HuH-7 may not be related to FGFR1 gene expression.更多还原