摘要
目的:通过Raf激酶抑制蛋白(Raf kinase inhibitor protein,RKIP)过表达腺病毒感染肝星状细胞(hepatic stellate cells,HSC),模拟肝纤维化病理状态下细胞外基质(extracellular matrix,ECM)成分的改变,探讨RKIP对HSC黏附功能的影响.方法:实验共分两组,分别是RKIP过表达组(RKIP-AD)和阴性对照组(GFP-AD).首先确定细胞感染病毒的最适感染强度(multiplicity of infection,MOI)值,在倒置荧光显微镜下观察细胞状态,计数细胞荧光数,筛选出最适MOI值.Western blot方法确定病毒感染后验证外源RKIP在细胞内表达情况.已干预好的细胞稀释成1.0×105/mL的浓度种在盖玻片和已准备好的由matrigel、Ⅰ型胶原及纤维连接蛋白包被的24孔板中,37℃培养1h后,甲醛固定,结晶紫染色.200×纤维镜下观察并计数黏附的细胞数,资料数据以mean±SD表示,采用t检验进行统计分析.结果:人源HSC细胞株LX-2的腺病毒载体感染率可达到80%-90%,适宜的MOI值为5.RKIP过表达组较对照组蛋白表达增多(3.47±0.02vs1.74±0.13,P<0.05).与GFP-AD对照组相比,RKIP在HSC的过表达抑制细胞在matrigel上的黏附(80±14vs152±42,P<0.05).与GFP-AD对照组相比,RKIP在HSC的过表达抑制细胞在Ⅰ型胶原上的黏附(70±13vs138±36,P<0.05).与GFP-AD对照组相比,RKIP在HSC的过表达抑制细胞在纤维连接蛋白上的黏附(133±27vs276±106,P<0.05).结论:体外过表达RKIP可以抑制HSC在胶原基质上的黏附.
AIM: To investigated the role of Raf kinase inhibi- tor protein (RKIP) in the adhesion of hepatic stel- late cells (HSCs) to extracellular matrix (ECM). METHODS: HSCs (LX-2 cell line) were cultured and infected with an adenovirus vector carrying RKIP (RKIP-AD) or the control adenovirus vec- tor (GFP-AD). Green fluorescence was detected under an inverted fluorescence microscope to assess the infection efficiency. The number of vi- rus particles required was calculated as the cell count x multiplicity of infection (MOI). RKIPprotein expression was investigated by Western blot. Infected cells were adjusted to a density of μ10s cells/mL, plated in dishes cultured with matrigel, type I collagen, and fibronectin. LX-2 cells were cultured for I h in a humidified atmo- sphere of 5% CO2 and 95% air at 37 ℃. The cells were fixed in 4% paraformaldehyde and stained with crystal violet. Cells in each field were viewed under light microscopy and positively stained cells were counted. RESULTS: The infection efficiency of RKIP-AD or GFP-AD reached 80%-90%. Adenovirus-me- diated gene transfection at a MOI of 5 was em- ployed for subsequent experiments. In the RKIP- AD group, RKIP expression was significantly higher than that in the control group (3.47 ± 0.02 vs 1.74 ±0.13, P 〈 0.05). RKIP overexpression reduced the number of HSC adhered to matrigel (80 ±14 vs 152 ± 4, P 〈 0.05), type I collagen (70 ± 13 vs 138 ± 36, P 〈 0.05) and fibronectin (133±27 vs 276 ±106, P 〈 0.05) compared to the control group.CONCLUSION: RKIP overexpression inhibits HSC adhesion to ECM.
出处
《世界华人消化杂志》
CAS
北大核心
2013年第14期1303-1308,共6页
World Chinese Journal of Digestology
基金
国家自然科学基金资助项目
Nos.81200311
81170411
河北省自然科学基金资助项目
No.C2010000530~~
关键词
肝纤维化
肝星状细胞
RAF激酶抑制蛋白
细胞外基质
黏附
Hepatic fibrosis
Hepatic stellate cells
Raf kinase inhibitor protein
Extracellular matrix
Adhesion