化学合成的Ac-SDKP类似物FAM-Aca-SDKP与HSC-T6细胞表面结合特征分析  

Binding characteristics of chemosynthetic Ac-SDKP analogue FAM-Aca-SDKP to hepatic stellate cell-T6 cells

作　　者：周达[1] 何玲楠 王晶[1] 丁永年[2] 陈源文[1] 范建高[1] 

机构地区：[1]上海交通大学医学院附属新华医院消化内科,200092 [2]新疆医科大学第二附属医院消化内科,乌鲁木齐830063

出　　处：《中华肝脏病杂志》2016年第3期186-190,共5页Chinese Journal of Hepatology

基　　金：国家自然科学基金项目（81170410,81260081）;新疆维吾尔自治区高校科研计划重点项目（XJEDU20121001）;上海市卫生系统优秀青年人才培养计划项目（XYQ2011010）

摘　　要：目的 利用化学合成N-乙酰基-丝氨酸-天冬氨酸-赖氨酸-脯氨酸（Ac-SDKP）类似物FAM-Aca-SDKP,探讨Ac-SDKP与肝星状细胞株（HSC-T6细胞）的结合及其结合的基本物理特征.方法 化学合成携带绿色荧光的Ac-SDKP类似物短肽[5-FAM]-酪氨酸-氨基己酸-丝氨酸-天冬氨酸-赖氨酸-脯氨酸（FAM-Aca-SDKP）,实时定量PCR检测其对HSC胶原分泌影响验证其生物学效应与Ac-SDKP一致性,采用荧光显微镜观察FAM-Aca-SDKP与HSC-T6结合,采用流式细胞术检测FAM-Aca-SDKP与HSC-T6结合的时间-浓度效应.据资料不同采用t检验或秩和检验进行统计学分析.结果 不同浓度的Ac-SDKP及FAM-Aca-SDKP与HSC-T6孵育24 h后,均观察到HSC-T6表达Ⅰ型前胶原增加,而作用时间为0.5h时,Ac-SDKP和FAM-Aca-SDKP导致Ⅰ型胶原表达分别下降30％ ～ 50％;FAM-Aca-SDKP与HSC-T6共孵育后,在荧光显微镜下观察到细胞表面出现显著绿色荧光,给予Ac-SDKP竞争抑制可显著减少细胞表面的荧光强度;流式细胞术检测显示,在FAM-Aca-SDKP浓度为0 ～ 50 μ mol/L时,荧光阳性细胞率从0快速上升至约12％,而在浓度为50 ～ 100μ mol/L,阳性细胞率仅从约12％上升至约14％,并且给予Ac-SDKP共孵育可显著降低阳性细胞百分率;FAM-Aca-SDKP阳性细胞数在孵育45 min达到高峰,随后逐渐降低. 结论 FAM-Aca-SDKP可结合于HSC-T6细胞表面,表现出竞争性抑制、可饱和、时间-浓度效应等配体-受体结合特征.Objective To investigate the binding of the chemosynthetic analogue of N-acetyl-seryl-aspartyl-lysyl-proline （Ac-SDKP） FAM-Aca-SDKP to hepatic stellate cell-T6 （HSC-T6） cells and basic physical characteristics.Methods The Ac-SDKP analogue short-peptide FAM-Aca-SDKP carrying green fluorescence was synthesized chemically.Quantitative real-time PCR was used to evaluate its effect on the secretion of HSC collagen and verify the consistency in the biological effect between FAM-Aca-SDKP and Ac-SDKP.A fluorescence microscope was used to observe the binding between FAM-Aca-SDKP and HSC-T6,and flow cytometry was used to evaluate the time-concentration effect of the binding between FAM-Aca-SDKP and HSC-T6.The t-test or rank sum test was used for the statistical analysis of different types ofdata.Results After HSC-T6 was incubated with Ac-SDKP or FAM-Aca-SDKP for 24 hours,the expression of type Ⅰ collagen in HSC-T6 was increased,when the action time was 0.5 hour,Ac-SDKP and FAM-Aca-SDKP caused a 30%-50% reduction in the expression of type Ⅰ collagen.After HSC-T6 was incubated with FAM-Aca-SDKP,strong green fluorescence was observed on cell surface under a fluorescence microscope,and after Ac-SDKP was added,Ac-SDKP significantly reduced the fluorescence intensity on cell surface due to competitive inhibition.Flow cytometry showed that when the concentration of FAM-Aca-SDKP was 0-50 μmol/L,the rate of fluorescence-positive cells rapidly increased from 0 to 12%;when the concentration was 50-100 μmol/L,the rate of fluorescence-positive cells only increased from 12% to 14%;co-incubation with Ac-SDKP significantly reduced the rate of fluorescence-positive cells.The number of positive cells reached the peak at the 45-minute point of the incubation and then decreased gradually.Conclusions FAM-Aca-SDKP can bind to the surface of HSC-T6 cells,and this process has ligand-receptor binding characteristics such as competitive inhibition,saturability,and time-concentration effect.

关 键 词：肝硬化 肝星状细胞 受体 配体 Ac-SDKP 结合特性 

分 类 号：R914.5[医药卫生—药物化学]