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目的探讨人工合成的含有精-甘-天冬氨酸(Arg-Gly-Asp,RGD)序列的环八肽(cRGD) 与大鼠肝星状细胞(HSC)体外的结合特点。方法采用异硫氢酸荧光素(FITC)标记环八肽(FITC- cRGD);流式细胞仪测定其与大鼠HSC体外结合的荧光强度;荧光显微镜观察FITC-cRGD在HSC内的定位;用氚标记的cRGD(3H-cRGD)与HSC进行受体的放射性配基结合分析,Scatchard作图法计算二者结合的平衡解离常数(Kd)和每个细胞上的最大结合位点数(Bmax)。同时,FITC标记的环肽(cAGA)作为阴性对照肽。结果FITC-cRGD与活化的大鼠HSC的结合具有可饱和性、浓度与时间依赖性以及未标记的cRGD对FITC-cRGD具有竞争性抑制作用。FITC-cRGD荧光主要集中在HSC胞浆中,尤其是胞浆近核周区。受体放射性配基结合分析经Scatchard作图提示Kd为7.05×109mol/L,Bmax约为6.79 ×105。与阴性对照肽相比,RGD序列对识别HSC起重要作用。结论HSC表面具有可供含有RGD序列人工合成环肽结合的位点;制备的含有RGD序列的环肽符台特异性配体的标准;FITC标记不影响环肽生物学活性。
OBJECTIVE: To study the in vitro binding characteristics of synthetic cGG with arg-Gly-Asp (RGD) sequence and rat hepatic stellate cells (HSC). Methods FITC-cRGD was labeled with FITC-cRGD. The fluorescence intensity of HSC in vitro was detected by flow cytometry. The localization of FITC-cRGD in HSC was observed by fluorescence microscopy. Radioligand binding analysis of tritium-labeled cRGD (3H-cRGD) and HSCs was performed using a Scatchard plot. The equilibrium dissociation constants (Kd) and maximum binding sites per cell (Bmax) . Meanwhile, FITC-labeled cyclic peptide (cAGA) served as a negative control peptide. Results The binding of FITC-cRGD to activated rat HSC was saturable, concentration-dependent and time-dependent, as well as competitive inhibition of FITC-cRGD by unlabeled cRGD. FITC-cRGD fluorescence mainly concentrated in the cytoplasm of HSC, especially in the cytoplasm near the perinuclear region. Radioligand binding assay by Scatchard suggested Kd was 7.05 × 109mol / L, Bmax was about 6.79 × 105. Compared with the negative control peptide, the RGD sequence plays an important role in recognizing HSC. CONCLUSION: The site of HSC on the surface of HSC is suitable for the synthesis of cyclic peptide containing the RGD sequence. The prepared cyclic RGD sequence-containing cyclobutaxel-specific ligand standard does not affect the biological activity of the cyclic peptide.