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为了探讨神经生长因子(NGF)及其活化受体磷酸化的酪氨酸蛋白激酶A(p-TrkA)在人脑胶质瘤细胞株U251细胞周期中的多靶点分布模式及其生物学意义,采用免疫荧光双标记技术对处于细胞周期不同时相细胞内的NGF和p-TrkA的动态分布进行亚细胞定位,并观察了抗癌药物羟基脲、紫杉醇和秋水仙素及抗NGF-β中和血清对NGF和p-TrkA在细胞分布的影响;用免疫印迹技术检测细胞核和细胞质内NGF和p-TrkA的相对含量以及细胞培养液内的分泌性NGF。免疫荧光染色结果表明:分裂相细胞在重新贴壁生长6h后,NGF主要分布于核周区,p-TrkA主要定位于细胞膜;培养12h后,NGF和p-TrkA共同转位至细胞核内;在M期,NGF主要定位于中心体,p-TrkA主要定位于纺锤丝;用羟基脲将细胞阻滞于G1/S期后,NGF和p-TrkA主要积聚在细胞核内;用紫杉醇或秋水仙素处理后,NGF与γ-Tubulin仍然共定位于中心体,p-TrkA与α-Tubulin共定位于异形纺锤丝上或弥散分布于细胞质内。用兔抗人NGF-β抗血清中和培养基中分泌性的NGF后,细胞内NGF和p-TrkA免疫荧光强度明显减弱。免疫印迹结果显示:G1/S期细胞核内的NGF和p-TrkA蛋白条带明显浓于细胞质内的蛋白条带。上述结果提示:人脑胶质瘤U251细胞高表达NGF及其高亲和力受体TrkA,并将NGF分泌至细胞外;胞外NGF与细胞膜上TrkA结合后形成NGF/p-TrkA复合物内化入胞内;NGF/p-TrkA在细胞内的分布具有细胞周期性特征;NGF/p-TrkA可通过多靶点作用模式调控U251细胞的生物学行为。上述多靶点分布模式为研制NGF修饰的抗肿瘤靶向药物提供了细胞生物学基础。
To investigate the multi-target distribution pattern of nerve growth factor (NGF) and its activated receptor phosphorylation tyrosine kinase A (p-TrkA) in human glioma cell line U251 and its biological significance , The dynamic distribution of NGF and p-TrkA in cells with different phases in the cell cycle was subcellularly localized by immunofluorescence double labeling technique and the anti-cancer drug hydroxyurea, paclitaxel and colchicine and anti-NGF-β And serum on the distribution of NGF and p-TrkA; Western blotting was used to detect the relative content of NGF and p-TrkA in the nucleus and cytoplasm and secreted NGF in the cell culture medium. The results of immunofluorescence staining showed that NGF mainly distributed in the perinuclear zone and the p-TrkA mainly located in the cell membrane after re-adherent growth for 6h. After cultured for 12h, NGF and p-TrkA translocated to the nucleus; M phase, NGF mainly located in the centrosome, p-TrkA mainly located in the spindle wire; with hydroxyurea cells arrested in G1 / S phase, NGF and p-TrkA mainly in the nucleus; with paclitaxel or colchicine After treatment, NGF and γ-Tubulin still co-localized in the centrosome, p-TrkA and α-Tubulin co-located on the shaped spindle or diffusely distributed in the cytoplasm. After the secreted NGF was neutralized with rabbit anti-human NGF-β antiserum, the immunofluorescence intensity of NGF and p-TrkA in cells was significantly weakened. Immunoblotting showed that the bands of NGF and p-TrkA in G1 / S phase were obviously thicker than those in cytoplasm. The above results suggest that NGF and its high-affinity receptor TrkA are highly expressed in human glioma U251 cells and NGF is secreted into the extracellular domain. NGF / p-TrkA complex is formed after extracellular NGF is bound to TrkA on the cell membrane Intracellular distribution of NGF / p-TrkA in cells was characterized by cell cycle. NGF / p-TrkA could regulate the biological behavior of U251 cells through multi-target mode of action. The above multi-target distribution model provides a cellular biology foundation for the development of NGF-modified anti-tumor targeted drugs.