NGF及其活化受体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

THE MULTI-TARGET-DISTRIBUTION MODE OF NGF WITH ITS ACTIVATING RECEPTOR p-TrkA IN THE CELL CYCLE OF HUMAN GLIOMA CELL LINE U251

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 explore the mode and the cytobiological biologic significance of multi-target-distribution of nerve growth factor （NGF） and its activating receptor phosphorylated tyrosine kinase A （p-TrkA） in cell cycle of glioma cell line U251, the immanolluorescence double staining was used to localize the dynamic distributions of NGF and p-TrkA in different cell cycle phases of the cells. The influences of hydroxyurea, paclitaxel, colchicine and the serum of anti-NGF on the distribution of NGF and p-TrkA were observed as well. Western blotting was used to detect the secretory NGF in the medium and the contents of NGF and p-TrkA in cytoplasmic extractions with those in nuclear extractions of the cells. The results of immunofluorescence showed that after the mitotic cells were cultured for 6 h, the NGF was distributed in peri-nuclei and the p-TrkA was located on the membrane, then the NGF and the p-TrkA were gradually translocated to nuclei after cultured for 12 h; In the mitotic cells, the NGF was localized on the centrosomes, the p-TrkA was distributed on spindles; In the cells arrested at G1/S phase by treatment with hydroxyurea, the NGF and p-TrkA were localized in the nuclei; After treated with paclitaxel or colchicine, NGF was still co-localized with T-Tubulin at centrosomes and p-TrkA was co-localized with α-Tubulin on the abnormal spindies or distributed diffusely in the cytoplasm. After cultured with the medium containing the serum of anti-NGF to neutralize the secretory NGF, the immunofluorescence density of NGF and p-TrkA were weakened. The results of Western blotting showed that, in the medium cultured GI/S phase cells, the bands of NGF and p-TrkA in nuclear were thicker than those in the cytoplasmic extractions of the cells. This study suggested that the glioma U251 cells could overexpress NGF and secret it to the medium, then activate its receptor TrkA; The internalized NGF/p-TrkA could distribute to different targets in U251 cells according to the cell cycle phases, NGF/p-TrkA would modulated the biological behaviors of the glioma cells by multi-target activation mode; The multi-target distribution mode of NGF and p-TrkA would support the therapeutical strategy by targeted drugs modified with NGF to treat glioma.