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目的探讨微管相关蛋白2(MAP-2)基因对恶性黑素瘤细胞生长的抑制作用。方法扩增腺病毒-微管相关蛋白2(Ad-MAP-2)载体,空斑法测定Ad-MAP-2的滴度后,将Ad-MAP-2基因转染入鼠侵袭性恶性黑素瘤细胞B16C29细胞内。应用直接免疫荧光法测定细胞内MAP-2的表达,Ad-MAP-2对B16C29细胞生长的抑制作用,电镜观察细胞内微管形态,用流式细胞仪检测细胞凋亡。结果Ad-MAP-2基因转染后,B16C29细胞胞体延伸出长而细的树枝状突起,近似于较成熟的色素细胞。电镜下见转染后细胞内微管含量增加,微管变长、变粗,延伸至树突,部分微管集合成束。转染后细胞增殖速度减缓,并出现凋亡。结论MAP-2基因的表达可导致恶性黑素瘤细胞形态及微管状态的变化,抑制细胞增殖,诱导凋亡。
Objective To investigate the inhibitory effect of MAP-2 gene on the growth of malignant melanoma cells. Methods The Ad-MAP-2 vector was amplified by adenovirus-Ad-MAP-2 vector and the titer of Ad-MAP-2 was determined by plaque assay. Ad-MAP-2 gene was transfected into murine malignant melanocytes Tumor cells B16C29 cells. The expression of intracellular MAP-2, the inhibition of Ad-MAP-2 on the growth of B16C29 cells were detected by direct immunofluorescence. The morphology of intracellular microtubules was observed by electron microscope and the apoptosis was detected by flow cytometry. Results After transfection of Ad-MAP-2 gene, the long and thin dendrites of B16C29 cells were elongated, which was similar to that of mature pigmented cells. Under electron microscope, the content of intracellular microtubules was increased after transfection. The microtubules became longer and thicker, extending to the dendrites. Some microtubules gathered into bundles. After transfection, the proliferation of cells slowed down and apoptosis occurred. Conclusion The expression of MAP-2 gene may lead to the change of morphology and microtubule state of malignant melanoma cells, inhibiting cell proliferation and inducing apoptosis.