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目的:利用我们已经成功沉默前列腺特异性膜抗原(PSMA)的LNCaP前列腺癌细胞株,探讨PS-MA对LNCaP细胞磷酸化胞外信号调节激酶(ERK)及细胞生长、迁移的影响,为进一步研究PSMA在前列腺癌发展中的作用提供理论基础。方法:实验对象包括携带可稳定抑制PSMA表达siRNA慢病毒的LNCaP细胞组(实验组),携带对任何基因无干扰作用siRNA慢病毒的LNCaP细胞组(空转组),同时建立未进行处理的普通LNCaP细胞组(对照组),分别对在一般培养基及添加ERK蛋白上游抑制剂的3组细胞,使用Western blotting和细胞免疫化学的方法检测MAPK/ERK蛋白活性,并用MTT描绘细胞生长曲线、Transwell观察细胞迁移情况。结果:在一般培养基的3组细胞中,Western blotting提示实验组磷酸化ERK蛋白表达明显低于对照组和空转组;免疫细胞化学结果显示实验组染色明显比对照组和空转组弱,阳性细胞数较少;MTT绘制生长曲线,得到实验组细胞的增殖生长能力较对照组、空转组降低;Transwell结果提示实验组较对照组和空转组细胞的增殖迁移能力降低。在ERK磷酸化被抑制的情况下,3组细胞磷酸化ERK蛋白均低表达,MTT及Transwell检测显示其生长迁移能力都处于低水平,且与在一般培养基中实验组的效应类似。结论:初步发现PSMA可能通过上调前列腺癌LNCaP细胞ERK蛋白的活性,从而在其生长、迁移中起正向调节作用。
OBJECTIVE: To investigate the effect of PS-MA on the phosphorylation of extracellular signal-regulated kinase (ERK) and cell growth and migration in LNCaP cells by using LNCaP prostate cancer cell line which we have successfully silenced prostate-specific membrane antigen (PSMA) The role of PSMA in the development of prostate cancer provides a theoretical basis. Methods: The experimental group included LNCaP cell group (experimental group) which could stably inhibit PSMA-expressing siRNA lentivirus, LNCaP cell group (idling group) which carried siRNA lentivirus without interfering with any gene, and established normal LNCaP cell without treatment Cell group (control group). The activity of MAPK / ERK protein was detected by Western blotting and immunocytochemistry in three groups of cells in general medium and inhibitor of upstream ERK. MTT was used to characterize the cell growth curve. Transwell observation Cell migration. Results: Western blotting showed that the expression of phosphorylated ERK protein in experimental group was significantly lower than that in control group and idling group. Immunocytochemistry results showed that the expression of ERK protein in experimental group was weaker than that in control group and idling group The number of cells in the experimental group was lower than that in the control group and the idling group. The results of Transwell assay showed that the ability of proliferation and migration of the experimental group was lower than that of the control group and the idling group. In ERK phosphorylation was inhibited, the three groups of cells phosphorylated ERK protein were low expression, MTT and Transwell assay showed that their ability to grow and migrate at low levels, and with the general effect of the experimental group of similar effects. It is concluded that PSMA may play a positive regulatory role in the growth and migration of human LNCaP cells by up-regulating the activity of ERK protein.