【摘要】 目的 探讨神经表皮生长因子样分子1(NELL1)低表达是否通过黏着斑激酶（FAK）信号通路影响胶质母细胞瘤（GBM）细胞的恶性生物学行为。方法 培养人脑星形胶质细胞（SVGP12细胞）、人GBM细胞（A172、U251细胞），用Western blotting法检测不同类型细胞中NELL1蛋白表达以筛选实验细胞。取筛选后的实验细胞随机分为阴性对照组及NELL1敲低1组、NELL1敲低2组，分别感染阴性对照病毒及NELL1干扰慢病毒颗粒（Sh1、Sh2），实时荧光定量聚合酶链反应（qRT-PCR）和Western blotting法检测NELL1敲低效率。克隆形成实验、CCK-8实验检测细胞增殖[克隆形成数、光密度（OD）值]，Annexin V-APC/PI双染法检测细胞凋亡，Traswell实验检测细胞迁移，Western blotting法检测磷酸化FAK(p-FAK)及上皮间质转化（EMT）相关蛋白E-钙黏蛋白（E-cadherin）、N-钙黏蛋白（N-cadherin）、Snail表达。结果 与SVGP12细胞比较，A172、U251细胞中NELL1蛋白相对表达量高（P均<0.05）,A172细胞中NELL1蛋白相对表达量低于U251细胞，为保证实验的科学性选择A172细胞进行后续实验。与阴性对照组比较，NELL1敲低1、2组NELL1 mRNA及蛋白相对表达量低（P均<0.05），克隆形成数少（P均<0.05），培养24、48、72 h的OD值低（P均<0.05），凋亡率高（P均<0.05），穿膜细胞数少（P均<0.05）,p-FAK、E-cadherin、N-cadherin、Snail蛋白表达低（P均<0.05）。结论 敲低NELL1可能通过调控FAK信号通路促进EMT，从而抑制GBM细胞增殖与迁移，并诱导细胞凋亡。更多还原

【Abstract】 Objective To investigate whether low expression of neural epidermal growth factor-like 1(NELL1) influences the malignant biological behavior of glioblastoma(GBM) cells through the focal adhesion kinase(FAK) signaling pathway. Methods Human astrocytes(SVGP12 cells) and human GBM cells(A172, U251 cells) were cultured. Western blotting was used to detect NELL1 protein expression in different cell types for cell selection. The selected experimental cells were randomly divided into the negative control group, NELL1-knockdown group 1, and NELL1-knockdown group 2. These groups were infected with negative control lentivirus or NELL1-interfering lentiviral particles(Sh1, Sh2), respectively. Knockdown efficiency was verified by quantitative real-time polymerase chain reaction(qRT-PCR) and Western blotting. Cell proliferation was assessed by colony formation assay and CCK-8 assay. The apoptosis was detected by Annexin V-APC/PI double staining. Cell migration was evaluated by Transwell assay. The expression of phosphorylated FAK(pFAK) and epithelial-mesenchymal transition(EMT)-related proteins(E-cadherin, N-cadherin, and Snail) was measured by Western blotting. Results Compared with SVGP12 cells, the relative expression of NELL1 protein was higher in both A172 and U251 cells(both P<0. 05). Because NELL1 expression was lower in A172 cells than in U251 cells, A172 cells were selected for subsequent experiments to ensure scientific rigor. Compared with the negative control group, the relative expression levels of NELL1 mRNA and protein were lower(both P<0. 05), the number of colonies formed was smaller(both P<0. 05), the optical density(OD) values at 24, 48, and 72 hours of culture were lower(all P<0. 05), the apoptosis rates were higher(both P<0. 05), the number of transmembrane cells was smaller(both P<0. 05), and the protein expression of p-FAK, E-cadherin, N-cadherin, and Snail was lower in the NELL1-knockdown groups 1 and 2(all P<0. 05). Conclusion Knockdown of NELL1 may inhibit the proliferation and migration of GBM cells and induce apoptosis, potentially by regulating the FAK signaling pathway to promote EMT.更多还原