Zhang Luyi, Li Xiaoxia, Lin Xiaoqin, Wu Miaoqin
Department of Ophthalmology, Zhejiang Provincial People's Hospital, People's Hospital of Hangzhou Medical College, 158 Shangtang Road Hangzhou, Zhejiang, 310014, China.
BMC Ophthalmol. 2019 Jun 17;19(1):130. doi: 10.1186/s12886-019-1142-x.
Nerve growth factor (NGF), produced by Müller cells, and internal limiting membrane (ILM) have fundamental roles in the development of full-thickness macular hole (FTMH). However, the potential crosstalk between NGF and ILM in FTMH is unclear. This study aimed to explore the mechanism and effects of NGF on the proliferation of Müller cells co-cultured with ILM.
Primary Müller cells and ILM from New Zealand rabbits were extracted and authenticated with specific staining. Müller cells co-cultured with or without ILM were exposed to NGF and then analysed. Müller cell viability was estimated using cell counting kit-8. Cell cycle analysis was performed by flow cytometry. The levels of cell cycle-related gene were detected using qRT-PCR. The TrK-A/Akt signal axis and downstream signaling cascades such as p21, CyclinE, CDK2, CyclinD1, and CDK4 were investigated by western blotting.
ILM treatment alone induced the proliferation of Müller cells following the promotion of phosphorylated Akt, while growth of Müller cells was enhanced by activation of the Trk-A/Akt pathway under the stimulation of NGF or NGF + ILM. Additionally, the ratio of S-phase cells was increased, while G2-phase cells decreased upon the treatment with either ILM or NGF alone, or with NGF + ILM co-treatment. Cell cycle-related genes such as CyclinD1, CyclinE, CDK2, and CDK4 were all upregulated, but p21 expression was downregulated in the presence of NGF, ILM, or NGF + ILM. There was an additive effect on cell proliferation and cell cycle in the group of Müller cells exposed to NGF co-cultured with ILM compared with either NGF or ILM treatment alone. However, both K252ɑ (inhibitors of Trk-A) and LY294002 (inhibitor for Akt) counteracted the effect of NGF or NGF + ILM on the protein levels of Trk-A, Akt, CyclinD1, CyclinE, CDK2, and p21.
Müller cells co-cultured with ILM or NGF promoted cell proliferation by regulating cell cycle-correlated proteins via the PI3K/Akt pathway. ILM + NGF further amplified the PI3K/Akt signaling pathway by binding to Trk-A, leading to more cell growth. This study provides new insight into the potential mechanism of NGF-mediated proliferation of Müller cells co-cultured with or without ILM, which may have considerable impact on therapies for FTMH.
由穆勒细胞产生的神经生长因子（NGF）和内界膜（ILM）在全层黄斑裂孔（FTMH）的形成中起重要作用。然而，FTMH中NGF与ILM之间潜在的相互作用尚不清楚。本研究旨在探讨NGF对与ILM共培养的穆勒细胞增殖的作用机制及影响。
提取新西兰兔的原代穆勒细胞和ILM，并用特异性染色进行鉴定。将与ILM共培养或未与ILM共培养的穆勒细胞暴露于NGF后进行分析。使用细胞计数试剂盒-8评估穆勒细胞活力。通过流式细胞术进行细胞周期分析。使用qRT-PCR检测细胞周期相关基因的水平。通过蛋白质印迹法研究TrK-A/Akt信号轴及下游信号级联反应，如p21、细胞周期蛋白E、细胞周期蛋白依赖性激酶2（CDK2）、细胞周期蛋白D1和CDK4。
单独的ILM处理通过促进磷酸化Akt诱导穆勒细胞增殖，而在NGF或NGF + ILM刺激下，Trk-A/Akt途径的激活增强了穆勒细胞的生长。此外，单独用ILM或NGF处理，或用NGF + ILM联合处理后，S期细胞比例增加，G2期细胞减少。细胞周期相关基因如细胞周期蛋白D1、细胞周期蛋白E、CDK2和CDK4均上调，但在存在NGF、ILM或NGF + ILM时，p21表达下调。与单独用NGF或ILM处理相比，与ILM共培养并暴露于NGF的穆勒细胞组在细胞增殖和细胞周期方面具有相加作用。然而，K252ɑ（Trk-A抑制剂）和LY294002（Akt抑制剂）均抵消了NGF或NGF + ILM对Trk-A、Akt、细胞周期蛋白D1、细胞周期蛋白E、CDK2和p21蛋白水平的影响。
与ILM或NGF共培养的穆勒细胞通过PI3K/Akt途径调节细胞周期相关蛋白来促进细胞增殖。ILM + NGF通过与Trk-A结合进一步放大PI3K/Akt信号通路，导致更多细胞生长。本研究为NGF介导的与ILM共培养或未与ILM共培养的穆勒细胞增殖的潜在机制提供了新的见解，这可能对FTMH的治疗产生重大影响。
