南方医科大学学报

南方医科大学学报 ›› 2022, Vol. 42 ›› Issue (3): 354-359.doi: 10.12122/j.issn.1673-4254.2022.03.06

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Leptin对缺氧状态下大鼠视网膜祖细胞体外增殖和分化的影响

邢 瑶，刘子瑶，张晓辉，王建明   

  1. 西安交通大学第二附属医院眼科，陕西 西安 710004
  • 出版日期:2022-03-20 发布日期:2022-04-12

Effects of leptin on proliferation and differentiation of hypoxic rat retinal progenitor cells in vitro

XING Yao, LIU Ziyao, ZHANG Xiaohui, WANG Jianming   

  1. Department of Ophthalmology, Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710004, China
  • Online:2022-03-20 Published:2022-04-12

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摘要： 目的 探讨Leptin对体外培养的缺氧状态下视网膜祖细胞（RPCs）增殖、分化的影响及细胞中PTEN蛋白表达的变化。方法 原代培养大鼠RPCs，不同浓度Leptin作用RPCs细胞，分为：常氧培养组（Control组）、缺氧培养（Hypoxia）+0、0.3、1.0、3.0、10、30 nmol/L Leptin组，分别培养至12、24、48 h，CCK-8法检测细胞增殖活性。将原代培养的RPCs细胞分为：Control组、Hypoxia组、Hypoxia+Leptin组，培养48 h后，转换为分化培养基继续培养6 d，采用免疫荧光染色法对GFAP阳性细胞比例和β-tubulin III阳性细胞比例进行统计分析。Control组、Hypoxia组、Hypoxia+Leptin组细胞培养48 h后，采用Western blot法检测各组RPCs中PTEN蛋白表达水平。结果 原代培养的P0代RPCs为圆形悬浮生长，培养2 d后细胞可聚集成神经球。低浓度Leptin（0.3、1.0、3.0 nmol/L）作用RPCs 48 h，细胞增殖活性较Control组及Hypoxia组增强，RPCs细胞增殖活性随Leptin浓度增高呈递增趋势，其中，3.0 nmol/L Leptin作用48 h细胞增殖活性最强（P<0.05）；高浓度Leptin（10 nmol/L，30 nmol/L）组细胞增殖活性较对照组减弱（P<0.05）。细胞培养48 h，与Control组及Hypoxia组相比，Hypoxia+Leptin组β-tubulin III阳性细胞比例和GFAP阳性细胞比例均无显著性差异（P>0.05），而PTEN蛋白表达量较对照组显著下调（P<0.05）。结论 低浓度Leptin可促进缺氧状态下大鼠RPCs细胞增殖，抑制细胞中PTEN蛋白表达，对细胞分化无明显影响。

关键词: Leptin；缺氧；视网膜祖细胞；细胞增殖；细胞分化

Abstract: Objective To investigate the the effects of leptin on the proliferation, differentiation and PTEN expression of rat retinal progenitor cells (RPCs) cultured under hypoxic condition. Methods SD rat RPCs were cultured in normoxic conditions or exposed to hypoxia in the presence of 0, 0.3, 1.0, 3.0, 10, and 30 nmol/L leptin for 12, 48 and 72 h, and the cell viability was assessed using cell counting kit 8 (CCK 8) assay. The RPCs in primary culture were divided into control group, hypoxia group, and hypoxia+leptin group, and after 48 h of culture, the cell medium was replaced with differentiation medium and the cells were further cultured for 6 days. Immunofluorescence staining was employed to detect the cells positive for β-tubulin III and GFAP, and Western blotting was used to examine the expression of PTEN at 48 h of cell culture. Results The first generation of RPCs showed suspended growth in the medium with abundant and bright cellular plasma and formed mulberry like cell spheres after 2 days of culture. Treatment with low-dose leptin (below 3.0 nmol/L) for 48 h obviously improved the viability of RPCs cultured in hypoxia, while at high concentrations (above 10 nmol/L), leptin significantly suppressed the cell viability (P<0.05). The cells treated with 3.0 nmol/L leptin for 48 h showed the highest viability (P<0.05). After treatment with 3.0 nmol/L leptin for 48 h, the cells with hypoxic exposure showed similar GFAP and β-tubulin Ⅲ positivity with the control cells (P>0.05), but exhibited an obvious down-regulation of PTEN protein expression compared with the control cells (P<0.05). Conclusion In rat RPCs with hypoxic exposure, treatment with low dose leptin can promote the cell proliferation and suppress cellular PTEN protein expression without causing significant effects on cell differentiation.

Key words: leptin; hypoxia; retinal progenitor cells; cell proliferation; cell differentiation