目的：探讨胰岛素样生长因子-1(IGF-1)对人巩膜成纤维细胞(HSF)分泌转化生长因子-β2(TGF-β2)、基质金属蛋白酶-2(MMP-2)、缺氧诱导因子-1α(HIF-1α)的影响及其机制。

方法：分别使用IGF-1和PI3K/AKT通路抑制剂LY294002培养细胞，采用CCK-8法检测细胞活力，确定药物的最佳作用浓度和时间。采用细胞划痕法观察细胞迁移活性。为明确IGF-1对HSF细胞的影响以及PI3K/AKT通路在其中的调控作用，将细胞分为对照组(不含药物)、IGF-1(80 μg/L)组、IGF-1+LY294002(80 μg/L+5 mmol/L)组、LY294002(5 mmol/L)组，培养24 h； Western blot检测细胞中TGF-β2、MMP-2、HIF-1α、PI3K、AKT的蛋白表达水平； 细胞免疫荧光检测TGF-β2、MMP-2、HIF-1α的荧光表达。

结果：CCK-8结果显示不同浓度IGF-1培养的细胞活力以80 μg/L IGF-1组最高(均P<0.05)，且在不同培养时间下，以24 h时80 μg/L IGF-1组细胞活力最高，因此确定后续实验中IGF-1的浓度为80 μg/L，作用时间为24 h。不同浓度LY294002培养的细胞活力从6 h起逐渐降低(均P<0.05)，根据IC50值确定后续实验中LY294002浓度为5 mmol/L，作用时间为24 h。细胞划痕结果显示，与对照组相比40、80 μg/L IGF-1组细胞迁移率均升高(均P<0.05)。与对照组相比2.5、5 mmol/L LY294002组细胞迁移率均降低(均P<0.05)。Western blot结果显示，与对照组相比，IGF-1组细胞TGF-β2、MMP-2、HIF-1α、PI3K、AKT的蛋白表达量均升高，LY294002组的蛋白表达量均下降(均P<0.05)； 与IGF-1组比较，IGF-1+LY294002组的TGF-β2、MMP-2、HIF-1α、PI3K、AKT蛋白表达量表达均下降(均P<0.05)。细胞免疫荧光结果显示，与对照组相比，IGF-1组细胞TGF-β2、MMP-2、HIF-1α的荧光表达均升高，LY294002组的荧光表达均下降(均P<0.05)； 与IGF-1组比较，IGF-1+LY294002组的TGF-β2、MMP-2、HIF-1α荧光表达均显著下降(均P<0.05)。

结论：IGF-1促进了人HSF细胞增殖与迁移； IGF-1可能通过PI3K/AKT信号通路上调人HSF细胞中TGF-β2、MMP-2、HIF-1α的表达，参与近视的发生与发展。

METHODS: The cells were cultured with IGF-1 and PI3K/AKT pathway inhibitor LY294002, respectively. CCK-8 method was used to detect cell viability and determine the optimal concentration and time of drug action. Cell migration activity was observed by cell scratch method. To determine the effects of IGF-1 on HSF cells and the regulatory role of PI3K/AKT pathway, HSF cells were divided into control group(without drugs), IGF-1(80 μg/L)group, IGF-1+LY294002(80 μg/L+5 mmol/L)group, and LY294002(5 mmol/L)group, and were cultured for 24 h; the protein expression levels of TGF-β2, MMP-2, HIF-1α, PI3K and AKT were detected by Western blot; the fluorescence expression of TGF-β2, MMP-2 and HIF-1α was detected by cellular immunofluorescence.

RESULTS: The results of CCK-8 showed that the cell viability of the 80 μg/L IGF-1 group cultured with different concentrations of IGF-1 was the highest(all P<0.05), and the cell viability of the 80 μg/L IGF-1 group at 24 h was the highest under different culture times. Therefore, the concentration of IGF-1 was selected as 80 μg/L for 24 h. The viability of cells cultured with different concentrations of LY294002 gradually decreased from 6 h(all P<0.05). According to the IC50 value, therefore, the concentration of LY294002 was selected as 5 mmol/L for 24 h. The cell scratch results showed that compared with the control group, the cell mobility of 40 μg/L and 80 μg/L IGF-1 groups was increased(all P<0.05). Compared with the control group, cell mobility in the 2.5 and 5 mmol/L LY294002 groups was decreased(all P<0.05). Western blot results showed that compared with the control group, the protein expressions of TGF-β2, MMP-2, HIF-1α, PI3K and AKT in the IGF-1 group were increased, while those in the LY294002 group were decreased(all P<0.05). Compared with the IGF-1 group, the expression levels of TGF-β2, MMP-2, HIF-1α, PI3K and AKT in the IGF-1+LY294002 group were decreased(all P<0.05). The results of cell immunofluorescence showed that compared with the control group, the fluorescence expressions of TGF-β2, MMP-2 and HIF-1α in the IGF-1 group were increased, while those in the LY294002 group were decreased(all P<0.05). Compared with the IGF-1 group, the fluorescence expressions of TGF-β2, MMP-2 and HIF-1α in the IGF-1+LY294002 group were significantly decreased(all P<0.05).

CONCLUSION: IGF-1 promoted the proliferation and migration of human HSF. IGF-1 may up-regulate the expression of TGF-β2, MMP-2 and HIF-1α in HSF through the PI3K/AKT signaling pathway, and participate in the occurrence and development of myopia.