紫杉醇对视网膜Müller细胞的影响

Effects of paclitaxel on Müller cells in retina

摘要:

目的：探究抗肿瘤药物紫杉醇(PTX)对Müller细胞增殖、凋亡、细胞周期、细胞形态与相关蛋白表达的影响以研究其对视网膜的潜在毒性。

方法：体外培养Müller细胞并分成两组：对照组(正常培养基)、加药物组(PTX)。不同浓度PTX(0.005、0.05、0.5、5mg/L)处理视网膜Müller细胞12、24、36、48、72h，CCK8法检测不同浓度PTX、刺激不同时间对Müller细胞增殖的影响； 流式细胞术检测不同浓度PTX对Müller细胞凋亡的影响及细胞周期的阻滞作用； 免疫荧光观察Müller细胞的形态变化； Western blot及qRT-PCR检测PTX对Müller细胞凋亡相关蛋白表达以及水通道蛋白的影响。

结果：PTX可以抑制体外培养的Müller细胞增殖能力，药物浓度越高，刺激时间越久，细胞增殖能力越弱； 此外，PTX还能促进Müller细胞的凋亡，越高的药物浓度和更长的刺激时间会导致更高的细胞凋亡率； 流式细胞检测细胞周期表明，PTX将Müller细胞阻滞于G2-M期。而细胞形态也由形态清晰、细长纤维状的正常形态趋于圆形，且细胞数量显著减少； 药物处理后的细胞炎症因子较对照组出现一过性的高表达，但这种高表达可通过停止药物刺激缓解； 药物处理后的细胞中的CA XIV蛋白表达较对照组上调，VEGF的表达较对照组下调(P<0.05)； PTX组中炎症因子的表达较对照组显著升高(P<0.05)； 表明PTX会破坏视网膜屏障功能。

结论：PTX抑制Müller细胞的增殖、促进Müller细胞的凋亡，且细胞的增殖、凋亡与刺激时间和药物浓度相关； 此外，PTX将Müller细胞周期阻滞于G2-M期，并会改变细胞形态，使细胞炎症因子出现短暂性的高表达，对视网膜屏障产生影响。揭示抗肿瘤药物PTX对视网膜具有潜在毒性。

Abstract:

AIM: To investigate the effects of antitumor drug paclitaxel(PTX)on the proliferation, apoptosis, cell cycle, cell morphology, and related protein expression of Müller cells, and to evaluate its potential toxicity to the retina.

METHODS:Müller cells were cultured in vitro and divided into two groups: control group(normal medium)and PTX group. Retinal Müller cells were treated with different concentrations of PTX(0.005, 0.05, 0.5 and 5mg/L)for varying durations(12, 24, 36, 48 and 72h). The CCK8 method was used to assess the effects of different concentrations of PTX and treatment duration on the proliferation Müller cells. Flow cytometry was employed to investigate the impact of different concentrations of PTX on Müller cells apoptosis and cell cycle arrest. Immunofluorescence was used to observe morphological changes in Müller cells. The effects of PTX on the expression of apoptosis-related proteins and aquaporins were analyzed by Western blot and qRT-PCR.

RESULTS: PTX exhibits the ability to inhibit the proliferation of Müller cells when cultured in vitro. The efficacy of this inhibition was found to be dependent on both the concentration of the drug and the duration of the stimulation. Higher concentrations of the drug and longer stimulation times resulted in a weaker ability of the cells to proliferate. Additionally, PTX also induces apoptosis in Müller cells, with increased drug concentrations and longer stimulation times leading to higher apoptosis rates. Flow cytometry analysis demonstrates that PTX arrests Müller cells in the G2-M phase of the cell cycle. Moreover, there is a distinct change in cell morphology, with a shift from the typical appearance characterized by clear and slender fibrous structures to a rounder morphology, accompanied by a significant decrease in cell numbers. Further, our findings reveal that there is a transient increase in the expression of cytoinflammatory factors following drug treatment compared to the control group. However, discontinuation of drug stimulation can alleviate this heightened expression. In treated cells, the expression of the CA XIV protein is upregulated compared to the control group, while the expression of vascular endothelial growth factor(VEGF)is downregulated(P<0.05). Additionally, the levels of inflammatory factors in the PTX group are significantly higher than those in the control group(P<0.05), suggesting that PTX has the potential to disrupt the retinal barrier function.

CONCLUSION: PTX affects the proliferation and apoptosis of Müller cells, with the effects dependent on stimulation duration and drug concentration. In addition, PTX blocks the Müller cell cycle at the G2-M phase and alters cell morphology, leading to a transient upregulation of inflammatory factors and affecting the integrity of the retinal barrier. These findings indicate the potential toxicity of the antitumor drug PTX to the retina.