脂肪间充质干细胞外泌体对体外培养压力损伤的大鼠RGCs的保护作用
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云南省高层次人才培养支持计划青年拔尖人才专项(No.YNWR-QNBJ-2020-237); 云南省自然科学基金项目(No.202301AT070190); 昆医联合专项基金项目(No.202301AY070001-222); 云南省眼科疾病防治研究重点实验室项目(No.2017DG008); 云南省科技计划项目(No.202105AF150067)


Protective effects of adipose tissue-derived stromal cells exosomes against pressure-induced injury in retinal ganglion cells of cultured rat in vitro
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Yunnan Provincial High-level Talent Cultivation Support Program for Young Top-notch Talents(No.YNWR-QNBJ-2020-237); Yunnan Natural Science Foundation(No.202301AT070190); Kunming Medical University Joint Special Fund(No.202301AY070001-222); Project of Key Laboratory for Prevention and Treatment of Ophthalmic Diseases in Yunnan Province(No.2017DG008); Science and Technology Project of Yunnan Province(No.202105AF150067)

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    摘要:

    目的:通过体外建立大鼠视网膜神经节细胞(RGCs)压力损伤模型,研究脂肪间充质干细胞(ADSCs)外泌体对损伤的RGCs的保护作用。

    方法:培养ADSCs收集上清提取并鉴定外泌体,将体外培养大鼠RGCs分为正常培养的RGCs对照组、不同压力(40、80、120 mmHg)培养的RGCs模型组、不同压力培养的RGCs加入外泌体治疗组,通过CCK-8法检测各组RGCs细胞增殖活力,qPCR法检测各组RGCs中BDNF、Caspase-3的mRNA表达水平,Western Blot检测各组RGCs中BDNF、Caspase-3的蛋白表达水平。

    结果:CCK-8法检测发现,在对照组中,与24 h的细胞增殖活力相比,48 h时细胞增殖活力上升(P<0.05)。在48 h时,与加压40、80、120 mmHg模型组相比,加入外泌体后细胞活力均上升(均P<0.05)。qPCR法检测发现,与对照组比较,40 mmHg组中BDNF mRNA表达下降,但无差异(P>0.05),80、120 mmHg组中BDNF mRNA表达下降(均P<0.05)。加压40、80 mmHg组加入外泌体后RGCs的BDNF mRNA表达均上升(均P<0.05),加压120 mmHg组加入外泌体后RGCs的BDNF mRNA表达上升,但无差异(P>0.05)。与对照组比较,加压40 mmHg组中Caspase-3的mRNA表达上升,但无差异(P>0.05),加压80、120 mmHg组中Caspase-3 mRNA表达均上升(均P<0.05)。加压40、80 mmHg组加入外泌体治疗后RGCs的Caspase-3 mRNA表达下降(P<0.05),加压120 mmHg组加入外泌体治疗后RGCs Caspase-3的mRNA表达下降,但无差异(P>0.05)。Western Blot检测发现,与对照组比较,加压40 mmHg组中BDNF蛋白表达下降,但无差异(P>0.05),加压80、120 mmHg组中BDNF蛋白表达下降(均P<0.001)。与模型组相比,加入外泌体治疗后BDNF蛋白表达均上升(均P<0.05)。与对照组比,加压后各模型组中Caspase-3蛋白表达均上升(均P<0.05); 与模型组相比,加入外泌体治疗后各组Caspase-3蛋白表达均下降(均P<0.05)。

    结论:ADSCs来源的外泌体能够增加体外培养不同压力损伤的大鼠RGCs的细胞增殖活力,提高BDNF的mRNA及蛋白表达水平,降低Caspase-3的mRNA及蛋白表达水平,说明ADSCs来源的外泌体对体外培养压力损伤的大鼠RGCs具有保护作用。

    Abstract:

    AIM: To assess the protective effect of adipose tissue-derived mesenchymal stem cells(ADSCs)exosomes on injured retinal ganglion cells(RGCs)by establishing an in vitro rat RGC pressure injury model.

    METHODS: ADSCs were cultured, and exosomes were extracted from the supernatant and identified. Rat RGCs were divided into a control group, pressure model groups(40, 80, 120 mmHg), and exosome-treated groups under different pressures. Cell proliferation activity was assessed using the CCK-8 assay. The mRNA expression levels of brain-derived neurotrophic factor(BDNF)and Caspase-3 in RGCs were detected by qPCR, and protein levels were measured by Western Blot.

    RESULTS: The CCK-8 assay showed that cell proliferation activity in the control group increased significantly at 48 h compared to 24 h(P<0.05). At 48 h, cell viability in the exosome-treated groups increased significantly compared to the 40, 80, and 120 mmHg pressure model groups(all P<0.05). qPCR results indicated that BDNF mRNA expression decreased in the 40 mmHg pressure model group without statistical significance(P>0.05), and significantly decreased in the 80 and 120 mmHg pressure model groups(all P<0.05). BDNF mRNA expression significantly increased in the 40 and 80 mmHg pressure model groups after exosome treatment(both P<0.05), and increased in the 120 mmHg pressure model group without statistical significance(P>0.05). Caspase-3 mRNA expression increased in the 40 mmHg pressure model group without statistical significance(P>0.05), and significantly increased in the 80 and 120 mmHg pressure model groups(all P<0.05). Caspase-3 mRNA expression significantly decreased in the 40 and 80 mmHg pressure model groups after exosome treatment(P<0.05), and decreased in the 120 mmHg pressure model group without statistical significance(P>0.05). Western Blot analysis showed that BDNF protein expression decreased in the 40 mmHg pressure model group without statistical significance(P>0.05), and significantly decreased in the 80 and 120 mmHg pressure model groups(all P<0.001). After exosome treatment, BDNF protein expression significantly increased compared to the pressure model groups(all P<0.05). Caspase-3 protein expression increased significantly in all pressure model groups compared to the control group(all P<0.05), and significantly decreased in all exosome-treated groups compared to the model groups(all P<0.05).

    CONCLUSION: ADSCs-derived exosomes enhance cell proliferation and viability in cultured rat RGCs in vitro under different pressure-induced injuries, enhance BDNF mRNA and protein expression levels, and reduce Caspase-3 mRNA and protein expression levels, suggesting that ADSCs-derived exosomes have a protective effect on pressure-injured in cultured rat RGCs in vitro.

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陈一丹,戴敏,郑志坤.脂肪间充质干细胞外泌体对体外培养压力损伤的大鼠RGCs的保护作用.国际眼科杂志, 2024,24(11):1695-1700.

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  • 收稿日期:2024-03-18
  • 最后修改日期:2024-09-27
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  • 在线发布日期: 2024-10-18
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