外泌体对SH—SY5Y细胞缺氧再灌注损伤的影响

Effect of exsomes on SH-SY5Y cells after hypoxic ischemia/reperfusion injury

目的探讨外泌体对人神经母细胞瘤细胞株（SH—SY5Y）缺氧再灌注损伤的影响。方法构建人脐静脉血管内皮细胞（mJVEC）缺氧再灌注损伤（HUVECI／R1模型．从中提取外泌体并予以鉴定。构建SH．SY5Y细胞缺氧再灌注损伤（SH．SY5YI／R）模型并分为2组．其中外泌体组于再灌注同时予以外泌体刺激，对照组于再灌注同时予以无外泌体血清培养基培养。采用CCK8法检测2组细胞培养24h、48h、72h时细胞活力，采用Hochest33258染色检测细胞凋亡情况，采用流式细胞仪检测细胞凋亡情况及细胞周期，采用划痕实验、Transwell侵袭实验分别检测细胞迁移能力和侵袭能力，采用RT．qPCR、Westernblotting检测细胞中Caspase-3、Bax、Bcl-2mRNA和蛋白表达。结果与对照组比较，外泌体组细胞培养48h、72h时细胞活力明显提高（48h：0．70±0．05vs0．94±0．08；72h：0．83±0．05vs1．02±0．06），G0／G1期细胞比例明显降低（71．26％±5．24％vs566．87％±4．23％）．S期细胞比例明显增高（14．39％±4．11％vs20．54％±3．46％），相对划痕损伤面积明显减少（0．61±0．07vs50．52±0．10），穿膜细胞数量明显增多（44．00±6．56vs70．67±6．11），细胞中Bax、Caspase-3mRNA表达水平明显降低、Bcl-2mRNA表达水平明显增高，差异均有统计学意义（P〈0．05）。结论HUVECI／R模型来源外泌体可在SH-SY5Y细胞缺氧再灌注损伤后发挥神经保护作用。

Objective To study the effect of exsomes in SH-SY5Y cells after hypoxic ischemia/reperfusion injury. Methods Human umbilical vein endothelial cell hypoxic ischemia/reperfusion （HUVEC I/R） injury models were established, and the exosomes derived from HUVEC FR were extracted and identified. SH-SY5Y cell hypoxic ischemia/reperfusion injury models （SH-SY5Y UR） were established, and cells from SH-SY5Y UR were divided into control group and exosomes-treated group. The proliferation of SH-SY5Y cells was evaluated by CCK-8 assay 24, 48 and 72 h after cell inoculation. Transwell assay and wound-healing assay were used to examine the invasion and migration. Hochest33258 staining and Flow cytometry were used to monitor the changes of cell cycle and apoptosis. Expressions of Caspase-3, Bax and Bcl-2 were measured by real-time fluorescence quantificative-PCR and Western blotting. Results As compared with those in the control group, the proliferation abilities of SH-SY5Y cells in exosomes-treated group were significantly promoted （48 h： 0.70±0.05 vs. 0.94±0.08; 72 h： 0.83±0.05 vs. 1.02±0.06）, the cell cycle rate of S phase was significantly increased （14.39%±4.11% vs. 20.54%±3.46%）, and G0/G1 phase was statistically decreased （71.26%± 5.24% vs. 66.87%±4.23%, P〈0.05）. What＇ s more, cell invasive was significantly promoted （44.00±6.56 vs. 70.67±6.11）, and relative wound injury area was significantly reduced in the exosomes treated group （0.61±0.07 vs. 0.52±0.10）; significant differences were noted between the two groups （P〈0.05）. The mRNA expressions of Box and Caspase-3 were significantly decreased and the mRNA expressions of Bcl-2 was significantly increased in the exosomes-treated group as compared with those in the control group （P〈0.05）. Conclusion HUVEC I/R-derived exosomes play neuro-protective role in human SH-SY5Y cells after hypoxic lfR injury.