Objective Blood-brain barrier(BBB)may stop over 95%of the drugs delivered from entering the brain.This study aimed to establish a BBB model in vitro,detect the ability of the nano drug delivery system to penetrate the BBB,and observe its effect on angiogenesis and neuron cell proliferation after cerebral infarction. Methods A BBB model was established in vitro and the penetrability of PHRO through the BBB was detected by transwell assay.PBS,Rg1,and PHRO were placed in the upper chamber,and the content of Rg1 in the lower chamber was measured by HPLC.The effect of PHRO on angiogenesis was assessed with the in vitro tube formation model and the expression levels of the angiogenesis -related genes VEGFA and Dll4 determined by real time fluorescence quantitative PCR.Brain endothelial cells were incubated with 10 μL PBS(the control group),10 μmol/L Rg1(the Rg1 group),and PHRO(containing 10 μmol/L Rg1,the PHRO group)for 24 hours,and the SH5Y cells incubated the same way in the three groups for 72 hours.The effects of PHRO on the proliferation and apoptosis of the SH5Y cells were detected by MTT assay and flow cytometry respectively.The SH5Y cells were treated with 10 μL PBS(the PBS con-trol group),1 mmol/L Na2S2O4(the hypoxia-induction group),1 mmol/L Na2S2O4plus 10 μmol/L Rg1(the hypoxia-induction +Rg1 group),and 1 mmol/LNa2S2O4plus PHRO(including10 μmol/L Rg1,the hypoxia-induction +PHRO group), respectively. Results The content of Rg1 was 3.18%in the Rg1 group,28.8%in the PHRO group,and 0 in the control group.The angiogenesis of endothelial cells was markedly increased in the Rg 1 group as compared with the control(P<0.05), and even more significantly in the PHRO than in the Rg1 group(P<0.05).In comparison with the control group,the expressions of VEGFA and Dll 4 and the prolif-eration of the SH5Y cells were remarkably elevated in the Rg 1 group(P<0.05)and even more significantly in the PHRO than in the Rg1 group(P<0.05).The apoptosis rate of neurons was the lowest in PBS control(1.2%)and the highest in the hypoxia-induction group(21.6%),decreased to 13.14%and 8.25%in the hypoxia-induction +Rg1 and hypoxia-induction +PHRO group,respectively. Conclusion PHRO nanomedicine could penetrate the blood-brain barrier in vitro, promote angiogenesis and neuronal proliferation, reduce the apoptosis of neurons under hypoxia,and up-regulate the expressions of angiogenesis-related genes.%目的 血脑屏障能阻止95%以上的药物入脑发挥治疗作用.建立体外血脑屏障模型,观察人参皂苷Rg1纳米载药系统穿透体外血脑屏障的能力,以及其对脑梗死后血管、神经新生的影响. 方法 建立体外血脑屏障模型,利用Tran-swell嵌套小室检测PHRO穿越体外血脑屏障模型的能力.10 μL PBS(对照组),100 μmol/L Rg1(Rg1组)和PHRO(含有100 μmol/L Rg1,PHRO组)分别添加到嵌套小室的上室,HPLC检测下室的Rg1含量.利用体外成管模型检测PHRO对血管新生的影响.对照组,Rg1组和PHRO组分别与脑内皮细胞RBE4共同孵育24 h,同时在细胞水平上利用实时荧光定量PCR检测血管新生相关基因VEGFA和Dll4的表达量.利用MTT实验检测PHRO对神经细胞SH5Y增殖的影响.SH5Y细胞分别与对照组,Rg1组和PHRO组共同孵育72 h.利用流式细胞术检测PHRO对缺氧条件下SH5Y细胞凋亡的影响.SH5Y细胞分别与10 μL PBS(PBS处理组),1 mmol/L的Na2S2O4(缺氧诱导组),1 mmol/L的Na2S2O4+10 μmol/L Rg1(缺氧诱导+Rg1组), 1 mmol/L的Na2S2O4+PHRO(缺氧诱导+PHRO组,含有10 μmol/L的 Rg1)孵育24 h. 结果 对照组中未检测到Rg1的含量.Rg1组中检测到3.18%的Rg1的含量,PHRO组可检测到28.8%的Rg1含量.对照组内皮细胞成管能力最差,Rg1组内皮细胞的成管能力显著增强(P<0.05).与Rg1组相比,PHRO组展现出更加明显的成管能力(P<0.05).与对照相比,Rg1组、PHRO组VEGFA和Dll4的表达量以及SH5Y细胞的增殖显著上调(P<0.01),与Rg1组相比,PHRO组VEGFA和Dll4的表达量以及SH5Y细胞的增殖明显升高(P<0.05).PBS处理组细胞凋亡数量最少(1.2%),缺氧诱导组的凋亡率最高(21.6%),缺氧诱导+Rg1组细胞凋亡减少(13.14%);而缺氧诱导+PHRO处理组细胞凋亡的数量降低为8.25%. 结论 PHRO纳米药物能够穿越体外血脑屏障模型,促进血管、神经细胞增殖,同时能够降低缺氧条件下神经细胞的凋亡,并且促进血管生成相关基因的表达.
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