| 6-磷酸果糖激酶-2/果糖-2，6-二磷酸酶3对人脐静脉内皮细胞管腔形成的影响 |
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| 引用本文： | 曹阳,胡萍,田敏,韦芳,顾青,吕红彬. 6-磷酸果糖激酶-2/果糖-2，6-二磷酸酶3对人脐静脉内皮细胞管腔形成的影响[J]. 中国组织工程研究, 2020, 24(14): 2217-2222. DOI: 10.3969/j.issn.2095-4344.2599 |
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| 作者姓名： | 曹阳 胡萍 田敏 韦芳 顾青 吕红彬 |
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| 作者单位： | 西南医科大学附属医院眼科，四川省泸州市 646000；上海市眼底病重点实验室，上海市 200080 |
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| 基金项目： | 四川省教育厅资助项目(17ZA0428)，项目负责人：吕红彬;2017年度泸州市人民政府-西南医科大学科技战略合作项目(2017LZXNYD-J01)，项目负责人：吕红彬~~ |
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| 摘 要： | 文题释义：6-磷酸果糖激酶-2/果糖-2，6-二磷酸酶3(PFKFB3)：糖酵解作为血管内皮细胞中提供能量的主要方式，而6-磷酸果糖激酶-2/果糖-2，6-二磷酸酶作为糖酵解中重要的刺激剂，在肿瘤新生血管方面具有重要作用，在此次实验中主要用于说明在高糖环境下对血管内皮的作用。糖尿病性视网膜病变：是糖尿病中微血管严重并发症之一，一旦产生视网膜新生血管，将严重影响工作人群的视力，此次实验主要研究脐静脉内皮细胞在高糖环境中PFKFB3的作用，以期找到缓解视网膜新生血管的新靶点。背景：糖尿病性视网膜病变中关于新生血管的研究多局限于血管内皮生长因子，而6-磷酸果糖激酶-2/果糖-2，6-二磷酸酶(phosphofructokinase-2/fructose-2，6-bisphosphatase，PFKFB3)也有一定的作用。目的：探讨PFKFB3小干扰RNA(siRNA)对高糖环境下人脐静脉内皮细胞的作用及机制。方法：将人脐静脉内皮细胞分为4组：正常糖组(5.5 mmol/L 葡萄糖)、正常糖+PFKFB3-siRNA组、高糖组(30 mmol/L葡萄糖)、高糖+PFKFB3-siRNA组。正常糖+PFKFB3-siRNA组和高糖+PFKFB3-siRNA组以PFKFB3-siRNA转染人脐静脉内皮细胞。采用Western blot检测PFKFB3的沉默表达效果，并选取沉默效果最佳者进行后续实验。体外管腔形成实验检测细胞成管能力，实时荧光定量PCR检测PFKFB3的mRNA表达，Western blot检测PFKFB3及磷酸化AKT/AKT的蛋白表达。结果与结论：①PFKFB3-siRNA能明显抑制PFKFB3的蛋白表达(P < 0.01)；②与正常糖组相比，正常糖+ PFKFB3-siRNA组管腔形成总长度增加(P < 0.05)，高糖组管腔形成总长度明显减少(P < 0.01)，高糖+ PFKFB3-siRNA组管腔形成总长度无明显变化(P > 0.05)；与高糖组相比，高糖+PFKFB3-siRNA组管腔形成能力明显增加(P < 0.05)；与正常糖组相比，高糖组中PFKFB3的mRNA及蛋白表达均明显升高(均P < 0.01)；与高糖组相比，高糖+PFKFB3-siRNA组的PFKFB3蛋白表达降低(P < 0.05)；③与正常糖组相比，正常糖+PFKFB3-siRNA组与高糖组中磷酸化AKT/AKT比值均降低(P < 0.01)，而高糖组+PFKFB3-siRNA组磷酸化AKT/AKT蛋白比值无明显变化(P > 0.05)；与高糖组相比，高糖+PFKFB3-siRNA组磷酸化AKT/AKT蛋白比值升高(P < 0.01)。提示采用siRNA沉默PFKFB3基因表达可抑制人脐静脉内皮细胞中PFKFB3的表达，促进管腔形成能力，其机制可能与下调AKT表达有关。ORCID: 0000-0001-9070-3292(曹阳)中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程
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| 关 键 词： | PFKFB3 高血糖状态 血管生成 磷酸化AKT siRNA-MateTM 糖尿病性视网膜病变 人脐静脉内皮细胞 管腔形成 |
| 收稿时间： | 2019-09-21 |
Effects of 6-phosphofructokinase-2/fructose-2,6-bisphosphatase 3 on tubule formation of human umbilical vein endothelial cells |
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| Cao Yang,Hu Ping,Tian Min,Wei Fang,Gu Qing,Lü Hongbin. Effects of 6-phosphofructokinase-2/fructose-2,6-bisphosphatase 3 on tubule formation of human umbilical vein endothelial cells[J]. Chinese Journal of Tissue Engineering Research, 2020, 24(14): 2217-2222. DOI: 10.3969/j.issn.2095-4344.2599 |
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| Authors: | Cao Yang Hu Ping Tian Min Wei Fang Gu Qing Lü Hongbin |
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| Affiliation: | Departmentof Ophthalmology, Affiliated Hospital of Southwest Medical University, Luzhou646000, Sichuan Province, China; Shanghai Key Laboratory of FundusDisease, Shanghai 200080, China |
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| Abstract: | BACKGROUND:The research on neovascularization in diabetic retinopathy is mostly limited to vascular endothelial growth factor,but 6-phosphofructokinase-2/fructose-2,6-diphosphatase(PFKFB3)also plays a certain role.OBJECTIVE:To investigate the effect of PFKFB3-siRNA on human umbilical vein endothelial cells(HUVECs)in high glucose environment.METHODS:HUVECs were divided into four groups:normal glucose control group(5.5 mmol/L glucose),normal glucose+PFKFB3-siRNA group(5.5 mmol/L glucose+PFKFB3-siRNA),high glucose group(30 mmol/L glucose),high glucose+PFKFB3-siRNA group(30 mmol/L glucose+PFKFB3-siRNA).Western blot assay was used to detect the silencing effect of PFKFB3 expression.PFKFB3 with optimal silencing effect was selected for subsequent experiments.The tubule formation was detected by in vitro tubule formation assay.The expression of PFKFB3 mRNA was detected by real-time fluorescent quantitative PCR.The expression of PFKFB3 and AKT protein was detected by western blot.RESULTS AND CONCLUSION:PFKFB3-siRNA significantly inhibited the expression of PFKFB3(P<0.01).Compared with the normal glucose control group,the total length of tube formation was increased in the normal glucose+PFKFB3-siRNA group(P<0.05),and was significantly decreased in the high glucose group(P<0.01).There was no significant change in the total length of tube formation between normal glucose control group and high glucose+PFKFB3-siRNA group(P>0.05).Compared with the high glucose group,the tube formation ability of the high glucose+PFKFB3-siRNA group was significantly increased(P<0.05).Compared with the normal glucose control group,the mRNA and protein expression of PFKFB3 in the high glucose group were significantly increased(both P<0.01).Compared with the high glucose group,the expression of PFKFB3 protein in the high glucose+PFKFB3-siRNA group was decreased(P<0.05).Compared with the normal glucose control group,the ratio of p-AKT/AKT in the normal glucose+PFKFB3-siRNA group and the high glucose group was decreased(both P<0.01),while there was no significant difference in the ratio p-AKT/AKT protein between the high glucose group+PFKFB3-siRNA group and the normal glucose control group(P>0.05).Compared with the high glucose group,the ratio of p-AKT/AKT protein in the high glucose+PFKFB3-siRNA group was increased(P<0.01).To conclude,siRNA silencing of PFKFB3 gene expression can inhibit the expression of PFKFB3 and improve tube formation in HUVECs.The mechanism may be related to the down-regulation of AKT expression. |
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| Keywords: | PFKFB3 hyperglycemic condition angiogenesis p-Akt siRNA-MateTM diabetic retinopathy human umbilical vein endothelial cells tube formation |
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