CAJ | 학술논문

背景研究表明,微小RNA-184(miR-184)可能抑制血管内皮细胞的生物学功能,在眼科研究领域,研究miR-184对新生血管的抑制作用对角膜炎性病变、视网膜和脉络膜新生血管性病变的预防和治疗具有一定的意义. 目的验证miR-184对体外培养的人脐静脉血管内皮细胞(HUVECs)生物学功能的影响,探讨其对体外新生血管形成的作用和机制.方法体外培养HUVECs株,将对数生长期的细胞分为4个组.空白对照组细胞培养基中不添加脂质体和RNA分子;空白转染组仅添加脂质体,但无RNA分子;阴性对照组添加hsa-miR-184和脂质体;miR-184转染组添加hsa-miR-184 miRNA mimic和脂质体.采用MTT法检测各组HUVECs的增生值;采用Transwell小室法观察各组HUVECs的迁移数;构建HUVECs的体外三维培养体系,用Matrigel胶体外管腔形成实验观察各组HUVECs的体外形成管腔的数目. 结果 Cy3标记siR-RiboTM脂质体转染后阳性HUVECs呈红色荧光,转染剂浓度＞100 nmol/L时荧光最强,阳性细胞数最多,为最优转染浓度.MiR-184转染组和阴性对照组HUVECs中miR-184的相对表达量分别为1 524.10±385.89和1.00±0.05,差异有统计学意义(t=-7.894,P＜0.01).MiR-184转染组转染后48 h细胞吸光度(A490)值为0.50±0.04,转染后24 h迁移过膜的细胞数为(55.40±5.86)个/视野,分别少于阴性对照组的0.62±0,04和83,40±5.59个/视野,差异均有统计学意义(t=5.639、7.730,均P＜0.01).MiR-184转染组形成管腔样结构分支点数为(13.33±2.08)个/视野,明显少于阴性对照组的(22.00±2.00)个/视野,差异有统计学意义(t=5.511,P＜0.05). 结论 MiR-184转染后能抑制体外培养的HUVECs的生长和迁移能力,减弱HUVECs基质胶上体外管腔的形成能力,可能参与或调控血管的新生.
배경 연구표명,미소RNA-184(miR-184)가능억제혈관내피세포적생물학공능,재안과연구영역,연구miR-184대신생혈관적억제작용대각막염성병변、시망막화맥락막신생혈관성병변적예방화치료구유일정적의의. 목적 험증miR-184대체외배양적인제정맥혈관내피세포(HUVECs)생물학공능적영향,탐토기대체외신생혈관형성적작용화궤제.방법 체외배양HUVECs주,장대수생장기적세포분위4개조.공백대조조세포배양기중불첨가지질체화RNA분자;공백전염조부첨가지질체,단무RNA분자;음성대조조첨가hsa-miR-184화지질체;miR-184전염조첨가hsa-miR-184 miRNA mimic화지질체.채용MTT법검측각조HUVECs적증생치;채용Transwell소실법관찰각조HUVECs적천이수;구건HUVECs적체외삼유배양체계,용Matrigel효체외관강형성실험관찰각조HUVECs적체외형성관강적수목. 결과 Cy3표기siR-RiboTM지질체전염후양성HUVECs정홍색형광,전염제농도＞100 nmol/L시형광최강,양성세포수최다,위최우전염농도.MiR-184전염조화음성대조조HUVECs중miR-184적상대표체량분별위1 524.10±385.89화1.00±0.05,차이유통계학의의(t=-7.894,P＜0.01).MiR-184전염조전염후48 h세포흡광도(A490)치위0.50±0.04,전염후24 h천이과막적세포수위(55.40±5.86)개/시야,분별소우음성대조조적0.62±0,04화83,40±5.59개/시야,차이균유통계학의의(t=5.639、7.730,균P＜0.01).MiR-184전염조형성관강양결구분지점수위(13.33±2.08)개/시야,명현소우음성대조조적(22.00±2.00)개/시야,차이유통계학의의(t=5.511,P＜0.05). 결론 MiR-184전염후능억제체외배양적HUVECs적생장화천이능력,감약HUVECs기질효상체외관강적형성능력,가능삼여혹조공혈관적신생.
Background Previous studies showed that microRNA-184 (miR-184) might inhibit the biological function of vascular endothelial cells.In ophthalmology,to determine the inhibitory effect of miR-184 on angiogenesis is of clinical significance for the prevention and treatment of corneal inflammation,retinal and choroidal neovascularization.Objective This study was to verify the influence of miR-184 transfection on the proliferation,migration and angiogenic ability of HUVECs and explore its mechanism.Methods HUVECs line was cultured in vitro and divided into 4 groups.No transfection regent and RNA molecule were added in the medium of the blank control group;only transfection regent was added in the blank transfection group; hsa-miR-184 mimic/negative was added in the negative control group,and Cy3 labeled hsa-miR-184 mimic-lipofectamine 2 000 mixture was added in the miR-184 transfection group.The proliferation of HUVECs (absorbance,A490) was detected by MTT.The transmembrane cell number was counted by Transwell insert to evaluate the migration ability of HUVECs.A three dimensional cultural system of cells was constructed on the matrigel,and tube formation number of HUVECs was assessed.Results Positive HUVECs for Cy3 labeled siR-RiboTM + hposome showed red fluorescence,with the optimal transfecting concentration ＞ 100 nmol/L.The relative expression levels of miR-184 were 1 524.10±385.89 and 1.00±0.05 in the miR-184 transfection group and the negative control group,showing a significant difference (t=-7.894,P＜0.01).The A490value of HUVECs was 0.50±0.04 and the number of migrating cells through the transwell membrane was (55.40±5.86)/field in the miR-184 transfection group,and they are significantly reduced in comparison with (0.62±0.04) and (83.40±5.59)/field in the negative control group (t =5.639,7.730,all at P＜ 0.01).A significant difference was found in the total branch points of capillary tubes between the miR-184 transfection group and negative control group ([13.33 ± 2.08]/field versus [22.00 ± 2.00]/field) (t =5.511,P＜ 0.05).Conclusions MiR-184 can suppress the proliferation,migration and tubing of HUVECs after transfection in vitro.MiR-184 participates in the regulation of angiogenesis.