摘要
目的体外观察血管紧张素(1-7)[Ang(1-7)]对口腔黏膜下纤维性变(OSF)成纤维细胞凋亡与血管生成的影响,并初步探究作用机制。方法从人颊黏膜组织分离培养成纤维细胞,倒置显微镜观察细胞形态,免疫荧光染色检测波形蛋白(Vimentin)表达;以槟榔提取液(ANE)诱导人成纤维细胞模拟OSF内成纤维细胞体外模型,实验分组包括对照组(正常培养的细胞)、ANE组[100μg/ml ANE培养细胞48 h、ANE+低剂量Ang(1-7)组(100μg/ml ANE+10^(-7)mol/L Ang(1-7)培养细胞48 h]、ANE+高剂量Ang(1-7)组[100μg/ml ANE+10^(-5)mol/L Ang(1-7)培养细胞48 h],免疫荧光染色检测α-平滑肌肌动蛋白(α-SMA)表达,ELISA法检测细胞培养上清液中Ⅰ型胶原(CollagenⅠ)和Ⅲ型胶原(CollagenⅢ)的含量,MTT法检测细胞增殖活性,流式细胞术检测细胞凋亡,小管形成实验检测人脐静脉内皮细胞(HUVEC)的血管形成情况,将mRFP-GFP-LC3病毒转染至细胞后通过免疫荧光染色检测细胞自噬水平,Western blot检测自噬相关蛋白Beclin-1表达及LC3-Ⅱ/LC3-Ⅰ比值。结果分离培养的细胞呈长梭形,Vimentin呈阳性表达,说明成功分离到成纤维细胞;与ANE组比较,ANE+低剂量Ang(1-7)组和ANE+高剂量Ang(1-7)组的细胞内α-SMA蛋白荧光表达明显减弱,培养上清液中Collagen Ⅰ和Collagen Ⅲ的含量减少(P<0.05),细胞增殖活性降低(P<0.05),细胞凋亡率则升高(P<0.05),两组的细胞培养上清液均抑制了HUVEC的血管形成(P<0.05),细胞内自噬小体减少(P<0.05),Beclin-1蛋白表达降低(P<0.05),LC3-Ⅱ/LC3-Ⅰ比值下调(P<0.05);此外,ANE+高剂量Ang(1-7)组作用效果均强于ANE+低剂量Ang(1-7)组作用效果(P<0.05)。结论Ang(1-7)能够抑制ANE诱导下成纤维细胞的活化,促进细胞凋亡,并降低HUVEC的血管形成。该机制可能与调控细胞自噬水平有关。
Objective To observe the effect of angiotensin(1-7)[Ang(1-7)]on the apoptosis and angiogenesis of fibroblasts in the oral submucosal fibrosis(OSF),and to explore the effect preliminarily mechanism.Methods Fibroblasts were isolated and cultured from human buccal mucosal tissue,the cell morphology was observed by inverted microscope,and the expression of vimentin was detected by immunofluorescence staining;areca nut extract(ANE)was used to induce human fibroblasts to simulate the in vitro model of fibroblasts in OSF,the experimental groups included control group(normally cultured cells),ANE group(100μg/ml ANE cultured cells for 48 hours),ANE+low-dose Ang(1-7)group(100μg/ml ANE+10-7 mol/L Ang(1-7)cultured cells for 48 h),ANE+high-dose Ang(1-7)group(100μg/ml ANE+10-5 mol/L Ang(1-7)cultured cells for 48 h),immunofluorescence staining detected the expression ofα-smooth muscle actin(α-SMA),ELISA method detected the con tent of Collagen I and Collagen Ⅲ in the cell culture supernatant,MTT method detected cell proliferation activity,flow cytometry detected cell apoptosis,the tubule formation experiment detected the vascularization of human umbilical vein endothelial cell(HUVEC);After the mRFP-GFP-LC3 virus was transferred to the cells,the level of autophagy was detected by immunofluorescence staining,Western blot detected the expression of autophagy-related protein Beclin-1 and the ratio of LC3-Ⅱ/LC3-Ⅰ.Results The isolated and cultured cells were in a long spindle shape,and Vimentin was positively expressed,indicating that fibroblasts were successfully isolated;Compared with the ANE group,the fluorescence expression of α-SMA protein in ANE low dose Ang(1-7)group and ANE+high dose Ang(1-7)group significantly decreased,the contents of Collagen I and Collagen Ⅲ in the culture supernatant were reduced(P<0.05),cell proliferation activity decreased(P<0.05),and cell apoptosis rate increased(P<0.05),the cell culture supernatants of the two groups inhibited the angiogenesis of HUVEC(P<0.05),endophagosomes were reduce
作者
邱乐宏
邓伟
甘成文
孙莹
Qiu Lehong;Deng Wei;Gan Chengwen;Sun Ying(Dept of Stomatology,Hainan General Hospital,Hainan Hospital Affiliated to Hainan Medical College,Haikou 570311)
出处
《安徽医科大学学报》
CAS
北大核心
2023年第3期457-464,共8页
Acta Universitatis Medicinalis Anhui
基金
海南省自然科学基金高层次人才项目(编号:821RC681)
海南省自然科学基金面上项目(编号:821MS132)
海南省卫生健康行业科研项目(编号:22A200205)。
