敲低细丝蛋白B对小鼠颅顶成骨前体细胞增殖、迁移及凋亡的影响  

Effects of filament B knockdown on proliferation,migration and apoptosis of mouse MC3T3-E1 cells

作　　者：王茜 俞丽[1] 贾麒钰 黄金勇 刘泽彪 张俊 加依达尔•地力木拉提 谢增如 马海蓉[1] Wang Xi;Yu Li;Jia Qiyu;Huang Jinyong;Liu Zebiao;Zhang Jun;Jiayidaer•Dilimulati;Xie Zengru;Ma Hairong(Research Institute of Clinical Medicine,The First Affiliated Hospital of Xinjiang Medical University,Urumqi 830054,Xinjiang Uygur Autonomous Region,China;Department of Orthopaedic Trauma,The First Affiliated Hospital of Xinjiang Medical University,Urumqi 830054,Xinjiang Uygur Autonomous Region,China;Department of Orthopedics,Affiliated Hospital of North China University of Science and Technology,Tangshan 063000,Hebei Province,China)

机构地区：[1]新疆医科大学第一附属医院临床医学研究院,新疆维吾尔自治区乌鲁木齐市830054 [2]新疆医科大学第一附属医院创伤骨科,新疆维吾尔自治区乌鲁木齐市830054 [3]华北理工大学附属医院骨科,河北省唐山市063000

出　　处：《中国组织工程研究》2024年第32期5177-5181,共5页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金项目(82060411),项目负责人:马海蓉;国家自然科学基金项目(82260409),项目负责人:谢增如;新疆维吾尔自治区自然科学基金重点项目(2021D01D21),项目负责人:马海蓉;新疆维吾尔自治区自然科学基金重点项目(2021D01D19),项目负责人:谢增如;自治区研究生创新项目(XJ2023G166),项目负责人:王茜。

摘　　要：背景:细丝蛋白B(FLNB)能将肌动蛋白细胞骨架交联成动态结构,对细胞的定向移动至关重要,可调控软骨细胞的增殖、分化及凋亡的发生。然而,细丝蛋白B对成骨细胞增殖、迁移及凋亡的影响尚未报道。目的:探究细丝蛋白B对MC3T3-E1细胞的增殖、迁移及凋亡的影响。方法:构建敲低细丝蛋白B表达的腺病毒载体(sh-FLNB1、sh-FLNB2、sh-FLNB3),感染小鼠颅顶成骨前体细胞(MC3T3-E1),嘌呤霉素药筛,通过Western Blot以及RT-PCR技术检测敲低效率,选取敲低细丝蛋白B效率最佳的MC3T3-E1细胞株作为稳转细胞株。将细胞分为Blank组、mc3t3组、sh-NC组(空载体)、sh-FLNB组(sh-FLNB慢病毒),Blank组为α-MEM完全培养基无细胞;mc3t3组为单纯α-MEM完全培养基培养;sh-NC组、sh-FLNB组为含有嘌呤霉素2.5μg/mL的α-MEM完全培养基培养。培养3 d采用CCK-8法和划痕实验检测MC3T3-E1细胞增殖和迁移能力;流式细胞仪检测细胞凋亡情况;RT-PCR进一步验证细胞凋亡相关基因的表达。结果与结论:①Western Blot及RT-PCR结果显示,sh-FLNB3组细丝蛋白B敲低效率最佳,差异有显著性意义(P<0.0001),以此作为后续实验稳转细胞株;②CCK-8数据显示,敲低细丝蛋白B后MC3T3增殖能力显著减弱(P<0.05);③划痕实验显示,敲低细丝蛋白B后MC3T3的迁移能力明显下降;④流式细胞仪及RT-PCR显示,敲低细丝蛋白B后,MC3T3-E1细胞凋亡率增加,促凋亡因子Bax表达显著上升,抑凋亡因子Bcl-2表达下降(P<0.05);⑤结果说明,细丝蛋白B敲低可降低MC3T3-E1细胞的增殖能力,细胞迁移能力下降,并增加细胞凋亡的发生。BACKGROUND:Filamin B(FLNB)can crosslink the actin cytoskeleton into a dynamic structure that is essential for the directional movement of cells.It can regulate the proliferation,differentiation and apoptosis of chondrocytes.However,the effect of FLNB on osteoblast proliferation,migration and apoptosis has not been reported.OBJECTIVE:To investigate the effect of FLNB on the proliferation,migration and apoptosis of MC3T3-E1 cells.METHODS:The adenoviral vectors for knockdown of FLNB expression(sh-FLNB1,sh-FLNB2,sh-FLNB3)were constructed and infected with MC3T3-E1 cells.After screened by puromycin drug,the efficiency of FLNB knockdown was detected by western blot and RT-PCR.The MC3T3-E1 cell line with the best efficiency of FLNB knockdown was selected as the stable transient cell line of MC3T3-E1 for subsequent experiments.The cells were divided into blank group,mc3t3 group,sh-NC group(empty vector),and sh-FLNB group(sh-FLNB lentivirus).The blank group was cultured in cell-freeα-MEM complete medium;the mc3t3 group was cultured inα-MEM complete medium alone;and the sh-NC and sh-FLNB groups were cultured withα-MEM medium containing 2.5μg/mL puromycin.After 3 days of culture,cell counting kit-8 assay and cell scratch assay were used to detect the proliferation and migration ability of MC3T3-E1;flow cytometry was used to detect cell apoptosis;and RT-PCR was used to detect the expression of apoptosis-related genes.RESULTS AND CONCLUSION:Western blot and RT-PCR results showed that the efficiency of FLNB knockdown was the best in the sh-FLNB3(P<0.0001),which was used as a stable cell line for subsequent experiments.Cell counting kit-8 data showed that the proliferative ability of MC3T3 cells was significantly weakened after knockdown of FLNB(P<0.05).Cell scratch assay results showed that the migration ability of MC3T3 cells was significantly decreased after knockdown of FLNB.Flow cytometry and RT-PCR results showed that the apoptotic rate of MC3T3-E1 cells increased after knockdown of FLNB,the expression of pro-apoptoti

关 键 词：细丝蛋白B MC3T3-E1细胞 增殖 迁移 凋亡 

分 类 号：R496[医药卫生—康复医学] R318[医药卫生—临床医学] R446