Caveolae/Caveolin-1与膜胆固醇共同调控小鼠BMSCs成骨分化  

Caveolae/Caveolin-1 regulates osteogenic differentiation of mouse BMSCs together with membrane cholesterol

作　　者：杨阳 王琤 周文土 周冰 Yang Yang;Cheng Wang;Wentu Zhou;Bing Zhou(Department of Stomatology 900th Hospital of Joint Logistic Support Force,Fuzhou 350025,China)

机构地区：[1]联勤保障部队第九〇〇医院口腔科,福州350025

出　　处：《中华细胞与干细胞杂志（电子版）》2024年第3期137-142,共6页Chinese Journal of Cell and Stem Cell（Electronic Edition）

基　　金：联勤保障部队第九〇〇医院2021年度院杰青项目(2021JQ11)。

摘　　要：目的研究小鼠骨髓间充质干细胞(BMSCs)细胞膜上Caveolae/Caveolin-1与膜胆固醇(Chol)对BMSCs成骨分化的影响。方法体外培养BMSCs,未处理细胞作为对照,对细胞进行以下干预:用甲基-β-环糊精(MβCD)处理耗竭细胞膜Chol,添加Chol补充细胞膜Chol,转染非特异性小干扰siRNA(si Ctrl)和转染Caveolin-1 siRNA(si CAV-1)。通过Amplex Red法检测各处理组细胞的Chol含量;RT-PCR与Western blot检测各组Caveolin-1表达;碱性磷酸酶活性测定和茜素红染色观察成骨细胞形成情况。多组间比较采用单因素方差分析,组间两两比较采用LSD-t检验。结果与对照相比,MβCD处理细胞后Chol含量[(59.33±11.24)比(127.00±12.45)mmol/L]、Caveolin-1 mRNA(0.52±0.05比1.00±0.15)下降,ALP活性[(18.37±0.31)比(10.13±0.41)U/L]增强,添加Chol后细胞Chol含量[(228.21±10.38)比(127.00±12.45)mmol/L]、Caveolin-1 mRNA(4.58±0.25比1.00±0.15)和Caveolin-1蛋白表达(184.00±10.25比51.25±8.12)增加,ALP活性[(6.32±0.54)比(10.13±0.41)U/L]减弱(P均<0.05)。与转染si Ctrl相比,转染si CAV-1的细胞Chol含量[(95.35±14.05)比(136.00±15.52)mmol/L]、Caveolin-1 mRNA(0.10±0.21比1.00±0.45)和Caveolin-1蛋白表达(6.12±0.35比13.06±1.52)下降,ALP活性[(19.28±0.34)比(10.15±0.43)U/L]增强(P均<0.05)。茜素红染色显示,MβCD处理和转染si CAV-1使BMSCs基质矿化增加,而添加Chol使BMSCs基质矿化减少。结论Caveolae/Caveolin-1与膜Chol相互影响并共同调控小鼠BMSCs成骨分化。Objective To investigate the effect of Caveolae/Caveolin-1 and membrane cholesterol on the osteogenic differentiation of mouse bone marrow mesenchymal stem cells(BMSCs).Methods BMSCs were cultured in vitro and untreated cells as the control.The following interventions were performed on the cells:depletion of membrane cholesterol using methyl-β-cyclodextrin(MβCD),supplementation of membrane cholesterol,transfection with non-specific small interfering RNA(si Ctrl)and Caveolin-1 siRNA(si CAV-1).The Amplex Red method was used to detect the Chol content in the cells of each treatment group.RT-PCR and Western blot were used to detect the expression of Caveolin-1 in each group.Alkaline phosphatase(ALP)activity assays and alizarin red staining were performed to observe osteoblast formation.One-way analysis of variance was used for multi-group comparisons,and the LSD-t test was used for pairwise comparisons between groups.P<0.05 was considered statistically significant.Results Compared to untreated cells,treatment with MβCD led to a decrease in cholesterol[(59.33±11.24)vs(127.00±12.45)mmol/L],Caveolin-1 mRNA(0.52±0.05 vs 1.00±0.15),as well as an increase in ALP activity[(18.37±0.31)vs(10.13±0.41)U/L](P<0.05).Addition of cholesterol resulted in an increase in cholesterol[(228.21±10.38)vs(127.00±12.45)mmol/L],Caveolin-1 mRNA(4.58±0.25 vs 1.00±0.15),and Caveolin-1 protein expression(184.00±10.25 vs 51.25±8.12),as well as a decrease in ALP activity[(6.32±0.54)vs(10.13±0.41)U/L](P<0.05).Compared to cells transfected with si Ctrl,cells transfected with si CAV-1 showed a decrease in cholesterol[(95.35±14.05)vs(136.00±15.52)mmol/L],Caveolin-1 mRNA(0.10±0.21 vs 1.00±0.45),and Caveolin-1 protein expression(6.12±0.35 vs 13.06±1.52),as well as an increase in ALP activity[(19.28±0.34)vs(10.15±0.43)U/L](P<0.05).Alizarin red staining showed that treatment with MβCD and transfection with si CAV-1 increased mineralization of the BMSCs matrix,while the addition of cholesterol decreased mineralization.Conclusion Ca

关 键 词：小鼠 骨髓间充质干细胞 小窝蛋白-1 胆固醇 成骨分化 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]