ox-LDL对骨髓干细胞分化的平滑肌细胞泡沫化的影响

【作者】 李世煌；

【导师】 严鹏科；

【作者基本信息】 南华大学 ， 药理学， 2007， 硕士

【摘要】 目的:建立体外诱导骨髓间质干细胞分化的平滑肌细胞模型,比较氧化低密度脂蛋白对骨髓间质干细胞分化的平滑肌细胞及血管平滑肌细胞泡沫化的影响,探索分化的平滑肌细胞在动脉粥样硬化发生发展过程中的作用。方法:采用贴壁法从SD大鼠骨髓中分离骨髓间质干细胞(bone mesenchymal stem cell,BMSC),从胸主动脉分离血管平滑肌细胞(vascular smooth muscle cells,VSMC)。运用条件培养基诱导BMSC分化成SMC,采用免疫荧光法分析骨髓间质干细胞及其分化后的细胞特异性标志物。80mg/L ox-LDL分别处理骨髓间质干细胞分化的平滑肌细胞和VSMC 0、12、24、48、72小时;0、20、40、80mg/L ox-LDL分别处理骨髓间质干细胞分化的平滑肌细胞和血管平滑肌细胞72小时,油红O染色观察细胞内脂滴,HPLC检测细胞内的总胆固醇、胆固醇酯含量。Western bloting检测细胞清道夫受体CD36的表达变化。结果:1、分离的BMSC呈长梭形,免疫荧光检测细胞表达CD44,但不表达CD34,呈骨髓间质干细胞表型。用含碱性成纤维细胞生长因子(b-FGF)和转化生长因子(TGF-β)的条件培养基诱导分化15天后,诱导分化的细胞形态呈梭形平滑肌样。表达平滑肌细胞特异性蛋白标志物α-肌动蛋白和平滑肌肌球蛋白重链1,呈平滑肌细胞表型,命名为骨髓间质干细胞分化的平滑肌细胞(smooth muscle cell differentiated from bone mesen- chymal stem cell, BMSC-SMC)。2、骨髓间质干细胞分化的平滑肌细胞(BMSC-SMC )经80mg/L的ox-LDL处理0、12、24、48、72小时,随着处理时间延长,细胞内总胆固醇含量、胆固醇酯含量逐渐增加,到72小时细胞内总胆固醇含量达304.3±26.4μg/mg蛋白,较未处理时的28.8±3.6μg/mg蛋白增加了9.5倍,差异有显著性(P〈0.05);胆固醇酯/总胆固醇值为70.1%,较未处理时增加了2.2倍,差异有显著性(P〈0.05);符合泡沫细胞特征。相同处理的VSMC(80mg/L ox-LDL,处理72小时)细胞内总胆固醇含量为215.7±18.9μg/ mg蛋白,胆固醇酯/总胆固醇值为60.1 %,与BMSC-SMC比较,差异均有显著性(P〈0.05)。在量效关系方面,0、20、40、80mg/L ox-LDL,处理BMSC-SMC72小时,细胞内总胆固醇含量分别为29.4±2.2、137.3±7.3、171.3±11.7、295.9±20.3μg/mg;胆固醇酯/总胆固醇值分别为23.6%、38.4%、48.8%、68.5%,提示随着ox-LDL浓度增高,细胞内总胆固醇、胆固醇酯含量逐渐增加。BMSC-SMC与相同处理的VSMC细胞内总胆固醇、胆固醇酯/总胆固醇值比较,增幅较大,差异均有显著性(P〈0.05)。80mg/L ox-LDL作用细胞72小时,油红O染色观察到BMSC-SMC体积增大、细胞内出现大量脂滴,呈典型的泡沫细胞形态,且细胞内脂滴较VSMC明显增多。3、Western bloting检测发现,未处理BMSC-SMC少量表达清道夫受体CD36,经80mg/L ox-LDL处理72小时后,CD36表达显著增加,相同处理VSMC的CD36表达亦增高,但其增加幅度远较BMSC-SMC为小(P〈0.05)。结论:1、在ox-LDL的作用下骨髓细胞干细胞分化的平滑肌细胞能大量蓄积胆固醇酯,形成泡沫细胞,且这一表现较主动脉中膜分离而来VSMC更为明显,提示骨髓干细胞分化的平滑肌细胞可能参与和促进动脉粥样硬化的发生和发展。2、在ox-LDL的作用下,骨髓干细胞分化的平滑肌细胞大量蓄积胆固醇酯和泡沫化,与细胞清道夫受体CD36表达上调有关。更多还原

【Abstract】 Objective:To establish the model of smooth muscle cell differentiated from bone mesenchymal stem cell (BMSC-SMC) in vitro, compare the foam effects of oxidized low density lipoprotein (ox-LDL) on BMSC-SMC and VSMC, investigate the roles of BMSC- SMC in the occurrence and development of artherosclerosis(As).Methods: BMSC was isolated from the femoral bone of SD rats by adherent method, and VSMC from thoracic aorta. BMSC-SMC was differentiated from BMSC by special condition medium. The specific markers of BMSC and smooth muscle cell differentiated from bone mesenchymal stem cell (BMSC-SMC) was identified by immunofluorescence (IF) staining. After treated with 80mg/Lox-LDL for 0、12、24、48、72h and with0、20、40 and 80 mg/Lox-LDL for 72h, Oil Red O staining was adopted to observe the lipid droplet and high performance liquid chromatography(HPLC) to detect the contents of total cholesterol and cholesterol ester in BVSMC and VSMC. After treated by ox-LDL with 80mg/L for 72h, Western blotting was performed to detect the different expression of scavenger receptor CD36 in BMSC-SMC and VSMC.Results:1、The isolated BMSC present a slim-spindle appearance. Immunofluorescence (IF) staining showed that the cells expressed CD44,which was the specific markers of BMSC, but not CD34.the cells(BMSC-SMC) induced by condition medium containing b-FGF and TGF-βfor 15day present slim-spindle smooth muscle-like cells. Immuno-fluorescence (IF) staining showed that the cells expressed smooth muscleα-actin and smooth muscle Myosin heavy chain I,which were the specific markers of SMC. the cells are named smooth muscle cell differentiated from bone mesenchymal stem cell, (BMSC- SMC)。2、After treated by ox-LDL with 80mg/L for0、12、24、48、72h, the contents of total cholesterol and cholesterol ester in BMSC-SMC increased gradually with time prolonged, and the contents of total cholesterol reached 304.3±26.4μg/mg in 72h, compared with untreated cells 28.8±3.6μg/mg. which raised 9.5times(P〈0.05);the ratio of cholesterol ester to total cholesterol was 70.1% and raised 2.2times(P〈0.05),which correspond to the characteristic of foam cells.compared with that of VSMC treated with the same method. After treated by ox-LDL with 80mg/L for 72h,the contents of total cholesterol reached 215.7.3±18.9μg/mg,the ratio of cholesterol ester to total cholesterol in VSMC was 60.1%. compared with BMSC-SMC, variance is significance(P<0.05)After treated by ox-LDL with 0、20、40、80mg/L for 72h, the contents of total cholesterol in BMSC-SMC reached29.4±2.2、137.3±7.3、171.3±11.7、295.9±20.3μg/mg; the ratio of cholesterol ester to total cholesterol was 23.6%、38.4%、48.8%、68.5%,suggest increased gradually with concentration of ox-LDL , Cellular totol cholesteroland and Ester cholesterol also increased gradually.After treated by ox-LDL with 80mg/L for 72h, Oil Red O staining showed that the volume of BMSC-SMC was augmentation and a lot of lipid droplet appeared in cells, which was the typical form of foam cells. Lipid droplet in cells was significantly increased compared with that of VSMC.3、Western blotting analysis showed that the expression of CD36 was low in BSMC-MSC. After treated by ox-LDL with 80mg/L for 72h, the expression of CD36 was significantly increased,the same treated in VSMC the expression of CD36 was increased too, but the extent of increasion compared with that of VSMC is weak(P〈0.05).Conclusion 1、Compared with VSMC that isolated from Aortic tunica media, BMSC-SMC can accumulate a lot of lipid droplet and form foam cells after treated by ox-LDL, suggested that BMSC-SMC maybe participate and facilitate the occurrence and development of artherosclerosis(As)2、The accumulation of cholesterol ester and foam in BVSMC after treated by ox-LDL is relate with the up-regulation of scavenger receptor CD36 expression.更多还原