ox-LDL对VSMC表达SDF-1α的影响及其对VSMC和SPC趋化作用的研究
发布时间：2018-08-26 16:42
【摘要】：目的 1、探讨氧化低密度脂蛋白(ox-LDL)对小鼠血管平滑肌细胞表达基质细胞衍生因子1α(SDF-1α)的影响; 2、比较VSMC的条件培养液及SDF-1α对血管平滑肌细胞(VSMC)和平滑肌祖细胞(SPC)趋化迁移的影响。 方法 1、组织块贴壁法培养小鼠主动脉平滑肌细胞,用免疫组织化学方法对培养的平滑肌细胞进行鉴定; 2、ox-LDL与VSMC共培养,用免疫组织化学方法、酶联免疫吸附试验(ELISA)测定VSMC表达SDF-1α蛋白的变化,用逆转录-聚合酶链式反应(RT-PCR)测定VSMC表达SDF-1αmRNA的变化,观察剂量-时间效应; 3、趋化迁移实验:将含有趋化因子SDF-1α的培养液置于Transwell小室下室,将VSMC或SPC悬液置于其上室,共孵育6h后,乙醇固定,高倍镜下计数滤膜背面(下室面)的细胞数,检测VSMC的条件培养液及SDF-1α对VSMC和SPC的趋化迁移的影响。 结果 1、培养的小鼠血管平滑肌细胞的纯度可以达到约90%。 2、小鼠血管平滑肌细胞有基础水平的SDF-1α的表达。ox-LDL的浓度在0～60ng/ml范围内,作用24h,SDF-1α蛋白的表达随ox-LDL浓度的增加而变化,SDF-1α蛋白表达的峰值在ox-LDL浓度为45ng/ml时,是ox-LDL浓度为0ng/ml时的1.4倍。在ox-LDL浓度为45ng/ml时,0～48h范围内,SDF-1α蛋白的表达变化随时间的延长而变化, SDF-1α蛋白表达的峰值在24h,是0h的1.5倍。在ox-LDL浓度为45ng/ml时,0～30h范围内,SDF-1αmRNA的表达变化随时间的延长而变化,SDF-1α基因mRNA的峰值在10h,是0h的3.2倍。 3、VSMC的条件培养液及SDF-1α对VSMC和SPC都具有明显趋化作用。而且VSMC的条件培养液及SDF-1α对SPC的趋化作用强于对VSMC的趋化作用。 结论 1.、ox-LDL可增强小鼠血管平滑肌细胞SDF-1α的表达。 2、VSMC的条件培养液及SDF-1α对SPC的趋化作用明显强于对VSMC的趋化作用。
[Abstract]:Objective 1 to investigate the effect of oxidized low density lipoprotein (ox-LDL) on the expression of stromal cell-derived factor-1 伪 (SDF-1 伪) in mouse vascular smooth muscle cells (VSMCs), and to compare the effects of conditioned medium of VSMC and SDF-1 伪 on the expression of (VSMC) and SDF-1 伪 in VSMCs. Effects of (SPC) chemotactic migration on smooth muscle progenitor cells. Methods 1. Smooth muscle cells of mouse aorta were cultured by tissue mass adherence method, and the cultured smooth muscle cells were identified by immunohistochemical method, 2the cultured smooth muscle cells were co-cultured with VSMC. The changes of SDF-1 伪 protein expression in VSMC and SDF-1 伪 mRNA in VSMC were detected by enzyme linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) respectively. (3) chemotactic migration experiment: the culture medium containing chemokine SDF-1 伪 was placed under the Transwell chamber, and the VSMC or SPC suspension was placed in the upper chamber. After incubation for 6 h, ethanol was fixed. The number of cells on the back of filter membrane (lower chamber surface) was counted under high power microscope, and the effects of conditioned medium of VSMC and SDF-1 伪 on chemotaxis migration of VSMC and SPC were detected. Results 1. The purity of cultured mouse vascular smooth muscle cells was about 90.2. The expression of SDF-1 伪 in vascular smooth muscle cells of mice was at a basic level. The concentration of ox-LDL was in the range of 0~60ng/ml. The expression of SDF-1 伪 protein changed with the increase of ox-LDL concentration at 24 h. The peak expression of SDF-1 伪 protein was 1.4 times higher than that of 0ng/ml when ox-LDL concentration was 45ng/ml. The expression of SDF-1 伪 protein changed with the prolongation of time when the concentration of ox-LDL was 45ng/ml. The peak expression of SDF-1 伪 protein at 24 h was 1.5 times higher than that at 0 h. The expression of SDF-1 伪 mRNA changed with the prolongation of time when the concentration of ox-LDL was 45ng/ml. The peak value of mRNA of SDF-1 伪 gene was at 10 h, which was 3.2 times of that of 0 h. The conditioned medium of VSMC and SDF-1 伪 had obvious chemotaxis on VSMC and SPC. Moreover, the conditioned medium of VSMC and SDF-1 伪 had stronger chemotaxis on SPC than on VSMC. Conclusion 1. Ox-LDL can enhance the expression of SDF-1 伪 in mouse vascular smooth muscle cells. (2) the conditioned medium of VSMC and SDF-1 伪 have stronger chemotaxis on SPC than on VSMC. 2.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363
本文编号：2205523
[Abstract]:Objective 1 to investigate the effect of oxidized low density lipoprotein (ox-LDL) on the expression of stromal cell-derived factor-1 伪 (SDF-1 伪) in mouse vascular smooth muscle cells (VSMCs), and to compare the effects of conditioned medium of VSMC and SDF-1 伪 on the expression of (VSMC) and SDF-1 伪 in VSMCs. Effects of (SPC) chemotactic migration on smooth muscle progenitor cells. Methods 1. Smooth muscle cells of mouse aorta were cultured by tissue mass adherence method, and the cultured smooth muscle cells were identified by immunohistochemical method, 2the cultured smooth muscle cells were co-cultured with VSMC. The changes of SDF-1 伪 protein expression in VSMC and SDF-1 伪 mRNA in VSMC were detected by enzyme linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) respectively. (3) chemotactic migration experiment: the culture medium containing chemokine SDF-1 伪 was placed under the Transwell chamber, and the VSMC or SPC suspension was placed in the upper chamber. After incubation for 6 h, ethanol was fixed. The number of cells on the back of filter membrane (lower chamber surface) was counted under high power microscope, and the effects of conditioned medium of VSMC and SDF-1 伪 on chemotaxis migration of VSMC and SPC were detected. Results 1. The purity of cultured mouse vascular smooth muscle cells was about 90.2. The expression of SDF-1 伪 in vascular smooth muscle cells of mice was at a basic level. The concentration of ox-LDL was in the range of 0~60ng/ml. The expression of SDF-1 伪 protein changed with the increase of ox-LDL concentration at 24 h. The peak expression of SDF-1 伪 protein was 1.4 times higher than that of 0ng/ml when ox-LDL concentration was 45ng/ml. The expression of SDF-1 伪 protein changed with the prolongation of time when the concentration of ox-LDL was 45ng/ml. The peak expression of SDF-1 伪 protein at 24 h was 1.5 times higher than that at 0 h. The expression of SDF-1 伪 mRNA changed with the prolongation of time when the concentration of ox-LDL was 45ng/ml. The peak value of mRNA of SDF-1 伪 gene was at 10 h, which was 3.2 times of that of 0 h. The conditioned medium of VSMC and SDF-1 伪 had obvious chemotaxis on VSMC and SPC. Moreover, the conditioned medium of VSMC and SDF-1 伪 had stronger chemotaxis on SPC than on VSMC. Conclusion 1. Ox-LDL can enhance the expression of SDF-1 伪 in mouse vascular smooth muscle cells. (2) the conditioned medium of VSMC and SDF-1 伪 have stronger chemotaxis on SPC than on VSMC. 2.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363
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