IGF-1对血管平滑肌细胞增殖和凋亡的影响
发布时间：2018-11-08 12:44
【摘要】：目的:探讨在炎症状态下胰岛素样生长因子(insulin like growth factor-1,IGF-1)对血管平滑肌细胞增殖和凋亡的影响。方法:1.取雄性SD大鼠,断颈后分离主动脉,超净台内去除内膜和外膜,按组织贴块法+酶消化法获取原代血管平滑肌细胞并传代,实验取用第4-8代细胞,α-肌动蛋白阳性表达的细胞鉴定为平滑肌细胞。2.不同浓度的脂多糖(lipopolysaccharide,LPS)诱导RAW264.7细胞制备炎症模型,ELISA法检测各组上清液中IL-6的水平,与对照组相比差异有统计学意义为造模成功,即条件培养基(conditioned medium,CM),未加LPS诱导组为对照组即非条件培养基(nonconditioned medium,nCM)。3.平滑肌细胞分别与nCM、CM、CM+IGF-1 60ng/ml、CM+IGF-1 90ng/ml、CM+IGF-1 120ng/ml共培养,用MTT法检测各组OD值,以OD值反应各组VSMC的数量,用流式法检测各组平滑肌细胞的凋亡率。4.统计学方法:以SPSS17.0软件进行统计,计量资料用均数±标准差表示,两组间比较用t检验,多组间比较用方差分析,P0.05有统计学差异。结果:1.使用组织贴块法+酶消化法可成功获取实验所需血管平滑肌细胞。2.一定浓度的LPS诱导RAW264.7细胞后上清液中IL-6的表达量显著升高。RAW264.7细胞分别在LPS 0ng/ml,10ng/ml,100ng/ml,1ug/ml,10ug/ml浓度刺激下各组上清液中IL-6的浓度分别是(6.75±0.12)pg/ml;(7.82±1.53)pg/ml;(44.09±1.58)pg/ml;(155.71±23.93)pg/ml;(436.59±3.15)pg/ml,与对照组相比,10ng/ml组差异无统计学意义(P=0.916);后三组与对照组相比,差异有统计学意义(P=0.009,P=0.000,P=0.000)。3.CM组能抑制血管平滑肌细胞增殖,加入一定浓度的IGF-1可逆转其对血管平滑肌细胞增殖的抑制。VSMC在五组不同干预下各组OD值分别是(0.53±0.07)、(0.26±0.06)、(0.30±0.10)、(0.62±0.09)、(0.55±0.05)。与nCM组相比,CM组OD值明显降低(p=0.000),两组间比较有统计学意义。与CM组相比,CM+IGF-160ng/ml组、CM+IGF-190ng/ml组、CM+IGF-1120ng/ml组OD值升高(p=0.993,p=0.001,p=0.000),后两组与CM组之间有统计学意义,以90ng/ml组效果最好。4.CM组可促进血管平滑肌细胞凋亡,加入一定浓度的IGF-1可逆转其对血管平滑肌细胞的促凋亡作用。五组干预下VSMC凋亡率分别是(4.89±0.09)%;(10.86±0.15)%;(9.64±0.65)%;(3.85±0.51)%;(4.82±0.08)%。与nCM组相比,CM组凋亡率明显增加(p=0.000),两组间差异有统计学意义;与CM组相比,CM+IGF-160ng/ml组、CM+IGF-190ng/ml组、CM+IGF-1120ng/ml组凋亡率显著降低(p=0.045,p=0.000,p=0.000),差异有统计学意义,以90ng/ml组效果最好。结论:LPS可诱导RAW264.7细胞极化为M1型巨噬细胞。M1型巨噬细胞所致的炎症状态可促进血管平滑肌细胞凋亡,抑制血管平滑肌细胞增殖,给予一定浓度的IGF-1后可发现凋亡减少,增殖增加。
[Abstract]:Aim: to investigate the effects of insulin-like growth factor (insulin like growth factor-1,IGF-1) on proliferation and apoptosis of vascular smooth muscle cells (VSMCs) in inflammatory condition. Methods: 1. The primary vascular smooth muscle cells (VSMCs) were obtained from male SD rats after cervical dissection, and the intima and adventitia were removed from the ultraclean platform, and the primary vascular smooth muscle cells were obtained by enzyme digestion method, and the 4-8 passage cells were used in the experiment. 伪-actin positive cells were identified as smooth muscle cells. 2. 2. Different concentrations of lipopolysaccharide (lipopolysaccharide,LPS) induced inflammatory model of RAW264.7 cells. ELISA assay was used to detect the level of IL-6 in supernatant of each group. Compared with the control group, the difference was that the model was successful, that is, conditioned medium (conditioned medium,CM). The unconditioned medium (nonconditioned medium,nCM) was the control group without LPS induction. 3. Smooth muscle cells were co-cultured with nCM,CM,CM IGF-1 60ng / ml CM IGF-1 90ng / ml IGF-1 120ng/ml respectively. The OD values of each group were detected by MTT method, and the number of VSMC in each group was measured by OD value. The apoptosis rate of smooth muscle cells in each group was detected by flow cytometry. Statistical methods: SPSS17.0 software was used to analyze the statistical data. The measurement data were expressed as mean 卤standard deviation. The comparison between the two groups was by t test, and the analysis of variance was used by the analysis of variance between the two groups. There was a statistical difference between the two groups (P0.05). The result is 1: 1. The vascular smooth muscle cells needed in the experiment can be successfully obtained by enzyme digestion with tissue patch method. 2. 2. The expression of IL-6 in the supernatant of RAW264.7 cells was significantly increased by a certain concentration of LPS. The expression of IL-6 in the supernatant of LPS 0ng / ml 10ng / ml 10 ng / ml RAW264.7 cells was 100 ng / ml / ml, respectively. The concentration of IL-6 in supernatant stimulated by 10ug/ml was (6.75 卤0.12) pg/ml;. (7.82 卤1.53) pg/ml; (44.09 卤1.58) pg/ml; (155.71 卤23.93) pg/ml; (436.59 卤3.15) pg/ml, there was no significant difference between 10ng/ml group and control group (P0. 916). There was significant difference between the latter three groups and the control group (P0. 009 P0. 000). 3.CM group could inhibit the proliferation of vascular smooth muscle cells. The inhibitory effect of IGF-1 on the proliferation of vascular smooth muscle cells was reversed by adding a certain concentration of IGF-1. The OD value of VSMC in different intervention groups was (0.53 卤0.07), () 0.26 卤0.06), (0.30 卤0.10), (0.62 卤0.09, respectively. (0.55 卤0.05). Compared with nCM group, the OD value of CM group was significantly lower than that of nCM group (p0. 000). Compared with CM group, OD value in CM IGF-160ng/ml group, CM IGF-190ng/ml group and CM IGF-1120ng/ml group was increased (p0.993P 0.001P 0.000), and there was significant difference between the latter two groups and CM group. The effect of 90ng/ml group was the best. 4.CM group could promote the apoptosis of vascular smooth muscle cells. Adding a certain concentration of IGF-1 could reverse the apoptosis of vascular smooth muscle cells. The apoptotic rates of VSMC were (4.89 卤0.09)%, (10.86 卤0.15)%, (9.64 卤0.65)%, (3.85 卤0.51)% and (4.82 卤0.08)%, respectively. Compared with nCM group, the apoptotic rate in CM group was significantly increased (p0. 000), and the difference between the two groups was statistically significant. Compared with CM group, the apoptotic rate of CM IGF-160ng/ml group, CM IGF-190ng/ml group and CM IGF-1120ng/ml group was significantly lower (P < 0.05). The difference was statistically significant, especially in 90ng/ml group. Conclusion: LPS can induce RAW264.7 cells to become M1 macrophages. The inflammatory state induced by M1 macrophages can promote the apoptosis of vascular smooth muscle cells and inhibit the proliferation of vascular smooth muscle cells. After given a certain concentration of IGF-1, apoptosis was decreased and proliferation was increased.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R54
本文编号：2318541
[Abstract]:Aim: to investigate the effects of insulin-like growth factor (insulin like growth factor-1,IGF-1) on proliferation and apoptosis of vascular smooth muscle cells (VSMCs) in inflammatory condition. Methods: 1. The primary vascular smooth muscle cells (VSMCs) were obtained from male SD rats after cervical dissection, and the intima and adventitia were removed from the ultraclean platform, and the primary vascular smooth muscle cells were obtained by enzyme digestion method, and the 4-8 passage cells were used in the experiment. 伪-actin positive cells were identified as smooth muscle cells. 2. 2. Different concentrations of lipopolysaccharide (lipopolysaccharide,LPS) induced inflammatory model of RAW264.7 cells. ELISA assay was used to detect the level of IL-6 in supernatant of each group. Compared with the control group, the difference was that the model was successful, that is, conditioned medium (conditioned medium,CM). The unconditioned medium (nonconditioned medium,nCM) was the control group without LPS induction. 3. Smooth muscle cells were co-cultured with nCM,CM,CM IGF-1 60ng / ml CM IGF-1 90ng / ml IGF-1 120ng/ml respectively. The OD values of each group were detected by MTT method, and the number of VSMC in each group was measured by OD value. The apoptosis rate of smooth muscle cells in each group was detected by flow cytometry. Statistical methods: SPSS17.0 software was used to analyze the statistical data. The measurement data were expressed as mean 卤standard deviation. The comparison between the two groups was by t test, and the analysis of variance was used by the analysis of variance between the two groups. There was a statistical difference between the two groups (P0.05). The result is 1: 1. The vascular smooth muscle cells needed in the experiment can be successfully obtained by enzyme digestion with tissue patch method. 2. 2. The expression of IL-6 in the supernatant of RAW264.7 cells was significantly increased by a certain concentration of LPS. The expression of IL-6 in the supernatant of LPS 0ng / ml 10ng / ml 10 ng / ml RAW264.7 cells was 100 ng / ml / ml, respectively. The concentration of IL-6 in supernatant stimulated by 10ug/ml was (6.75 卤0.12) pg/ml;. (7.82 卤1.53) pg/ml; (44.09 卤1.58) pg/ml; (155.71 卤23.93) pg/ml; (436.59 卤3.15) pg/ml, there was no significant difference between 10ng/ml group and control group (P0. 916). There was significant difference between the latter three groups and the control group (P0. 009 P0. 000). 3.CM group could inhibit the proliferation of vascular smooth muscle cells. The inhibitory effect of IGF-1 on the proliferation of vascular smooth muscle cells was reversed by adding a certain concentration of IGF-1. The OD value of VSMC in different intervention groups was (0.53 卤0.07), () 0.26 卤0.06), (0.30 卤0.10), (0.62 卤0.09, respectively. (0.55 卤0.05). Compared with nCM group, the OD value of CM group was significantly lower than that of nCM group (p0. 000). Compared with CM group, OD value in CM IGF-160ng/ml group, CM IGF-190ng/ml group and CM IGF-1120ng/ml group was increased (p0.993P 0.001P 0.000), and there was significant difference between the latter two groups and CM group. The effect of 90ng/ml group was the best. 4.CM group could promote the apoptosis of vascular smooth muscle cells. Adding a certain concentration of IGF-1 could reverse the apoptosis of vascular smooth muscle cells. The apoptotic rates of VSMC were (4.89 卤0.09)%, (10.86 卤0.15)%, (9.64 卤0.65)%, (3.85 卤0.51)% and (4.82 卤0.08)%, respectively. Compared with nCM group, the apoptotic rate in CM group was significantly increased (p0. 000), and the difference between the two groups was statistically significant. Compared with CM group, the apoptotic rate of CM IGF-160ng/ml group, CM IGF-190ng/ml group and CM IGF-1120ng/ml group was significantly lower (P < 0.05). The difference was statistically significant, especially in 90ng/ml group. Conclusion: LPS can induce RAW264.7 cells to become M1 macrophages. The inflammatory state induced by M1 macrophages can promote the apoptosis of vascular smooth muscle cells and inhibit the proliferation of vascular smooth muscle cells. After given a certain concentration of IGF-1, apoptosis was decreased and proliferation was increased.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R54
【参考文献】
相关期刊论文 前2条
1 王华;方芳;柴坷;罗瑶;刘兵;刘东戈;何淑蓉;刘德平;杨杰孚;;80岁及以上老年冠心病患者临床病理特点分析[J];中华心血管病杂志;2015年11期
2 李悦梅,冯大明,万载阳,王双,沈丹彤,赵桂玲,杨永宗;组织块法培养大鼠肠系膜小动脉的平滑肌细胞[J];南华大学学报(医学版);2003年03期
，本文编号：2318541
本文链接：https://www.wllwen.com/yixuelunwen/xxg/2318541.html
最近更新
教材专著
