五羟色胺对人骨髓间充质干细胞增殖的影响
发布时间：2018-11-05 18:11
【摘要】：目的： 探讨五羟色胺(serotonin,5-HT)对人骨髓间充质干细胞(Bone Mesenchymal Stem Cells,BMSCs)体外增殖的影响。 方法： 从捐赠者髂骨穿刺抽取督髓细胞,采用密度梯度离心和贴壁培养法进行培养和纯化BMSCs,取生长良好的P3或P4代BMSCs,通过用含不同浓度5-HT的条件培养基分组对BMSCs进行干预。光学显微镜下观察细胞24、48及72小时的形态变化；MTS检测评估细胞增殖情况；流式细胞仪(FCM)检测BMSCs凋亡率。 结果： 1、细胞形态学观察：刚接种细胞时,BMSCs呈圆形,胞体透亮,折光性好。常规培养24、48、72小时后,BMSCs开始贴壁,细胞分布均匀,轮廓清楚,形态均-,呈多突状或长梭状,细胞增殖速度加快并逐渐相互融合。加入5-HT培养24、48、72小时后,观察细胞形态逐渐发生改变,细胞变圆、皱缩,与邻近细胞分离；核染色质固缩集聚、呈块或碎裂状改变；胞质浓缩,中心透光度降低。2、MTS法检测：在不同5-HT(0.01,0.1,1,10,100,500,1000μM)浓度作用下,在血清浓度分别为0%和10%时,与对照组相比,酶标仪检测OD值均有所降低。检测结果显示：在24、48小时时,5-HT(0.01,0.1,1,10,100μM)浓度对BMSCs的作用与对照组比较P0.05,5-HT浓度在500μM和1000μM时,对BMSCs的作用与对照组比较P0.05；在72小时时,5-HT(0.01,0.1,1,10μM)浓度对BMSCs的作用与对照组比较P0.05,5-HT浓度在100μM、500μM和1000μM时,对BMSCs的作用与对照组比较P0.05。检测结果显示随着观察时间延长,5-HT浓度增加,细胞存活率逐渐降低。在无血清情况下,存活率最低可达59.5%(P0.05)。3、流式细胞仪检测：BMSCs在各组5-HT(1,10,100,1000μM)药物浓度作用48小时后,与对照组相比,检测结果显示5-HT浓度在10μM、100μM和1000μM时,随着浓度增加,细胞凋亡率升高(P0.05),在5-HT为1000μM时,凋亡率为21.42%。 结论： 5-HT对体外培养的人BMSCs的增殖具有凋亡作用,无论在有血清或无血清情况下,5-HT作用时间越长,细胞存活率越低；5-HT药物浓度越高,细胞凋亡率越大；且在无血清这种低营养环境下,5-HT后期的凋亡作用愈明显。
[Abstract]:Aim: to investigate the effect of serotonin (serotonin,5-HT) on the proliferation of human bone marrow mesenchymal stem cells (Bone Mesenchymal Stem Cells,BMSCs) in vitro. Methods: medullary cells were extracted from donor iliac bone puncture and cultured with density gradient centrifugation and adherent culture. The BMSCs, of P3 or P4 passages were obtained from BMSCs, by using the method of density gradient centrifugation and adherent culture. BMSCs was treated with conditioned medium containing different concentrations of 5-HT. The morphological changes of the cells at 48 and 72 hours were observed under optical microscope, the proliferation of cells was evaluated by MTS assay, and the apoptosis rate of BMSCs was detected by flow cytometry (FCM). Results: 1. Observation of cell morphology: when the cells were first inoculated, the BMSCs was round, the cell body was bright, and the refraction was good. After 72 hours of conventional culture, BMSCs began to adhere to the wall. The cells were evenly distributed, with clear outline, uniform shape, multi-protruding or long fusiform, and the proliferation rate of the cells was accelerated and gradually fused with each other. After being cultured with 5-HT for 72 hours, the morphology of the cells was gradually changed, the cells became round, shrinked and separated from the adjacent cells, the chromatin was pyknosis and accumulated, and the nuclear chromatin was changed in the shape of block or fragmentation. The concentration of cytoplasm and the decrease of central transmittance. 2Detection by 5-HT: under the action of different concentration of 5-HT (0.01 ~ (0.1) ~ (0.1) ~ (10 ~ (10) ~ (100) ~ (500) 渭 M, when the serum concentration was 0% and 10 ~ (5) 渭 M, compared with the control group, the value of OD detected by enzymatic marker was lower than that of the control group. The results showed that the effect of 5-HT on BMSCs was significantly higher than that of the control group (P 0.05 渭 M and 1000 渭 M), and the effect of BMSCs was higher than that of the control group (P 0.05 渭 M and P 0.05 渭 M) at 24 h for 48 h. The effect of 5-HT (0.01g / ml) on BMSCs was higher than that of the control group (P 0.05 渭 M and 1000 渭 M). At 72 hours, the effect of 5-HT (0.01 渭 m) on BMSCs was higher than that of control group (100 渭 M, 1000 渭 M), and that of BMSCs was higher than that of control group (P 0.05). The results showed that the concentration of 5-HT increased and the cell survival rate decreased with the increase of observation time. In serum-free condition, the lowest survival rate was 59.5% (P0.05). Flow cytometry showed that the concentration of BMSCs was 10 渭 m after 48 hours of exposure to the drug concentration of 5-HT (100 渭 M). Compared with the control group, the results showed that the concentration of 5-HT was 10 渭 m. When the concentration of 5-HT was 1000 渭 M, the apoptotic rate was 21.42 渭 M, and the apoptosis rate increased with the increase of the concentration of 100 渭 M and 1000 渭 M (P0.05). Conclusion: 5-HT has apoptotic effect on the proliferation of human BMSCs in vitro. The longer the 5-HT is, the lower the cell survival rate is, and the higher the concentration of 5-HT is, the higher the apoptosis rate is. Moreover, in the low nutrition environment of serum-free, the effect of apoptosis in the late stage of 5-HT was more obvious.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329
本文编号：2312919
[Abstract]:Aim: to investigate the effect of serotonin (serotonin,5-HT) on the proliferation of human bone marrow mesenchymal stem cells (Bone Mesenchymal Stem Cells,BMSCs) in vitro. Methods: medullary cells were extracted from donor iliac bone puncture and cultured with density gradient centrifugation and adherent culture. The BMSCs, of P3 or P4 passages were obtained from BMSCs, by using the method of density gradient centrifugation and adherent culture. BMSCs was treated with conditioned medium containing different concentrations of 5-HT. The morphological changes of the cells at 48 and 72 hours were observed under optical microscope, the proliferation of cells was evaluated by MTS assay, and the apoptosis rate of BMSCs was detected by flow cytometry (FCM). Results: 1. Observation of cell morphology: when the cells were first inoculated, the BMSCs was round, the cell body was bright, and the refraction was good. After 72 hours of conventional culture, BMSCs began to adhere to the wall. The cells were evenly distributed, with clear outline, uniform shape, multi-protruding or long fusiform, and the proliferation rate of the cells was accelerated and gradually fused with each other. After being cultured with 5-HT for 72 hours, the morphology of the cells was gradually changed, the cells became round, shrinked and separated from the adjacent cells, the chromatin was pyknosis and accumulated, and the nuclear chromatin was changed in the shape of block or fragmentation. The concentration of cytoplasm and the decrease of central transmittance. 2Detection by 5-HT: under the action of different concentration of 5-HT (0.01 ~ (0.1) ~ (0.1) ~ (10 ~ (10) ~ (100) ~ (500) 渭 M, when the serum concentration was 0% and 10 ~ (5) 渭 M, compared with the control group, the value of OD detected by enzymatic marker was lower than that of the control group. The results showed that the effect of 5-HT on BMSCs was significantly higher than that of the control group (P 0.05 渭 M and 1000 渭 M), and the effect of BMSCs was higher than that of the control group (P 0.05 渭 M and P 0.05 渭 M) at 24 h for 48 h. The effect of 5-HT (0.01g / ml) on BMSCs was higher than that of the control group (P 0.05 渭 M and 1000 渭 M). At 72 hours, the effect of 5-HT (0.01 渭 m) on BMSCs was higher than that of control group (100 渭 M, 1000 渭 M), and that of BMSCs was higher than that of control group (P 0.05). The results showed that the concentration of 5-HT increased and the cell survival rate decreased with the increase of observation time. In serum-free condition, the lowest survival rate was 59.5% (P0.05). Flow cytometry showed that the concentration of BMSCs was 10 渭 m after 48 hours of exposure to the drug concentration of 5-HT (100 渭 M). Compared with the control group, the results showed that the concentration of 5-HT was 10 渭 m. When the concentration of 5-HT was 1000 渭 M, the apoptotic rate was 21.42 渭 M, and the apoptosis rate increased with the increase of the concentration of 100 渭 M and 1000 渭 M (P0.05). Conclusion: 5-HT has apoptotic effect on the proliferation of human BMSCs in vitro. The longer the 5-HT is, the lower the cell survival rate is, and the higher the concentration of 5-HT is, the higher the apoptosis rate is. Moreover, in the low nutrition environment of serum-free, the effect of apoptosis in the late stage of 5-HT was more obvious.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329
【参考文献】
相关期刊论文 前5条
1 沈亚莉;徐酉华;罗庆;郭振华;郑改焕;郭玉霞;;wnt5a基因修饰bMSCs对HL60细胞生长分化影响的研究[J];四川大学学报(医学版);2010年06期
2 李小川,李雷,王欢,崔少千,白伦浩;Fas/FasL基因在退变腰椎间盘组织中的表达及诱导凋亡作用[J];中华医学杂志;2005年24期
3 赵梓汝;吴小涛;祁亚斌;付文芹;王刚;;TGF-β1干预下体内兔骨髓间充质干细胞对退变椎间盘治疗的实验研究[J];中国矫形外科杂志;2006年13期
4 易岂建,杨默,庞雅轩,李桂霞,夏文杰,霍泰辉,宋银子;五羟色胺促进骨髓基质细胞生长初探[J];重庆医科大学学报;2005年02期
5 张琦;钱济先;文艳华;裘秀春;马保安;范清宇;;SOX9基因真核表达载体转染诱导BMSCs分化为类髓核细胞[J];中国修复重建外科杂志;2010年07期
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