镁离子对体外培养血管平滑肌细胞增殖和迁移的影响

发布时间：2018-05-02 22:51

本文选题：镁离子 + 血管平滑肌细胞　；参考：《河北医科大学》2007年硕士论文

【摘要】： 目的:镁在细胞内液中含量仅次于钾,是调节机体循环系统功能的重要金属元素,参与许多重要酶系如激酶、环化酶、腺苷三磷酸、鸟嘌呤三磷酸等的生化反应。同时作为钙离子的天然拮抗剂,具有调节血管舒缩强度、降低动脉压力及改善外周血液循环的作用。因此镁对心血管系统如心脏舒缩功能,心脏节律调节,血管紧张度及血管平滑肌的增殖能够产生重要影响。有研究表明细胞浆液中的镁只占全身镁总量的很少一部分,主要以蛋白结合形式、螯合形式及离子形式存在,其中离子形式是具有生物活性的形式。在循环中具有生物活性的镁离子能够对血管内膜及血管功能产生积极的影响。本实验通过镁离子对体外培养血管平滑肌细胞的干预作用,探讨其与平滑肌细胞增殖和迁移的相互关系,从而研究镁离子对血管内膜损伤的保护和修复作用。方法:SD大鼠主动脉血管平滑肌细胞(VSMC)的培养。SD大鼠颈椎脱臼法处死后,无菌条件下取出胸主动脉,将血管平滑肌层切割成1.0mm3的小块采用组织块贴壁法进行原代培养。实验第一部分:1、以体外培养的大鼠血管平滑肌细胞为实验对象,分为对照组和实验组,其中实验组根据镁离子浓度不同分为2mmol/L、4mmol/L、8mmol/L、12mmol/L及16mmol/L组,各组细胞在0.5%血清培养液, 37℃、50ml/LCO2环境中培养48小时后用CCK-8法分析细胞增殖活力。2、将平滑肌细胞分为对照组及实验组,实验组加入含4mmol/L镁离子的0.5%血清培养液,对照组加入等量的0.5%血清培养液,37℃、50ml/LCO2条件下培养,分别于24h、48h、72h后用CCK-8法分析细胞增殖活力。3、流式细胞仪(flow cytometry, FCM)检测细胞增殖周期。将体外培养的血管平滑肌细胞分为对照组及实验组,实验组加入含4mmol/L镁离子的0.5%血清培养液,对照组加入等量的0.5%血清培养液, 37℃、50ml/LCO2条件下培养48h,经消化离心等处理后用Epics-XLⅡ型流式细胞仪测定细胞周期。实验第二部分:将血管平滑肌细胞分为对照组、FN组及Mg2+-FN组,应用改良的boyden chamber小室,分析镁离子干预后的血管平滑肌细胞迁移情况,并在显微镜下对迁移细胞进行计数。结果:在实验第一部分,经镁离子作用体外培养的平滑肌细胞CCK-8值明显高于对照组(8mmol/L Mg2+组:0.805±0.021,对照组:0.534±0.081, P0.01),并且在一定范围内(0-8mmol/L)呈剂量依存效应;但当镁离子浓度过高时(12mmol/L),CCK-8值开始下降(0.742±0.040),提示细胞生长出现减缓趋势。在对镁离子作用下细胞增殖的时间相关性研究中发现,镁离子对血管平滑肌细胞增殖的促进作用具有时间依存性,细胞增殖程度随镁离子作用时间的延长而增加。流式细胞分析显示通过镁离子的干预作用,血管平滑肌细胞G1期细胞比例减少(65.15%下降至46.66%),S期细胞比例增加(21.49%上升为25.25%),提示细胞处于增殖状态。在实验第二部分,通过应用改良的boyden chamber小室进行细胞迁移实验发现Mg2+-FN组迁移细胞数量(Mg2+-FN组:53.78±8.05,P0.01)明显超过FN组(FN组:40.39±6.60)及对照组(对照组:25.17±4.67),可以推断镁离子能够促进体外培养血管平滑肌细胞的迁移。结论:通过本次实验证明镁离子能够促进体外培养的血管平滑肌细胞增殖,并且这种促进作用存在剂量-时间依存关系,同时镁离子对体外培养血管平滑肌细胞的迁移也具有积极的推动作用,从而可以推断镁离子对血管内膜的损伤具有一定的保护和修复作用。
[Abstract]:Objective: magnesium is second only to potassium in the intracellular liquid. It is an important metal element that regulates the function of the organism's circulatory system. It participates in many important enzymes such as kinase, cyclase, adenosine three phosphoric acid, guanine three phosphoric acid and other biochemical reactions. Meanwhile, as a natural antagonist of calcium ion, it can regulate vascular systolic and contractile intensity, reduce arterial pressure and improve The effect of magnesium on the cardiovascular system, such as cardiac contractile and contractile function, cardiac rhythm regulation, vascular tension and vascular smooth muscle proliferation, can have important effects on the cardiovascular system. Some studies have shown that magnesium in the cell size is only a small part of the total amount of magnesium, mainly in the form of protein binding, chelation and ion forms. In this experiment, the ionic form is a bioactive form. The bioactive magnesium ions in the circulation can have a positive effect on the vascular intima and vascular function. The interaction of magnesium ions on the culture of vascular smooth muscle cells in vitro was studied in this experiment, and the relationship between the proliferation and migration of smooth muscle cells was investigated. Protective and repairing effects of magnesium ions on intimal injury.
Methods: after the SD rat aorta vascular smooth muscle cells (VSMC) were cultured, the.SD rats were killed by the dislocated cervical vertebra. The thoracic aorta was removed under aseptic conditions and the vascular smooth muscle layer was cut into the small block of 1.0mm3 by the tissue block adherence method for primary culture.
The first part of the experiment: 1, the rat vascular smooth muscle cells cultured in vitro were divided into the control group and the experimental group. The experimental group was divided into 2mmol/L, 4mmol/L, 8mmol/L, 12mmol/L and 16mmol/L groups according to the different concentration of magnesium ions. The cells in each group were analyzed by CCK-8 in the 0.5% serum culture solution, 37, and 50ml/LCO2 environment. The cell proliferation activity was.2. The smooth muscle cells were divided into the control group and the experimental group. The experimental group was added to the 0.5% serum culture medium containing 4mmol/L magnesium ion. The control group was added to the same amount of 0.5% serum culture solution, 37 and 50ml/LCO2, and the cell proliferation activity.3 was analyzed by CCK-8 method after 24h, 48h, 72h, and the flow cytometry (flow cytometry, FCM) was used. The cell proliferation cycle was measured. The cultured vascular smooth muscle cells were divided into the control group and the experimental group. The experimental group was added to the 0.5% serum culture medium containing 4mmol/L magnesium ion. The control group was added to the same amount of 0.5% serum culture solution, 37 C and 50ml/LCO2 under the condition of 48H, and the cell cycle was measured by Epics-XL II flow cytometry after digestion and centrifugation. Period.
The second part of the experiment: the vascular smooth muscle cells were divided into control group, FN group and Mg2+-FN group, and the modified Boyden chamber chamber was used to analyze the migration of vascular smooth muscle cells after the magnesium ion dry, and the number of migratory cells was counted under the microscope.
Results: in the first part of the experiment, the CCK-8 value of smooth muscle cells cultured in vitro by magnesium ion was significantly higher than that of the control group (8mmol/L Mg2+ group: 0.805 + 0.021, 0.534 + 0.081, P0.01), and a dose dependent effect in a certain range (0-8mmol/L), but when the concentration of magnesium ions was too high (12mmol/L), the CCK-8 value began to decline (0.742 + 0.040). In the study of the time dependence of cell proliferation under the action of magnesium ion, it was found that the promoting effect of magnesium ions on the proliferation of vascular smooth muscle cells was time dependent, and the proliferation of cells increased with the prolongation of the time of action of magnesium ions. Flow cytometry showed the intervention of magnesium ion. The percentage of G1 phase cells in vascular smooth muscle cells decreased (65.15% to 46.66%), and the proportion of S phase cells increased (21.49% to 25.25%), suggesting that the cells were in proliferative state.
In the second part of the experiment, the number of migratory cells in group Mg2+-FN (group Mg2+-FN: 53.78 + 8.05, P0.01) was obviously higher than that of group FN (group FN: 40.39 + 6.60) and control group (control group: 25.17 + 4.67) in the cell migration test of the modified Boyden cell (Mg2+-FN group, P0.01). It can be concluded that the magnesium ion can promote the transfer of vascular smooth muscle cells in vitro.
Conclusion: this experiment shows that magnesium ions can promote the proliferation of vascular smooth muscle cells in vitro, and this promotion has a dose time dependence. Meanwhile, magnesium ions can also promote the migration of vascular smooth muscle cells in vitro. It can be deduced from the effect of magnesium ions on the vascular intima damage. Certain protective and repair functions.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329.2

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