本文選題：鎂離子 + 血管平滑肌細(xì)胞��； 參考：《河北醫(yī)科大學(xué)》2007年碩士論文

【摘要】： 目的:鎂在細(xì)胞內(nèi)液中含量僅次于鉀,是調(diào)節(jié)機(jī)體循環(huán)系統(tǒng)功能的重要金屬元素,參與許多重要酶系如激酶、環(huán)化酶、腺苷三磷酸、鳥嘌呤三磷酸等的生化反應(yīng)。同時作為鈣離子的天然拮抗劑,具有調(diào)節(jié)血管舒縮強(qiáng)度、降低動脈壓力及改善外周血液循環(huán)的作用。因此鎂對心血管系統(tǒng)如心臟舒縮功能,心臟節(jié)律調(diào)節(jié),血管緊張度及血管平滑肌的增殖能夠產(chǎn)生重要影響。有研究表明細(xì)胞漿液中的鎂只占全身鎂總量的很少一部分,主要以蛋白結(jié)合形式、螯合形式及離子形式存在,其中離子形式是具有生物活性的形式。在循環(huán)中具有生物活性的鎂離子能夠?qū)ρ軆?nèi)膜及血管功能產(chǎn)生積極的影響。本實(shí)驗(yàn)通過鎂離子對體外培養(yǎng)血管平滑肌細(xì)胞的干預(yù)作用,探討其與平滑肌細(xì)胞增殖和遷移的相互關(guān)系,從而研究鎂離子對血管內(nèi)膜損傷的保護(hù)和修復(fù)作用。 方法:SD大鼠主動脈血管平滑肌細(xì)胞(VSMC)的培養(yǎng)。SD大鼠頸椎脫臼法處死后,無菌條件下取出胸主動脈,將血管平滑肌層切割成1.0mm3的小塊采用組織塊貼壁法進(jìn)行原代培養(yǎng)。 實(shí)驗(yàn)第一部分:1、以體外培養(yǎng)的大鼠血管平滑肌細(xì)胞為實(shí)驗(yàn)對象,分為對照組和實(shí)驗(yàn)組,其中實(shí)驗(yàn)組根據(jù)鎂離子濃度不同分為2mmol/L、4mmol/L、8mmol/L、12mmol/L及16mmol/L組,各組細(xì)胞在0.5%血清培養(yǎng)液, 37℃、50ml/LCO2環(huán)境中培養(yǎng)48小時后用CCK-8法分析細(xì)胞增殖活力。2、將平滑肌細(xì)胞分為對照組及實(shí)驗(yàn)組,實(shí)驗(yàn)組加入含4mmol/L鎂離子的0.5%血清培養(yǎng)液,對照組加入等量的0.5%血清培養(yǎng)液,37℃、50ml/LCO2條件下培養(yǎng),分別于24h、48h、72h后用CCK-8法分析細(xì)胞增殖活力。3、流式細(xì)胞儀(flow cytometry, FCM)檢測細(xì)胞增殖周期。將體外培養(yǎng)的血管平滑肌細(xì)胞分為對照組及實(shí)驗(yàn)組,實(shí)驗(yàn)組加入含4mmol/L鎂離子的0.5%血清培養(yǎng)液,對照組加入等量的0.5%血清培養(yǎng)液, 37℃、50ml/LCO2條件下培養(yǎng)48h,經(jīng)消化離心等處理后用Epics-XLⅡ型流式細(xì)胞儀測定細(xì)胞周期。 實(shí)驗(yàn)第二部分:將血管平滑肌細(xì)胞分為對照組、FN組及Mg2+-FN組,應(yīng)用改良的boyden chamber小室,分析鎂離子干預(yù)后的血管平滑肌細(xì)胞遷移情況,并在顯微鏡下對遷移細(xì)胞進(jìn)行計(jì)數(shù)。 結(jié)果:在實(shí)驗(yàn)第一部分,經(jīng)鎂離子作用體外培養(yǎng)的平滑肌細(xì)胞CCK-8值明顯高于對照組(8mmol/L Mg2+組:0.805±0.021,對照組:0.534±0.081, P0.01),并且在一定范圍內(nèi)(0-8mmol/L)呈劑量依存效應(yīng);但當(dāng)鎂離子濃度過高時(12mmol/L),CCK-8值開始下降(0.742±0.040),提示細(xì)胞生長出現(xiàn)減緩趨勢。在對鎂離子作用下細(xì)胞增殖的時間相關(guān)性研究中發(fā)現(xiàn),鎂離子對血管平滑肌細(xì)胞增殖的促進(jìn)作用具有時間依存性,細(xì)胞增殖程度隨鎂離子作用時間的延長而增加。流式細(xì)胞分析顯示通過鎂離子的干預(yù)作用,血管平滑肌細(xì)胞G1期細(xì)胞比例減少(65.15%下降至46.66%),S期細(xì)胞比例增加(21.49%上升為25.25%),提示細(xì)胞處于增殖狀態(tài)。 在實(shí)驗(yàn)第二部分,通過應(yīng)用改良的boyden chamber小室進(jìn)行細(xì)胞遷移實(shí)驗(yàn)發(fā)現(xiàn)Mg2+-FN組遷移細(xì)胞數(shù)量(Mg2+-FN組:53.78±8.05,P0.01)明顯超過FN組(FN組:40.39±6.60)及對照組(對照組:25.17±4.67),可以推斷鎂離子能夠促進(jìn)體外培養(yǎng)血管平滑肌細(xì)胞的遷移。 結(jié)論:通過本次實(shí)驗(yàn)證明鎂離子能夠促進(jìn)體外培養(yǎng)的血管平滑肌細(xì)胞增殖,并且這種促進(jìn)作用存在劑量-時間依存關(guān)系,同時鎂離子對體外培養(yǎng)血管平滑肌細(xì)胞的遷移也具有積極的推動作用,從而可以推斷鎂離子對血管內(nèi)膜的損傷具有一定的保護(hù)和修復(fù)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.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R329.2

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