丁香醛代谢物对小鼠胚胎成纤维细胞及小鼠骨髓辐射损伤的防护作用

发布时间：2018-03-29 01:30

本文选题：丁香醛代谢物　切入点：辐射防护剂　出处：《安徽医科大学》2013年硕士论文

【摘要】：在人类生存环境中，电离辐射时刻威胁着人们的健康，人们一直在寻求有效的防护药物,目前广泛应用于临床且效果满意的辐射防护药物并不多,寻找高效低毒活性高，不良反应少的辐射防护剂变得日益重要。国内外对辐射防护剂的研究都比较重视，除含硫化合物外，，细胞生长因子、天然药物等以外，对细胞活素类，中草药及复方、雌激素的抗辐射作用研究也较多。天然化合物香兰素，研究证实其具有抗氧化和抗诱变活性，清除电离辐射产生的自由基，抑制射线诱导的DNA和染色体损伤等，但其作用浓度高，活性相对较低。本实验室筛选出辐射防护作用更好的香兰素衍生物--丁香醛，已证实，丁香醛清除电离辐射产生的自由基的效果更好，可降低DNA损伤，提高DNA损伤修复能力以及辐射细胞存活率。丁香醛合成方便，理化性质稳定，具有开发成为新型辐射防护药的价值。药物代谢动力学发现，丁香醛进入动物体内10～15min即可被机体吸收，达到血液循环的量明显低于给药剂量，在血浆里检测到大量的丁香酸和少量的丁香醇，我们推测丁香醛进入体内发挥辐射防护作用的是其主要代谢物--丁香酸或者两种代谢物（丁香酸和丁香醇的混合物，以下简称混合物）协同发挥作用。本课题将从药效学部分研究，利用细胞实验观察和比较丁香醛主要代谢物--丁香酸和混合物的辐射防护作用。利用动物实验对丁香醛的主要代谢物--丁香酸的放射性氧化损伤和放射性造血损伤的防护作用进行研究，为丁香醛的进一步临床应用提供实验依据。主要研究结果如下： 1.MTT方法观察丁香醛代谢物对辐射后MEFs细胞效应，结果显示：和0μmol/L组相比，各加药组的OD值明显增加（p0.01），说明丁香醛代谢物能有效减轻辐射对MEF细胞的损伤。 2.MTT方法观察丁香醛代谢物对照射后的MEFs细胞活力，结果显示：和IR组相比，各加药组的OD值明显增加（p0.01），说明丁香醛代谢物能够提高辐射后的MEF细胞活力。 3.细胞增殖实验观察丁香醛代谢物对照射后的MEFs细胞生长的影响，通过绘制细胞生长曲线显示，和IR组相比，丁香醛代谢物能促进照射后MEFs细胞增殖。 4.细胞克隆形成实验检测丁香醛代谢物对照射后的MEFs细胞克隆形成率的影响，结果显示：和IR组相比，各加药组都能够提高细胞克隆形成率，差异有统计学意义（p0.01），说丁香醛代谢物能提高照后MEFs细胞克隆形成率。 5．小鼠骨髓有核细胞计数，观察丁香酸对小鼠骨髓造血损伤的作用，与IR组相比较，差异有统计学意义（p0.01），说明丁香酸对照后小鼠骨髓有核细胞有一定的辐射保护作用，能够有效减轻小鼠骨髓造血损伤。 6.流式细胞术检测小鼠骨髓有核细胞凋亡，结果显示，与IR组相比较，差异有统计学意义（p0.01），说明丁香酸可降低辐射诱导的小鼠骨髓有核细胞凋亡。 7.照后取小鼠骨髓和脾脏组织，处理后光镜下观察各组动物的骨髓和脾脏的组织病理学变化，结果显示：和IR组相比，丁香酸组小鼠的骨髓和脾脏组织损伤明显减轻。 8.利用试剂盒检测丁香酸对照后小鼠抗氧化损伤作用，与IR组相比较，丁香酸组在照后3d和6d，SOD活性的恢复都较显著，差异有统计学意义(p0.01)，说明丁香酸可促进小鼠血清SOD活性的恢复，同时也降低了血清MDA的含量，与IR组比较有显著的差异(p0.01)，丁香酸能够增强细胞清除自由基的能力，减轻辐射诱发的膜脂质过氧化损伤程度，减轻细胞的辐射损伤。综上所述，丁香醛代谢物对MEFs细胞具有辐射损伤防护作用，丁香醛主要代谢物--丁香酸对辐射小鼠也具有抗氧化损伤和造血损伤防护作用，为丁香醛的进一步应用于临床提供实验依据。
[Abstract]:In the human survival environment, ionizing radiation, threatening people's health, people have been seeking the protection of effective drugs, radiation protection drugs currently used in clinical and the effect is not satisfactory, for high efficiency and low toxicity and high activity, less adverse reaction of the radiation protective agent becomes more and more important. The domestic and foreign research on radiation protection agent are more seriously, except sulphur compounds, cell growth factor, other than natural drugs, on cytokines, Chinese herbal medicine and compound, more research on anti radiation effect of estrogen.
Natural compounds vanillin, proved that it has antioxidant and antimutagenic activity, scavenging of ionizing radiation generated free radicals, inhibition of radiation-induced DNA and chromosomal damage, but the effect of high concentration of activity is relatively low. Our laboratory screened better radiation protective effect of vanillin derivatives -- syringaldehyde have confirmed that better effect of clove formaldehyde removal of ionizing radiation generated free radicals, can reduce DNA damage, improve the ability to repair DNA damage and radiation. The cell survival rate lilac aldehyde synthesis is convenient, stable physicochemical property, has developed as a new type of radiation protection drug found value. Pharmacokinetics of lilac aldehyde into animal body 10 ~ 15min can be absorbed by the body. Reach the blood circulation was significantly lower than the dosage, to a large number of syringic acid and a small amount of clove alcohol detection in the plasma, we speculate that syringaldehyde into play Radiation protection is the main metabolite -- syringic acid or two metabolites (Ding Xiangsuan and syringic alcohol mixture, hereinafter referred to as the synergistic mixture). This paper will study some research from the effect of radiation protective effect by cell experiment to observe and compare the main metabolites -- Ding Xiangsuan and lilac aldehyde mixture. The protective effect of the use of animal experiments the main metabolites of lilac aldehyde -- syringic acid radioactiveoxidative damage and radioactive hematopoietic injury were studied for further clinical application of syringaldehyde to provide experimental basis. The main results are as follows:
1.MTT method to observe the syringaldehyde metabolites on MEFs cells after radiation effect, the results show that compared with the 0 mol/L group, the OD values of each dosing group was significantly increased (P0.01), that syringaldehyde can effectively reduce the radiation of metabolites on MEF cell injury.
2.MTT method to observe the syringaldehyde metabolites on MEFs cell viability after irradiation, the results showed that compared with the IR group, the OD values of each dosing group was significantly increased (P0.01), that syringaldehyde metabolites can improve the viability of MEF cells after radiation.
3. cell proliferation experiments were conducted to observe the effects of syringal metabolites on the growth of MEFs cells after irradiation. By drawing cell growth curves, Ding Xiangquan metabolites could promote the proliferation of MEFs cells after irradiation compared with group IR.
4. cell clone formation experiment to detect the formation rate of syringaldehyde metabolites influence on MEFs cell clones after irradiation results show that compared with IR group, the experimental groups can improve cell colony formation rate, the difference was statistically significant (P0.01), said Ding Xiangquan metabolite MEFs after irradiation can improve the cell colony formation rate.
5. mice bone marrow nucleated cell count, observe the syringic acid injury of mouse bone marrow hematopoietic function, compared with the IR group, the difference was statistically significant (P0.01), indicates syringic acid in mouse bone marrow cells have some radiation protection effect, can effectively reduce the bone marrow hematopoietic damage in mice.
6. flow cytometry was used to detect the apoptosis of mouse bone marrow nucleated cells. The results showed that compared with group IR, the difference was statistically significant (P0.01), indicating that syringic acid could reduce the apoptosis of bone marrow nucleated cells induced by radiation.
The bone marrow and spleen tissues of mice after exposure to 7., changes of pathology were observed in animal bone marrow and spleen treatment under light microscope results showed that compared with the IR group, the bone marrow and spleen tissue injury mice syringic acid significantly reduced.
8. using the kit of syringic acid in mouse anti oxidative effect, compared with group IR, group 3D after irradiation of syringic acid and 6D, SOD activity recovery significantly, the difference was statistically significant (P0.01), indicates syringic acid can promote the activity of SOD in serum of mice recovered, while also reducing the content of the serum levels of MDA, there is a significant difference compared with IR group (P0.01), syringic acid can enhance the ability of the cells to eliminate free radicals, reducing lipid peroxidation induced by radiation damage, reduce the radiation injury of the cells.
In conclusion, Ding Xiangquan metabolites have protective effects on MEFs cells, and the main metabolite of Ding Xiangquan, syringic acid, has protective effects against oxidative damage and hematopoietic injury in radiation mice, providing experimental evidence for further application of syringaldehyde in clinic.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R818

【共引文献】