地塞米松对小鼠成骨细胞凋亡影响及其机制的实验研究
发布时间：2018-11-08 10:12
【摘要】：目的：研究观察不同剂量地塞米松诱导成骨细胞调亡形态、DNA断裂及caspase-3表达产生的影响；并探讨地塞米松诱导成骨细胞凋亡的机制。 实验方法：⑴培养并保存MC3T3-E1小鼠成骨细胞。⑵取对数生长期中的MC3T3-E1小鼠颅骨成骨细胞进行分组。实验组：培养基中加入不同浓度(10~(-8)、10~(-7)、10~(-6)、10~(-5)、10~(-4)mol/L)地塞米松；拮抗剂组：于10~(-4)mol/LDex中加入10~(-5)mol/L RU486；空白对照组：不设干预。⑶三组细胞共同培养72小时后，分别留置细胞行MTT比色试验检测细胞存活率与杀伤率；应用荧光染料Hoechst33258染色观察细胞凋亡形态及数量；留置细胞行流式细胞术检测细胞凋亡率；留置细胞行底物酶检测技术检测caspase-3的活性。 结果：⑴MTT比色试验结果显示：与空白对照组及拮抗剂组相比，实验组细胞随着加入地塞米松药物浓度的增加，，细胞生长抑制作用明显增强，杀伤率增高尤以加入地塞米松浓度为10~(-5)-10~(-4)mol/L时，细胞杀伤率最高。 ⑵荧光染色法观察细胞凋亡形态：与空白对照组及拮抗剂组比较，加入Hoechst33258荧光剂后，随着地塞米松干预浓度的增加，荧光显微镜下观察实验组细胞其细胞核内出现的浓染致密的颗粒状亮蓝色荧光逐渐数量增多亮度增强。说明Dex对MC3T3-E1细胞的促进凋亡作用逐渐增强。 ⑶流式细胞术检测细胞凋亡率结果：与空白组及拮抗组相比，实验组随着地塞米松干预浓度的增高，细胞凋亡加剧，凋亡率逐渐增高并呈现浓度依赖性。 ⑷酶联免疫测定实验(ELISA)检测caspase-3活性结果：各浓度Dex诱导MC3T3-E1细胞时，其caspase-3活性表达均随Dex浓度增加而增加，并均强于空白对照组；在相同浓度Dex诱导时，caspase-3活性表达明显强于加入GR拮抗剂组。 结论：1、DEX能够促进小鼠成骨细胞凋亡；2、随着DEX干预浓度的增加，小鼠成骨细胞凋亡率增高；3、随着DEX浓度的增加，小鼠成骨细胞内caspase-3活性增高；4、DEX可能通过激活caspase-3途径诱导并促进小鼠成骨细胞凋亡。
[Abstract]:Aim: to investigate the effects of dexamethasone on apoptosis of osteoblasts, DNA fragmentation and caspase-3 expression in osteoblasts induced by dexamethasone, and to explore the mechanism of apoptosis of osteoblasts induced by dexamethasone. Methods: 1 osteoblasts of MC3T3-E1 mice were cultured and preserved. 2 osteoblasts of MC3T3-E1 mice in logarithmic growth period were divided into groups. Experimental group: dexamethasone (10 ~ (-8), 10 ~ (-7), 10 ~ (-6), 10 ~ (-5), 10 ~ (-4) mol/L) was added to the culture medium. Antagonist group: 10 ~ (-5) mol/L RU486; blank control group was added to 10 ~ (-4) mol/LDex: there was no intervention. 3 after 72 hours of co-culture of the cells in the three groups, the survival rate and the killing rate of the cells were detected by MTT colorimetric assay. Fluorescence dye Hoechst33258 staining was used to observe the morphology and number of apoptosis, flow cytometry was used to detect the apoptosis rate, and substrate enzyme assay was used to detect the activity of caspase-3. Results: the results of 1MTT colorimetric test showed that compared with the control group and the antagonist group, the cell growth inhibition of the experimental group increased with the increase of dexamethasone concentration. When the concentration of dexamethasone was 10 ~ (-5) -10 ~ (-4) mol/L, the cytotoxicity was the highest. 2the morphology of apoptosis was observed by fluorescence staining: compared with the control group and the antagonist group, the concentration of dexamethasone increased with the addition of Hoechst33258 fluorescence. Under fluorescence microscope, the number of dense granular bright blue fluorescence in the nucleus of the experimental group was gradually increased and the brightness was enhanced. The results showed that the effect of Dex on promoting apoptosis of MC3T3-E1 cells was increased gradually. 3The results of flow cytometry: compared with the control group and the antagonistic group, the apoptosis rate of the experimental group increased with the increase of dexamethasone concentration, and the apoptosis rate gradually increased in a concentration-dependent manner. 4 the results of caspase-3 activity detected by (ELISA): when MC3T3-E1 cells were induced by Dex, the expression of caspase-3 activity increased with the increase of Dex concentration, and was stronger than that of the control group. At the same concentration of Dex, the expression of caspase-3 was significantly higher than that of GR antagonist. Conclusion: (1) DEX can promote osteoblast apoptosis in mice; (2) with the increase of DEX concentration, the apoptosis rate of osteoblasts increases; (3) with the increase of DEX concentration, the activity of caspase-3 in osteoblasts increases. 4DEX may induce and promote the apoptosis of mouse osteoblasts by activating caspase-3 pathway.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R96;R-332
本文编号：2318161
[Abstract]:Aim: to investigate the effects of dexamethasone on apoptosis of osteoblasts, DNA fragmentation and caspase-3 expression in osteoblasts induced by dexamethasone, and to explore the mechanism of apoptosis of osteoblasts induced by dexamethasone. Methods: 1 osteoblasts of MC3T3-E1 mice were cultured and preserved. 2 osteoblasts of MC3T3-E1 mice in logarithmic growth period were divided into groups. Experimental group: dexamethasone (10 ~ (-8), 10 ~ (-7), 10 ~ (-6), 10 ~ (-5), 10 ~ (-4) mol/L) was added to the culture medium. Antagonist group: 10 ~ (-5) mol/L RU486; blank control group was added to 10 ~ (-4) mol/LDex: there was no intervention. 3 after 72 hours of co-culture of the cells in the three groups, the survival rate and the killing rate of the cells were detected by MTT colorimetric assay. Fluorescence dye Hoechst33258 staining was used to observe the morphology and number of apoptosis, flow cytometry was used to detect the apoptosis rate, and substrate enzyme assay was used to detect the activity of caspase-3. Results: the results of 1MTT colorimetric test showed that compared with the control group and the antagonist group, the cell growth inhibition of the experimental group increased with the increase of dexamethasone concentration. When the concentration of dexamethasone was 10 ~ (-5) -10 ~ (-4) mol/L, the cytotoxicity was the highest. 2the morphology of apoptosis was observed by fluorescence staining: compared with the control group and the antagonist group, the concentration of dexamethasone increased with the addition of Hoechst33258 fluorescence. Under fluorescence microscope, the number of dense granular bright blue fluorescence in the nucleus of the experimental group was gradually increased and the brightness was enhanced. The results showed that the effect of Dex on promoting apoptosis of MC3T3-E1 cells was increased gradually. 3The results of flow cytometry: compared with the control group and the antagonistic group, the apoptosis rate of the experimental group increased with the increase of dexamethasone concentration, and the apoptosis rate gradually increased in a concentration-dependent manner. 4 the results of caspase-3 activity detected by (ELISA): when MC3T3-E1 cells were induced by Dex, the expression of caspase-3 activity increased with the increase of Dex concentration, and was stronger than that of the control group. At the same concentration of Dex, the expression of caspase-3 was significantly higher than that of GR antagonist. Conclusion: (1) DEX can promote osteoblast apoptosis in mice; (2) with the increase of DEX concentration, the apoptosis rate of osteoblasts increases; (3) with the increase of DEX concentration, the activity of caspase-3 in osteoblasts increases. 4DEX may induce and promote the apoptosis of mouse osteoblasts by activating caspase-3 pathway.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R96;R-332
【参考文献】
相关期刊论文 前4条
1 马大龙;新细胞因子及细胞凋亡基因的发现与功能研究[J];北京大学学报(医学版);2002年05期
2 赵建宁,包倪荣,王与荣,吴苏稼,周利武,曾晓峰,王骏骅;地塞米松诱导原代培养的成骨样细胞凋亡的分子细胞学研究[J];医学研究生学报;2004年01期
3 苏欣;廖二元;罗湘杭;;甲状旁腺激素对MG63细胞OPG、OPGL及其相关因子的调节[J];中国骨质疏松杂志;2006年04期
4 孟凡东;隋承光;王晓华;王扬;蒋涛;付立业;姜又红;;体外培养的树突状细胞与细胞因子诱导的杀伤细胞相互作用的研究[J];中国老年学杂志;2006年06期
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