【摘要】：目的：研究觀察不同劑量地塞米松誘導(dǎo)成骨細(xì)胞調(diào)亡形態(tài)、DNA斷裂及caspase-3表達(dá)產(chǎn)生的影響；并探討地塞米松誘導(dǎo)成骨細(xì)胞凋亡的機(jī)制。 實(shí)驗(yàn)方法：⑴培養(yǎng)并保存MC3T3-E1小鼠成骨細(xì)胞。⑵取對(duì)數(shù)生長(zhǎng)期中的MC3T3-E1小鼠顱骨成骨細(xì)胞進(jìn)行分組。實(shí)驗(yàn)組：培養(yǎng)基中加入不同濃度(10~(-8)、10~(-7)、10~(-6)、10~(-5)、10~(-4)mol/L)地塞米松；拮抗劑組：于10~(-4)mol/LDex中加入10~(-5)mol/L RU486；空白對(duì)照組：不設(shè)干預(yù)。⑶三組細(xì)胞共同培養(yǎng)72小時(shí)后，分別留置細(xì)胞行MTT比色試驗(yàn)檢測(cè)細(xì)胞存活率與殺傷率；應(yīng)用熒光染料Hoechst33258染色觀察細(xì)胞凋亡形態(tài)及數(shù)量；留置細(xì)胞行流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡率；留置細(xì)胞行底物酶檢測(cè)技術(shù)檢測(cè)caspase-3的活性。 結(jié)果：⑴MTT比色試驗(yàn)結(jié)果顯示：與空白對(duì)照組及拮抗劑組相比，實(shí)驗(yàn)組細(xì)胞隨著加入地塞米松藥物濃度的增加，，細(xì)胞生長(zhǎng)抑制作用明顯增強(qiáng)，殺傷率增高尤以加入地塞米松濃度為10~(-5)-10~(-4)mol/L時(shí)，細(xì)胞殺傷率最高。 ⑵熒光染色法觀察細(xì)胞凋亡形態(tài)：與空白對(duì)照組及拮抗劑組比較，加入Hoechst33258熒光劑后，隨著地塞米松干預(yù)濃度的增加，熒光顯微鏡下觀察實(shí)驗(yàn)組細(xì)胞其細(xì)胞核內(nèi)出現(xiàn)的濃染致密的顆粒狀亮藍(lán)色熒光逐漸數(shù)量增多亮度增強(qiáng)。說明Dex對(duì)MC3T3-E1細(xì)胞的促進(jìn)凋亡作用逐漸增強(qiáng)。 ⑶流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡率結(jié)果：與空白組及拮抗組相比，實(shí)驗(yàn)組隨著地塞米松干預(yù)濃度的增高，細(xì)胞凋亡加劇，凋亡率逐漸增高并呈現(xiàn)濃度依賴性。 ⑷酶聯(lián)免疫測(cè)定實(shí)驗(yàn)(ELISA)檢測(cè)caspase-3活性結(jié)果：各濃度Dex誘導(dǎo)MC3T3-E1細(xì)胞時(shí)，其caspase-3活性表達(dá)均隨Dex濃度增加而增加，并均強(qiáng)于空白對(duì)照組；在相同濃度Dex誘導(dǎo)時(shí)，caspase-3活性表達(dá)明顯強(qiáng)于加入GR拮抗劑組。 結(jié)論：1、DEX能夠促進(jìn)小鼠成骨細(xì)胞凋亡；2、隨著DEX干預(yù)濃度的增加，小鼠成骨細(xì)胞凋亡率增高；3、隨著DEX濃度的增加，小鼠成骨細(xì)胞內(nèi)caspase-3活性增高；4、DEX可能通過激活caspase-3途徑誘導(dǎo)并促進(jìn)小鼠成骨細(xì)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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R96;R-332

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