【摘要】：目的:探討在機(jī)械應(yīng)力誘導(dǎo)軟骨細(xì)胞凋亡的過(guò)程中,細(xì)胞內(nèi)活性氧(reactive oxygen species,ROS)與內(nèi)質(zhì)網(wǎng)應(yīng)激(endoplasmic reticulum stress,ERS)表達(dá)情況及兩者在此作用過(guò)程中的相互作用關(guān)系方法:分離培養(yǎng)人膝關(guān)節(jié)軟骨細(xì)胞,用應(yīng)力加載系統(tǒng)分別將培養(yǎng)的細(xì)胞加載0h,6h,12h,24h和48h,分別在各受力組中加入抗氧化劑N-乙酰半胱氨酸(NAC)和半胱氨酸蛋白酶12(caspase12)特異性抑制劑Z-ATAD-FMK作為抑制劑組,應(yīng)用RT-PCR檢測(cè)各組細(xì)胞中凋亡相關(guān)基因Bcl-2、Bcl-2相關(guān)x蛋白(Bcl,associated X protein,Bax)及caspase12 m RNA表達(dá)量;western blot檢測(cè)各組細(xì)胞中Caspase-12蛋白表達(dá)量;DCFH-DA法檢測(cè)細(xì)胞中ROS的含量;流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡率。結(jié)果:RT-PCR檢測(cè)6 h受力組Bax和caspase-12相對(duì)表達(dá)量增加,24 h達(dá)峰值,48 h有所回降,而B(niǎo)cl-2相對(duì)表達(dá)量6 h后開(kāi)始下降,12 h降到最低,24 h開(kāi)始回升;ROS含量結(jié)果檢測(cè)6h受力組表達(dá)量增加,24h達(dá)到峰值,48h有所回落;NAC抑制劑組ROS、Bax、caspase-12表達(dá)量均下降,Bcl-2升高。Z-ATAD-FMK抑制劑組caspase-12表達(dá)量下降,而ROS表達(dá)量不變,Western-Blot檢測(cè)caspase-12蛋白表達(dá)與相應(yīng)基因表達(dá)趨勢(shì)相一致。凋亡檢測(cè)結(jié)果與受力組caspase12表達(dá)趨勢(shì)相似:6 h受力組凋亡率開(kāi)始增加,24 h達(dá)峰值,48 h凋亡率有所回降;結(jié)論:機(jī)械應(yīng)力可激活內(nèi)質(zhì)網(wǎng)應(yīng)激途徑介導(dǎo)軟骨細(xì)胞凋亡,而且在此過(guò)程中,活性氧可能作為內(nèi)質(zhì)網(wǎng)應(yīng)激上游信號(hào)分子參與其中。
[Abstract]:Objective: to investigate the intracellular reactive oxygen species (reactive oxygen species,ROS) and endoplasmic reticulum stress (endoplasmic reticulum stress, in the process of mechanical stress-induced apoptosis of cartilage cells. The expression of ERS) and the interaction between them in this process: human knee cartilage cells were isolated and cultured. The cultured cells were loaded with stress loading system for 0 h, 6 h, 12 h, 24 h and 48 h, respectively. the cultured cells were loaded with stress loading system for 0 h, 6 h, 12 h, 24 h and 48 h, respectively. Antioxidants N-acetylcystine (NAC) and Z-ATAD-FMK, a specific inhibitor of cysteinase 12 (caspase12), were added to each stress group as inhibitors. RT-PCR was used to detect apoptosis-related gene Bcl-2, in each group. Bcl-2 associated x protein (Bcl,associated X protein,Bax) and caspase12 m RNA expression; The expression of Caspase-12 protein in each group was detected by western blot, the content of ROS in cells was detected by DCFH-DA assay, and the apoptosis rate was detected by flow cytometry. Results: the relative expression of Bax and caspase-12 in the stress group increased at 6 h by RT-PCR, reached the peak at 24 h and decreased at 48 h, while the relative expression of Bcl-2 began to decrease after 6 h, reached the lowest level at 12 h, and began to rise at 24 h. The results of ROS content showed that the expression increased at 6 h, reached the peak at 24 h, and decreased at 48 h. The expression of ROS,Bax,caspase-12 in NAC inhibitor group decreased and Bcl-2 increased. In Z-ATAD-FMK inhibitor group, the expression of caspase-12 decreased, but the expression of ROS remained unchanged. The expression of caspase-12 protein detected by Western-Blot was consistent with the corresponding gene expression trend. The expression trend of caspase12 in the stress group was similar to that in the stress group: the apoptosis rate began to increase at 6 h, reached the peak at 24 h, and decreased at 48 h. Conclusion: mechanical stress can activate endoplasmic reticulum stress pathway to mediated apoptosis of chondrocytes, and reactive oxygen species may be involved as upstream signal molecules of endoplasmic reticulum stress.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R684.3

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本文編號(hào)：2487048