發(fā)布時間：2018-08-20 07:40

【摘要】： 目的本實驗建立酒精誘導(dǎo)PC12細(xì)胞凋亡模型,應(yīng)用此模型觀察PC12細(xì)胞凋亡過程中神經(jīng)鞘磷脂循環(huán)相關(guān)酶活性及mRNA表達(dá)量的改變,分析該循環(huán)在神經(jīng)細(xì)胞凋亡中的作用,為進(jìn)一步研究神經(jīng)細(xì)胞的凋亡機(jī)制提供依據(jù)。 方法MTT法測定酒精對PC12細(xì)胞增殖的抑制作用；Hoechst33258染色熒光顯微鏡觀察PC12細(xì)胞凋亡形態(tài)學(xué)變化；DNA瓊脂糖凝膠電泳檢測細(xì)胞凋亡梯狀DNA條帶；RT-PCR法檢測酒精對PC12細(xì)胞SMS1、SMS2和N-SMase mRNA表達(dá)的影響；薄層層析方法測定SMS的活性；熒光分光光度法檢測N-SMase的活性。 結(jié)果MTT法結(jié)果顯示,去血清培養(yǎng)的PC12細(xì)胞24h,酒精濃度在100mmol/L、200 mmol／L、400 mmol/L和800 mmol/L時,細(xì)胞存活率分別是單純?nèi)パ宓?7.54%、70.73%、57.89%和51.70%,表現(xiàn)出較強(qiáng)的細(xì)胞增殖抑制作用,與去血清對照組比較差異顯著(P0.05),呈濃度依賴性。含10%胎牛血清的細(xì)胞培養(yǎng)組,酒精濃度達(dá)到200mmol/L時對細(xì)胞的增殖抑制作用不明顯,與正常培養(yǎng)組相比無顯著性差異(P0.05)。Hoechst 33258熒光染色觀察細(xì)胞核形態(tài)學(xué)變化,結(jié)果顯示,不同濃度的酒精作用PC12細(xì)胞24h,酒精處理組凋亡細(xì)胞增多,表現(xiàn)染色質(zhì)凝集,細(xì)胞核變小、核碎裂成碎片等典型細(xì)胞凋亡特征性變化,凋亡率隨著酒精濃度的增大而升高,去血清組的酒精濃度為100mmoL／L、200mmoL／L和300mmoL/L時,細(xì)胞凋亡率分別是19.21±3.2%(P0.05)、28.39±5.11%(P0.05)和38.68±4.28%(P0.01),呈劑量依賴關(guān)系。瓊脂糖凝膠電泳可見100-300mmoL／L濃度酒精處理組有不同程度的DNA斷裂,顯示凋亡細(xì)胞典型的梯狀DNA。RT-PCR檢測酒精對PC12細(xì)胞SMS和N-SMase基因在轉(zhuǎn)錄水平表達(dá)的影響,結(jié)果顯示,不同濃度酒精作用于PC12細(xì)胞0.5h,SMS1表達(dá)量無顯著變化,當(dāng)作用時間達(dá)1h和2h, SMS1表達(dá)量顯著增加,并呈劑量依賴性,而SMS2的mRNA表達(dá)則不受酒精作用的影響；不同濃度酒精作用PC12細(xì)胞0.5h和1h,N-SMase表達(dá)量無顯著變化,作用2h時表達(dá)升高。以NBD-神經(jīng)酰胺為底物,用薄層層析法檢測細(xì)胞內(nèi)總SMS活性,顯示不同濃度酒精作用PC12細(xì)胞2h,SMS活性隨酒精濃度增加而升高。熒光分光光度法檢測N-SMase的活性,顯示酒精作用PC12細(xì)胞0.5h時N-SMase活性變化不顯著(P0.05),當(dāng)作用時間達(dá)2h時酶活性升高,與去血清組相比差異顯著(P0.05)。 結(jié)論： 1.酒精可導(dǎo)致PC12細(xì)胞凋亡,凋亡率與酒精濃度呈正相關(guān)。 2.酒精可致PC12細(xì)胞SMS1和N-SMase的mRNA表達(dá)量增高,酶活性增強(qiáng)。
[Abstract]:Objective to establish PC12 cell apoptosis model induced by alcohol, observe the activity of neurilipid circulation-related enzymes and the expression of mRNA during apoptosis of PC12 cells, and analyze the role of this cycle in neuronal apoptosis. It provides evidence for further study of apoptosis mechanism of nerve cells. Methods the inhibitory effect of alcohol on the proliferation of PC12 cells was determined by MTT method. The morphological changes of apoptosis of PC12 cells were observed by fluorescence microscope with Hoechst33258 staining. DNA agarose gel electrophoresis was used to detect the ladder DNA bands of apoptosis. The effects of alcohol on the expression of SMS1, SMS2 and N-SMase mRNA in PC12 cells were detected by RT-PCR assay, the activity of SMS by TLC and the activity of N-SMase by fluorescence spectrophotometry. Results the results of MTT assay showed that the survival rate of PC12 cells cultured with serum free for 24 h and the concentration of alcohol at 100 mmol / L ~ 200 mmol / L for 400 mmol/L and 800 mmol/L were 87.54% and 57.89%, respectively, and 51.70%, respectively, showing a strong inhibitory effect on cell proliferation. Compared with the control group, the difference was significant (P0.05), in a concentration-dependent manner. In the culture group containing 10% fetal bovine serum, the inhibitory effect on cell proliferation was not obvious when alcohol concentration reached 200mmol/L, but there was no significant difference compared with the normal culture group (P0.05). Hoechst 33258 fluorescence staining was used to observe the morphological changes of the cell nucleus. The apoptosis rate of PC12 cells increased with the increase of alcohol concentration, chromatin agglutination, nuclear degeneration, nuclear fragmentation and fragmentation, when alcohol was treated with different concentration of alcohol for 24 h, and the apoptotic rate increased with the increase of alcohol concentration, and the apoptosis rate increased with the increase of alcohol concentration, and the apoptosis rate increased with the increase of alcohol concentration. In the serum free group, the apoptotic rate was 19.21 鹵3.2% (P0.05) 28.39 鹵5.11% (P0.05) and 38.68 鹵4.28% (P0.01) in a dose-dependent manner. Agarose gel electrophoresis showed that there was different degree of DNA fragmentation in the alcohol treated group with 100-300mmoL/L concentration. The typical ladder DNA.RT-PCR of apoptotic cells was used to detect the effect of alcohol on the expression of SMS and N-SMase genes in PC12 cells at the transcriptional level. There was no significant change in the expression of SMS1 in PC12 cells treated with different concentrations of alcohol for 0.5 h. When the exposure time was 1 h and 2 h, the expression of SMS1 was significantly increased in a dose-dependent manner, while the mRNA expression of SMS2 was not affected by alcohol. The expression of N-SMase in PC12 cells did not change significantly at 0. 5 h and 1 h after exposure to different concentrations of alcohol, but increased at 2 h. The total SMS activity of PC12 cells was detected by thin layer chromatography with NBD- ceramide as substrate. The results showed that the activity of PC12 cells increased with the increase of alcohol concentration. The activity of N-SMase was detected by fluorescence spectrophotometry. The results showed that the N-SMase activity of PC12 cells did not change significantly at 0.5 h after alcohol treatment (P0.05), but the enzyme activity increased when the time reached 2 h, which was significantly different from that of serum removal group (P0.05). Conclusion: 1. Alcohol can induce apoptosis of PC12 cells, and the apoptotic rate is positively correlated with alcohol concentration. 2. 2. Alcohol could increase the mRNA expression of SMS1 and N-SMase and increase the activity of enzyme in PC12 cells.
【學(xué)位授予單位】：河南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R363

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相關(guān)期刊論文 前1條

1 周濤,許百男,陳德蕙,闕海萍,林秋霞,呂雙紅,劉少君;PC12細(xì)胞分化的神經(jīng)元與離體培養(yǎng)的大鼠原代皮質(zhì)神經(jīng)元之間功能性突觸的形成[J];中華神經(jīng)科雜志;2005年03期

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本文編號：2192905