溴苯腈对SH-SY5Y细胞毒性及NF-κB、MAPKs通路的影响

发布时间：2019-06-03 14:04

【摘要】：第一部分溴苯腈对SH-SY5Y细胞增殖与凋亡的影响目的：探讨溴苯腈对SH-SY5Y细胞增殖、凋亡的影响方法：用噻唑蓝(MTT)法检测溴苯腈细胞毒性，根据细胞存活率筛选合适剂量浓度；以选定的溴苯腈剂量组，处理时间为24h，倒置显微镜下观察细胞形态及数目变化；用Hoechst33342/PI双染法和流式细胞术检测溴苯腈对细胞凋亡形态及凋亡率的影响；用噻唑蓝(MTT)法检测溴苯腈分别处理24h和48h后对细胞抑制率及IC50的影响，用细胞计数法测定溴苯腈对SH-SY5Y细胞生长曲线及倍增时间的影响。结果：MTT法测定细胞存活率筛选的合适剂量浓度为0、10、50、100μmol/L。按确定的剂量组处理SH-SY5Y细胞24h，在倒置显微镜下观察细胞形态发现，阴性对照组细胞呈多角形，胞体大，排列紧密，缝隙小；高剂量组细胞呈长梭形状改变，胞体变小，稀疏。溴苯腈对细胞凋亡形态及凋亡率的影响：阴性对照组细胞核染成淡蓝色,形态饱满，核膜完整，核质着色深，密度均匀一致；10、50μmol/L处理组与阴性对照组比较未见异常；100μmol/L可见少量核皱缩变小，染色质浓缩边集，核形态多样，荧光增强，可见少量形似块状的凋亡细胞碎片的特征。与阴性对照组比较，各剂量组溴苯腈对SH-SY5Y细胞早期和晚期凋亡率均无明显影响，差异均无统计学意义（P＞0.05）。检测细胞增殖活力发现,与阴性对照组比较,染毒24h和48h后，50、100μmol/L溴苯腈组的细胞增殖抑制率均增加,差异有统计学意义(P＜0.05)；与24h各组比,处理48h后50、100μmol/L组的细胞增殖抑制率显著增加,差异有统计学意义(P＜0.05)。不同浓度溴苯腈处理SH-SY5Y细胞24h和48h后，其IC50分别是267.00±29.54μmol/L，95%CI（193.06～340.40μmol/L)和54.33±8.08μmol/L，95%CI（34.25～74.41μmol/L)，IC50随着时间的延长而减小，差异有统计学意义(P＜0.05)。生长曲线实验结果显示，随着染毒剂量的增加，细胞数目开始增加的时间后延，细胞倍增时间增加，差异均有统计学意义(P＜0.05)；100μmol/L组在0～96h生长曲线中细胞数目无明显变化（P＞0.05）；各组细胞数目在溴苯腈处理24h开始出现差异，组间差异随着培养时间延长更加明显，差异有统计学意义(P＜0.05)。结论：在本实验条件下，根据细胞数目形态、增殖抑制率、生长曲线结果均提示，溴苯腈可抑制SH-SY5Y细胞的增殖；细胞凋亡形态及凋亡率未观察到明显变化。第二部分NF-κB、MAPKs等在溴苯腈致SH-SY5Y细胞增殖抑制中表达及作用初探目的：探讨溴苯腈作用SH-SY5Y细胞后对NF-κB、MAPKs通路的影响。方法：以0、10、50、100μmol/L溴苯腈分别处理SH-SY5Y细胞24h，蛋白免疫印迹法（Western blot）检测NF-κB、IκBα、Bcl-2、XIAP、p-p38、p-JNK的表达；免疫细胞化学法（immunocytochemistry）检测NF-κB、Cytc-c的表达。结果：溴苯腈处理SH-SY5Y细胞24h，与阴性对照组比较,随着染毒剂量的增加，NF-κB、p-p38、Cytc-c表达增加，IκBα、Bcl-2表达降低，差异均有统计学意义(P＜0.05)；XIAP、p-JNK表达与阴性对照组比较，差异无统计学意义（P＞0.05）。结论：在本实验条件下，观察到溴苯腈处理后，，有NF-κB、p38的激活、Bcl-2表达抑制等表现，其可能与抑制细胞的增殖有关。
[Abstract]:Effect of the first part of bromobenzonitrile on the proliferation and apoptosis of SH-SY5Y cells Objective: To study the effect of bromobenzonitrile on the proliferation and apoptosis of SH-SY5Y cells. Methods: The cytotoxicity of bromobenzonitrile was detected by MTT method, and the appropriate dose concentration was selected according to the cell survival rate; the selected bromobenzonitrile was selected. The effect of bromobenzonitrile on the morphology and apoptosis rate of the cells was examined by Hoechst33342/ PI double-staining and flow cytometry. The inhibition rate and the IC50 of bromobenzonitrile after 24 h and 48 h were measured by the method of Hoechst33342/ PI double-staining and flow cytometry. Effect of bromobenzonitrile on SH-SY5Y cell growth curve and doubling time by cell counting method Results: The appropriate dose of MTT assay for cell survival was 0,10,50,100. m mol/ L. The SH-SY5Y cells were treated with the determined dose group for 24 h, and the cell morphology was observed under the inverted microscope. The cells of the negative control group were polygonal, the body was large, the arrangement was tight, the gap was small, and the cells of the high-dose group were changed in the shape of the long shuttle and the cell body Small, sparse. The effect of bromobenzonitrile on the morphology and apoptosis rate of the cells: the nucleus of the negative control group was stained with light blue, the shape was full, the nuclear membrane was intact, the color of the core was deep and the density was uniform;10,50 & mu; mol/ L treatment group was not found to be abnormal compared with the negative control group; a small amount of 100 & mu; mol/ L was found The nuclear shrinkage is small, the chromatin condensation side set, the nuclear form is various, the fluorescence is enhanced, and a small amount of the like-shaped apoptotic cells can be seen. There was no significant difference in the early and late apoptotic rates of SH-SY5Y cells in each dose group compared with the negative control group (P> The results showed that the inhibition rate of cell proliferation was increased in 50 and 100. m u.mol/ L bromobenzene group after 24 h and 48 h after 24 h and 48 h, and the cell proliferation of 50 and 100. m mol/ L group was inhibited after 48 h. The rate was significantly increased and the difference was statistically significant (P < The IC50 was 267.00, 29.54. mu.mol/ L,95% CI (193.06-340.40. mu.mol/ L) and 54.33-8.08. m u.mol/ L,95% CI (34.25-74.41. mu.mol/ L), and the IC50 decreased with the extension of time and the difference was statistically significant (P < The results of the growth curve showed that with the increase of the dose, the number of cells increased, the number of cells increased, the number of cells increased, the difference was statistically significant (P <0.05), and the number of cells in the 100. mu.mol/ L group was not significantly changed in the 0-96h growth curve (P> 0.05); the number of cells in each group started to be different in the treatment of bromobenzonitrile for 24 hours, and the difference between the groups was more obvious with the increase of culture time, and the difference was statistically significant (P < The results showed that bromobenzonitrile could inhibit the proliferation of SH-SY5Y cells according to the number of cells, the inhibition rate of proliferation and the results of the growth curve. Obvious changes were observed. The second part of NF-SYB, MAPKs, and the like in bromobenzonitrile-induced SH-SY5Y cell proliferation Objective: To study the expression and effect of bromobenzonitrile in SH-SY5Y cells, and to investigate the expression of NF-SY5Y. Methods: The expression of NF-SY5Y cells, I, B, Bcl-2, XIAP, p-p38, p-JNK, and the expression of Bcl-2, XIAP, p-p38 and p-JNK were detected by Western blot respectively. Results: The expression of cyctc-c in the SH-SY5Y cells was treated with bromobenzonitrile. The expression of NF-SYB, p-p38, Cyc-c increased, and the expression of Bcl-2 and Bcl-2 decreased with the increase of the dose, and the expression of Bcl-2 decreased and the difference was significant (P <0.05); XIAP, Comparison of p-JNK expression with negative control group, poor Conclusion: Under the condition of this experiment, the activation of NF-B, p38 and the expression of Bcl-2 in the treatment of bromobenzonitrile were observed.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R114

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