【摘要】：第一部分溴苯腈對(duì)SH-SY5Y細(xì)胞增殖與凋亡的影響 目的：探討溴苯腈對(duì)SH-SY5Y細(xì)胞增殖、凋亡的影響 方法：用噻唑藍(lán)(MTT)法檢測(cè)溴苯腈細(xì)胞毒性，根據(jù)細(xì)胞存活率篩選合適劑量濃度；以選定的溴苯腈劑量組，處理時(shí)間為24h，倒置顯微鏡下觀察細(xì)胞形態(tài)及數(shù)目變化；用Hoechst33342/PI雙染法和流式細(xì)胞術(shù)檢測(cè)溴苯腈對(duì)細(xì)胞凋亡形態(tài)及凋亡率的影響；用噻唑藍(lán)(MTT)法檢測(cè)溴苯腈分別處理24h和48h后對(duì)細(xì)胞抑制率及IC50的影響，用細(xì)胞計(jì)數(shù)法測(cè)定溴苯腈對(duì)SH-SY5Y細(xì)胞生長(zhǎng)曲線及倍增時(shí)間的影響。 結(jié)果：MTT法測(cè)定細(xì)胞存活率篩選的合適劑量濃度為0、10、50、100μmol/L。按確定的劑量組處理SH-SY5Y細(xì)胞24h，在倒置顯微鏡下觀察細(xì)胞形態(tài)發(fā)現(xiàn)，陰性對(duì)照組細(xì)胞呈多角形，胞體大，排列緊密，縫隙��；高劑量組細(xì)胞呈長(zhǎng)梭形狀改變，胞體變小，稀疏。溴苯腈對(duì)細(xì)胞凋亡形態(tài)及凋亡率的影響：陰性對(duì)照組細(xì)胞核染成淡藍(lán)色,形態(tài)飽滿，核膜完整，核質(zhì)著色深，密度均勻一致；10、50μmol/L處理組與陰性對(duì)照組比較未見異常；100μmol/L可見少量核皺縮變小，染色質(zhì)濃縮邊集，核形態(tài)多樣，熒光增強(qiáng)，可見少量形似塊狀的凋亡細(xì)胞碎片的特征。與陰性對(duì)照組比較，各劑量組溴苯腈對(duì)SH-SY5Y細(xì)胞早期和晚期凋亡率均無(wú)明顯影響，差異均無(wú)統(tǒng)計(jì)學(xué)意義（P＞0.05）。檢測(cè)細(xì)胞增殖活力發(fā)現(xiàn),與陰性對(duì)照組比較,染毒24h和48h后，50、100μmol/L溴苯腈組的細(xì)胞增殖抑制率均增加,差異有統(tǒng)計(jì)學(xué)意義(P＜0.05)；與24h各組比,處理48h后50、100μmol/L組的細(xì)胞增殖抑制率顯著增加,差異有統(tǒng)計(jì)學(xué)意義(P＜0.05)。不同濃度溴苯腈處理SH-SY5Y細(xì)胞24h和48h后，其IC50分別是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隨著時(shí)間的延長(zhǎng)而減小，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05)。生長(zhǎng)曲線實(shí)驗(yàn)結(jié)果顯示，隨著染毒劑量的增加，細(xì)胞數(shù)目開始增加的時(shí)間后延，細(xì)胞倍增時(shí)間增加，差異均有統(tǒng)計(jì)學(xué)意義(P＜0.05)；100μmol/L組在0～96h生長(zhǎng)曲線中細(xì)胞數(shù)目無(wú)明顯變化（P＞0.05）；各組細(xì)胞數(shù)目在溴苯腈處理24h開始出現(xiàn)差異，組間差異隨著培養(yǎng)時(shí)間延長(zhǎng)更加明顯，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05)。 結(jié)論：在本實(shí)驗(yàn)條件下，根據(jù)細(xì)胞數(shù)目形態(tài)、增殖抑制率、生長(zhǎng)曲線結(jié)果均提示，溴苯腈可抑制SH-SY5Y細(xì)胞的增殖；細(xì)胞凋亡形態(tài)及凋亡率未觀察到明顯變化。 第二部分NF-κB、MAPKs等在溴苯腈致SH-SY5Y細(xì)胞增殖抑制中表達(dá)及作用初探 目的：探討溴苯腈作用SH-SY5Y細(xì)胞后對(duì)NF-κB、MAPKs通路的影響。 方法：以0、10、50、100μmol/L溴苯腈分別處理SH-SY5Y細(xì)胞24h，蛋白免疫印跡法（Western blot）檢測(cè)NF-κB、IκBα、Bcl-2、XIAP、p-p38、p-JNK的表達(dá)；免疫細(xì)胞化學(xué)法（immunocytochemistry）檢測(cè)NF-κB、Cytc-c的表達(dá)。 結(jié)果：溴苯腈處理SH-SY5Y細(xì)胞24h，與陰性對(duì)照組比較,隨著染毒劑量的增加，NF-κB、p-p38、Cytc-c表達(dá)增加，IκBα、Bcl-2表達(dá)降低，差異均有統(tǒng)計(jì)學(xué)意義(P＜0.05)；XIAP、p-JNK表達(dá)與陰性對(duì)照組比較，差異無(wú)統(tǒng)計(jì)學(xué)意義（P＞0.05）。 結(jié)論：在本實(shí)驗(yàn)條件下，觀察到溴苯腈處理后，，有NF-κB、p38的激活、Bcl-2表達(dá)抑制等表現(xiàn)，其可能與抑制細(xì)胞的增殖有關(guān)。
[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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R114

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