本文選題：活性氧自由基 + 鈣內(nèi)流��； 參考：《蘭州大學(xué)》2008年碩士論文

【摘要】： 本文通過正常細(xì)胞(HL-7702)和腫瘤細(xì)胞(Raji)的體外實(shí)驗(yàn)，研究鎘誘導(dǎo)下活性氧自由基(ROS)堆積，細(xì)胞內(nèi)鈣離子變化和細(xì)胞凋亡之間的關(guān)系。細(xì)胞凋亡水平通過Hoechst33258染色實(shí)驗(yàn)來檢測。細(xì)胞內(nèi)鈣離子變化和細(xì)胞內(nèi)活性氧自由基堆積分別采用熒光染料Flu-3/AM和DCFH-DA染色后通過流式細(xì)胞儀檢測熒光強(qiáng)度來獲得。在實(shí)驗(yàn)中我們加入了可以專一螯合細(xì)胞外鈣離子的EGTA(20mM)和能進(jìn)入細(xì)胞內(nèi)螯合細(xì)胞內(nèi)鈣離子的BAPTA-AM(10μM)處理細(xì)胞，以研究在鎘離子誘導(dǎo)下細(xì)胞內(nèi)濃度明顯上升的鈣離子主要來源細(xì)胞內(nèi)還是細(xì)胞外；通過加入BAPTA-AM(10μM)和可以清除細(xì)胞內(nèi)ROS的NAC(15mM)后檢測細(xì)胞凋亡率變化，，以進(jìn)一步研究細(xì)胞內(nèi)鈣離子和ROS堆積在細(xì)胞凋亡途徑中的作用。本實(shí)驗(yàn)結(jié)果發(fā)現(xiàn)HL-7702和Raji細(xì)胞在用鎘處理以后細(xì)胞凋亡率會明顯上升，在提高了細(xì)胞凋亡水平的同時(shí)，細(xì)胞內(nèi)鈣離子濃度和ROS堆積也明顯增加。當(dāng)細(xì)胞內(nèi)鈣離子和ROS被清除以后，細(xì)胞凋亡率也顯著下降。BAMTA/AM可以降低了由鎘誘導(dǎo)產(chǎn)生的細(xì)胞鈣離子升高(p0．001)，ROS積累增加(p0．001)和細(xì)胞凋亡水平升高(p0．001)等效應(yīng)。EGTA可以降低了由鎘離子誘導(dǎo)產(chǎn)生的細(xì)胞內(nèi)鈣離子升高(pO．01)，ROS積累增加(p0．001)但是不能降低細(xì)胞凋亡水平。以上結(jié)果證明了由鎘誘導(dǎo)的細(xì)胞內(nèi)鈣離子上升的鈣離子主要來源細(xì)胞內(nèi)(HL-7702細(xì)胞中約占78％，Raji細(xì)胞中約占59％)，另外少部分來源細(xì)胞外。實(shí)驗(yàn)結(jié)果同時(shí)顯示，在鎘離子的誘導(dǎo)下細(xì)胞凋亡和ROS堆積(HL-7702細(xì)胞中r=0．8781；Raji細(xì)胞中r=0．6919)和細(xì)胞內(nèi)鈣離子濃度上升(HL-7702細(xì)胞中r=0．9073；Raji細(xì)胞中r=0．8692)均呈顯著正相關(guān)。而ROS堆積和細(xì)胞內(nèi)鈣離子濃度上升也呈顯著正相關(guān)(HL-7702細(xì)胞中r=0．8619；Raji細(xì)胞中r=0．9061)。通過本實(shí)驗(yàn)我們可以得出兩個(gè)結(jié)論：1)在鎘離子誘導(dǎo)下，細(xì)胞在6h出現(xiàn)的濃度上升的細(xì)胞內(nèi)鈣離子來源于細(xì)胞內(nèi)部；2)鎘離子誘導(dǎo)下細(xì)胞內(nèi)ROS積累和細(xì)胞內(nèi)鈣動員是兩個(gè)同時(shí)發(fā)生的事件，并且在細(xì)胞凋亡途徑中起關(guān)鍵作用。
[Abstract]:The relationship between the accumulation of reactive oxygen free radicals (Ros), intracellular calcium and apoptosis in normal cells (HL-7702) and tumor cells (Raji) induced by cadmium was studied in vitro. Apoptosis level was detected by Hoechst 33258 staining. The changes of intracellular calcium ions and the accumulation of reactive oxygen free radicals in cells were stained with fluorescent dye Flu-3 / AM and DCFH-DA, respectively. The fluorescence intensity was measured by flow cytometry. In our experiment, we added EGTA (20mm), which can specifically chelate extracellular calcium, and BAPTA-AM (10 渭 M), which could enter the intracellular chelate cells. In order to study the intracellular or extracellular sources of Ca ~ (2 +), BAPTA-AM (10 渭 M) and NAC (15mm) which can scavenge the intracellular Ros, the apoptosis rate of the cells was determined by the addition of BAPTA-AM (10 渭 M) and NAC (15mm) which could scavenge the intracellular Ros. To further study the role of intracellular calcium and Ros accumulation in apoptosis pathway. The results showed that the apoptosis rate of HL-7702 and Raji cells increased significantly after cadmium treatment, and the intracellular Ca ~ (2 +) concentration and Ros accumulation also increased. When intracellular calcium and Ros are removed, Apoptosis rate also decreased significantly. BAMTA / AM decreased the effects of cadmium induced increase in calcium ion accumulation (p0.001), increased Ros accumulation (p0.001) and apoptosis level (p0.001). Increased intracellular calcium (pO.01) increased Ros accumulation (p0.001) but did not decrease apoptosis. The results showed that the intracellular calcium ion increased by cadmium (about 59% of HL-7702 cells) and a small number of extracellular sources. The results also showed that there was a significant positive correlation between the apoptosis induced by cadmium ion and the accumulation of Ros in HL-7702 cells (rn 0.6919 in Raji cells) and the increase in intracellular Ca ~ (2 +) concentration (rn 0.9073) in Raji cells (HL-7702 cells). There was also a positive correlation between the accumulation of Ros and the increase of intracellular Ca ~ (2 +) concentration in HL-7702 cells. Through this experiment, we can draw two conclusions: 1) the intracellular calcium ion which appeared in the cell at 6 h after the cadmium ion induction came from the inside of the cell. 2) intracellular Ros accumulation and intracellular calcium mobilization are two simultaneous events induced by cadmium ions and play a key role in the apoptosis pathway.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R363

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