镉诱导下活性氧自由基、细胞内钙离子和细胞凋亡三者间的关系

发布时间：2018-06-23 02:32

本文选题：活性氧自由基 + 钙内流　；参考：《兰州大学》2008年硕士论文

【摘要】： 本文通过正常细胞(HL-7702)和肿瘤细胞(Raji)的体外实验，研究镉诱导下活性氧自由基(ROS)堆积，细胞内钙离子变化和细胞凋亡之间的关系。细胞凋亡水平通过Hoechst33258染色实验来检测。细胞内钙离子变化和细胞内活性氧自由基堆积分别采用荧光染料Flu-3/AM和DCFH-DA染色后通过流式细胞仪检测荧光强度来获得。在实验中我们加入了可以专一螯合细胞外钙离子的EGTA(20mM)和能进入细胞内螯合细胞内钙离子的BAPTA-AM(10μM)处理细胞，以研究在镉离子诱导下细胞内浓度明显上升的钙离子主要来源细胞内还是细胞外；通过加入BAPTA-AM(10μM)和可以清除细胞内ROS的NAC(15mM)后检测细胞凋亡率变化，，以进一步研究细胞内钙离子和ROS堆积在细胞凋亡途径中的作用。本实验结果发现HL-7702和Raji细胞在用镉处理以后细胞凋亡率会明显上升，在提高了细胞凋亡水平的同时，细胞内钙离子浓度和ROS堆积也明显增加。当细胞内钙离子和ROS被清除以后，细胞凋亡率也显著下降。BAMTA/AM可以降低了由镉诱导产生的细胞钙离子升高(p0．001)，ROS积累增加(p0．001)和细胞凋亡水平升高(p0．001)等效应。EGTA可以降低了由镉离子诱导产生的细胞内钙离子升高(pO．01)，ROS积累增加(p0．001)但是不能降低细胞凋亡水平。以上结果证明了由镉诱导的细胞内钙离子上升的钙离子主要来源细胞内(HL-7702细胞中约占78％，Raji细胞中约占59％)，另外少部分来源细胞外。实验结果同时显示，在镉离子的诱导下细胞凋亡和ROS堆积(HL-7702细胞中r=0．8781；Raji细胞中r=0．6919)和细胞内钙离子浓度上升(HL-7702细胞中r=0．9073；Raji细胞中r=0．8692)均呈显著正相关。而ROS堆积和细胞内钙离子浓度上升也呈显著正相关(HL-7702细胞中r=0．8619；Raji细胞中r=0．9061)。通过本实验我们可以得出两个结论：1)在镉离子诱导下，细胞在6h出现的浓度上升的细胞内钙离子来源于细胞内部；2)镉离子诱导下细胞内ROS积累和细胞内钙动员是两个同时发生的事件，并且在细胞凋亡途径中起关键作用。
[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.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R363

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