细胞生物学技术-细胞微量元素含量测定

细胞中微量元素含量测定
原子吸收光谱法测定细胞
中微量元素
原子吸收光谱分析
基本原理
基于从光源辐射出待测元素的特征谱线的光，通过试样蒸气时，被蒸气中待测元素的基态原子所吸收，根据特征谱线的光减弱的程度来测定试样中待测元素含量的方法。

共振线：在原子吸收光谱中能被基态原子吸收的谱线。

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2017/11/16单光束原子吸收光谱仪示意图
高压电源
读数器
放大器光电倍增管单色器光源
原子化器
分光系统燃料气载气检测系统
试样
光源（空心阴极灯）工作原理示意图
A = log I0
I
= k N l
光的吸收定律
I0 —光源所发射的待测元素“共振线”的强度；
A —吸光度；
I —被火焰中待测元素吸收后的透光强度；
k —原子吸收系数；
N —蒸汽中基态原子的浓度；
l —“共振线”所通过的火焰长度；
A = k C
（原子吸收光谱进行定量分析的基本公式）
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原子吸收光谱仪
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原子吸收分光光度计
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自动进样器
方法特点及应用
（1）干扰少准确度高
（2）仪器简单操作方便
（3）灵敏度高
（4）测定元素范围广
应用：痕量分析
冶金、地质、采矿、石油、化工、环境保护、医药卫生等
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实例
火焰原子吸收光谱法测定淋巴细胞中微量的铜和锌
1. 淋巴细胞的分离
抽取外用静脉血3.0 ml，以等渗氯化理溶液4׃1稀释后，以Ficoll分离液分离淋巴细胞，以转速2000 rpm，分离30 min。

将分离的淋巴细胞层吸出，加4倍等渗氯化锂后以转速1500 rpm，离心分离10 min，弃去上清液，加1.0 ml 超纯水轻轻振摇1 min，即加高渗氯化锂1.0 ml，振摇1 min，再加等渗氯化锂，清洗3遍，每次离心10 min。

用等渗氯化锂0.12 ml加入经离心沉淀的淋巴细胞中轻轻振摇，使其成为均匀的悬浮液，从中吸取0.02 ml计数，另吸出0.1 ml置于康氏管，加等量胃蛋白酶消化液置于37℃水浴，18 h后，加0.4％硝酸至2.0 ml标定，并按公式换算每个淋巴细胞的铜、锌浓度。

2. 标准加入法曲线制作
由于淋巴细胞内铜、锌的含量极低，且灵敏
度受到共存物的影响，使用标准加入法可以
对基体影响加以校正。

即在等量同一样品中
加入标准溶液使铜浓度分别为0、0.1、0.2、0.5、1.0、2.0 mg/ml，绘制标准加入法曲线。

线性方程Cu：Y＝0.021X + 0.001，r＝1.000；Zn：Y＝0.100X + 0.004，r＝0.9992。

3. 精密度
按上述方法步骤对同一份淋巴细胞样品进行8次平行试验，结果见表2。

4. 回收率
采用样品加标准测定其回收率，来验证方法的准确度。

回收率见表3。

5．应用
采用上述方法对克山病区、非病区正常人及潜在、慢性克山病人淋巴细胞内铜、锌进行了调查，结果见下表6。

Pro-inflammatory effects and oxidative stress in lung macrophages and epithelial cells
induced by ambient particulate matter
Environmental Pollution. 2013
Fig. 1. Illustration of PM-induced intracellular and extracellular toxicological mechanisms in the lung.
Fig. 2. Experimental procedure for PM preparation and cell exposure.
ICP-MS
Fig. 3. Cell viability [%] of A549 and RAW264.7 cells during 0e96 h incubation with PM10 samples from urban traffic and rural sites. Each measuring point shows mean standard deviation of one experiment in triplicate. Statistically significant difference between exposed and unexposed cells (*) p < 0.01, (**) p < 0.001, (***) p < 0.0001.
Fig. 4. D total GSH content [nmol/106 cells] of A549 and RAW264.7 cells after 0e96 h incubation with urban traffic and rural PM10. The data described as D total GSH content (measured values without control values), below the x-axis the graphic shows the control values. Each bar shows mean
standard deviation of one experiment in triplicate. Statistically significant differences between exposed and unexposed cells (*) p < 0.01, (**) p < 0.001, (***) p < 0.0001.
Fig. 5. SOD activity [mmol/min/106 cells] of A549 and RAW264.7 cells after 0-96 h incubation with urban traffic and rural PM10. The data described as D SOD activity (measured values without control values), below the x-axis the graphic shows the control values. Each bar shows mean standard deviation of one experiment in triplicate. Statistically significant differences between exposed and unexposed cells (*) p < 0.01, (**) p < 0.001, (***) p < 0.0001.
Fig. 6. CAT activity [nmol/min/106 cells] of A549 and RAW264.7 cells after 0-96 h incubation with urban traffic and rural PM10. The data described as D CAT activity (measured values without control values), below the x-axis the graphic shows the control values. Each bar shows mean standard deviation of one experiment in triplicate. Statistically significant differences between exposed and unexposed cells (*) p < 0.01, (**) p < 0.001, (***) p < 0.0001.
Fig. 7. IL-6 release [ng/ml] of A549 and RAW264.7 cells after 0-96 h incubation with urban traffic and rural PM10. The data described as D IL-6 release (measured values without control values), below the x-axis the graphic shows the control values. Each bar shows mean standard deviation of one experiment in triplicate. Statistically significant differences between exposed and unexposed cells (*) p < 0.01, (**) p < 0.001, (***) p < 0.0001.
Fig. 7. IL-8 and TNF-ɑrelease [ng/ml] of A549 and RAW264.7 cells after 0-96 h incubation with urban traffic and rural PM10. The data described as D IL-6 release (measured values without control values), below the x-axis the graphic shows the control values. Each bar shows mean standard deviation of one experiment in triplicate. Statistically significant differences between exposed and unexposed cells (*) p < 0.01, (**) p < 0.001, (***) p < 0.0001.。