【作者】 段蓉；

【导师】 丁国武； 郭红云；

【作者基本信息】 兰州大学 ， 劳动卫生与环境卫生学， 2008， 硕士

【摘要】 目的：本研究以黄河水中的有机污染物（Organic Pollutants）为研究对象，通过检测人乳腺癌MCF-7细胞生长增殖抑制率、细胞周期、端粒酶活性的表达和细胞内游离钙离子的浓度([Ca²⁺]i)，探讨有机污染物的毒性作用及其机制。方法：分别在黄河兰州段中山桥（污染断面）、什川桥（自净断面）和新城桥（清洁断面）三个断面进行采样，采用固相萃取法（SPE）对三个断面水样中的有机污染物进行富集萃取。然后将MCF-7细胞分为不加任何处理的对照组和经不同浓度的有机污染物处理的实验组，应用噻唑蓝（MTT）比色法检测不同浓度有机污染物对细胞增殖活性及IC₅₀的影响。在此基础上，利用流式细胞仪检测有机污染物处理细胞48h后细胞周期分布和细胞凋亡率；逆转录端粒重复序列扩增（PCR-TRAP）法检测有机污染物对MCF-7细胞端粒酶活性表达的影响；激光共聚焦显微镜法检测细胞内[Ca²⁺]i的浓度。结果：1、有机污染物对MCF-7细胞生长增殖的影响用SPE法提取中山桥、什川桥和新城桥三个断面水样中有机污染物的浓度分别为46．00mg·ml^-1、35．80mg·ml^-1、26．40mg·ml^-1；各断面有机污染物分别作用MCF-7细胞48h后，MTT检测结果表明，在0-0．50mg·ml^-1范围内，实验组MCF-7细胞的细胞增殖抑制率（CPIR）与对照组无明显差别，各组之间变化亦不明显，说明在此浓度范围内有机污染物对细胞生长增殖基本无抑制作用；在1．00-5．00mg·ml^-1范围内，各实验组的CPIR值均高于对照组（P<0．01）；不同浓度组中，中山桥组的CPIR值最高；经相关性分析，各实验组的有机污染物浓度与MCF-7的CPIR呈明显正相关（P<0．01），相关系数分别为0．903、0．766、0．820，从中得到中山桥、什川桥、新城桥三个断面的IC₅₀分别为3．37±0．02mg·ml^-1、4．03±0．01mg·ml^-1、4．34±0．03mg·ml^-1，说明相同剂量下，中山桥断面的有机污染物对细胞的增殖抑制作用更为明显。在10．00-40．00mg·ml^-1浓度，各实验组对细胞的生长抑制作用出现饱和，随有机污染物浓度的升高，其细胞生长增殖抑制率逐渐下降，中山桥组更加显著（P<0．01）。2、有机污染物对MCF-7细胞周期和凋亡的影响各断面的有机污染物以1／2IC₅₀处理MCF-7细胞48h后，各组细胞凋亡率明显高于对照组（P<0．01）；细胞凋亡率由高到低依次为中山桥组31．43±3．00％、新城桥组17．63±0．47％、什川桥组13．37±1．43％和8．10±0．25％，三个断面相互比较均有统计学意义（P<0．05）；不同断面各组的细胞周期分布显示：G₁期细胞均值百分比由高到低依次为：新城桥组84．47±1．67％、什川桥组77．86±0．14％、中山桥组55．03±3．63％，其百分比均大于55％，提示细胞主峰在G₁期积聚。3、有机污染物对MCF-7细胞端粒酶活性表达的影响PCR-TRAP法检测端粒酶的表达结果表明，不同断面有机污染物分别作用细胞24h、48h和72h后，不同时点各组的端粒酶灰度值与对照组相比显著降低（P<0．01）；随各自作用时间的延长，各组及相应对照组的端粒酶条带灰度值均降低，呈时间依赖性（P<0．05）。4、细胞内游离钙离子([Ca²⁺]i)浓度的检测本研究利用激光共聚焦显微镜检测细胞内[Ca²⁺]i的浓度结果显示，有机污染物分别作用MCF-7细胞24h、48h和72h后，可使各组不同时点细胞内[Ca²⁺]i浓度升高；随着各断面有机污染物作用时间的延长，细胞内[Ca²⁺]i的浓度随之降低，呈时间依赖性。含钙离子细胞外液组和不含钙离子细胞外液组之间在不同时点分别比较均有统计学差异（P<0．05）。实验中将检测时的无Ca²⁺Hank’s（D-Hank’s）缓冲液换成Hank’s缓冲液时，有机污染物诱导的MCF-7细胞质内[Ca²⁺]i浓度的升高被抑制，提示[Ca²⁺]i的增加不是来源于外钙内流。结论：黄河兰州段水体中有机污染物在一定浓度范围对细胞有损伤作用，初步探讨其毒作用机制可能有以下几个方面：（1）黄河兰州段水体中有机污染物在中剂量浓度时抑制人乳腺癌MCF-7细胞增殖，并诱导其凋亡。（2）TRAP-银染法可检测到MCF-7细胞端粒酶活性的表达，黄河兰州段水体中有机污染物在中剂量浓度时下调体外MCF-7细胞端粒酶活性的表达，其作用机制需进一步探讨。（3）黄河兰州段水体中有机污染物可诱导MCF-7细胞内[Ca²⁺]i浓度升高。随作用时间，其诱导[Ca²⁺]i浓度升高的作用减弱。更多还原

【Abstract】 Objective: The organic pollutants were extracted in Lanzhou reach of the Yellow River in this study, the cellular proliferation inhibition of MCF-7 cells, cell cycles, telomerase expression and free cytoplasmic calcium of cells ([Ca²⁺]i) concentration were observed with the extracted organic pollutants treated MCF-7 cells, the mechanisms and effects of the extracted organic pollutants on cell toxicity were investigated.Methods: Water samples were collected from three sections （the pollution section is Zhongshan bridge, the self-purification section is Shichuan bridge and the clean section is Xincheng bridge） in Lanzhou reach of the Yellow River and the trace organic pollutants were concentrated in samples by Solid-Phase Extraction （SPE） method. The proliferation of MCF-7 cells treated by different concentrations of the extracted organic pollutants for 48h at three sections by MTT assay, the 50% inhibitory concentrations (IC₅₀) of the extracted organic pollutants groups were measured. Based on the IC₅₀, assessment of cell cycles and apoptosis were performed by Flow cytometry; Telomerase activity expression was measured by the telomeric repeat amplification protocol （TRAP） method and [Ca²⁺]i concertration was detected by the Laser confocal microscope method.Results:（1） Analysis of the extracted organic pollutants on MCF-7 cells proliferation by MTT assaySPE method was used to extract the organic pollutants in Zhongshan, Shichuan and Xincheng bridge at three sections, the concentration of three sections is 46.00mg·ml^-1, 35.80mg·ml^-1 and 26.40mg·ml^-1 respectively. Compared with the control group, in the range of 0-0.50mg·ml^-1 no significant inhibitory effects was observed at three sections with the MTT detection, while in the range of 1.00-5.00mg·ml^-1 the cellular proliferation inhibitory effects of MCF-7 were observed, there was a linear correlation between CPIR of MCF-7 and different concentration （P<0.05）, with correlation coefficients （r²） is 0.903, 0.766 and 0.820 respectively, the IC50 in Zhongshan, Shichuan and Xincheng bridge were 3.37±0.02mg·ml^-1, 4.03±0.01mg·ml^-1, 4.34±0.03mg·ml^-1 respectively. It showed that the inhibitory effect in Zhongshan bridge was highest among three sections under the same concentration （P<0.05）. In the range of 10.00-40.00mg·ml^-1 the cellular proliferation inhibitory effects of MCF-7 declined, especially in Zhongshan bridge （P<0.01）.（2） Analysis of MCF-7 cell cycles and apoptosis in 48hCompared with the control group, the flow cytometry analysis showed that the apoptosis rates in MCF-7 cells [31.43±3.00% （Zhongshan bridge）, 17.63±0.47% （Xincheng bridge） and 13.37±1.43% （Shichuan bridge）] increased significantly after exposure to the extracted organic pollutants at the concentration of half IC₅₀ for 48h （P<0.01）. There have significant effects when compared with treated groups （P<0.05）. The cell cycles distribution revealed that the mean percentage of G₁ phase （84.47±3.63） in Xincheng bridge was the highest, secondly Shichuan bridge and Zhongshan bridge as compared with the control group （P<0.01）. The mean percent of G₁ was more than 55% at three sections, so cells were accumulated in the G₁ phase.（3） Influences of the extracted organic pollutants on telomerase activityCompared with the control group, telomerase expression declined in the organicpollutants group at three sections （P<0.01）; telomerase expression had declined in a time-dependent manner （P<0.05）.（4） Changes of free intracellular calcium ([Ca²⁺]i) concentrationThe [Ca²⁺]i concentrations were detected after the extracted organic pollutants treated MCF-7 cells for 24h, 48h and 72h by Laser scanning microscope （LSCM）, it showed that [Ca²⁺]i concentrations were increased in each group as compared with the control group in different times. Furthermore, [Ca²⁺]i concentration was decreased in a time-dependent manner. It was also found that there have significant changes between the calcic groups and non-calcium groups （P<0.05）. When the D-Hank’s buffer was changed into Hank’s buffer, [Ca²⁺]i was detected again, the increasing of cytoplasm [Ca²⁺]i had been inhibited. It suggested that the increasing was not derived from the flow of extracellular calcium. Conclusions:The extracted organic pollutants in Lanzhou reach of the Yellow River can lead to cells impairmed, apoptosis or death, the probable mechanisms were as follows:（1） The extracted organic pollutants in Lanzhou reach of the Yellow River inhibit MCF-7 cellular proliferation through blocking cell cycles and inducing apoptosis. （2）Expressions of telomerase are detected in human breast cancer MCF-7 cells by PCR-TRAP-silver staining method. The extracted organic pollutants may inhibit their expressions in vitro MCF-7 cells in Lanzhou reach of the Yellow River, but the inhibition is declining with the time, the mechanism needs further exploration. （3） The [Ca²⁺]i concentration in MCF-7 cells increased by the extracted organic pollutants with treated time it declined.更多还原