量子点诱导Hela细胞凋亡分子机制的研究
发布时间：2018-11-25 06:58
【摘要】：研究目的： 1、通过研究硫化锌包被硒化镉量子点（CdSe/ZnS QDs）基本物理及光学特征，验证其是否为一种适用于生物医学的荧光染料。 2、通过检测CdSe/ZnS QDs及氯化镉（CdCl2）对Hela细胞活性及凋亡率的影响，探究CdSe/ZnS QDs是否具有细胞毒性作用。 3、通过比较空白对照组、CdSe/ZnS QDs组及CdCl2组Hela细胞内ROS产量，探究CdSe/ZnS QDs诱导Hela细胞凋亡可能的机制。 4、通过对凋亡相关蛋白的检测，进一步阐述CdSe/ZnS QDs诱导细胞凋亡的机制。 方法： 本实验采用透射电子显微镜（Transmission electron microscope，TEM）、紫外分光光度计（Ultraviolet spectrophotometer）及荧光光谱仪（Fluorescencespectrometer）对CdSe/ZnS QDs进行表征。透射电子显微镜用于了解CdSe/ZnSQDs的大小、形状、外观均一程度，紫外分光光度计用于检测CdSe/ZnS QDs的吸收光波长范围，荧光光度计用于检测CdSe/ZnS QDs所发射荧光波长范围。 在建立稳定Hela传代细胞株，，通过MTT实验比较不同浓度CdSe/ZnS QDs及CdCl2孵育Hela细胞24h后细胞活性的改变，再通过流式细胞术比较不同浓度CdSe/ZnS QDs及CdCl2对Hela细胞凋亡率的影响。通过对比空白对照组、CdSe/ZnS QDs组及CdCl2组Hela细胞活性及凋亡率差异，初步判断CdSeCdSe/ZnS QDs是否具有细胞毒性。 通过DCFH-DA荧光探针检测不同浓度CdSe/ZnS QDs及CdCl2孵育Hela细胞24h后细胞内ROS量，并比较不同组之间ROS量的差异，探究CdSe/ZnSQDs对Hela细胞内ROS量的影响。 最后通过Western blot检测不同浓度CdSe/ZnS QDs及CdCl2孵育Hela细胞24h后细胞内凋亡相关蛋白Bcl-2与Bax表达量，对比不同组间Bcl-2与Bax蛋白表达量差异分析CdSe/ZnS QDs对细胞内Bcl-2与Bax蛋白表达的影响并分析其可能的原因。 结果： 1、本实验所用CdSe/ZnS QDs颗粒均匀呈球形，直径小，具有良好的单分散性和晶体结构，激发光谱宽，发射光谱连续且对称。 2、CdSe/ZnS QDs对Hela细胞的毒性作用远强于CdCl2，且其细胞毒性作用与给药浓度呈正比。 3、 CdSe/ZnS QDs较CdCl2诱导Hela内产生更多的ROS，且CdSeQDs/ZnS对ROS的诱导效应与给药浓度成正比。 4、CdSe/ZnS QDs较CdCl2诱导Hela细胞凋亡效应更强，且其诱导凋亡效应与给药浓度成正比。 5、CdSe/ZnS QDs处理组细胞内Bcl-2蛋白表达量较空白对照组及相应CdCl2处理组少，而Bax蛋白表达较空白对照组及相应CdCl2处理组多。 结论： 1、CdSe/ZnS QDs直径小，光学特性优，适用于生物医学研究。 2、CdSe/ZnS QDs具有细胞毒性作用，其毒性作用强于CdCl2，且毒性作用与给药浓度成正比。 3、CdSe/ZnS QDs能通过促进细胞内ROS生成而诱导Hela细胞凋亡，且CdSe/ZnS QDs能通过诱导Bax表达增加、Bcl-2表达减少而诱导Hela细胞凋亡。
[Abstract]:Objective: 1. By studying the basic physical and optical properties of zinc sulfide coated cadmium selenide quantum dots (CdSe/ZnS QDs), it is proved that it is a kind of fluorescent dye suitable for biomedicine. 2. By examining the effects of CdSe/ZnS QDs and cadmium chloride (CdCl2) on the activity and apoptosis rate of Hela cells, we investigated the cytotoxicity of CdSe/ZnS QDs. 3. By comparing ROS production in Hela cells of blank control group, CdSe/ZnS QDs group and CdCl2 group, the possible mechanism of Hela cell apoptosis induced by CdSe/ZnS QDs was explored. 4. Through the detection of apoptosis-related proteins, the mechanism of apoptosis induced by CdSe/ZnS QDs was further explained. Methods: CdSe/ZnS QDs was characterized by transmission electron microscope (Transmission electron microscope,TEM), ultraviolet spectrophotometer (Ultraviolet spectrophotometer) and fluorescence spectrometer (Fluorescencespectrometer). Transmission electron microscopy (TEM) is used to understand the size, shape and appearance of CdSe/ZnSQDs. Ultraviolet spectrophotometer is used to detect the absorption wavelength range of CdSe/ZnS QDs, and fluorescence photometer is used to detect the range of fluorescence wavelength emitted by CdSe/ZnS QDs. After establishing stable Hela cell lines, the changes of cell activity of Hela cells incubated with different concentrations of CdSe/ZnS QDs and CdCl2 for 24 hours were compared by MTT assay, and the effects of different concentrations of CdSe/ZnS QDs and CdCl2 on the apoptosis rate of Hela cells were compared by flow cytometry. By comparing the difference of Hela cell activity and apoptosis rate among control group, CdSe/ZnS QDs group and CdCl2 group, the cytotoxicity of CdSeCdSe/ZnS QDs was preliminarily determined. The amount of ROS in Hela cells incubated with different concentrations of CdSe/ZnS QDs and CdCl2 for 24 hours was detected by DCFH-DA fluorescence probe, and the difference of ROS content among different groups was compared to explore the effect of CdSe/ZnSQDs on the quantity of ROS in Hela cells. Finally, Western blot was used to detect the expression of Bcl-2 and Bax in Hela cells incubated with different concentrations of CdSe/ZnS QDs and CdCl2 for 24 hours. The effect of CdSe/ZnS QDs on the expression of Bcl-2 and Bax in different groups was analyzed and the possible causes were analyzed. Results: 1. The CdSe/ZnS QDs particles used in this experiment are spherical and small in diameter, with good monodispersity and crystal structure, wide excitation spectrum, continuous and symmetrical emission spectrum. 2 the cytotoxic effect of CDS / ZnS QDs on Hela cells was much stronger than that on CdCl2, cells, and its cytotoxicity was proportional to the drug concentration. 3. More ROS, was produced in Hela induced by CdSe/ZnS QDs than that induced by CdCl2, and the effect of CdSeQDs/ZnS on ROS was directly proportional to the drug concentration. 4CdSee / ZnS QDs was more effective than CdCl2 in inducing apoptosis of Hela cells, and the apoptotic effect was in direct proportion to the drug concentration. 5The expression of Bcl-2 protein in CDSe-ZnS QDs treated group was lower than that in control group and CdCl2 treatment group, but the expression of Bax protein was higher than that in blank control group and CdCl2 treatment group. Conclusion: 1 CdSeR / ZnS QDs has small diameter and excellent optical properties, which is suitable for biomedical research. 2CdSe- / ZnS QDs has cytotoxic effect, which is stronger than CdCl2, and is directly proportional to the concentration of the drug. 3CdSee / ZnS QDs could induce apoptosis of Hela cells by promoting the production of intracellular ROS, and CdSe/ZnS QDs could induce apoptosis of Hela cells by increasing the expression of Bax and decreasing the expression of Bcl-2.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.3
本文编号：2355188
[Abstract]:Objective: 1. By studying the basic physical and optical properties of zinc sulfide coated cadmium selenide quantum dots (CdSe/ZnS QDs), it is proved that it is a kind of fluorescent dye suitable for biomedicine. 2. By examining the effects of CdSe/ZnS QDs and cadmium chloride (CdCl2) on the activity and apoptosis rate of Hela cells, we investigated the cytotoxicity of CdSe/ZnS QDs. 3. By comparing ROS production in Hela cells of blank control group, CdSe/ZnS QDs group and CdCl2 group, the possible mechanism of Hela cell apoptosis induced by CdSe/ZnS QDs was explored. 4. Through the detection of apoptosis-related proteins, the mechanism of apoptosis induced by CdSe/ZnS QDs was further explained. Methods: CdSe/ZnS QDs was characterized by transmission electron microscope (Transmission electron microscope,TEM), ultraviolet spectrophotometer (Ultraviolet spectrophotometer) and fluorescence spectrometer (Fluorescencespectrometer). Transmission electron microscopy (TEM) is used to understand the size, shape and appearance of CdSe/ZnSQDs. Ultraviolet spectrophotometer is used to detect the absorption wavelength range of CdSe/ZnS QDs, and fluorescence photometer is used to detect the range of fluorescence wavelength emitted by CdSe/ZnS QDs. After establishing stable Hela cell lines, the changes of cell activity of Hela cells incubated with different concentrations of CdSe/ZnS QDs and CdCl2 for 24 hours were compared by MTT assay, and the effects of different concentrations of CdSe/ZnS QDs and CdCl2 on the apoptosis rate of Hela cells were compared by flow cytometry. By comparing the difference of Hela cell activity and apoptosis rate among control group, CdSe/ZnS QDs group and CdCl2 group, the cytotoxicity of CdSeCdSe/ZnS QDs was preliminarily determined. The amount of ROS in Hela cells incubated with different concentrations of CdSe/ZnS QDs and CdCl2 for 24 hours was detected by DCFH-DA fluorescence probe, and the difference of ROS content among different groups was compared to explore the effect of CdSe/ZnSQDs on the quantity of ROS in Hela cells. Finally, Western blot was used to detect the expression of Bcl-2 and Bax in Hela cells incubated with different concentrations of CdSe/ZnS QDs and CdCl2 for 24 hours. The effect of CdSe/ZnS QDs on the expression of Bcl-2 and Bax in different groups was analyzed and the possible causes were analyzed. Results: 1. The CdSe/ZnS QDs particles used in this experiment are spherical and small in diameter, with good monodispersity and crystal structure, wide excitation spectrum, continuous and symmetrical emission spectrum. 2 the cytotoxic effect of CDS / ZnS QDs on Hela cells was much stronger than that on CdCl2, cells, and its cytotoxicity was proportional to the drug concentration. 3. More ROS, was produced in Hela induced by CdSe/ZnS QDs than that induced by CdCl2, and the effect of CdSeQDs/ZnS on ROS was directly proportional to the drug concentration. 4CdSee / ZnS QDs was more effective than CdCl2 in inducing apoptosis of Hela cells, and the apoptotic effect was in direct proportion to the drug concentration. 5The expression of Bcl-2 protein in CDSe-ZnS QDs treated group was lower than that in control group and CdCl2 treatment group, but the expression of Bax protein was higher than that in blank control group and CdCl2 treatment group. Conclusion: 1 CdSeR / ZnS QDs has small diameter and excellent optical properties, which is suitable for biomedical research. 2CdSe- / ZnS QDs has cytotoxic effect, which is stronger than CdCl2, and is directly proportional to the concentration of the drug. 3CdSee / ZnS QDs could induce apoptosis of Hela cells by promoting the production of intracellular ROS, and CdSe/ZnS QDs could induce apoptosis of Hela cells by increasing the expression of Bax and decreasing the expression of Bcl-2.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.3
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