CdTe量子点对人正常肝细胞HL-7702的细胞毒性
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摘要
量子点由于其具有独特的光学特性,在离体细胞标记、活体细胞成像、细胞结构与受体标记、免疫荧光标记、活体动物荧光显微成像和寻找药物靶点等生物医药领域具有广阔的应用前景。随着量子点的应用,人体暴露机会日益增加,其安全性问题也日益受到关注。
本研究以人正常肝细胞HL-7702为模型,研究TGA包覆的CdTe量子点对HL-7702细胞的细胞毒性。采用MTT法检测CdTe量子点对细胞增殖的影响;单细胞凝胶电泳和琼脂糖凝胶电泳检测CdTe量子点对细胞DNA损伤情况;PI单染法检测CdTe量子点对细胞周期的影响;AO/EB双荧光染色法和AnnexinⅤ- FITC/PI双染流式细胞术检测CdTe量子点对细胞凋亡的影响。结果表明:CdTe量子点可抑制HL-7702细胞的增殖,随着作用时间的延长,细胞存活率下降并存在剂量-效应关系;CdTe量子点可引起细胞DNA损伤,随着浓度的增加,DNA损伤率逐渐增高;CdTe量子点可影响细胞周期进程,出现S期和G2/M期阻滞;CdTe量子点可诱导细胞凋亡。
本研究对探讨CdTe量子点对人正常肝细胞的毒性及其作用机制具有重要理论意义,同时为量子点的安全性评价提供有价值的实验依据。
Quantum dots (QDs) are now becoming widely used in biotechnology and medical applications because of their unique optical properties. Consequently, the professional and public exposure to QDs is supposed to increase dramatically in the coming years. Therefore, QDs are attracting considerable and increasing concern of the public and government worldwide. In this study, we selected human liver HL-7702 cell line as cell model, cytotoxicity of CdTe QDs was measured by MTT assay, DNA damage was detected by SCGE assay and gel electrophoresis, changes of cell cycle were observed by flow cytometry after PI staining, cell apoptosis was examined by AO/EB double fluorescent dye staining and AnnexinⅤ-FITC/PI dying methods. The results of this study were shown as follow:
1. Cytotoxicity of HL-7702 cells treated with CdTe QDs
MTT assays were performed to examine the viability of HL-7702 cells treated with various doses of CdTe QDs (0, 6.25, 12.50, 25.00 and 50.00μg/mL). Our results revealed that CdTe QDs induced a time-dependent increase in cytotoxicity. When HL-7702 cells were exposed to CdTe QDs for 6 h, there were no obvious changes in cell viability compared with control group (P > 0.05). After 12 h of exposure, with the increase of exposure dosage, the survival rate rose first, and then fell. After 24 h of exposure, QDs caused a decrease in cell metabolic activity that was significant at 12.50-50.00μg/mL groups (P < 0.05 or P < 0.001). When HL-7702 cells were exposed to CdTe QDs for 48 h, with the increase of exposure dosage, cell viability significantly decreased (P < 0.001). CdTe QDs could increase cell inhibitory rate to about 55% after been treated with 50μg/mL CdTe QDs for 24 h. And after 48 h of exposure at the same dose, CdTe QDs exerted more toxic effect by decreasing the cell viability to approximately 80%. The results suggest that CdTe QDs had the inhibitory effect on the proliferation of HL-7702 cells.
2. DNA damage in HL-7702 cells treated with CdTe QDs
DNA damage was measured by the single cell gel electrophoresis (SCGE). Our results showed that in the comet assay, after 24h of exposure, with the increase of exposure dosage, the rate of DNA damage was significantly increased(P < 0.05), and the length of comet tail was elevated. At the dose of 12.5, 25 and 50μg/mL, CdTe QDs can cause significant DNA damage compared with control group (P < 0.05). It indicated that the rate of DNA damage induced by CdTe QDs was in a dose-dependent manner.
Gel electrophoresis was used to detect the DNA fragment. After 12 and 24 h of exposure, an obvious and continual tape was seen in agarose gel electrophoresis. When HL-7702 cells were treated with CdTe QDs for 48 h, no tailing tapes were found at both 6.25 and 12.5μg/mL exposure groups in agarose gel electrophoresis. No typical DNA ladder was observed in all groups.
3. Cell cycle changes of HL-7702 cells treated with CdTe QDs
Flow cytometry with PI staining was performed to detect the cell cycle changes. We analyzed the cell cycle phases by collecting more than 10 000 cells and classified them into G0/G1, S or G2/M phases of cell cycle. The results showed that as the dosage of CdTe QDs increased, the percentage of G0/G1 phase cells were significantly decreased (P < 0.001), while the percentage of S and G2/M phases cells were significantly increased (P < 0.001), which indicated that CdTe QDs can induce S and G2/M phases arrest in HL7702 cells.
4. Apoptosis in HL-7702 cells induced by CdTe QDs
AO/EB double fluorescent dye staining and FCM with AnnexinⅤ-FITC/PI dying methods were performed to examine apoptosis. The results of AO/EB staining showed that distinctive cell apoptosis could be observed by fluorescence microscope after exposure to CdTe QDs. When HL-7702 cells were exposed to 6.25μg/mL CdTe QDs for 24 h, cell apoptotic rate increased to 20%, and there was significant difference compared with control groups (P < 0.001). As the dosage of CdTe QDs increased, cell apoptotic rate only increased slightly and necrotic rate increased significantly.
The results of flow cytometry showed that when HL-7702 cells were exposed to 6.25μg/mL CdTe QDs for 24 h, cell apoptotic rate increased to 24%, and there was significant difference compared with control groups (P < 0.001). But there was no significantly difference in apoptotic rate between 12.5μg/mL and 50μg/mL CdTe QDs group. In this study, two methods were applied to detect cell apoptosis and both indicated that CdTe QDs could induce apoptosis in HL-7702 cells.
In conclusion, CdTe QDs could inhibit cell viability, induce DNA damage, cause S and G2/M phases arrest and induce cell apoptosis in HL-7702 cells.
本研究以人正常肝细胞HL-7702为模型,研究TGA包覆的CdTe量子点对HL-7702细胞的细胞毒性。采用MTT法检测CdTe量子点对细胞增殖的影响;单细胞凝胶电泳和琼脂糖凝胶电泳检测CdTe量子点对细胞DNA损伤情况;PI单染法检测CdTe量子点对细胞周期的影响;AO/EB双荧光染色法和AnnexinⅤ- FITC/PI双染流式细胞术检测CdTe量子点对细胞凋亡的影响。结果表明:CdTe量子点可抑制HL-7702细胞的增殖,随着作用时间的延长,细胞存活率下降并存在剂量-效应关系;CdTe量子点可引起细胞DNA损伤,随着浓度的增加,DNA损伤率逐渐增高;CdTe量子点可影响细胞周期进程,出现S期和G2/M期阻滞;CdTe量子点可诱导细胞凋亡。
本研究对探讨CdTe量子点对人正常肝细胞的毒性及其作用机制具有重要理论意义,同时为量子点的安全性评价提供有价值的实验依据。
Quantum dots (QDs) are now becoming widely used in biotechnology and medical applications because of their unique optical properties. Consequently, the professional and public exposure to QDs is supposed to increase dramatically in the coming years. Therefore, QDs are attracting considerable and increasing concern of the public and government worldwide. In this study, we selected human liver HL-7702 cell line as cell model, cytotoxicity of CdTe QDs was measured by MTT assay, DNA damage was detected by SCGE assay and gel electrophoresis, changes of cell cycle were observed by flow cytometry after PI staining, cell apoptosis was examined by AO/EB double fluorescent dye staining and AnnexinⅤ-FITC/PI dying methods. The results of this study were shown as follow:
1. Cytotoxicity of HL-7702 cells treated with CdTe QDs
MTT assays were performed to examine the viability of HL-7702 cells treated with various doses of CdTe QDs (0, 6.25, 12.50, 25.00 and 50.00μg/mL). Our results revealed that CdTe QDs induced a time-dependent increase in cytotoxicity. When HL-7702 cells were exposed to CdTe QDs for 6 h, there were no obvious changes in cell viability compared with control group (P > 0.05). After 12 h of exposure, with the increase of exposure dosage, the survival rate rose first, and then fell. After 24 h of exposure, QDs caused a decrease in cell metabolic activity that was significant at 12.50-50.00μg/mL groups (P < 0.05 or P < 0.001). When HL-7702 cells were exposed to CdTe QDs for 48 h, with the increase of exposure dosage, cell viability significantly decreased (P < 0.001). CdTe QDs could increase cell inhibitory rate to about 55% after been treated with 50μg/mL CdTe QDs for 24 h. And after 48 h of exposure at the same dose, CdTe QDs exerted more toxic effect by decreasing the cell viability to approximately 80%. The results suggest that CdTe QDs had the inhibitory effect on the proliferation of HL-7702 cells.
2. DNA damage in HL-7702 cells treated with CdTe QDs
DNA damage was measured by the single cell gel electrophoresis (SCGE). Our results showed that in the comet assay, after 24h of exposure, with the increase of exposure dosage, the rate of DNA damage was significantly increased(P < 0.05), and the length of comet tail was elevated. At the dose of 12.5, 25 and 50μg/mL, CdTe QDs can cause significant DNA damage compared with control group (P < 0.05). It indicated that the rate of DNA damage induced by CdTe QDs was in a dose-dependent manner.
Gel electrophoresis was used to detect the DNA fragment. After 12 and 24 h of exposure, an obvious and continual tape was seen in agarose gel electrophoresis. When HL-7702 cells were treated with CdTe QDs for 48 h, no tailing tapes were found at both 6.25 and 12.5μg/mL exposure groups in agarose gel electrophoresis. No typical DNA ladder was observed in all groups.
3. Cell cycle changes of HL-7702 cells treated with CdTe QDs
Flow cytometry with PI staining was performed to detect the cell cycle changes. We analyzed the cell cycle phases by collecting more than 10 000 cells and classified them into G0/G1, S or G2/M phases of cell cycle. The results showed that as the dosage of CdTe QDs increased, the percentage of G0/G1 phase cells were significantly decreased (P < 0.001), while the percentage of S and G2/M phases cells were significantly increased (P < 0.001), which indicated that CdTe QDs can induce S and G2/M phases arrest in HL7702 cells.
4. Apoptosis in HL-7702 cells induced by CdTe QDs
AO/EB double fluorescent dye staining and FCM with AnnexinⅤ-FITC/PI dying methods were performed to examine apoptosis. The results of AO/EB staining showed that distinctive cell apoptosis could be observed by fluorescence microscope after exposure to CdTe QDs. When HL-7702 cells were exposed to 6.25μg/mL CdTe QDs for 24 h, cell apoptotic rate increased to 20%, and there was significant difference compared with control groups (P < 0.001). As the dosage of CdTe QDs increased, cell apoptotic rate only increased slightly and necrotic rate increased significantly.
The results of flow cytometry showed that when HL-7702 cells were exposed to 6.25μg/mL CdTe QDs for 24 h, cell apoptotic rate increased to 24%, and there was significant difference compared with control groups (P < 0.001). But there was no significantly difference in apoptotic rate between 12.5μg/mL and 50μg/mL CdTe QDs group. In this study, two methods were applied to detect cell apoptosis and both indicated that CdTe QDs could induce apoptosis in HL-7702 cells.
In conclusion, CdTe QDs could inhibit cell viability, induce DNA damage, cause S and G2/M phases arrest and induce cell apoptosis in HL-7702 cells.
引文
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