无机纳米粒子进入肝癌细胞的过程及肝癌细胞的变化
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摘要
本研究的目的是为了确认癌细胞质内的颗粒物质是否为无机纳米粒子,并探讨无机纳米粒子进入癌细胞的途径、过程,及无机纳米粒子和癌细胞的变化规律。
研究将Bel—7402肝癌细胞与HAP纳米粒子共同孵育8小时后,电镜观察到癌细胞内有与HAP纳米粒子形貌相似的颗粒物质;能谱和电子衍射图检测表明:细胞质内外的的颗粒物质均为含钙磷成分的物质,且具有典型的HAP晶体结构特征。证实了HAP纳米粒子进入到癌细胞内。
实验发现:与DMSO处理后的肝癌细胞共同孵育后,大部分HAP纳米粒子进入到细胞核。表明生理状态下的核膜可阻挡纳米粒子,自由扩散进入细胞的可能性较小。与细胞松弛素B处理过的癌细胞共同孵育后,仅有少量处理1小时的癌细胞内有纳米粒子,而处理12小时的癌细胞内均未发现纳米颗粒。证明了HAP纳米粒子被细胞吞噬进入的,吞噬的过程需要微丝参与。与尼莫地平处理过的肝癌细胞共同孵育,HAP纳米粒子同样进入癌细胞质内,主动运输的方式的可能性较小。与肝癌细胞分别孵育0.5小时到40小时,均发现HAP纳米粒子进入癌细胞的细胞质内。说明细胞吞噬纳米粒子的速度很快。
经过高温高压消毒后,无机纳米晶体粒子的形貌和粒径均无明显影响。说明高温高压法是简便易行的消毒方法。在生理环境中,无机纳米粒子仍能保持分散的晶体粒子状态,但其平均粒径减少约50%。低速离心对其粒径无明显影响。因此,本研究中的无机纳米粒子仍为分散的纳米颗粒,不影响实验结果。
进入癌细胞后,TiO_2纳米粒子逐渐被浓缩,团聚,但不能被细胞代谢分解,因此不能作为理想的材料。而HAP纳米粒子的尺寸变小,颗粒边缘变模糊圆钝,晶体的结晶度逐渐不完全,说明它被细胞降解。
与无机纳米粒子共同孵育后,发现肝癌细胞发生纳米粒子周围细胞质的溶解,内质网过度肿胀,线粒体肿胀崩解、嵴结构紊乱,核膜周间隙扩大、核固
It had been reported that inorganic crystal nanoparticles (ICN) could inhibit the proliferation of cancer cells. The purpose of this study is to confirm that ICN could be absorbed into cytoplasm, exam the approach and process of absorption, and investigate change of nanoparticles and cancer cell.Bel-7402 cells, a hepatoma cell line, were selected to incubation with HAP nanoparticles for 8 hours. Then many particles in shape similar to HAP were found in cytoplasm under Transmission Electronic Microscope (TEM ) . By energy dispersing spectrum (EDS) analysis, the intracellular particles contained calcium (Ca) and phosphorus (P) . The classic rings of HAP crystal appeared in the electronic diffraction (ED) pictures of these particles. So these particles were confirmed as HAP nanoparticles. Thus, it was concluded that HAP nanoparticles coulc be absorbed by cancer cells as the crystal particles.To investigate the approach of nanoparticles absorption, the Bel-7402 cells were treated before incubation with HAP nanoparticles. After incubation with the Bel-7402 cells treated by DMSO, most of HAP nanoparticles appeared in the nuclear area. This illustrated that nuclear envelope could block off free diffusion of nanoparticles. After treated by Cytochalasin B for 1 hour, only few nanoparticles could be absorbed into cytoplasm. In treated by Cytochalasin B for 12 hour group, no HAP nanoparticle was found in cells. This indicated cancer cells phagocytosised nanoparticles by microfiber assistance. After treated by Nimodipine, HAP nanoparticles were absorbed into cytoplasma as before. So calcium channel block had nothing to do with the absorption process. Another, the fact that intracellular HAP nanoparticles were found after incubation with Bel-7402 cells for 0.5 hour, indicated the phagocytosis process finished fast.After high temperature and press disinfection, no change in the shape and size of ICN stated that this disinfection way was fitted to nanoparticles. When mixed with medium, the ICN dispersed but about 50% reducing in size. However, the ICN size did not change after low speed centrifugation. So those nanoparitcles in this study still remained dispersing and nano-size, and the results did not affected by disinfection,
mix in medium and centrifugation.After phagocytosised into cytoplasm, TiC>2 could not be decompounded in cancer cells. On the contrary, HAP nanoparticles could be digested in cells so that they should be the ideal biomaterials.Endocytosised ICN, the Bel-7402 cells were examined under TEM: disruption of cytoplasm around nanoparticles, expansion of endoplasmic reticulums, swelling and collapse of most of mitochondrias, disorder of mitochondrias crista, expansion of nuclear space, shrinkage of nuclear envelope, pycnosis of karyotin. Even apoptosis body was found. By TUNEL technology, more brown signals were detected in the HAP nanoparticle group than the control group. After incubation with HAP nanoparticles, the DNA of Bel-7402 cells appeared 'ladder'. So it could be concluded that HAP nanoparticles induced apoptosis of Bel-7402 cells.
研究将Bel—7402肝癌细胞与HAP纳米粒子共同孵育8小时后,电镜观察到癌细胞内有与HAP纳米粒子形貌相似的颗粒物质;能谱和电子衍射图检测表明:细胞质内外的的颗粒物质均为含钙磷成分的物质,且具有典型的HAP晶体结构特征。证实了HAP纳米粒子进入到癌细胞内。
实验发现:与DMSO处理后的肝癌细胞共同孵育后,大部分HAP纳米粒子进入到细胞核。表明生理状态下的核膜可阻挡纳米粒子,自由扩散进入细胞的可能性较小。与细胞松弛素B处理过的癌细胞共同孵育后,仅有少量处理1小时的癌细胞内有纳米粒子,而处理12小时的癌细胞内均未发现纳米颗粒。证明了HAP纳米粒子被细胞吞噬进入的,吞噬的过程需要微丝参与。与尼莫地平处理过的肝癌细胞共同孵育,HAP纳米粒子同样进入癌细胞质内,主动运输的方式的可能性较小。与肝癌细胞分别孵育0.5小时到40小时,均发现HAP纳米粒子进入癌细胞的细胞质内。说明细胞吞噬纳米粒子的速度很快。
经过高温高压消毒后,无机纳米晶体粒子的形貌和粒径均无明显影响。说明高温高压法是简便易行的消毒方法。在生理环境中,无机纳米粒子仍能保持分散的晶体粒子状态,但其平均粒径减少约50%。低速离心对其粒径无明显影响。因此,本研究中的无机纳米粒子仍为分散的纳米颗粒,不影响实验结果。
进入癌细胞后,TiO_2纳米粒子逐渐被浓缩,团聚,但不能被细胞代谢分解,因此不能作为理想的材料。而HAP纳米粒子的尺寸变小,颗粒边缘变模糊圆钝,晶体的结晶度逐渐不完全,说明它被细胞降解。
与无机纳米粒子共同孵育后,发现肝癌细胞发生纳米粒子周围细胞质的溶解,内质网过度肿胀,线粒体肿胀崩解、嵴结构紊乱,核膜周间隙扩大、核固
It had been reported that inorganic crystal nanoparticles (ICN) could inhibit the proliferation of cancer cells. The purpose of this study is to confirm that ICN could be absorbed into cytoplasm, exam the approach and process of absorption, and investigate change of nanoparticles and cancer cell.Bel-7402 cells, a hepatoma cell line, were selected to incubation with HAP nanoparticles for 8 hours. Then many particles in shape similar to HAP were found in cytoplasm under Transmission Electronic Microscope (TEM ) . By energy dispersing spectrum (EDS) analysis, the intracellular particles contained calcium (Ca) and phosphorus (P) . The classic rings of HAP crystal appeared in the electronic diffraction (ED) pictures of these particles. So these particles were confirmed as HAP nanoparticles. Thus, it was concluded that HAP nanoparticles coulc be absorbed by cancer cells as the crystal particles.To investigate the approach of nanoparticles absorption, the Bel-7402 cells were treated before incubation with HAP nanoparticles. After incubation with the Bel-7402 cells treated by DMSO, most of HAP nanoparticles appeared in the nuclear area. This illustrated that nuclear envelope could block off free diffusion of nanoparticles. After treated by Cytochalasin B for 1 hour, only few nanoparticles could be absorbed into cytoplasm. In treated by Cytochalasin B for 12 hour group, no HAP nanoparticle was found in cells. This indicated cancer cells phagocytosised nanoparticles by microfiber assistance. After treated by Nimodipine, HAP nanoparticles were absorbed into cytoplasma as before. So calcium channel block had nothing to do with the absorption process. Another, the fact that intracellular HAP nanoparticles were found after incubation with Bel-7402 cells for 0.5 hour, indicated the phagocytosis process finished fast.After high temperature and press disinfection, no change in the shape and size of ICN stated that this disinfection way was fitted to nanoparticles. When mixed with medium, the ICN dispersed but about 50% reducing in size. However, the ICN size did not change after low speed centrifugation. So those nanoparitcles in this study still remained dispersing and nano-size, and the results did not affected by disinfection,
mix in medium and centrifugation.After phagocytosised into cytoplasm, TiC>2 could not be decompounded in cancer cells. On the contrary, HAP nanoparticles could be digested in cells so that they should be the ideal biomaterials.Endocytosised ICN, the Bel-7402 cells were examined under TEM: disruption of cytoplasm around nanoparticles, expansion of endoplasmic reticulums, swelling and collapse of most of mitochondrias, disorder of mitochondrias crista, expansion of nuclear space, shrinkage of nuclear envelope, pycnosis of karyotin. Even apoptosis body was found. By TUNEL technology, more brown signals were detected in the HAP nanoparticle group than the control group. After incubation with HAP nanoparticles, the DNA of Bel-7402 cells appeared 'ladder'. So it could be concluded that HAP nanoparticles induced apoptosis of Bel-7402 cells.
引文
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