纳米颗粒对血管内皮细胞的毒性作用研究
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摘要
自上世纪90年代初起,纳米科技得到了迅速发展,已广泛应用于电子、信息、半导体、生物和医药等领域。越来越多的国家开始增加在该领域研究的投资,以期在这个孕育着巨大商机的领域中夺得先机。
然而,随着研究的深入和发展,纳米材料的生物安全性问题引起越来越多的关注,已有的一些研究证据也表明某些纳米材料对人类的健康的确存在潜在的危害。为保证纳米科技的健康发展,进行纳米技术的生物安全性评估及研究已迫在眉睫。
本论文以目前大量生产及应用的多壁碳纳米管(MWNTs)和在基因治疗中具有良好应用前景的精氨酸修饰壳聚糖(Arg-CS)基因纳米粒子(ACGN)作为纳米材料模型,选择人体中发挥重要生理功能的血管内皮细胞(VEC)作为研究对象,研究纳米材料与血管内皮细胞相互作用及机制,为纳米材料生物学效应的深入开展提供新的理论依据和实验基础。具体内容主要包括以下几个方面:
1.血管内皮细胞的分离与培养:自人脐静脉分离血管内皮细胞,采用光镜、电镜和免疫组化等方法进行内皮细胞的形态观察和鉴定。结果表明光镜下内皮细胞呈铺路石状镶嵌排列;免疫组化可见内皮细胞胞浆中人vWF相关抗原呈阳性反应;电镜下可见胞浆中有W-P小体,证实培养的细胞为内皮细胞。
2.MWNTs的表面效应对VEC的影响:噻唑蓝实验(MTT比色法)结果显示功能化多壁碳纳米管(f-CNTs)与原始多壁碳纳米管(p-CNTs)均对VEC产生一定的毒性作用;与细胞共育24h后,p-CNTs的毒性大于f-CNTs;而共育48及72h后,f-CNTs的毒性大于p-CNTs。透射电镜实验表明,f-CNTs共育细胞内空腔明显多于p-CNTs,而空腔内f-CNTs聚集体紧密程度明显小于p-CNTs。由此推断,表面效应可能是MWNTs对VEC造成毒性损伤的作用机制之一;纳米材料的毒性可能受团聚、形状、化学组成等许多因素的影响。
3.MWNTs长度对VEC的影响:选择50μm-MWNTs及0.5μm-MWNTs作为实验材料,采用透射电镜、扫描电镜及激光粒度测定仪对碳纳米管进行表征;MTT结果显示,50μm-MWNTs的细胞毒性明显大于0.5μm-MWNTs;流式细胞分析显示长碳纳米管诱导的细胞凋亡率明显高于短碳纳米管,电镜观察显示长碳纳米管对超微结构损伤严重。提示尺寸效应是导致细胞毒性差异的主要原因,其毒性作用可能受其团聚程度影响。
4.研究Arg-CS介导的TFPI基因转染与血管内皮细胞的相互作用:RT-PCR观察显示Arg-CS可介导TFPI基因进入内皮细胞并得到表达,MTT结果显示Arg-CS/TFPI基因纳米粒子对血管内皮细胞没有明显毒性作用。提示精氨酸修饰的壳聚糖作为基因载体可成功转染脐静脉内皮细胞(HUVEC),基本无毒,是较理想的基因载体。
Since the beginning of 1990's,nano-science and technology gained rapid development,and has been widely applied in the areas of electronics,information and communication,semiconductor,biology and medicine.More and more countries have been putting huge investment in the research and development of nanotechnology for the purpose of taking advantage of the opportunities which could bring huge commercial benefits.
However,the bio-safety of nanomaterials has been drawing increasing attention of scientists and environmental protection organization.Preliminary studies showed that some kinds of nano-scale materials might have negative impact on human beings.Greater effort is urgently needed on the evaluation of the bio-safety of nano-scale materials for the healthy development of nanotechnology.The current study was aimed at investigating the interactions between nanomaterials and the vascular endothelial cells,which includes the following four parts:
1.Isolation and identification of vascular endothelial cells(VECs) from human umbilical vein.The morphologic observation and evaluation of ECs were made with light microscopy,electron microscopy and immunohistochemistry.The results showed that the isolated VECs arrayed like pitching stones under light microscope.W-P corpuscles were observed in the cytoplasm under electron microscope and immunostaining of the cells with antibody againstⅧfactor demonstrated positive reaction,suggesting that the isolated cells were VECs.
2.Study on the effect of surface chemistry of MWNT on the cytotoxicity.It was demonstrated that the cytotoxicity of p-CNTs was greater than that of f-CNTs at 24 h of exposure.However it was on the contrary at 48 h and 72 h of exposure.Transmission electron microscopic observation showed that there were much more CNT aggregates in the cells in contact with f-CNTs,but the p-CNT aggregates were much more condensed than f-CNTs aggregates.These observations suggested that the modification of CNTs have effects on cellular function that can not be attribute to one factor alone,but are more likely the combined result of several unfavorable interactions.
3.Study of the effect of the length of MWCNTs on the cytotoxicity.(MWCNTs). The results showed that the 50μm-long MWCNTs induced more severe damages to the vascular endothelial cells(VECs) in comparison to the 0.5μm-long MWCNTs.It was also demonstrated that the long CNTs at 50μg/mL induced significantly higher number of apoptotic cells than the short MWCNTs.It provided the first evidence that CNT aggregates with a dimension of up to 10μm×6.25μm were internalized by VECs and caused severe damages,which is postulated as a new mechanism underlying the cellular injuries induced by aggregated CNTs.
4.Evaluation of the arginine modified chitosan(Arg-CS) as gene carriers for introducing tissue factor pathway inhibitor(TFPI) gene into VECs.The level of TFPI mRNA was tested using RT-PCR ant the in vitro cytotoxicity of Arg-CS/DNA complexes was determined by MTT assay.The results showed that Arg-CS/pIRES-TFPI DNA complexes were nontoxic to the VECs and successfully mediated the expression of TFPI gene in the cells.
然而,随着研究的深入和发展,纳米材料的生物安全性问题引起越来越多的关注,已有的一些研究证据也表明某些纳米材料对人类的健康的确存在潜在的危害。为保证纳米科技的健康发展,进行纳米技术的生物安全性评估及研究已迫在眉睫。
本论文以目前大量生产及应用的多壁碳纳米管(MWNTs)和在基因治疗中具有良好应用前景的精氨酸修饰壳聚糖(Arg-CS)基因纳米粒子(ACGN)作为纳米材料模型,选择人体中发挥重要生理功能的血管内皮细胞(VEC)作为研究对象,研究纳米材料与血管内皮细胞相互作用及机制,为纳米材料生物学效应的深入开展提供新的理论依据和实验基础。具体内容主要包括以下几个方面:
1.血管内皮细胞的分离与培养:自人脐静脉分离血管内皮细胞,采用光镜、电镜和免疫组化等方法进行内皮细胞的形态观察和鉴定。结果表明光镜下内皮细胞呈铺路石状镶嵌排列;免疫组化可见内皮细胞胞浆中人vWF相关抗原呈阳性反应;电镜下可见胞浆中有W-P小体,证实培养的细胞为内皮细胞。
2.MWNTs的表面效应对VEC的影响:噻唑蓝实验(MTT比色法)结果显示功能化多壁碳纳米管(f-CNTs)与原始多壁碳纳米管(p-CNTs)均对VEC产生一定的毒性作用;与细胞共育24h后,p-CNTs的毒性大于f-CNTs;而共育48及72h后,f-CNTs的毒性大于p-CNTs。透射电镜实验表明,f-CNTs共育细胞内空腔明显多于p-CNTs,而空腔内f-CNTs聚集体紧密程度明显小于p-CNTs。由此推断,表面效应可能是MWNTs对VEC造成毒性损伤的作用机制之一;纳米材料的毒性可能受团聚、形状、化学组成等许多因素的影响。
3.MWNTs长度对VEC的影响:选择50μm-MWNTs及0.5μm-MWNTs作为实验材料,采用透射电镜、扫描电镜及激光粒度测定仪对碳纳米管进行表征;MTT结果显示,50μm-MWNTs的细胞毒性明显大于0.5μm-MWNTs;流式细胞分析显示长碳纳米管诱导的细胞凋亡率明显高于短碳纳米管,电镜观察显示长碳纳米管对超微结构损伤严重。提示尺寸效应是导致细胞毒性差异的主要原因,其毒性作用可能受其团聚程度影响。
4.研究Arg-CS介导的TFPI基因转染与血管内皮细胞的相互作用:RT-PCR观察显示Arg-CS可介导TFPI基因进入内皮细胞并得到表达,MTT结果显示Arg-CS/TFPI基因纳米粒子对血管内皮细胞没有明显毒性作用。提示精氨酸修饰的壳聚糖作为基因载体可成功转染脐静脉内皮细胞(HUVEC),基本无毒,是较理想的基因载体。
Since the beginning of 1990's,nano-science and technology gained rapid development,and has been widely applied in the areas of electronics,information and communication,semiconductor,biology and medicine.More and more countries have been putting huge investment in the research and development of nanotechnology for the purpose of taking advantage of the opportunities which could bring huge commercial benefits.
However,the bio-safety of nanomaterials has been drawing increasing attention of scientists and environmental protection organization.Preliminary studies showed that some kinds of nano-scale materials might have negative impact on human beings.Greater effort is urgently needed on the evaluation of the bio-safety of nano-scale materials for the healthy development of nanotechnology.The current study was aimed at investigating the interactions between nanomaterials and the vascular endothelial cells,which includes the following four parts:
1.Isolation and identification of vascular endothelial cells(VECs) from human umbilical vein.The morphologic observation and evaluation of ECs were made with light microscopy,electron microscopy and immunohistochemistry.The results showed that the isolated VECs arrayed like pitching stones under light microscope.W-P corpuscles were observed in the cytoplasm under electron microscope and immunostaining of the cells with antibody againstⅧfactor demonstrated positive reaction,suggesting that the isolated cells were VECs.
2.Study on the effect of surface chemistry of MWNT on the cytotoxicity.It was demonstrated that the cytotoxicity of p-CNTs was greater than that of f-CNTs at 24 h of exposure.However it was on the contrary at 48 h and 72 h of exposure.Transmission electron microscopic observation showed that there were much more CNT aggregates in the cells in contact with f-CNTs,but the p-CNT aggregates were much more condensed than f-CNTs aggregates.These observations suggested that the modification of CNTs have effects on cellular function that can not be attribute to one factor alone,but are more likely the combined result of several unfavorable interactions.
3.Study of the effect of the length of MWCNTs on the cytotoxicity.(MWCNTs). The results showed that the 50μm-long MWCNTs induced more severe damages to the vascular endothelial cells(VECs) in comparison to the 0.5μm-long MWCNTs.It was also demonstrated that the long CNTs at 50μg/mL induced significantly higher number of apoptotic cells than the short MWCNTs.It provided the first evidence that CNT aggregates with a dimension of up to 10μm×6.25μm were internalized by VECs and caused severe damages,which is postulated as a new mechanism underlying the cellular injuries induced by aggregated CNTs.
4.Evaluation of the arginine modified chitosan(Arg-CS) as gene carriers for introducing tissue factor pathway inhibitor(TFPI) gene into VECs.The level of TFPI mRNA was tested using RT-PCR ant the in vitro cytotoxicity of Arg-CS/DNA complexes was determined by MTT assay.The results showed that Arg-CS/pIRES-TFPI DNA complexes were nontoxic to the VECs and successfully mediated the expression of TFPI gene in the cells.
引文
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