PAMAM纳米材料及碳纳米管的毒性研究

【作者】 刘昊霖；

【导师】 蒋澄宇；

【作者基本信息】 中国协和医科大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 PAMAM G3通过AKT-TSC2-mTOR通路引起A549自噬性细胞死亡,并导致小鼠急性肺损伤聚酰胺-胺树枝状大分子纳米材料(PAMAM dendrimer)是一种具有高度支化、对称、呈辐射状的大分子。它由中心核、内层重复的分支亚单位以及表面的功能基团组成。整代的PAMAM纳米材料表面的功能基团为氨基,半代的PAMAM纳米材料表面的功能基团为羧基。由于氨基带有正电荷,整代的PAMAM常被用作载体,递送DNA,进行细胞或生物体内表达。然而,对于这些PAMAM材料的生物安全性却了解甚少。在本文中,我们测定了PAMAM G1-G8以及G3.5、G4.5、G5.5和G7.5对A549细胞的影响。发现从PAMAM G3开始,到PAMAM G8均会引起A549细胞的死亡。而表面基团为阴离子的半代PAMAM对A549的活性不会产生影响。我们对PAMAM G3引起的A549死亡进行了分析,发现PAMAMG3引起的A549死亡不是通过凋亡通路,而是引起A549自噬性细胞死亡。自噬的抑制剂3MA或是利用siRNA技术抑制自噬基因ATG 6的表达,可以有效地缓解PAMAM G3引起的A549细胞死亡。同时,自噬性细胞死亡也在PAMAM G4、G5、G6、G7和G8引起的A549细胞死亡中扮演着重要角色。同时,我们对PAMAM G3引起的自噬性细胞死亡的信号通路进行了分析。发现PAMAM G3可以通过AKT-TSC2-mTOR的信号通路引起A549发生自噬性细胞死亡。而且,我们发现PAMAM G3会导致小鼠死亡,引起小鼠急性肺损伤。而自噬的抑制剂3MA会有效地缓解PAMAM G3引起的小鼠死亡,改善小鼠急性肺损伤程度。说明自噬在PAMAM G3引起的小鼠死亡中发挥着重要作用。表面为COOH修饰的单壁碳纳米管引起A549发生自噬性细胞死亡碳纳米管(Carbon NanoTubes,CNT)是碳原子之间形成化学键组成的管状结构。CNT通常有两种形式:单壁碳管(single-walled carbon nanotubes,SWCNT),由一层碳原子网组成,直径范围0.6-2.4纳米;多壁碳管(multi-walled carbon nanotubes,MWCNT),由多于一层的同圆心的单壁碳管套叠在一起形成,直径范围10-200纳米。由于其表面受到不同功能基团所修饰,碳纳米管可能会表现出截然不同的性质。在本章中,我们发现表面功能基团为COOH的单壁碳管会引起A549细胞发生自噬,并导致A549细胞死亡。而自噬的抑制剂3MA会有效的抑制COOH单壁碳管引起的A549细胞死亡。而且,我们发现COOH单壁碳管也会引起A549细胞中磷酸化AKT/总AKT,以及磷酸化mTOR/总mTOR的比值出现下调,与PAMAM G3引起的A549细胞死亡有相同的信号通路。对COOH单壁碳管的进一步研究仍在进行之中。多壁碳纳米管通过不依赖于caspase-3的途径引起A549细胞凋亡我们研究发现多壁碳管可以引起A549细胞的死亡,而且通过细胞流式的方法判定多壁碳管可以引起A549细胞的凋亡。但是这种凋亡并不激活A549细胞的caspase-3的活性,同时,caspase的光谱抑制剂Z-VAD-fmk对多壁碳管引起的细胞凋亡无缓解作用,说明多壁碳管是通过一条不依赖于caspase-3的信号通路来引起A549细胞凋亡。刺突蛋白表达引起HEK 293T细胞内质网应激,并干扰胰岛素信号通路内质网是分泌性或是膜定位蛋白质进行修饰、折叠的场所。未折叠或是折叠错误的蛋白过量积累会引起内质网应激。有研究表明,全长的刺突蛋白表达会引起细胞产生内质网应激。在本文中,我们发现刺突蛋白的S2片段(S681-1190)的表达是引起HEK 293T细胞内质网应激的原因。而且,我们发现S2或全长刺突蛋白的表达会干扰胰岛素的信号通路,降低胰岛素引起的磷酸化AKT/总AKT的比值。我们推测S2或全长刺突蛋白表达引起的内质网应激是干扰胰岛素信号的原因,相关的研究正在进行之中。刺突蛋白中内毒素的去除以及刺突蛋白引起人外周血单核细胞的炎性因子的分析SARS冠状病毒的感染可以引起患者体内发生严重的炎性反应,引起血液中炎性因子的升高。我们用最初表达的刺突蛋白刺激人外周血单核细胞,发现可以引起大量TNF-a,IL-1β,IL-6以及IFN-γ的分泌。经内毒素检测试剂检测后,我们发现最初表达的刺突蛋白中含有大量的内毒素。经内毒素去除柱去除其中的内毒素后,内毒素指标合格的刺突蛋白也会引起人外周血单核细胞分泌TNF-a与IL-6,但分泌的量较内毒素去除前大为降低。同时,经内毒素去除的刺突蛋白未引起IFN-γ的分泌。更多还原

【Abstract】 PAMAM dendrimer(polyamidoamine dendrimer) is a polymer with dentritic branching and radial symmetry.It consists of three parts:a central core,an interior dendritic structure(the branches) and an exterior suface(the end group).PAMAM dendrimers can be divided in to the whole(cationic) or half (anionic) generation polymers.The surface of the whole generation is functionalized with primary amino surface groups and the half generation is carboxylate surface group.Due to the cationic surface,the whole generation PAMAM could be used for gene delivery for expression in cells or in animals. Therefore,the biosafety of these PAMAMs need to be determined.In this dissertation,we determine the cell viability after PAMAM G1 to G8 and G3.5,G4.5,G5.5 and G7.5 treatment.We find that treatment of A549 by cationic PAMAM,from G3 to G8 could cause cell death,while the anionic PAMAMs have no effect on cell viability.Furthermore,we find that PAMAM G3 causes A549 cell death through autophagy,but not through apoptosis.Treating cells with the autophagy inhibitor,3-methyladenine,or the knockdown of the autophagy related gene,beclin,could improve the cell viability treated by PAMAM G3.In the meanwhile,autophagic cell death happens in the PAMAM G4,G5,G6,G7 and G8 treated A549 cells.In the following step,we analyze the signal transduction pathways in the PAMAM G3 induced autphogic cell death.We find that PAMAM G3 induced A549 autophagic cell death through AKT-TSC2-mTOR pathway. Furthermore,we discover that PAMAM G3 could induce acute lung injury and cause death in mice,while the autophagy inhibitor 3MA could improve the situation.It is indicated that autophagy plays an important role in PAMAM G3 induced mice acute lung injury.COOH functionalized single walled carbon nanotube induces A549 autophagic cell deathCarbon NanoTube(CNT) is a special form of carbon in which chemical bonds of carbon form tubes form carbon atoms.Usually there are two kinds of CNT:single-walled carbon nanotube(SWCNT),containing only one tube in the CNT and multi-walled carbon nanotube(MWCNT),containing more than one concentric tube in the basic element of the CNT.The CNTs may have different properties with different surface functionalizations.In this dissertation,we find COOH functionalized SWCNT induces A549 autophagic cell death,while the autophagy inhibitor 3MA could improve the situation.Furthermore,we discover COOH functionalized SWCNT downregulates p-AKT/AKT and p-mTOR/mTOR ratio in A549 cells,similar with PAMAM G3 treated A549 cells.The research on the COOH functionalized SWCNT induced autophagy is still under way,both in cell and mice.MWCNT induces A549 cell apoptosis in a caspase-3 independent pathwayWe find that MWCNT could induce A549 apoptosis.But it fails to active the caspase-3 in A549 cell.In the meanwhile,a pancaspase inhibitor,Z-VAD-fmk could not rescue the MWCNT induced apoptosis,indicating that MWCNT induced A549 cell apoptosis is in a caspase-3 independent pathway. Spike expression induces ER stress and interferes with insulin signal transduction pathway in HEK 293T ceilsEndoplasmic Reticulum is the place where secretary and transmembrane proteins are properly folded and decorated.Too much unfolded or misfolded protein will induce ER stress,in which situation cells will take unfolded protein response to elimate those proteins.It is reported that the spike expression could induce ER stress in HEK 293 cells.In this dissertation,we find that it is S2 (S681-1190) that induces ER stress in HEK 293T cells.Furtermore,we find that expression of S2 interferes with insulin signal transduction pathway,with downregulation of phospho-AKT/total AKT ratio.We speculate that S2 induced ER stress could interfere with insulin signaling,and the related research is going on.Endotoxin removal from the expressed spike protein and analysis of inflammatory cytokines induced by spike proteinSARS-CoV infection could induce severe inflammatory reaction,with a elevation of inflammatory cytokine in the victims.We expresse and purify SARS-CoV spike protein in human cell line andfind that it could induce TNF-a, IL-1β,IL-6 and IFN-γsecretion in human monocytes from peripheral blood mononuclear cells.With the endotoxin detection test,we find there is a lot of endotoxin in the expressed spike protein.After endotoxin removal with endotoxin removal column, we find the spike protein could also induce TNF-a and IL-6 secretion,but to a much less degree compared with the previous protein before endotoxin removal. In the meanwhile,we could not detection IFN-γsecretion treated by spike protein after endotoxin removal.更多还原