KRAS突变胰腺癌细胞表面巨胞饮结构的表征以及介孔二氧化硅抗肿瘤药物载体入胞特征的研究
发布时间：2018-11-23 16:16
【摘要】：胰腺癌是死亡率较高的一种癌症,患病后平均存活时间只有6个月,5年生存率只有3%-5%。70%-95%的胰腺癌组织中都存在KRAS基因突变,胰腺癌肿瘤细胞所处的组织环境血管化程度低,营养较为贫瘠。已有研究报道证明,胰腺癌细胞可通过KRAS突变所诱导的巨胞饮作用摄取胞外蛋白质,以维持肿瘤细胞的生存和增殖,相应地这种细胞较少依赖网格蛋白介导的内吞途径内化胞外物质。近年来纳米抗肿瘤药物载体的研究逐渐受到了广泛的关注,针对于不同肿瘤细胞设计不同的纳米抗肿瘤药物载体也是研究的重点之一,本研究致力于KRAS突变胰腺癌细胞表面巨胞饮结构的确认,巨胞饮结构特点和发生位置的分析,巨胞饮组成的分析以及介孔二氧化硅抗肿瘤药物载体入胞特征等研究,为达到以上研究目标,本论文按如下方案实施:1、合成带有两端酶切位点的HSA基因全序列,连接到pPIC9K质粒中,并转入毕赤酵母菌株rHSA GS115,使HSA基因融入酵母基因组中,又通过表达、鉴定及摇瓶培养、蛋白纯化浓缩等步骤,得到具有一定浓度和纯度的重组人血清白蛋白,连接到红色荧光分子Alexa Fluor 568上,获得了本研究所需要的重要工具蛋白rHSA和rHSA-Alexa Fluor 568。2、利用含有十四烷酰佛波醇乙酸酯(TPA)的培养液孵育细胞,刺激细胞产生巨胞饮,观察到细胞表面出现大量“杯”状结构,分别给予细胞带有绿色荧光的巨胞饮标志物Dextran Alexa Fluor488,以及已被确认是通过巨胞饮进入细胞的白蛋白(rHSA-Alexa Fluor 568),都观察到了“杯”状结构和它们的共定位,在同时含有Dextran Alexa Fluor488、Dextran Alexa Fluor488两种培养液孵育细胞后我们也观察到了二者的共定位,确认了“杯”状结构为巨胞饮在细胞表面的瞬时状态,经过进一步的研究获得了巨胞饮结构的尺寸信息和大致形态,为方便理解,给出了巨胞饮的三种形态示意图。3、利用已知的板状伪足中微丝和细胞膜之间的关系作为参照,分析形成巨胞饮的膜皱褶的组成成分,通过超高分辨率显微镜所拍摄的图片,能够清楚地观察到微丝和细胞膜之间的位置关系,通过iMaris软件的拟合后所形成的模型,观察到具有一定直径和深度的巨胞饮“杯”状结构,通过动态拍摄,观察到经血清饥饿处理的胰腺癌细胞突变株KRASG12CMIA PaCa-2细胞,在加入白蛋白后细胞表面“杯”状结构随着时间发生明显变化,微丝在此期间变化明显,随着时间的变化贯穿状的微丝逐渐消失,呈现散在状态,随后贯穿状微丝又重新出现。4、利用本研究中建立的方法观察到人脐静脉内皮细胞(HUVEC)中巨胞饮标志物Dextran-568(红色荧光)与单壁碳纳米管(绿色荧光)的共定位,确定在HUVEC细胞中单壁碳纳米管通过巨胞饮内化,验证了本论文中所建立的纳米材料入胞试验系统所得结果的可信度和可靠性,从而对不同尺寸的介孔二氧化硅纳米颗粒的入胞方式进行分析,得出圆形介孔二氧化硅纳米颗粒作为抗肿瘤药物载体的直径不应超过400nm的结论。本研究中结构照明显微技术为我们的实验奠定了基础,利用基于结构照明显微技术的超高分辨率显微镜,本研究建立了一种实时观察细胞表面巨胞饮的方法,对KRAS突变胰腺癌细胞KRASG12CMIA PaCa-2表面的巨胞饮结构进行了表征,并实现了对介孔二氧化硅纳米颗粒入胞方式的研究,为胰腺癌的纳米靶向给药载体设计提供可借鉴的细胞生物学数据基础。
[Abstract]:Pancreatic cancer is a kind of cancer with higher mortality. The average survival time after the disease is only 6 months, and the 5-year survival rate is only 3% -5%. In 70%-95% of the pancreatic cancer tissues, the KRAS gene mutation is present, the degree of vascularization of the pancreatic cancer cells is low, and the nutrition is poor. It has been reported that pancreatic cancer cells can take the extracellular protein by the giant cell-drinking induced by the KRAS mutation to maintain the survival and proliferation of the tumor cells, and accordingly the cells are less dependent on the endocytosis-mediated endocytosis to internalize the extracellular substance. In recent years, the research of nanometer anti-tumor drug carrier has been widely concerned, and it is also one of the key points of the study to design different nanometer anti-tumor drug carriers for different tumor cells, and this study is devoted to the confirmation of KRAS mutant pancreatic cancer cell surface giant cell drink structure, In order to achieve the above research objectives, the whole sequence of HSA gene with both ends is synthesized by the following scheme: 1. the recombinant human serum albumin with a certain concentration and purity is obtained through the steps of connecting to the pPIC9K plasmid and transferring into the Pichia pastoris strain rHSA GS115, The important tool proteins rHSA and rHSA-Alexa Fluor 568.2, which were required by the study, were connected to the red fluorescent molecule Alexa Fluor 568. The cells were incubated with a culture solution containing tetradecanol acetate (TPA) to stimulate the cells to produce a giant cell drink, and a large number of cell surfaces were observed "Cup" The cell-like structure, the macrocytosis marker Dextran Alexa Fluor 488 with green fluorescence, and the albumin (rHSA-Alexa Fluor 568), which have been confirmed to enter the cells through the giant cell, were observed. "Cup" After incubation of the cells with both Dextran Alexa Fluori488 and Dextran Alexa Fluor488, we also observed a co-location of the two, and confirmed the 鈥淐up "The shape structure is the transient state of the giant cell drink on the surface of the cell, and the size information and the general form of the giant cell drink structure are obtained through further research, and the three forms of the giant cell drink are given for the convenience of understanding. 3, by using the relation between the micro-filament and the cell membrane in the known plate-like pseudo-foot as a reference, the composition component of the film fold forming the giant cell drink is analyzed, and the positional relationship between the micro-filament and the cell membrane can be clearly observed through the picture taken by the super-high-resolution microscope, With the model formed by the fitting of iMaris software, a giant cell drink with a certain diameter and depth was observed.鈥，
本文编号：2352033
[Abstract]:Pancreatic cancer is a kind of cancer with higher mortality. The average survival time after the disease is only 6 months, and the 5-year survival rate is only 3% -5%. In 70%-95% of the pancreatic cancer tissues, the KRAS gene mutation is present, the degree of vascularization of the pancreatic cancer cells is low, and the nutrition is poor. It has been reported that pancreatic cancer cells can take the extracellular protein by the giant cell-drinking induced by the KRAS mutation to maintain the survival and proliferation of the tumor cells, and accordingly the cells are less dependent on the endocytosis-mediated endocytosis to internalize the extracellular substance. In recent years, the research of nanometer anti-tumor drug carrier has been widely concerned, and it is also one of the key points of the study to design different nanometer anti-tumor drug carriers for different tumor cells, and this study is devoted to the confirmation of KRAS mutant pancreatic cancer cell surface giant cell drink structure, In order to achieve the above research objectives, the whole sequence of HSA gene with both ends is synthesized by the following scheme: 1. the recombinant human serum albumin with a certain concentration and purity is obtained through the steps of connecting to the pPIC9K plasmid and transferring into the Pichia pastoris strain rHSA GS115, The important tool proteins rHSA and rHSA-Alexa Fluor 568.2, which were required by the study, were connected to the red fluorescent molecule Alexa Fluor 568. The cells were incubated with a culture solution containing tetradecanol acetate (TPA) to stimulate the cells to produce a giant cell drink, and a large number of cell surfaces were observed "Cup" The cell-like structure, the macrocytosis marker Dextran Alexa Fluor 488 with green fluorescence, and the albumin (rHSA-Alexa Fluor 568), which have been confirmed to enter the cells through the giant cell, were observed. "Cup" After incubation of the cells with both Dextran Alexa Fluori488 and Dextran Alexa Fluor488, we also observed a co-location of the two, and confirmed the 鈥淐up "The shape structure is the transient state of the giant cell drink on the surface of the cell, and the size information and the general form of the giant cell drink structure are obtained through further research, and the three forms of the giant cell drink are given for the convenience of understanding. 3, by using the relation between the micro-filament and the cell membrane in the known plate-like pseudo-foot as a reference, the composition component of the film fold forming the giant cell drink is analyzed, and the positional relationship between the micro-filament and the cell membrane can be clearly observed through the picture taken by the super-high-resolution microscope, With the model formed by the fitting of iMaris software, a giant cell drink with a certain diameter and depth was observed.鈥，
本文编号：2352033
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