| Pancreatic cancer is a kind of cancers with a high mortality rate and a median survival of 6 months and a dismal 5-year survival rate of 3–5% and this data has remained relatively unchanged over years. 70%-95% of the pancreatic cancer exists KRAS mutations, the vascularization degree of the pancreatic cancer cells microenviroment is low and the nutrition is barren. It has been reported that pancreatic cancer cells with KRAS mutations scavenge extracellular protein by RAS-driven macropinocytosis in order to maintain the proliferation and survival of tumor cells. Correspondingly, such kind of cells less depends on clathrin-mediated endocytosis to internalize extracellular substance. Recent years, studies of anticancer drug nanocarriers have been widespread concerned, one important point is how to design different antineoplastic nanocarrier for different tumor cells. This study is to confirm macropinocytosis structures on the cell surface of KRAS mutations pancreatic cancer. And the studies are focused on analyzing the characteristics of macropinocytosis structures and location of macropinocytosis. More over, the internalization of mesoporous silica nanoparticles(MSNs) anticancer carriers into the cell are expected to be characterized. In order to achieve the above research purposes, the thesis is carried out according to the following scheme:1. HSA gene sequences with restriction sites at both terminus of them were synthesized, then connected to pPIC9 K plasmid, and transferred to Pichia yeast strain rHSA GS115. HSA gene was integrated into the yeast genome followed by the steps of expression, identification, flasks culture, protein purification and protein concentrated. The recombinant human serum albumin with certain concentration and purity was obtained. The red fluorescent probe, Alexa Fluor 568, is attached to the protein. The protein rHSA and rHSA-Alexa Fluor 568 are important tools in this study.2. We incubate the cells with culture fluid containing tetradecanoyl phorbolacetate(TPA) in order to stimulate macropinocytosis. We observed a large number of "cup"-like structures on the cell surface. The cells were then treated with a green fluorescent probe, Dextran Alexa Fluor488, which is the marker of macropinocytosis, and albumin(rHSA-Alexa Fluor 568) which has been recognized as a macropinocytosis-mediated protein. The co-localization of "cup"-like structures has been observed. While cells are incubated with both Dextran Alexa Fluor488 and rHSA-Alexa Fluor 568, co-localization of them was also observed. So the "Cup"-like structures are confirmed to be the transient state of macropinocytosis structures on the cell surface. Further study provided the information of the size and general morphology of the macropinocytosis structures to facilitate understanding.The pictures of three forms of macropinocytosis structures has been shown.3. According to the known relationship between actin and the cell membrane of lamellipodia, we can use it as a reference to analyze the formation of macropinocytosis structure. And we can analyse the components of membrane ruffles through the images taken by ultra-high resolution microscope. The position between the actin and membranes can be clearly observed. After reconstruction by the software i Maris, we can observe macropinocytosis "cup"-like structures with a certain diameter and depth through dynamic shooting. More over, we can observe that the "cup"-like structures of serum starved pancreatic cancer cells with KRAS mutation were changed significantly once albumin was added during this period.The integrated actin disappeared gradually and became scattered state. The integrated actin reoccured.4. According to the developed method in this study, we observed the co-localization between macropinocytosis marker Dextran-568(red fluorescence)and single-walled carbon nanotubes(green fluorescence) in human umbilical vein endothelial cells(HUVEC). And we confirmed that single walled carbon nanotubes are internalized via macropinocytosis. This further verified the method we established in this paper. Thus, the method can be applied to analyze internalizationmode of different sizes of MSNs. The conclusion is that circular MSNs as carriers of anticancer drugs should not exceed the diameter of 400 nm.In this study, the structured illumination microscopy is the foundation of our experiments. Utilizing ultra-high resolution microscopy based on microscope of structured illumination technology, we established a method for real-time observation of macropinocytosis on cell surface in this study and characterized the macropinocytosis structures on the suface of KRAS mutation pancreatic cancer.More importantly, we realised the study of the internalization of MSNs into the cells,and provided a reference for designing the nanocarriers as pancreatic cancer targeting drug delivery devices. |
PDF Full Text Request