Isolation And Analysis Of DNA Engineered Circulating Tumor Cells Based On Proximity-enhancement Effect

Circulating tumor cells(CTCs)are collective terms for various types of tumor cells that fall off from the primary tumor site and enter the peripheral blood.They are an important new type of markers in tumor research,which have high application value in cancer diagnosis and treatment effect evaluation,so they have become the object of widespread attention in tumor marker research.At present,some methods of isolation and analysis of circulating tumor cells have been developed,but these methods are not universal and do not have universality for different types of circulating tumor cells.In the work of this paper,we combine functionalized nanomaterials and engineered cell techniques to develop a method for capturing isolated circulating tumor cells.Among them,the functionalized nanomaterial is a magnetic bead modified by DNA tetrahedral nucleic acid framework,which is formed by the combination of biotin and streptavidin,and DNA tetrahedron as a rigid nucleic acid structure provides direction and adjacent positions for the capture of single-stranded DNA of engineered cells,and has better capture efficiency and biocompatibility compared with other nanomaterials.Engineered cells are through the cell sugar metabolism engineering,and then through the click chemical reaction to modify the DNA to the cell surface,because the tumor cells than the ordinary cells of the sugar metabolism rate is faster,DNA engineered tumor cells are far more than the engineered normal cells,so that the construction of DNA engineered tumor cells can be complementary to the functional beads through DNA complementary combination,and then through the magnetic absorption can easily be the circulating tumor cells from the cell population isolated.We used this method to successfully construct multiple DNA-engineered tumor cell lines and successfully isolate them by magnetic absorption.This method can isolate and capture a variety of circulating tumor cells,which has good universality and has high reference value for the isolation and analysis of circulating tumor cells in clinical research.The research content of the thesis mainly includes the following three aspects of work:1.Construction and characterization of DNA-functionalized magnetic beadsDNA functionalization beads are nanomaterials and tools used to isolate circulating tumor cells in this work,and in this chapter we first construct DNA functionalization beads and characterize and investigate them.We use streptavidin beads as the substrate,through the specific binding of avidin to biotin,DNA tetrahedral nucleic acid framework modified with biotin is bound to the surface of the magnetic beads to obtain DNA functionalization modification of the magnetic beads.DNA tetrahedral framework was chosen to functionalize the beads because the tetrahedron has excellent rigidity and stability,and the two single-stranded tails extending outward on its vertices are used to subsequently use the orthometric enhancement effect to bind to engineered cells.The assembly of the DNA tetrahedral nucleic acid framework is characterized by agarose gel electrophoresis,and the modification of the DNA tetrahedral nucleic acid framework on the bead is identified by fluorescent modification on the DNA strand.Experimental results show that DNA tetrahedron can be successfully and stably modified on the outer surface of the magnetic bead,providing a favorable precondition for subsequent capture of engineered cells.2.Construction and application of DNA engineered cellsThe successful construction of DNA engineered cells is important for the isolation and analysis of circulating tumor cells.The construction strategy is to allow cells to metabolize chemical sugars containing azide groups(tetraacylated-Nazidoacetyl-mannitolamine),so that the cell membrane protein glycosyl chains are banded with azide groups,and then the DNA strands are modified to the cell surface by clicking chemical reactions to construct DNA engineered cells.Due to tumor cells preference for glucose metabolism,the surface modified DNA strand density of circulating tumor cells in the blood will be larger than that of normal blood cells,making it possible for functionalized magnetic beads to capture engineered circulating tumor cells using the orthometric enhancement effect.We performed the same engineering treatment on three kinds of tumor cells(human cervical cancer cell Hela,human pancreatic cancer cell PATTU-8988 and human liver cancer cell HepG2)and two normal blood cells(red blood cells and white blood cells),and then used functional magnetic beads to bind these cells,and found that all three tumor cells can bind functional magnetic beads,while two normal blood cells cannot bind to functional magnetic beads.Such results indicate that tumor cells can be successfully engineered and can be tightly bound to functional magnetic beads,while normal blood cells cannot,which is the basis for subsequent isolation and analysis of circulating tumor cells in the blood.3.Detection of DNA-engineered circulating tumor cells based on proximityenhancement effectIn this chapter,DNA engineered circulating tumor cells are captured by using DNA functionalization beads as cell trappers,and then the isolated circulating tumor cells are cultured and analyzed.By lysing red blood cells in human whole blood,adding DNA engineered tumor cells to the resulting white blood cells to simulate the engineered circulating tumor cells in the peripheral blood,and then using functional magnetic beads and an applied magnetic field for capture and isolation.Experimental results show that DNA functionalized beads will bind to DNA engineered circulating tumor cells,flow cytometry can be combined with a small number of engineered circulating tumor cells bound to magnetic beads in a large number of white blood cells that have never bound magnetic beads;on the other hand,under the action of the applied magnetic field,circulating tumor cells can also be isolated from the cell mixture,and after routine cell culture of the isolated circulating tumor cells,it is found that the cell growth and proliferation are good,and the petri dish can be covered within 72 h.The viable cell rate reaches more than95%.The above results are applicable to two types of tumor cells of different types(human cervical cancer Hela cells and human liver cancer HepG2 cells),indicating that the DNA engineered circulating tumor cell isolation and analysis method we have developed has a certain universality.