Carbon Quantum Dots Based Nano-gene Delivery And Its Application In U251 Stem Cell Differentiation

Glioma(GBM)is one of the most aggressive and difficult tumors in adult tumor diseases.Glioma stem cells(GSC)are the basic factors leading to the development of GBM and subsequent recurrence.Based on this,the treatment of the tumor stem cells is actually the process of radical method of the disease.In recent years,with the rapid development of regenerative medicine and tissue engineering biotechnology,gene therapy has become a prelude for glioma treatment.During the treatment process,the viral vector was the most widely used gene delivery system,but it still has some limitations.In view of this,scientist turned their attention to non-viral vector system and combined gene therapy.As one of the hotspots in gene therapy research,there is the need to prepare the non-viral nanocarriers by using reasonable materials.Based on the abovementioned research problems,this topic focused on the preparation of a new type of non-viral nanocarrier Carbon quantum dots(CDs).On the basis of U251 neural differentiation of stem cells research,we verified relevant differentiation as well as the in vivo and mechanistic studies for clinical GBM treatment with the aim of contributing new methods and ideas to stem cell research.Chapter 1 ReviewBased on the global problems of GBM,the introduction of stem cell theory,the advantages of gene therapy and the diversification of gene vectors,this chapter is divided into two parts,namely the research progress of GBM and CDs.The disease status,research direction and the treatment methods of GBM were introduced,which included the advantages and disadvantages of each method,as well as the status of its application in the theoretical research,clinical research and clinical treatment.This thesis mainly introduces CDs as a new nano-gene carrier,from its development to preparation method,as well as wide application in gene delivery and prospect,which lays a solid theoretical foundation for this topic.Chapter 2 Preparation and characterization of CDsWith chitosan quaternary ammonium salt and anhydrous ethylenediamine(EDA)as raw materials and on the basis of preliminary experiment,experiments were designed with 16 kinds of CDs.Then,we examined the two aseptic methods such as filtration and heating,while with fluorescence intensity and carrying EGFP plasmid ability as an index,we obtained the best prescription of CDs from group 7 samples.The optimal reaction temperature was 160?,while the EDA was diluted five times with PBS and the system of volume ratio was 20%(the filtering aseptic method).The optimal gene transfection ratio of gene and CDs was 1:10,while the sample was used as the basis for subsequent transfection and induction in conducting a series of corresponding studies.Characteristics such as morphology(by transmission electron microscopy-TEM),particle size,zeta potential,linkage functional group(by infra-red-IR)and cytotoxicity of the optimal QDS were characterized.The results showed that the CDs were less toxic to the cells(almost non-toxic)and had bright green fluorescence,which could be used as a non-viral vector for tissue engineering research.Chapter 3 CDs-mediated differentiation of U251 stem cells in vitroThis chapter is mainly divided into the following three aspects:firstly,the U251cells were cultured in stem cell induction medium(ES culture medium),and CD133~+U251 cells were selected by flow cytometry,while the U251 stem cells were obtained as seed cells for subsequent experiments(source of experimental cells).Through the study of CDs mechanism of entering into the cells using eight kinds of related inhibitors,it was indicated that CDs-loaded genes could enter the cell through the endocytosis mediated by clathrin and vesicles.Subsequently,the free combination of the transcription factors such as Wnt3a,Brn2 and ascl-1 was used to select the optimal association of the factors.According to the immunofluorescence(IF)results,the combination of Wnt3a and Brn2 factors could achieve the differentiation of U251 stem cells into nerve cells in the shortest time,and this informed the selection of the optimal gene combination for further study.Then,U251 stem cells were transfected and induced by CDs-loaded optimal gene combination.After four times transfections,the cells first exhibited morphological structures similar to nerve cells.Subsequently,IF,enzyme-linked immunosorbent assay(ELISA),reverse transcription-PCR(QT-qPCR)and Western blotting(WB)assays proved that the expression of CD133,a stem cell marker protein,was significantly down-regulated from a qualitative and quantitative perspective.GFAP,a characteristic of glial cells,was down-regulated with the time of transfection induction,while the neurocyte related proteins(NeuN,?-tubulin III(Tuj1))were gradually up-regulated with the time of transfection to explore the efficacy of neural differentiation.In addition,the gene transcription and protein levels were also investigated with the result indicating that CDs-carrying genes Wnt3a and Brn2 were introduced into the cells for biological transcription and translation.Collectively,these findings suggest a possible differentiation into nerve cells,thereby providing a new idea for basic theoretical GBM research.Chapter 4 CDs-mediated differentiation of U251 stem cells in vivoThis chapter primarily studies the differentiation of U251 stem cells and its induction after in situ transplantation in nude mice.The survival status,weight change,tumorigenicity,HE staining,immunohistochemistry and gene microarray of nude mice were used to study the efficacy of the neural differentiation in vivo.The results showed that the survival status of nude mice in the experimental group was relatively well,and the body weight was comparatively stable.HE and immunohistochemical staining also demonstrated that the heteromorphism of the experimental group was less obvious than that of the control group,while the tumor cells instead showed a trend of neuronal differentiation.Through the analysis of gene chip data,it was found that the related proteins of p53 and other oncogene-related pathways showed a downregulation trend.This paper provides reliable preclinical data and basis for further studies of glial diseases,which is consistent with the data of in vitro studies.Chapter 5 Studies on Wnt and Notch pathwaysThis chapter studied the relationship between transcription factors of Wnt pathway and Notch pathway and their relations.First,QT-qPCR was performed to investigate the expression strength of Notch1 and HES-1,the key genes of Notch signaling pathway,and it was found that the decrease of pathway protein was closely related to the differentiation of U251 stem cells.Secondly,the expression level of the related proteins in the Wnt pathway was detected via immunohistochemistry,and the result indicated that the Wnt pathway plays an important role in the induction and differentiation of the U251 stem cells into nerve cells through the induction and differentiation of the CDs neuro-carrying transcription factors.