| Some of the diseases,such as malignant tumors,Alzheimer's disease,Parkinson's disease,congenital genetic defects and various tissues and organs damage,have complex etiology and are difficult to cure or achieve desired therapeutic effect using traditional medical means.Regenerative medicine came into being to solve this problem.With the renewal and development of life science and technology and therapeutic concepts,regenerative medicine has covered many aspects,including tissue engineering,cell and cytokine therapy and gene therapy treatment.Cell types in cell transplantation treatment vary from diseases to diseases,which is essential for establishing therapeutic schedule.Thus,cell source is extremely important.Somatic cell reprogramming techniques,which make it possible for the transformation between different cell lines,have perfectly solved the problem.In this study,human umbilical cord mesenchymal stem cells(HUCMSCs)were used to form induced pluripotent stem cells by polysaccharide-modified calcium phosphate nanoparticles carrier containing miRNA plasmid,in which cell reprogramming is mediated by miRNA.This may provide a theoretical basis for solving the problem of regenerative cell source.The full text consists mainly of five chapters:Chapter 1 ReviewThis chapter focuses on cell reprogramming techniques,and the more efficient methods are exogenous factor-inducing methods,including small molecule induction,miRNA induction,transposon,multicistron plasmids and non-viral vector-mediated transient transfection and etc.The advantages and disadvantages of the viral vector and non-viral vector application in cell reprogramming were compared.Taking the safety and biocompatibility into account,the advantages of non-viral gene vectors began to emerge.Natural polysaccharides represented by traditional Chinese medicine polysaccharides have a wide range of applications because of many advantages and prospect in the field of non-viral gene carrier materials.In order to achieve sustained and effective gene release,thereby enhancing gene transfection efficiency,the three-dimensional gene delivery system is applied to cell reprogramming.Chapter 2 Extraction and Purification of Polysaccharides from Traditional Chinese Medicine and Preparation and Characterization of Cationic PolysaccharidesIn this chapter,the polysaccharides of Salvia miltiorrhiza Bunge were isolated and purified by macroporous resin and obtained polysaccharide SMP with no protein and nucleic acid and high purity with 5% ethanol elution.High performance liquid chromatography(HPLC)showed that SMP polysaccharide had only one single peak,and the molecular weight of SMP was 11455 Da.The SMP polysaccharides were subjected to cationization(ethylene diamine modification)by Schiff base method.The elemental analysis of the cationized polysaccharides was carried out.The N content was 6.18% and the Fourier transform infrared spectroscopy(FT-IR)showed N-H bending vibration at 1630 cm-1.The zeta potential of the cationized polysaccharides was +7.29 m V.The results of gel electrophoresis showed that the cationized polysaccharide had the ability of gene carrying and the protective effect on the plasmid,and could protect the plasmid from DNase.Chapter 3 Preparation,Characterization and Transduction Efficiency of Calcium Phosphate Nanoparticles mixed with miRNA and PolysaccharideIn this chapter,the calcium phosphate nanoparticles containing miRNA plasmids were modified by cationic polysaccharide,and the mixed nanoparticles were characterized.The Zeta potential of the hybrid nanoparticles is +6.54 m V and +5.88 m V,and the particle size is within 200 nm.The electron microscopy shows that the nanoparticles are spherical.The results of infrared spectroscopy showed that the prepared calcium phosphate nanoparticles contained cationized Salvia miltiorrhiza polysaccharide,which indicated that the cationized Salvia miltiorrhiza polysaccharide was successfully linked/ encapsulated in the nanoparticles.The results of gel electrophoresis showed that the nanoparticles could contain the plasmid,they have low cytotoxicity and high transfection efficiency.The transduction of miRNA mixed nanoparticles in somatic cells involves the endocytosis pathway of the cytoplasm,the vesicular vesicle-mediated endocytosis pathway and the endosomal lysosomal system play an important role in the effective transfection.The cytoskeleton-mediated endocytosis pathway does not participate in the uptake of mixed nanoparticles.Cytoskeleton microfilaments mediate the intracellular transport of hybrid nanoparticles,but the inhibition of cytoskeleton is not conducive to the transfection of mixed nanoparticles.When the mass ratio of polysaccharide-calcium phosphate nanoparticles to miRNA plasmid was 160: 1,the number of cells was 2×106 cells /m L,and the transfection efficiency was the best.Chapter 4 Somatic cell reprogramming and its mechanism mediated by miRNA polysaccharide mixed calcium phosphate nanoparticleIn this chapter,we mainly introduced that the transfected HUCMSCs cells with miRNA mixed nanoparticles.After transfected for four times,the clones formed.The clones were characterized with small morphological,high cytoplasmic ratio,dense colonies and clear edges.Alkaline phosphatase staining,immunofluorescence and Western Blot showed that iPS overexpressed Sox2,Oct4,SSEA3,Tra-1-81,Nanog which are specific marker molecule of the pluripotent stem cells.The iPS has the ability of induction and differentiation to any cells belonging to any germ in vitro and in vivo.It can differentiate into ectodermal nerve cells,mesodermal chondrocytes,endodermal liver cells in vitro;form a variety of three layers of tissue,including ectodermal nerve,Mesodermal fat and muscle and endodermal glands in vivo.The teratoma formed from the iPS is a substantial tumor and has good clinical safet.The iPS has both pluripotent genes and three germ differentiation genes,the formation of embryoid bodies(EB)in vitro can not maintain its pluripotency and three germination ability for a long time.m RNA and protein expression of NR2F2 gene was down-regulated with NR2F2 gene suppressed by miRNA,antagonizing TGF-?2 and inhibiting the epithelialmesenchymal transition(EMT)of the cells.Thus cell reprogramming was triggered to induced pluripotent stem cells;miRNA can also directly inhibit TGF-?2 receptors,down-regulate the expression of smad2/3 and psmad2/3,thereby promoting somatic cell reprogramming.The prepared non-viral polysaccharide miRNA vectors have high biocompatibility,high safety and low risk of carcinogenesis,and can provide an ideal way for reprogramming human cells for therapeutic use.Chapter 5 Somatic cell reprogramming mediated by miRNA mixed nanoparticles in 3 dimensional fibrin scaffoldsIn this chapter,fibrinogen and thrombin were used to prepare three-dimensional scaffolds suitable for cell growth,and the best fibrin scaffolds were screened by scanning electron microscopy and used for three-dimensional somatic cell reprogramming.The preparation method is simple and the cell reprogramming efficiency is greatly improved.Fibrin scaffolds was prepared with 50 mg/m L fibrinogen plus 10 ?L 100 U/m L thrombin.The Fibrin scaffolds have loose porous structure,50-100 ?m pore size and suitable for cell growth.It was demonstrated that HUCMSCs were reprogrammed in fibrin scaffolds.The cells in fibrin scaffolds grow better and proliferate faster than those in two-dimensional cells.Cell clones formed in 2-4 days in fibrin scaffolds,alkaline phosphatase staining were positive.The positive expression of Nanog,Tra-1-81 and SSEA3 were identified by immunofluorescence which were the characteristics of iPS/ES cells.Sox2 and Oct4 were aslo identified by immunehistochemistry and paraffin-embedded sections and HE staining.The clones formed in the three-dimensional scaffolds were identified as iPS cells.The fibrin scaffolds are biocompatible and well sized to provide cells with a three-dimensional environment similar to that in vivo,and reprogramming efficiency are highly improved and require a short time for somatic cell reprogramming which may provide a new,efficient strategy for cell reprogramming in clinical applications. |
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