Gene Delivery Ang Cell Reprogramming Mediated By Polysaccharides Of Traditional Chinese Medicine

Linega reprogramming has been deeply explored in liver diseases therapeutic field,especially the therapy of End-stage liver disease(ESLD).Safty and efficient gene vector play a pivotal role in the application of direct reprogramming.Angelica sinensis polysaccharide(ASP)possesses numerous active groups,which are prone to reacting with cationic reagents.Thus,cationic ASP was considered as an emerging,low-toxicity,efficient gene vector.Herein,ASP was obtained by water extraction and ethanol precipitation method and cationized by polyethyleneimine(PEI).Then,cASP-pHNF1 A self-assembly nanoparticle was fabricated through electrostatic interaction between ASP-PEI and plasmid HNF1 A.Additionally,the hepatocytes reprogramming conducted by cASP-pHNF1 A nanoparticle was studied.cASP-pHNF1 A self-assembly nanoparticle was spherical,uniformly-distributed and possessed high transfected efficiency.Induced human hepatocytes(ihHeps)could express liver markers ALB,AAT,CK18,CK19 and drug metablic enzyme CYP450,UGT,GST,uptake and excrete Indocyanine Green(ICG),store glycogen and uptake Low Density Lipoprotein(LDL).Therefore,as a novel natural gene vector,catonic ASP could conducted cells reprogramming efficiently.Mature and functional ihHeps provide possibility for application of hepatocytes transplation and other cells-based therapies in ESLD.Chapter 1 ReviewGiving a simple overview of liver-related diseases;giving a demonstration of application of cell-based therapy in liver disease and its functional failure;giving a discussion for the advantages and disadvantages of viral and non-viral vectors respectively,and giving a conclusion of their application in hepatocytes reprogramming;giving a conclusion and expectation for the application of natural polysaccharides served as novel non-viral vector in cell reprogramming;giving the aim,significance and major content of this study,planning the design ideas of this study and providing theoretical supporting for the development of this work.Chapter 2 Isolation,purification and identification of ASPThis section develops around extraction of ASP.Crude polysaccharides was isolated by traditional hot water extraction and ethanol precipitation,deproteinated by trichloroacetic acid(TCA)method and purified by D315 weak-base Ion-exchange resin.The final purified fraction was designated as ASP.The uronic acid content was determined by carbazole and sulphuric acid method.Result of HPLC suggested that the molecular weight of ASP is 3255 Da.Result of congo red experiment identified that ASP possessed triple-helical structure.The major monosaccharide components of ASP are mannose,rhamnose,glucuronic acid,galactose,arabinose and xylose with weight ratio of 0.23:0.17:14.41:0.39:1.68.Chapter 3 Preparation and characteristic of ASP-PEIThis section develops around cationized of ASP.PEI is selected as cationized reagent.Under the catalytic of Triethylamine,PEI could generate cross-linking with ASP and achieve the cationized reaction.Results of Zeta potential assay,?-amino nitrogen determination and element analysis suggest that ASP-PEI possesses higher content of element N,C and H compared with ASP,which is initially identified that cationized of ASP is success.FT-IR and NMR are employed for the further structural analysis,compared with ASP,characteristic absorption peaks of ASP-PEI are changed which is consistent with cationized reaction.Chapter 4 Preparation and characteristic of cASP-pHNF1A nanoparticlesThis section develops around preparation and characteristic of cASP-pHNF1 A nanoparticles.ASP-PEI could condense with plasmid HNF1 A into nanoparticles.Results of Zeta potential assay?diameter assay and TEM suggest that cASP-pHNF1 A nanoparticles is positive charge,nano-scale,homogeneous distribution and spherical,which facilitate to cellular internalization and cell reprogramming.Result of MTT demonstrated that cytotoxicity of nanoparticles is lower that commercial golden transfection reagents PEI and Lipfectamine2000 and is nearly equal with Lipfectamine3000.GFP reporter and qRT-PCR are carried out for the determination of cells transduction efficiency,finally,the best weight ratio between ASP-PEI and plasmid were selected for further hepatocytes reprogramming.Chapter 5 Study of somatic cells reprogramming conducted by cASP-pHNF1A nanoparticlesThis section develops around study of somatic cells reprogramming conducted by cASP-pHNF1 A nanoparticles.HFFs could be effectively reprogrammed into mature functional human hepatocytes via cASP-pHNF1 A nanoparticles.Results of immunofluorescence staining and Western Blotting,ihHeps could express liver markers ALB?AAT?CK18 and CK19.Result of qRT-PCR revealed that ihHeps could secret phase?and?metabolic enzymes.Uptake and release indocyanine green(ICG),PAS staining and low-density lipoprotein(LDL)uptake experiments identify that ihHeps possessed liver-related functions including metabolism,secretion,glycogen storage and lipid regulation.