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Experimental Study on the in Vitro Transfection of Thiolated N-alkylated Chitosan Bearing Coexpression Plasmid pIRES-hVEGFl2lcDNA/hBMP4

Author: LuDi
Tutor: WangChunLan
School: Anhui Medical University,
Course: Surgery
Keywords: Chitosan Mesenchymal stem cells Vascular endothelial growth factor Bone morphogenetic protein Transfection
CLC: R68
Type: Master's thesis
Year: 2010
Downloads: 25
Quote: 0
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Abstract


Objective: to new mercaptoalkyl chitosan (TACS) as the carrier -mediated recombination total express plasmid pIRES-hVEGFl2lcDNA/hBMP4 vitro transfection of rat bone marrow mesenchymal stem cells ( MSCs ) to investigate the gene the TACS for bone tissue engineering the feasibility of the carrier , to provide an experimental basis for the further study of gene therapy of bone defects . : Whole bone marrow culture separated from cultured rat MSCs ; eukaryotic co-expression plasmid pIRES-hVEGFl2lcDNA/hBMP4 by restriction endonuclease complex coacervation the TACS- gene nanoparticles , their morphology and size were observed by transmission electron microscopy and characterization , gel retardation analysis of gene protection ; nanoparticles transfected rat 3 of MSCs and set chitosan group , liposome group , and the naked plasmid group were experimental control , positive control total RNA extracted MSCs 3,4 d respectively after transfection , total protein , reverse transcription - polymerase chain reaction (RT-PCR), Western and negative controls , thiazolyl blue (MTT) method to compare different cytotoxic gene vector difference ; blot to detect the expression of the target gene . Results: whole bone marrow culture method available to a large number of MSCs and has the ability to actively proliferating ; co-expression plasmid by restriction endonuclease , with the expected results ; TACS the / of pDNA composite nanoparticles morphology is not very uniform , an average particle size of about 264nm ; gel retardation the analysis demonstrates TACS parcels and protection co-expression plasmid from DNase Ⅰ enzyme digestion ; group of MSCs transfected index detection , TACS cell viability (73.18 ± 6.56)%, significantly higher than the liposome group (45.92 ± 4.93)% (P lt; 0.01); addition to the negative control group , RT-PCR and Western blot were detected in MSCs transfected with expression hVEGF121 and hBMP4 gray value of the resulting bands on Western blot semi-quantitative analysis shows that TACS group expression of the target protein is less than the liposome group (P lt; 0.05), but significantly higher than the chitosan group ( P lt; 0.01 ) . Conclusion: the expression plasmid pIRES-hVEGFl2lcDNA/hBMP4 TACS mediated successfully transfected rat MSCs and expression . TACS cell toxicity , and significantly improved compared to the unmodified chitosan transfection efficiency .

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CLC: > Medicine, health > Surgery > Orthopaedic Surgery ( movement system diseases,orthopedic surgery )
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