Experimental Study On The In Vitro Transfection Of Thiolated N-alkylated Chitosan Bearing Coexpression Plasmid PIRES-hVEGFl2lcDNA/hBMP4

Objective Recombinant coexpression plasmid of pIRES-hVEGFl2lcDNA/hBMP4 was transfected into rat bone marrow mesenchymal stem cells(MSCs) mediated by thiolated N-alkylated chitosan(TACS).To investigate the feasibility of using TACS as a gene vector in bone tissue engineering and build an experimental base on conbination gene therapy of bone defects.Methods MSCs were isolated and cultivated from the bone marrow of rat. Recombinant coexpression plasmid of pIRES-hVEGFl2lcDNA/hBMP4 was confirmed by restriction enzymolysis,then TACS-pDNA nanoparticles were prepared by complex coacervation.The morphology and particle size of nanoparticles were observed by transmission electronic microscopy and nanoparticle size analyzer. The protection of the nanoparticles for pDNA was observed by gel electrophoresis.TACS bearing pIRES-hVEGFl2lcDNA/hBMP4 was transfected into the third generation of MSCs.Meanwhile,Chitosan group, liposome group and naked pDNA group was respectively experimental control,positive control and negative control.Different effects of the nanoparticles on cell viability were illustrated by MTT assay.After 3 days and 4 days of transfection, the total RNA and total protein were extracted from MSCs respectively.The target gene expressions were detected by RT-PCR and Western blot . Results Lots of MSCs were separated from bone marrow of rat and the cells showed active proliferative ability.We got expected results on identification of pIRES-hVEGFl2lcDNA/hBMP4 by restriction enzymolysis.The shape of nanoparticles was not very homogeneous and the average particle size was about 264nm determined by nanoparticle size analyzer. TACS could protect encapsulated pDNA effectively from nuclease degradation as shown by electrophoretic mobility analysis.The related indexes of MSCs were inspected after transfection,and we found that cell viability of TACS group(73.18±6.56)% was significantly higher than liposome group(45.92±4.93)%(P<0.01).HVEGF121 and hBMP4 were expressed well in transfected MSCs detected by RT-PCR and Western blot except the negative control.We also conducted relative semi-quantitative analysis on the grey value of Western blot strips and got the result that the target protein expressions of TACS group were lower than liposome group(P<0.05) but significantly higher than chitosan group(P<0.01).Conclusion The coexpression plasmid was transfected into MSCs successfully by TACS. TACS has low cytotoxicity and its transfection efficiency is obviously higher than non-modified chitosan.