PEGFP-C3-CNTF Effect Of Neuron Differentiation Of Myoblasts And Its Compatibility With Acellular Allogeneic Nerve Study

Objective1. To construct the fusion expression plasmid of the rat ciliary ganglionnerve growth factor (CNTF) and enhanced green fluorescent protein(pEGFP-C3).2. To observe pEGFP-C3which marked CNTF expressed in muscle cellswith fluorescence microscopy,tracing the vesting conditions of myoblasts invitro.3. To research the compatibility of pEGFP-C3-CNTF which transfer themyoblasts after induced differentiation in vitro and cellular nerve that co-cultrer in vitro,using scanning electron microscope.MethodsUsing the method of Freezing and thawing to prepare acellular allogenicnerve.As a template with PDBLeu-CNTF,to gain full-length of rat CNTF mRNAwith PCR amplification,the directional fragments insert to pEGFP-C3whichexpressed in eukaryotic,then transfer to Escherichia coli and filter with antibiotictablets to gain positive clones,and then digestion,PCR and sequencing assay,respectively.Sampling, isolation, culture of rat Myoblast cells. To establish astable transfection of myoblasts of rat pEGFP-C3-CNTF,filtering with G418,weuse liposomes Lipofectin2000to recombine plasmid transfected into musclecells.Using the MTT assay cell growth via reverse transcription polymerasechain reaction (RT-PCR) to detect the CNTF mRNA expression,using pEGFP-C3with fluorescence microscope trace CNTF expression in Myoblast cells. Wedivided induced differentiation in vitro experiments into three group, A:pEGFP-C3-CNTF groups, B:pEGFP-C3into muscle cells turn into muscle cells group,C:did not transfer into muscle cells group.After7days to confirm whether the myoblast differentiation into neuron-like cells by the S-100induced immunohisto-chemical staining.Using Pathological Image Analyzer to analyze immunohisto-chemical slice and calculate the area distribution of S-100-positive,usingfluorescence microscopy to deserve pEGFP-C3-CNTF go into muscle cells. ThePEGFP-C3-CNTF induced in vitro transfer myoblasts to multi-point injectcellular allogeneic nerve and deserve the ultrastructure of nerve by scanningelectron microscope after3days.ResultsThrough HE staining we use Freezing and thawing chemical method toprepare acellular nerve allograft,the method can remove effectively thecomponents antigen of cell and retain completely the components of thebasement membrane.As the template with pDBLeu-CNTF mass to grain422bpfull length DNA fragment (cDNA),increasing the length of rat CNTF coding areaby PCR and inserting successfully to downstream of really nuclear expressioncarrier pEGFP-C3of CMV started child,to construct large rat CNTF gene ofrestructuring mass grain pEGFP-C3-CNTF by PCR and double enzyme to gain422bp of section. Confirmed that the recombinant plasmid with CNTF targetgene fragment, DNA sequencing to confirm the insertion sequences areaccurate.In accordance with the Lipofecamine2000, using cationic liposome totransfer into muscle cells, proving that CNTF expression in Myoblast cellstability and high by RT-PCR, proving the transfection of plasmid DNA intomuscle cells have a higher relative proliferation rate by MTT bypEGFP-C3-CNTF, pEGFP-C3have no effect on cell growth and proliferation ofMyoblast.Dserving cell with fluorescence microscope at24h and72h,the cell in72h increase in number,to fliter at6-8w by G418pEGFP-C3-CNTF establishedstable transfection of plasmid DNA into muscle cells. The resulting cells in vitroprove S-100staining positive,the cells are nerve-like cells.The transfection ofpEGFP-C3-CNTF are more significantly than muscle cells into muscle cellstransfected by pEGFP-C3group and did not transfer group, differences havesignificant statistically (p<0.01) and no statistically significant differencebetween two groups.After7d induced by using fluorescence microscopy,induced to differentiate into cells are still issued a green fluorescence, cells are polygonal, star, and the same as S-100staining cells. The pEGFP-C3-CNTFinto muscle cells and acellular allogeneic nerve scaffold in vitro co-culture byscanning electron microscope, we can see types of Neuron-like cells in acellularnerve Allograft gathering attachment on the basement membrane andadsorption of growth.Conclusions1.pEGFP-C3-CNTF transfer successfully into muscle cells, CNTF inmyoblast cells can express stablely.2. pEGFP-C3-CNTF transfer into muscle cells can differentiate intonerve-like cells in vitro.3. There are good compatibility between pEGFP-C3-CNTF transfer intomuscle cells and acellular nerve Allograft.