| Objective: Schwann cells plays an important role in regeneration of nerve repair. Invitro, schwann cells were cultured in a continuous passage way, the cells were gradualsenescence and apoptosis. Immortalized schwann cells have the function of the infiniteto proliferation, can solve the problem of schwann cells in nerve tissue engineering. Inthis experiment, To detect the changes of Schwann cells after transfection with hTERT.Lay the foundation of immortalized Schwann cells used in nerve tissue engineering.Methods:1.Select the SD rats7days after birth, frozen executed iodophor soakdisinfection, whichever is the sciatic nerve and brachial plexus, cut it into pieces to1mm3size of the organization, use dual-enzyme digestion method vitro culture, whenthe cell growth to85%confluence density use low concentrations of trypsin to digestionand passage rapidly,30minutes differential attachment wall method further purifiedSchwann cells, Schwann cells were observed by optical microscope and photographed,S-100protein was identified by immunofluorescence camera.2.The purified Schwanncells into two groups, a group of transfected Schwann cells by lipofection with theplasmid containing the hTERT gene, another group using the empty-free hTERTplasmid liposome transfection methodtransfected Schwann cells, with cells cultured inDMEM medium containing10%fetal bovine serum (FBS), until the cell growth to fulldensity of80%replacement of complete medium containing of G418(100ug/ml).Every3days change1times, screening2weeks, the transferred empty plasmid does notcontain hTERT was filtered out, transferred to the plasmid containing the hTERT gene,cells continue to pass filtered to obtain positive clones cultured, passaged cells forS-100protein identified by immunofluorescence. Schwann cells (5,10,20,30passage)trasfected with hTERT and five generations compared with primary culture cell wereused to detect the expressions of NGFã€BDNF and BFGF secretion by Real-time PCRand Western blot.Results:1. By double enzyme digestion method can get a lot of proliferation of Schwann cells, Schwann cells arranged side by side wall passages can be a typical longspindle radial differential adhesion, light microscope view snow Schwann cell puritycan be achieved more than90%of fibroblast cells less, measured by S-100proteinimmunofluorescence identification of Schwann cells for the pure snow.2lipofectiontransfected hTERT observed under optical microscope after transfection of Schwanncell body bulky, long spindle. Cytoplasm yellow, S-100immunofluorescence ofSchwann cells.3. Real-time PCR showed that there is a significantly increased in NGF,BDNF BFGF and mRNA expression of Primary schwann cells cultured five generationscompared with primary culture cell. After trasfection with hTERT, there were stableexpressions of NGF, BDNF and BFGF in Schwann cells. However, with continuouspassage culture, the expressions of NGF, BDNF and BFGF were decreasd gradually.4.Western Bloting results showed that there is a significantly increased in protein levels ofNGF, BFGF and BDNF of Primary schwann cells cultured five generations comparedwith primary culture cell, and have stable expressions of NGF, BDNF and BFGF inSchwann cells. However, with continuous passage culture, the expressions of NGF,BDNF and BFGF were also decreasd gradually.Conclusion:1. SD rats7days to take the ischium and the brachial plexus, adual-enzyme digestion, organization pulomonary, differential attachment of Schwanncells in the wall of passage can get more than90%purity.2. Schwann cells transfectedwith hTERT can be obtained pure Schwann cells and proliferation of Schwann cellsthan untransfected strong increase in function of secretion.3. After the success of thetransfection of schwann cells value-added ability enhancement, its ability to secreteNGF, BDNF, BFGF has been enhanced, can solve the limition of normal culturedschwann cells... |
