Experimental Study On The Affection Of The Schwann Cells Which Are Transfected By HTERT

Objective:1.Schwann cells of newborn SD rats7days hTERT transfected usingthe lipofection method, cultured in vitro, to explore the safe, efficient lipofectionmethod expect to get immortalized Schwann cells.2. To detect the change of Schwann cells after transfection in the cell cycle,morphology, secretory function, lay the foundation of immortalized Schwann cells usedin nerve tissue engineering.Method: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 Schwann cells into two groups, a group of transfected Schwanncells by lipofection with the plasmid containing the hTERT gene, another group usingthe empty-free hTERT plasmid liposome transfection methodtransfected Schwann cells,with cells cultured in DMEM medium containing10%fetal bovine serum (FBS), untilthe cell growth to full density of80%replacement of complete medium containing ofG418(100ug/ml).Every3days change1times, screening2weeks, the transferredempty plasmid does not contain hTERT was filtered out, transferred to the plasmidcontaining the hTERT gene, cells continue to pass filtered to obtain positive clonescultured, passaged cells for S-100protein identified by immunofluorescence.Schwanncells and tumor cells after transfection was detected by flow cytometry cell cycle, thecontrol group, the OD value of MTT assay at different time points and growth curve by Western blot were used to detect the transfection get up early and mid-termneurotrophic factor (NGF) secretion.Results:1. by double enzyme digestion method can get a lot of proliferation ofSchwann 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 snow2lipofection transfected hTERT observed under optical microscope after transfection ofSchwann cell body bulky, long spindle. Cytoplasm yellow, S-100immunofluorescenceof Schwann cells3.flow cytometry after transfection of Schwann cell cycleBy Western blot detection of transfer dye early (1-10generations) and transfected withthe medium-term Schwann cell NGF results show that NGF secretion showed anupward trend and contrast transfected with the Schwann cell NGF secretion aftertransfection, Schwann cells secrete amount more.Conclusions: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%purity2by the lipofection method hTERT transfected stained Schwann cells can beobtained pure Schwann cells and proliferation of Schwann cells than untransfectedstrong increase in NGF secretion.