Experiment Study Of Separating And Proliferation Of Cochlear Schwann Cells

Sensorineural hearing loss(SHL) is one of the most popular diseases whichaffect people’s normal life quality. Many studies have been carried out on thepathogenic mechanism and therapy method of SHL. The main diseased regionslocate at hair cells and primary afferent spiral ganglion neurons(SGN),whichcan’t be regenerated after injury. Therefore, it is very important to explore themethods to repair the injured SGN and improve its self-recoveryability.Although SCs are the most import supportive and myelinating cellswhich play a critical role in regeneration of peripheral nerves. My study focuseson purification and separation of cochlear Schwann cells by immunomagneticbeads method. This method can obtain massive purified normal Schwann cellsto permit a study of the molecular nature of interacting cochlear glia andauditory neurons．Part one：Purification and Separation of Cochlear Schwann Cellsby Immunomagnetic Beads Method[Objective] To introduce a method to obtain Schwann cells from newly born SD rats massively and purely by immunomagnetic beads method.[Method] SD rats, which were born1-3days, were chosen. Under sterileconditions, expose the bilateral auditory vesicles, dissect the snail shell at highmagnification, open cochlea and take out the cochlea completely, isolate andremove stria vascularis and basement membrane on the lateral wall ofmembranous cochlear duct, and cut in pieces.0.25%trypsin and0.2%collagenase I was used for digestion, FBS was used to suspend the digestion,and that was centrifuged with the addition of DMEM/F12medium. After3-5days, cells were purified using immunomagnetic microbeads, and cellpassage was done after culturing for2days. During the whole process，thechange of Schwann cells was observed under the inverted biological microscopeand vital force of Schwann cells was evaluated．The growth curve of Schwanncell was drawn with MTT． The Schwann cells was identified underimmunofluorescence test and record the purity．[Result] The cell separated from the SD rat’s spiral ganglia and cultured inincubator was affirmed as Schwann cells．Immunomagnetic beads can be used topurify the Schwann cells．The density of cochlear Schwann cells purified was97%．[Conclusion] We can get lots of purified normal cochlear Schwann cellsfrom this method to permit a study of the molecular nature of interactingcochlear glia and auditory neurons．Part two: Experiment study of proliferation of cochlearSchwann cells[Objective] To search a best culture solution prescription for proliferatingSchwann cells. [Method] Choose the third generation cochlear Schwann cells，make cellssuspension and make sure that the density of SCs is1×104/ml, and then make aninoculation into the96-well culture plates. There are6groups in the experiment.[Result] All of the B, C, D, E and F group are advantageous to the blankcontrol group, with significance in statistics; Group E and F are the best of thesegroups.[Conclusion] It is fast to proliferate Schwann cells with bFGF and HSPGin the culture solution; Group F has the best effect. At the7th day Group A, B, Cand D reach the top in cell fission, however, at the9th day Group E and Fexceed and get the top.