The Effects Of Rat Olfactory Ensheathing Cells On Spiral Ganglion Cells And The Relative Cytokines And Receptors

PartⅠCultivation, purification and identification ofolfactory ensheathing cellsObjectives: To develop a method of cultivation and purification of olfactory ensheathing cells (OECs), calculate the purity of OECs and observe the immunofluorescence properties under the laser confocal microscope.Methods: The olfactory bulbs from neonatal 2d SD rats were dissected. The tissue was dissociated by digestion and triturating and made into single cell suspension, which was cultured in DMEM/F12 media containing 10% FBS (FBS group), DMEM/F12 media containing 1% N2 supplement (N2 group) , DMEM/F12 media containing 1%B27 supplement (B27 group) and DMEM/F12 media containing 1%N2 and 1% B27 supplements (N2+B27 group), the growth and purity of OECs were investigated. OECs were purified by repetitious differential adhesion and rapid low concentration trpsinization. The cells were identified with p75NTR and GFAP immunofluorescence staining.Results: OECs can't survival in serum-free media containing 1% N2, 1%B27, or 1% N2 and 1% B27 supplements, but it can grow well in DMEM/F12 media containing 10% FBS, the main contaminate cells are fibroblast. OECs can be purified with the methods of repetitious differential adhesion and rapid low concentration trpsinization, the purity of OECs was 89．4±7．3% at 2 weeks. OECs were double labeled with p75NTR and GFAP, p75NTR was expressed in cellular body, and GFAP was expressed both in cellular body and prominence.Conclusions: OECs should be cultured in DMEM/F12 media containing 10% FBS, It can be purified with repetitious differential adhesion and rapid low concentration trpsinization, the methods to culture and purify OECs were repeatable.PartⅡThe effects of different culture media on the spiral ganglion cells in vitro Objectives: In order to develop a better SGCs culture method, this study investigated the effects of different culture media on the growth of SGCs in vitro.Methods: The Cort's tunnel from neonatal 2d SD rats was dissected. The tissues were dissociated by digestion and triturating and made into single cell suspension, which were cultured in DMEM/F12 media containing 10%FBS(FBS group), DMEM/F12 media containing 1%N2(N2 group), DMEM/F12 media containing 1%N2 and 1%B27 (N2+B27 group) and DMEM/F12 media containing 1% B27(B27 group) for 2 weeks. The cells were identified by Neurofilement immunofluorescence staining, the length of nervous process and the cellular number of SGCs were investigated.Results: The lengths of nervous process attained the peak at day 5 in vitro(div 5) in FBS group and N2 group, the average longest process were 111．24±28．61um and 113．15±17．70um, the lengths of two groups had no difference (P > 0．05) . At the same time, the lengths of N2+B27 group and B27 groups attained the longest at div7 with 184．26±30．06um and 193．50±29．13um, both lengths didn't have difference (P > 0．05) , but it had significant difference to the anterior two groups (P<0．01) . The cellular number decreased gradually during culture, it grew downwards fleetly to 7．07±2．55 /eyeshot and 4．34±2．63 /eyeshot in FBS group and N2 group at div 14, but it grew slowly to 115．60±16．33 /eyeshot and 117．8±15．52 /eyeshot in N2+B27 group and B27 group at div 14．The cellular number didn't have difference between FBS group and N2 group(P > 0．05) , it also had no difference between N2 + B27 group and B27 group (P > 0．05 ) . But the posterior two groups had significant difference to the anterior two groups from div3 (P＜0．01) .Conclusions: DMEM/F12 containing 1%B27 or containing 1%B27 and 1%N2 was better for SGCs culture.PartⅢThe effects of rat olfactory ensheathing cellson spiral ganglion cells in vitroObjectives: To investigate the effects of rat OECs on the growth, apoptosis and proliferation of SGCs in vitro.Methods: SGCs were cultured with OECs CM(OECs CM group), FB CM(FB CM group) and DF12 containing with FBS(FBS group), the growth and proliferation were observed by immunofluorescence method, the apoptosis was determined with TUNEL method. The cell population, ratios of apoptosis and proliferative SGCs were compared among the 3 groups on day1, 3, 5, 7．Results: The cell population is the highest and the ratio of apoptosis is the lowest in OECs CM group, both of the two index were significant statistical differences between OECs CM group and the other 2 groups (P<0．01) , and no statistical difference was analyzed between FB CM group and FBS group (P>0．05) . At day 3 of OECs CM group, a few proliferative SGCs were observed, whereas no proliferative SGCs were detected at the other times of OECs CM group and any time of the other 2 groups, there has significant statistical difference between OECs CM group d3 and the other times of OECs CM group as well as any time of the other 2 groups (P＜0．001) .Conclusions: The apoptosis of SGCs was inhibited in OECs CM which results in more SGCs survive. OECs CM can promote the proliferation of SGCs.PartⅣThe cytokines and receptors whichparticipate the effects of OECs on SGCsObjectives: To initially investigate which cytokines and receptors take part in the effects of the OECs on the survival, apoptosis and proliferation of SGCs by RT-PCR.Methods: The total RNA of olfactory bulb and OECs were extracted by TRIzol reagent, the expression of Laminin, NCAM, BMP-4, BMP-7, CNTF and bFGF were analysis by reverse transcriptase polymerase chain reaction; at the same time, the total RNA of SGCs and cochlea were extracted for investigating the expression ofα7 integrin,αV integrin, BMPR-1A, BMPR-1B, BMPR-Ⅱ, CNTFR and FGFR-1 by RT-PCR, the mRNA of these cytokines and receptors were reviewed by gel electrophoresis. Judging which cytokines and receptors participate the effects according the results of RT-PCR.Results: The cytokines, NCAM and BMP-4 were expressed in olfactory bulb and OECs, BMP-7 was slightly expressed in olfactory bulb, while none of these cytokines was expressed in FB. The cytokine receptors, BMPR-1A, NCAM andα7 integrin were expressed in both SGCs and cochlea, meanwhile BMPR-1B, BMPR-Ⅱand CNTFR were expressed in cochlea.Conclusion:α7 integrin,BMP4 and BMPR-1A as well as NCAM potential take part in the effects of OECs on SGCs.