Quantitative Study Of The High Proliferative Cells From The Inner Ear Of SD Rats

Objettive：To investigate the influence on the proliferation of high proliferative cellsfrom the inner ear of SD rats by different ages and growth factors in vitro．Methods：1．Organs of utricle or saccule or cochlear Corti were isolated fromdifferent days’ Sprague-Dawley rats(P1、P7、P14、P21、P30、P60)．We chose6rats(12ears) in each age group，with a total of72rats(144ears)．Cells were dissociated andcultivated under non-adherent conditions as single cell．Counting the number of cellspheres under inverted phase contrast microscope，which were statistically anayzedand depicted the quantity changed curve of the same organ in different age rats，untilthe single cells were cultured for7days；2．We isolated P1rats’ organs of utricle orsaccule or cochlear Corti from60rats(120ears)，which were randomly divided intocontrol and experimental group．Control groups were cultivated without growthfactor，as experimental groups were supplemented with epidermal growth factor(EGF) or basic fibroblast growth factor (bFGF) or insulin-like growth factor-1(IGF-1) or leukemia inhibitory factor (LIF)，under non-adherent conditions as singlecell．After7days in culture，cell spheres will be counted under inverted phasecontrast microscope，effect of the four growth factors on the proliferative cells werecompared by statistical analysis．3．Immunofluorescence assays were conducted forphenotype characterization．Result：1．Some new isolated single cells，labeled by nestin，were from each innerera organ of rats(P1)．During7days in culture，cells keeped proliferation and formedsuspended spheres，which were expressed BrdU and nestin positive．2．(1)Thenumber of cell spheres formed from each inner ear organ：A．Cochlear Corti：Theaverage50±23cell spheres were observed in a single Corti culture medium of P1rats，as the P7form average33±15．There is statistically significant differencebetween them(P<0.05)．In the same culture conditions，we can not oberve cellspheres， which were formed by a single Corti of rats(P14、 P21、 P30、P60)．B．Saccule：Cell spheres were observed in a single saccule culture medium of all rats(P1-P60)，each of the average number was：23±9、21±8、16±6、14±4、13±3、12±3．Statistically difference was not seen between P1and P7(P﹥0.05)，but wasfound as compared with others(P<0.05)．Also there was no difference between P7and P14(P﹥0.05)，but was seen as compared with P21、P30、P60(P<0.05)．We cannot see the statistically difference among P14、P21、P30、P60(P﹥0.05)．C．Utricle：Cell spheres were observed in the single cell culture medium of all rats(P1-P60)，each of the average number was：29±10、24±9、20±6、18±4、15±4、14±5．Statisticallydifference was not seen between P1and P7(P﹥0.05)，but was found as comparedwith others(P<0.05)．Also there was no difference between P7and P14、P21(P﹥0.05)，but was seen as compared with P30、P60(P<0.05)．Difference between P14and P21can not be found(P﹥0.05)， while it exists among P14、 P30、P60(P<0.05)．We can not see the statistically difference among P21、P30、P60(P﹥0.05)．(2)The statistical analysis of cell spheres from the single different inner earorgans of the same age group：①P1：Differences between cochlear Corti and sacculeor utricle are statistically significant(P<0.05)，while there is no difference betweensaccule an utricle(P﹥0.05)；②P7：There is no significant between Corti and utricle，which is same with saccule and utricle(P﹥0.05)，but can seen between Corti andsaccule(P<0.05)．③P14、P21、P30、P60：We can see the statistically differencebetween saccule and utricle of P21(P<0.05)，but can not find among P14、P30、P60(P﹥0.05)．(3)．The number of cell spheres，which were formed by the isolated cellsfrom inner ear organs of single utricle or saccule or cochlear Corti，increasedobviously in the presence of EGF or bFGF or IGF-1，but were not obviously inLIF．Statistically significant differences were seen between control group and everyexperimental group including EGF group、bFGF group、IGF-1group(P<0.01)，butwere not seen between control group and LIF group(P﹥0.05)．There were noStatistically significant difference among EGF group and bFGF group and IGF-1group(P﹥0.05)．Conclusions：1．The highly proliferative cells were present in the vestibularorgan(utricle or saccule) and cochlear Corti of SD rats．They could express thecharacterization of neural stem cell．As increase of the age，the overall trend ofproliferative capability is declining．2．Compared with the vestibular organs，the proliferative capacity of highly proliferative cells in the cochlea decreased faster，butis more powerful of P1rats．We could not find the proliferative cells in the cochleaof the P14rat．Proliferative capacity of highly proliferative cells in vestibular organsdeclined slowly，while they still could be seen in the P60rats．The decrease of highlyproliferative cells in the saccule focused on P1to P21，which in the utricle wasmainly concentrated on P1to P30．Except for the P21rats，the highly proliferativecells in the two vestibular organs of the same age have no difference in proliferativecapacity．3．Each of EGF and bFGF and IGF-1possess the promoting effects forproliferation on the highly proliferative cells alone，which is not obviously in presentof LIF．There is no difference among the effects of the three growth factor．...