CAJ | 학술논문

目的：探讨不同年龄及生长因子对大鼠内耳前体细胞体外增殖的影响。方法①分离取出出生后1天（P1）、7天（P7）、14天（P14）、21天（P21）、30天（P30）、60天（P60）各天龄12只（24耳）SD大鼠的球囊斑、椭圆囊斑、Corti器的前体细胞，在无血清条件下进行单细胞悬浮培养，至第七天，在倒置相差显微镜下对所形成的细胞球进行计数；②取P1大鼠球囊斑、椭圆囊斑、Corti器高增殖细胞分为实验组和对照组，实验组分为四组，培养液分别添加表皮生长因子（epidermal growth factor ，EGF）、碱性成纤维细胞生长因子（basic fibroblast growth factor ，bF-GF）、胰岛素样生长因子-1（insulin-like growth factor -1，IGF -1）、白血病抑制因子（leukemia inhibitory factor ， LIF），对照组不加生长因子，观察不同生长因子对细胞球数量的影响；③免疫荧光染色检测细胞球的表型特征。结果①P1SD大鼠内耳各器官单细胞表现为nestin阳性，培养7天后，可形成悬浮的细胞球，球体细胞免疫荧光表现为nestin和BrdU阳性；②耳蜗Corti器只在P1、P7可形成细胞球，而球囊斑、椭圆囊斑各天龄段均可形成细胞球；③培养液中添加EGF、bFGF、IGF-1组，培养形成的细胞球数量明显增多，与对照组比较，差异有显著统计学意义（P＜0．01）；添加LIF组则与对照组比较差异无统计学意义（P＞0．05）。结论大鼠内耳球囊斑、椭圆囊斑以及耳蜗Corti器内有高增殖能力细胞，能表达神经干细胞的特性；随着鼠龄的增长，这种增殖能力总体表现为减退趋势；EGF、bFGF、IGF-1均可单独促进P1大鼠内耳前体细胞的增殖，而LIF无促增殖作用。
목적：탐토불동년령급생장인자대대서내이전체세포체외증식적영향。방법①분리취출출생후1천（P1）、7천（P7）、14천（P14）、21천（P21）、30천（P30）、60천（P60）각천령12지（24이）SD대서적구낭반、타원낭반、Corti기적전체세포，재무혈청조건하진행단세포현부배양，지제칠천，재도치상차현미경하대소형성적세포구진행계수；②취P1대서구낭반、타원낭반、Corti기고증식세포분위실험조화대조조，실험조분위사조，배양액분별첨가표피생장인자（epidermal growth factor ，EGF）、감성성섬유세포생장인자（basic fibroblast growth factor ，bF-GF）、이도소양생장인자-1（insulin-like growth factor -1，IGF -1）、백혈병억제인자（leukemia inhibitory factor ， LIF），대조조불가생장인자，관찰불동생장인자대세포구수량적영향；③면역형광염색검측세포구적표형특정。결과①P1SD대서내이각기관단세포표현위nestin양성，배양7천후，가형성현부적세포구，구체세포면역형광표현위nestin화BrdU양성；②이와Corti기지재P1、P7가형성세포구，이구낭반、타원낭반각천령단균가형성세포구；③배양액중첨가EGF、bFGF、IGF-1조，배양형성적세포구수량명현증다，여대조조비교，차이유현저통계학의의（P＜0．01）；첨가LIF조칙여대조조비교차이무통계학의의（P＞0．05）。결론대서내이구낭반、타원낭반이급이와Corti기내유고증식능력세포，능표체신경간세포적특성；수착서령적증장，저충증식능력총체표현위감퇴추세；EGF、bFGF、IGF-1균가단독촉진P1대서내이전체세포적증식，이LIF무촉증식작용。
Objective To investigate the influence on the proliferation of the precursor cells from the inner ear of SD rats by different ages and growth factors in vitro .Methods The organs of utricle or saccule or cochlear Corti were isolated from different days’ SD rats born on different days(P1 ,P7 ,P14 ,P21 ,P30 ,P60) .From 12 SD rats of different age ,with 24 ears ,precursor cells were dissociated and cultivated under non -adherent conditions as single cell .We then counted the number of cell spheres under inverted phase contrast microscope .The results were statisti-cally anayzed and the quantity change curve of the same organ in different age rats were described until the single cells were cultured for 7 days .The culture medium was supplemented with defferent growth factor to observe the effects of the grow th factor on the number of cell spheres .Immunofluorescence assays were conducted for phenotype characterization .Results ① Some newly isolated single cells ,labeled by nestin ,were from each inner ear organ of rats(P1) .During 7 days in culture ,cells kept proliferation and formed suspended spheres ,which were expressed Br-dU and nestin positive .② The cell spheres could only formed from each cochlear Corti of rats (P1 ,P7) .But cell spheres that formed from each utricle or saccule were observed in a single saccule culture medium of all rats (P1~P60) .③The number of cell spheres ,which were formed by the isolated cells from inner ear organs of single utricle or saccule or cochlear Corti ,increased obviously in the presence of EGF or bFGF or IGF -1 ,but were not obviously in LIF .Statistically significant differences were seen between control group and every experimental group including EGF group ,bFGF group ,IGF-1 group(P<0 .01) ,but were not seen between control group and LIF group (P>0 .05) .Conclusion The highly proliferative cells were present in the vestibular organ (utricle or saccule) and cochle-ar Corti of SD rats .They could express the characterization of neural stem cell .As increase of the age ,the overall trend of proliferative capability is declining .Each of EGF and bFGF and IGF -1 possess the promoting effects for proliferation on the precursor cells alone ,which is not in present of LIF .