CAJ | 학술논문

目的：观察脑缺血再灌注大鼠内耳细胞损伤与内源性耳蜗干细胞的激活状态以及神经生长因子（N GF ）对内耳毛细胞凋亡的抑制作用及其发生机制。方法采用四动脉闭塞法制作大鼠全脑缺血再灌注损伤模型，将72只SD大鼠随机分为3组：A组：假手术组，B组：生理盐水组和C组：NGF组，每组再分24 h和72 h 2个时间段。在C组，我们用人NGF对脑缺血再灌注大鼠进行干预，用免疫荧光染色法检测耳蜗细胞巢蛋白（nestin）、酪氨酸激酶A（trkA）和NGF的表达，用原位末端标记法（TUNEL法）检测耳蜗毛细胞凋亡。用均数±标准差（x珋±s）表示计量资料，用单因素方差分析和LSD-t检验进行统计学分析。结果（1）与假手术组相比，生理盐水组内耳nestin阳性细胞数在再灌注24 h后增加，再灌注72 h后，耳蜗细胞nestin表达明显增强（t＝6.030、12.516，均P＜0.05），但trkA和NGF表达减低（t＝-6.667、-2.415，均P＜0.05），再灌注72 h后的耳蜗毛细胞凋亡率升高（t＝40.836，P＜0.05）。（2）与生理盐水组相比，NGF组在再灌注24 h和72 h后，耳蜗细胞nestin表达明显减低（t＝-4.732、-11.272，均P＜0.05），再灌注72 h后的trkA和NGF表达均显著增强（t＝15.663、17.932，均P＜0.05），再灌注72 h后毛细胞凋亡率明显下降（t＝-35.284，P＜0.05）。（3）与假手术组相比，NGF组在再灌注24 h和72 h后，nestin表达水平与假手术组接近（P＞0.05），但在再灌注72 h后的trkA和NGF表达均显著增强（t＝8.996、15.517，均P＜0.05）。结论脑缺血再灌注可引起大鼠耳蜗毛细胞凋亡，促进成熟大鼠内耳细胞nestin阳性表达，诱导内源性耳蜗干细胞的激活；给予NGF可下调nestin表达，明显抑制耳蜗毛细胞的凋亡，其机制可能与上调trkA和NGF的表达有关。
목적：관찰뇌결혈재관주대서내이세포손상여내원성이와간세포적격활상태이급신경생장인자（N GF ）대내이모세포조망적억제작용급기발생궤제。방법채용사동맥폐새법제작대서전뇌결혈재관주손상모형，장72지SD대서수궤분위3조：A조：가수술조，B조：생리염수조화C조：NGF조，매조재분24 h화72 h 2개시간단。재C조，아문용인NGF대뇌결혈재관주대서진행간예，용면역형광염색법검측이와세포소단백（nestin）、락안산격매A（trkA）화NGF적표체，용원위말단표기법（TUNEL법）검측이와모세포조망。용균수±표준차（x류±s）표시계량자료，용단인소방차분석화LSD-t검험진행통계학분석。결과（1）여가수술조상비，생리염수조내이nestin양성세포수재재관주24 h후증가，재관주72 h후，이와세포nestin표체명현증강（t＝6.030、12.516，균P＜0.05），단trkA화NGF표체감저（t＝-6.667、-2.415，균P＜0.05），재관주72 h후적이와모세포조망솔승고（t＝40.836，P＜0.05）。（2）여생리염수조상비，NGF조재재관주24 h화72 h후，이와세포nestin표체명현감저（t＝-4.732、-11.272，균P＜0.05），재관주72 h후적trkA화NGF표체균현저증강（t＝15.663、17.932，균P＜0.05），재관주72 h후모세포조망솔명현하강（t＝-35.284，P＜0.05）。（3）여가수술조상비，NGF조재재관주24 h화72 h후，nestin표체수평여가수술조접근（P＞0.05），단재재관주72 h후적trkA화NGF표체균현저증강（t＝8.996、15.517，균P＜0.05）。결론뇌결혈재관주가인기대서이와모세포조망，촉진성숙대서내이세포nestin양성표체，유도내원성이와간세포적격활；급여NGF가하조nestin표체，명현억제이와모세포적조망，기궤제가능여상조trkA화NGF적표체유관。
Objective To investigate the inner ear cells injury induced by cerebral ischemia reperfusion in rats,the activation status of endogenous cochlear stem cells,the inhibitory roles of nerve growth factor(NGF)on the inner ear hair cells apoptosis and its possible mechanism.Methods Rat models of whole cerebral ischemic reperfusion injury were established using the four artery occlusion method .A total of 72 Sprague-Dawley rats were randomly divided into 3 groups:Group A,the sham operation group,Group B, the normal saline group,and Group C,the NGF group,every group was randomly divided into the 24 h and 72 h corresponding time points.In Group C,we intervened the rats after cerebral ischemia reperfusion with exogenous human NGF,detected the expressions of nestin,tyrosine kinase A(trkA)and NGF in the cochlear cells with immunofluorescent staining,and examined the apoptotic hair cells in cochleae by the terminal deoxynucleotidyl transferase mediated d-UTP nick end labeling(TUNEL).We expressed the measurement data with mean ±standard deviation(x-±s)and adopted the single factor variance analysis and LSD-t test. Results (1 )Of Group B,compared with Group A,the number of nestin positive cells in the inner ear increased at the 24 h corresponding time point after reperfusion.At the 72 h corresponding time point after reperfusion,the nestin expression in the cochlea cells was markedly enhanced (t values were 6.030 and 12.5 16 respectively,both P<0.05 ),but the expressions of trkA and NGF both decreased (t values were-6.667 and-2.415 respectively,both P<0.05 ).At the 72 h corresponding time point after reperfusion, the apoptosis rate of the cochlear hair cells increased(t value was 40.836,P<0.05).(2)Of Group C, compared with Group B and at the 24 h and 72 h corresponding time points after reperfusion,the nestin expressions of the cochlear cells both obviously decreased(t values were respectively-4.732 and-1 1.272, both P<0.05 ).At the 72 h corresponding time point after reperfusion,the expressions of trkA and NGF both significantly increased(t values were 15.663 and 17.932 respectively,both P<0.05),at 72 h after cerebral ischemia-reperfusion,the apoptosis rate of cochlear hair cells obviously decreased(t value was-35.284,P<0.05).(3)Of Group C,compared with Group A,and at the 24 h and 72 h corresponding time points after reperfusion,the nestin expressions of the cochlear cells were really close (P>0.05 ),but the expressions of trkA and NGF at the 72 h corresponding time point after reperfusion both significantly increased (t values were 8.996 and 15.5 17 respectively,both P <0.05 ).Conclusions Cerebral ischemia reperfusion can induce the cochleae hair cells apoptosis in rats,promote the positive nestin expression in the inner ear cells of the mature rats,and induce the activation of endogenous cochlear stem cells.Giving NGF can lower the expression of nestin,and significantly inhibit the apoptosis of cochlear hair cells.The mechanism happened may be related with the increased trkA and the NGF expression.