摘要
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(NGF)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。
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 ceils 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 immunofluoreseent staining, and examined the apoptotic hair cells in cochleae by the terminal deoxytmcleotidyl transferase mediated d-UTP nick end labeling （TUNEL）. We expressed the measurement data-with mean ＋ standard deviation （ x^2 ± 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. 516 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 - 11. 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. 517 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.
出处
《中华脑科疾病与康复杂志（电子版）》
2014年第1期26-30,共5页
Chinese Journal of Brain Diseases and Rehabilitation（Electronic Edition）
