细胞周期蛋白依赖激酶5与皇家外科学院大鼠视网膜色素变性发病过程中细胞凋亡的关系

Relationship between cyclin dependent kinase 5 and apoptosis in the pathogenesis of retinitis pigmentosa inRoyal College of Surgeon rats

目的观察细胞周期蛋白依赖激酶5（Cdk5）和p25在不同病程皇家外科学院（RCS）大鼠视网膜色素变性（RP）模型视网膜中的表达及与细胞凋亡的关系，探讨Cdk5／p25介导的光感受器细胞凋亡在RP发病过程中的作用。方法取RCSRP大鼠（RCS大鼠）及正常对照RCS-rdy＋ 大鼠17、25、35、60日龄视网膜，光学显微镜下观察视网膜结构，测量外核层厚度；利用免疫组织化学染色法观察视网膜中Cdk5、p25和活化型半胱氨酸天冬氨酸蛋白酶3（cleave-caspase3）的表达和变化；采用Westernblot法测定视网膜中cleave-caspase3的蛋白质水平；原位末端标记（TUNEL）法检测视网膜中凋亡发生的情况，并对凋亡细胞平均吸光度EA，旧称光密度（OD）]值进行统计分析。采用SPSS17．0统计软件处理数据。结果随着出生日龄的增加，RCS大鼠视网膜厚度较RCS-rdy＋大鼠变薄，主要表现为视杆与视锥层和外核层明显变薄。在RCS大鼠17、25、35日龄组，Cdk5、p25和cleave-caspase3的表达逐渐增强，60日龄组表达下降；对照组RCS-rdy＋ 大鼠各日龄组表达无明显变化。Westernblot法检测结果显示，随着出生日龄的增加，RCS大鼠各日龄组视网膜cleave-easpase3的表达明显增加，以35日龄组增加最为明显，而RCS-rdy＋大鼠各日龄组视网膜cleave-caspase3的表达没有明显的变化。TUNEL法检测结果显示，凋亡细胞主要出现在外核层中，在RCS大鼠17、25、35日龄组，各组凋亡细胞呈逐渐增多的趋势，60日龄组凋亡细胞显著下降；RCS-rdy＋各日龄组凋亡细胞没有明显变化。多变量偏相关分析结果显示，Cdk5表达与p25表达、Cdk5表达与cleave-caspase3表达、Cdk5表达与凋亡细胞表达、p25表达与cleave-caspase3表达、p25表达与凋亡细胞表达、cleave-caspase3表达与凋亡细胞表达的偏相关系数分别为0．949、0．808、0．959、0．887、0．979、0．852，P值为0．000，两两变量之间均显著相关。结论17、25、35日龄RCS大鼠，随着出生日龄的增加，其视网膜凋亡细胞逐渐增多，而Cdk5、p25和eleave-caspase3的表达逐渐增强。RCS大鼠视网膜中凋亡细胞变化趋势与Cdk5、p25和cleave-caspase3的表达基本一致。提示RCS大鼠视网膜中光感受器细胞的凋亡与Cdk5、p25的表达水平升高有关，Cdk5可能参与RCS大鼠RP的发生与发展。

Objective To observe the expression of cyclin dependent kinase 5 （Cdk5） and p25 in the pathogenesis of retinitis pigmentosa （RP） in Royal College of Surgeon （RCS） rats and its relationships with apoptosis. To explore the mechanism of Cdk5 and p25 induced photoreceptor apoptosis in the pathogenesis of RP. Methods Retinas of RCS and RCS-rdy＋ rats were obtained at the ages of postnatal day 17, 25, 35, 60. The retinal structure and thickness of outer nuclear layer were measured by optical microscopy. The expression of Cdk5, p25, cleave-caspase 3 in the retina was evaluated by immunohistochemistry. The protein expression of cleave-caspase 3 in the retina was determined by Western blot. The apoptosis of retinal cells was examined by terminal-deoxynucleotidyl transferase mediated nick end labeling （TUNEL）. The mean absorbance value of apoptotic cells was analyzed by SPSS 17.0 software. Results The retinal thicknessof the RCS rats was significantly reduced in comparison to the RCS-rdy＋ rats as the postnatal days progressed, particularly in the layer of rods and cones and the outer nuclear tayer. The expression level of CdkS, p25, cleave-caspase 3 of RCS rats increased from postnatal 17 days to postnatal 35 days, while decreased on postnatal 60 days＋ but there was no obvious change of above indexes in RCS-rdy＋ rats. The protein expression of cleave-caspase 3 in＇ the RCS rats was significantly increased with progression of postnatal days to postnatal 353 but there was no obvious similar change in RCS-rdy＋ rats. The results of TUNEL showed that the apoptotic cells significantly increased in the outer nuclear layer of RCS rats from postnatal 17 days to postnatal 35 days, while decreased on postnatal 60 days; but there was no obvious change of above index in RCS-rdy＋ rats. This study showed that there were significant correlations between the following variables： Cdk5 expression and p25 expression, Cdk5 expression and cleave-caspase 3 expression, Cdk5 expression and apoptotic cells, p25 expression and cleave-caspase 3 expression, p25 expression and apoptotic cells, cleave-easpase 3 expression and apoptotic cells. The partial correlation coefficients were 0. 949, 0. 808, 0. 959, 0. 887, 0. 979, 0. 852, respectively and the P value was 0. 000. Conclusions The apoptotic cells significantly increases and the expression level of Cdk5, p25, cleave- caspase 3 of RCS rats increases from postnatal 17 days to postnatal 35 days. The tendency of apoptotic cells to increase is consistent with the change of Cdk5, p25, cleave-caspase 3 expression. The apoptosis of photoreceptor cells is related to increasing expression of Cdk5 and p25 in RCS rats. Cdk5 may be involved in the development of RP in RCS rats.