CAJ | 학술논문

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RCS RP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-caspase 3)的表达和变化；采用Western blot法测定视网膜中cleave-caspase 3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[A,旧称光密度(OD)]值进行统计分析.采用SPSS 17.0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-caspase 3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.Western blot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-caspase 3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-caspase 3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-caspase 3表达、Cdk5表达与凋亡细胞表达、p25表达与cleave-caspase 3表达、p25表达与凋亡细胞表达、cleave-caspase 3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cleave-caspase 3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-caspase 3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. 目的觀察細胞週期蛋白依賴激酶5(Cdk5)和p25在不同病程皇傢外科學院(RCS)大鼠視網膜色素變性(RP)模型視網膜中的錶達及與細胞凋亡的關繫,探討Cdk5/p25介導的光感受器細胞凋亡在RP髮病過程中的作用.方法取RCS RP大鼠(RCS大鼠)及正常對照RCS-rdy+大鼠17、25、35、60日齡視網膜,光學顯微鏡下觀察視網膜結構,測量外覈層厚度；利用免疫組織化學染色法觀察視網膜中Cdk5、p25和活化型半胱氨痠天鼕氨痠蛋白酶3(cleave-caspase 3)的錶達和變化；採用Western blot法測定視網膜中cleave-caspase 3的蛋白質水平；原位末耑標記(TUNEL)法檢測視網膜中凋亡髮生的情況,併對凋亡細胞平均吸光度[A,舊稱光密度(OD)]值進行統計分析.採用SPSS 17.0統計軟件處理數據.結果隨著齣生日齡的增加,RCS大鼠視網膜厚度較RCS-rdy+大鼠變薄,主要錶現為視桿與視錐層和外覈層明顯變薄.在RCS大鼠17、25、35日齡組,Cdk5、p25和cleave-caspase 3的錶達逐漸增彊,60日齡組錶達下降；對照組RCS-rdy+大鼠各日齡組錶達無明顯變化.Western blot法檢測結果顯示,隨著齣生日齡的增加,RCS大鼠各日齡組視網膜cleave-caspase 3的錶達明顯增加,以35日齡組增加最為明顯,而RCS-rdy+大鼠各日齡組視網膜cleave-caspase 3的錶達沒有明顯的變化.TUNEL法檢測結果顯示,凋亡細胞主要齣現在外覈層中,在RCS大鼠17、25、35日齡組,各組凋亡細胞呈逐漸增多的趨勢,60日齡組凋亡細胞顯著下降；RCS-rdy+各日齡組凋亡細胞沒有明顯變化.多變量偏相關分析結果顯示,Cdk5錶達與p25錶達、Cdk5錶達與cleave-caspase 3錶達、Cdk5錶達與凋亡細胞錶達、p25錶達與cleave-caspase 3錶達、p25錶達與凋亡細胞錶達、cleave-caspase 3錶達與凋亡細胞錶達的偏相關繫數分彆為0.949、0.808、0.959、0.887、0.979、0.852,P值為0.000,兩兩變量之間均顯著相關.結論 17、25、35日齡RCS大鼠,隨著齣生日齡的增加,其視網膜凋亡細胞逐漸增多,而Cdk5、p25和cleave-caspase 3的錶達逐漸增彊.RCS大鼠視網膜中凋亡細胞變化趨勢與Cdk5、p25和cleave-caspase 3的錶達基本一緻.提示RCS大鼠視網膜中光感受器細胞的凋亡與Cdk5、p25的錶達水平升高有關,Cdk5可能參與RCS大鼠RP的髮生與髮展.
목적 관찰세포주기단백의뢰격매5(Cdk5)화p25재불동병정황가외과학원(RCS)대서시망막색소변성(RP)모형시망막중적표체급여세포조망적관계,탐토Cdk5/p25개도적광감수기세포조망재RP발병과정중적작용.방법 취RCS RP대서(RCS대서)급정상대조RCS-rdy+대서17、25、35、60일령시망막,광학현미경하관찰시망막결구,측량외핵층후도；이용면역조직화학염색법관찰시망막중Cdk5、p25화활화형반광안산천동안산단백매3(cleave-caspase 3)적표체화변화；채용Western blot법측정시망막중cleave-caspase 3적단백질수평；원위말단표기(TUNEL)법검측시망막중조망발생적정황,병대조망세포평균흡광도[A,구칭광밀도(OD)]치진행통계분석.채용SPSS 17.0통계연건처리수거.결과 수착출생일령적증가,RCS대서시망막후도교RCS-rdy+대서변박,주요표현위시간여시추층화외핵층명현변박.재RCS대서17、25、35일령조,Cdk5、p25화cleave-caspase 3적표체축점증강,60일령조표체하강；대조조RCS-rdy+대서각일령조표체무명현변화.Western blot법검측결과현시,수착출생일령적증가,RCS대서각일령조시망막cleave-caspase 3적표체명현증가,이35일령조증가최위명현,이RCS-rdy+대서각일령조시망막cleave-caspase 3적표체몰유명현적변화.TUNEL법검측결과현시,조망세포주요출현재외핵층중,재RCS대서17、25、35일령조,각조조망세포정축점증다적추세,60일령조조망세포현저하강；RCS-rdy+각일령조조망세포몰유명현변화.다변량편상관분석결과현시,Cdk5표체여p25표체、Cdk5표체여cleave-caspase 3표체、Cdk5표체여조망세포표체、p25표체여cleave-caspase 3표체、p25표체여조망세포표체、cleave-caspase 3표체여조망세포표체적편상관계수분별위0.949、0.808、0.959、0.887、0.979、0.852,P치위0.000,량량변량지간균현저상관.결론 17、25、35일령RCS대서,수착출생일령적증가,기시망막조망세포축점증다,이Cdk5、p25화cleave-caspase 3적표체축점증강.RCS대서시망막중조망세포변화추세여Cdk5、p25화cleave-caspase 3적표체기본일치.제시RCS대서시망막중광감수기세포적조망여Cdk5、p25적표체수평승고유관,Cdk5가능삼여RCS대서RP적발생여발전.
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 thickness of 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 layer.The expression level of Cdk5,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 35; 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-caspase 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 ceils significantly increases and the expression level of Cdk5,p25,cleavecaspase 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.