CAJ | 학술논문

目的观察细胞周期依赖性蛋白激酶5(Cdk5)抑制剂Roscovtine对皇家外科学院(RCS)大鼠视网膜光感受器细胞凋亡的影响.方法 RCS雄性大鼠30只,随机数字表法随机分为早期干预组、中期干预组、晚期干预组,每组10只.分别于出生后17、25、35 d,给予右眼玻璃体腔注射Cdk5抑制剂Roscovitine 4μl作为实验眼;左眼玻璃体腔注射Roscovitine溶剂二甲基亚砜4μl作为对照眼.早期干预组、中期干预组、晚期干预组大鼠分别于出生后25、35、45 d处死,摘除眼球作视网膜切片,采用苏木精-伊红(HE)染色观察视网膜外核层厚度变化;采用免疫组织化学染色观察Cdk5、p25及活化型半胱氨酸天冬氨酸蛋白酶3 (cleave-caspase 3)的阳性表达;采用原位末端标记法(TUNEL)检测视网膜凋亡细胞的表达.取视网膜组织,采用蛋白免疫印迹法(Western blot)检测大鼠视网膜中Cdk5、p25及cleave-caspase 3的蛋白表达.对Cdk5、p25、cleave-caspase 3阳性表达与凋亡细胞表达两两变量之间行多变量偏相关分析.结果早期干预组、中期干预组大鼠实验眼外核层厚度均较对照眼厚,且早期干预组实验眼与对照眼外核层厚度的差异更明显.晚期干预组大鼠实验眼与对照眼的外核层厚度差异不明显.免疫组织化学染色结果显示,早期干预组、中期干预组实验眼外核层Cdk5、p25、cleave-caspase 3阳性表达均较对照眼降低,差异均有统计学意义(P＜0.05).晚期干预组实验眼与对照眼外核层Cdk5、p25、cleave-caspase 3阳性表达比较,差异无统计学意义(P＞0.05).TUNEL检测结果显示,早期干预组、中期干预组大鼠实验眼视网膜外核层凋亡细胞表达较对照眼明显降低;晚期干预组大鼠实验眼与对照眼视网膜外核层凋亡细胞表达差异不明显.多变量偏相关分析结果显示,Cdk5、p25、cleave-caspase 3阳性表达与凋亡细胞表达两两变量之间均呈明显相关性(P＜0.05).Western blot检测结果显示,早期干预组、中期干预组大鼠实验眼视网膜中cleave-caspase 3蛋白表达较对照眼明显降低,且早期干预组实验眼与对照眼视网膜中cleave-caspase 3蛋白表达之间的差异更明显.晚期干预组实验眼与对照眼视网膜中cleave-caspase 3蛋白表达的差异不明显.结论 Cdk5抑制剂Roscovtine可能对视网膜光感受器细胞的凋亡过程起到一定延缓作用. 目的觀察細胞週期依賴性蛋白激酶5(Cdk5)抑製劑Roscovtine對皇傢外科學院(RCS)大鼠視網膜光感受器細胞凋亡的影響.方法 RCS雄性大鼠30隻,隨機數字錶法隨機分為早期榦預組、中期榦預組、晚期榦預組,每組10隻.分彆于齣生後17、25、35 d,給予右眼玻璃體腔註射Cdk5抑製劑Roscovitine 4μl作為實驗眼;左眼玻璃體腔註射Roscovitine溶劑二甲基亞砜4μl作為對照眼.早期榦預組、中期榦預組、晚期榦預組大鼠分彆于齣生後25、35、45 d處死,摘除眼毬作視網膜切片,採用囌木精-伊紅(HE)染色觀察視網膜外覈層厚度變化;採用免疫組織化學染色觀察Cdk5、p25及活化型半胱氨痠天鼕氨痠蛋白酶3 (cleave-caspase 3)的暘性錶達;採用原位末耑標記法(TUNEL)檢測視網膜凋亡細胞的錶達.取視網膜組織,採用蛋白免疫印跡法(Western blot)檢測大鼠視網膜中Cdk5、p25及cleave-caspase 3的蛋白錶達.對Cdk5、p25、cleave-caspase 3暘性錶達與凋亡細胞錶達兩兩變量之間行多變量偏相關分析.結果早期榦預組、中期榦預組大鼠實驗眼外覈層厚度均較對照眼厚,且早期榦預組實驗眼與對照眼外覈層厚度的差異更明顯.晚期榦預組大鼠實驗眼與對照眼的外覈層厚度差異不明顯.免疫組織化學染色結果顯示,早期榦預組、中期榦預組實驗眼外覈層Cdk5、p25、cleave-caspase 3暘性錶達均較對照眼降低,差異均有統計學意義(P＜0.05).晚期榦預組實驗眼與對照眼外覈層Cdk5、p25、cleave-caspase 3暘性錶達比較,差異無統計學意義(P＞0.05).TUNEL檢測結果顯示,早期榦預組、中期榦預組大鼠實驗眼視網膜外覈層凋亡細胞錶達較對照眼明顯降低;晚期榦預組大鼠實驗眼與對照眼視網膜外覈層凋亡細胞錶達差異不明顯.多變量偏相關分析結果顯示,Cdk5、p25、cleave-caspase 3暘性錶達與凋亡細胞錶達兩兩變量之間均呈明顯相關性(P＜0.05).Western blot檢測結果顯示,早期榦預組、中期榦預組大鼠實驗眼視網膜中cleave-caspase 3蛋白錶達較對照眼明顯降低,且早期榦預組實驗眼與對照眼視網膜中cleave-caspase 3蛋白錶達之間的差異更明顯.晚期榦預組實驗眼與對照眼視網膜中cleave-caspase 3蛋白錶達的差異不明顯.結論 Cdk5抑製劑Roscovtine可能對視網膜光感受器細胞的凋亡過程起到一定延緩作用.
목적 관찰세포주기의뢰성단백격매5(Cdk5)억제제Roscovtine대황가외과학원(RCS)대서시망막광감수기세포조망적영향.방법 RCS웅성대서30지,수궤수자표법수궤분위조기간예조、중기간예조、만기간예조,매조10지.분별우출생후17、25、35 d,급여우안파리체강주사Cdk5억제제Roscovitine 4μl작위실험안;좌안파리체강주사Roscovitine용제이갑기아풍4μl작위대조안.조기간예조、중기간예조、만기간예조대서분별우출생후25、35、45 d처사,적제안구작시망막절편,채용소목정-이홍(HE)염색관찰시망막외핵층후도변화;채용면역조직화학염색관찰Cdk5、p25급활화형반광안산천동안산단백매3 (cleave-caspase 3)적양성표체;채용원위말단표기법(TUNEL)검측시망막조망세포적표체.취시망막조직,채용단백면역인적법(Western blot)검측대서시망막중Cdk5、p25급cleave-caspase 3적단백표체.대Cdk5、p25、cleave-caspase 3양성표체여조망세포표체량량변량지간행다변량편상관분석.결과 조기간예조、중기간예조대서실험안외핵층후도균교대조안후,차조기간예조실험안여대조안외핵층 후도적차이경명현.만기간예조대서실험안여대조안적외핵층후도차이불명현.면역조직화학염색결과현시,조기간예조、중기간예조실험안외핵층Cdk5、p25、cleave-caspase 3양성표체균교대조안강저,차이균유통계학의의(P＜0.05).만기간예조실험안여대조안외핵층Cdk5、p25、cleave-caspase 3양성표체비교,차이무통계학의의(P＞0.05).TUNEL검측결과현시,조기간예조、중기간예조대서실험안시망막외핵층조망세포표체교대조안명현강저;만기간예조대서실험안여대조안시망막외핵층조망세포표체차이불명현.다변량편상관분석결과현시,Cdk5、p25、cleave-caspase 3양성표체여조망세포표체량량변량지간균정명현상관성(P＜0.05).Western blot검측결과현시,조기간예조、중기간예조대서실험안시망막중cleave-caspase 3단백표체교대조안명현강저,차조기간예조실험안여대조안시망막중cleave-caspase 3단백표체지간적차이경명현.만기간예조실험안여대조안시망막중cleave-caspase 3단백표체적차이불명현.결론 Cdk5억제제Roscovtine가능대시망막광감수기세포적조망과정기도일정연완작용.
Objective To evaluate the photoreceptor-protective effects of Cdk5 inhibitor Roscovitine on retinal degeneration in Royal College of Surgeons (RCS) rat.Methods The RCS rats were divided into three groups according to postnatal days:the early (17 days),medium (25 days) and late intervention group (35 days).Cdk5 inhibitor Roscovitine were used in the right eyes by intravitreal injection as experimental eyes and Roscovitine solvent dimethylsulfoxide were used in the left as control at postnatal 17,25,35 days.Hematoxylin-eosin (HE) staining was used to observe the thickness of outer nuclear layer.The expression of Cdk5 P25 and 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.Results HE staining showed that thickness of outer nuclear layer in the early and medium intervention groups were significantly thicker than that in the control group (P＜0.05),particularly in the early intervention group.And there was no significant change in the late intervention group (P＞0.05).The expression level of Cdk5,p25,cleave-caspase 3 in the outer nuclear layer in three intervention groups were lower than that in the control group (P＜0.05),especially in the early intervention group.Conclusion Cdk5 inhibitor Roscovitine can delay the retinitis pigmentosa process in RCS rats by early,medium interventional therapy and may have a certain degree of photoreceptor-protective effects.