中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2013年
6期
546-550
,共5页
张金金%盛迅伦%任英华%容维宁%李慧平%刘雅妮
張金金%盛迅倫%任英華%容維寧%李慧平%劉雅妮
장금금%성신륜%임영화%용유저%리혜평%류아니
细胞周期依赖性蛋白激酶5%RCS大鼠%视网膜神经细胞%凋亡%P35%P25%Tau磷酸化
細胞週期依賴性蛋白激酶5%RCS大鼠%視網膜神經細胞%凋亡%P35%P25%Tau燐痠化
세포주기의뢰성단백격매5%RCS대서%시망막신경세포%조망%P35%P25%Tau린산화
Cyclin-dependent kinase 5%RCS rat%Retinal neural cell%Apoptosis%P35%P25%Tau phosphorylation
背景 视网膜色素变性(RP)是眼科常见的遗传性、致盲性眼病,可能与阿尔茨海默病等慢性神经退行性疾病具有共同的病理生理机制,细胞周期依赖性蛋白激酶5(Cdk5)与光感受器细胞及视网膜神经节细胞正常功能的维持相关,可能是引起RP光感受器细胞凋亡的途径,有可能成为新的治疗靶点. 目的 探讨Cdk5/P25激酶活性在RCS大鼠视网膜神经细胞凋亡中的作用.方法 SPF级RCS变性大鼠及RCS-rdy+正常对照大鼠各18只,按随机数字表法亚分至出生后17、25、35 d组,每亚组各6只大鼠.各组大鼠分别在相应时间点直接处死后摘除眼球,并取视网膜组织,以Western blot法检测大鼠视网膜组织中Cdk5、P35、P25蛋白的表达和tau蛋白磷酸化水平,并利用紫外分光光度计用光谱法测定两组不同日龄大鼠视网膜组织吸光度(A340)峰值的变化,定量分析各组Cdk5/P25激酶的活性. 结果 P35蛋白在17日龄的RCS大鼠和RCS-rdy+大鼠的视网膜组织中表达水平(A340)差异无统计学意义(t=0.52,P＞0.05),25日龄和35日龄RCS大鼠视网膜组织中P35蛋白表达水平分别为2.20±0.48、1.23±0.14,明显高于RCS-rdy+大鼠的1.43±0.13和0.93±0.10,差异均有统计学意义(t=3.78、4.28,P＜0.05)；P25蛋白在17日龄的RCS大鼠及RCS-rdy+大鼠的视网膜组织中均未检测到,但在25日龄和35日龄的RCS大鼠视网膜组织中表达水平(A340)为0.300±0.003、0.230±0.004,明显高于RCS-rdy+大鼠的0.040±0.004和0.070±0.004,差异均有统计学意义(t=121.81、77.51,P＜0.01)；Cdk5蛋白在各日龄的RCS大鼠视网膜组织中的表达水平与RCS-rdy+大鼠相比差异均无统计学意义(t=-0.60、0.19、1.62,P＞0.05)；17日龄RCS大鼠和RCS-rdy+大鼠视网膜组织中Cdk5/P25激酶活性差异无统计学意义(t=0.19,P＞0.05),25日龄和35日龄的RCS大鼠视网膜组织中Cdk5/P25激酶活性分别为0.0058±0.0005、0.0056±0.0004,明显高于RCS-rdy+大鼠的0.0038±0.0003和0.0032±0.0007,差异均有统计学意义(t=8.07、5.97,P＜0.01)；25日龄和35日龄RCS大鼠视网膜组织中tau蛋白磷酸化水平明显高于RCS-rdy+大鼠,差异均有统计学意义(t=4.71、3.17,P＜0.05). 结论 RCS大鼠视网膜组织中CdkS/P25激酶活性与P25蛋白表达水平和tau蛋白磷酸化水平的变化趋势一致,提示P25表达增加可能诱导Cdk5/P25激酶活性升高,并通过磷酸化其作用底物引起视网膜神经细胞凋亡.
揹景 視網膜色素變性(RP)是眼科常見的遺傳性、緻盲性眼病,可能與阿爾茨海默病等慢性神經退行性疾病具有共同的病理生理機製,細胞週期依賴性蛋白激酶5(Cdk5)與光感受器細胞及視網膜神經節細胞正常功能的維持相關,可能是引起RP光感受器細胞凋亡的途徑,有可能成為新的治療靶點. 目的 探討Cdk5/P25激酶活性在RCS大鼠視網膜神經細胞凋亡中的作用.方法 SPF級RCS變性大鼠及RCS-rdy+正常對照大鼠各18隻,按隨機數字錶法亞分至齣生後17、25、35 d組,每亞組各6隻大鼠.各組大鼠分彆在相應時間點直接處死後摘除眼毬,併取視網膜組織,以Western blot法檢測大鼠視網膜組織中Cdk5、P35、P25蛋白的錶達和tau蛋白燐痠化水平,併利用紫外分光光度計用光譜法測定兩組不同日齡大鼠視網膜組織吸光度(A340)峰值的變化,定量分析各組Cdk5/P25激酶的活性. 結果 P35蛋白在17日齡的RCS大鼠和RCS-rdy+大鼠的視網膜組織中錶達水平(A340)差異無統計學意義(t=0.52,P＞0.05),25日齡和35日齡RCS大鼠視網膜組織中P35蛋白錶達水平分彆為2.20±0.48、1.23±0.14,明顯高于RCS-rdy+大鼠的1.43±0.13和0.93±0.10,差異均有統計學意義(t=3.78、4.28,P＜0.05)；P25蛋白在17日齡的RCS大鼠及RCS-rdy+大鼠的視網膜組織中均未檢測到,但在25日齡和35日齡的RCS大鼠視網膜組織中錶達水平(A340)為0.300±0.003、0.230±0.004,明顯高于RCS-rdy+大鼠的0.040±0.004和0.070±0.004,差異均有統計學意義(t=121.81、77.51,P＜0.01)；Cdk5蛋白在各日齡的RCS大鼠視網膜組織中的錶達水平與RCS-rdy+大鼠相比差異均無統計學意義(t=-0.60、0.19、1.62,P＞0.05)；17日齡RCS大鼠和RCS-rdy+大鼠視網膜組織中Cdk5/P25激酶活性差異無統計學意義(t=0.19,P＞0.05),25日齡和35日齡的RCS大鼠視網膜組織中Cdk5/P25激酶活性分彆為0.0058±0.0005、0.0056±0.0004,明顯高于RCS-rdy+大鼠的0.0038±0.0003和0.0032±0.0007,差異均有統計學意義(t=8.07、5.97,P＜0.01)；25日齡和35日齡RCS大鼠視網膜組織中tau蛋白燐痠化水平明顯高于RCS-rdy+大鼠,差異均有統計學意義(t=4.71、3.17,P＜0.05). 結論 RCS大鼠視網膜組織中CdkS/P25激酶活性與P25蛋白錶達水平和tau蛋白燐痠化水平的變化趨勢一緻,提示P25錶達增加可能誘導Cdk5/P25激酶活性升高,併通過燐痠化其作用底物引起視網膜神經細胞凋亡.
배경 시망막색소변성(RP)시안과상견적유전성、치맹성안병,가능여아이자해묵병등만성신경퇴행성질병구유공동적병리생리궤제,세포주기의뢰성단백격매5(Cdk5)여광감수기세포급시망막신경절세포정상공능적유지상관,가능시인기RP광감수기세포조망적도경,유가능성위신적치료파점. 목적 탐토Cdk5/P25격매활성재RCS대서시망막신경세포조망중적작용.방법 SPF급RCS변성대서급RCS-rdy+정상대조대서각18지,안수궤수자표법아분지출생후17、25、35 d조,매아조각6지대서.각조대서분별재상응시간점직접처사후적제안구,병취시망막조직,이Western blot법검측대서시망막조직중Cdk5、P35、P25단백적표체화tau단백린산화수평,병이용자외분광광도계용광보법측정량조불동일령대서시망막조직흡광도(A340)봉치적변화,정량분석각조Cdk5/P25격매적활성. 결과 P35단백재17일령적RCS대서화RCS-rdy+대서적시망막조직중표체수평(A340)차이무통계학의의(t=0.52,P＞0.05),25일령화35일령RCS대서시망막조직중P35단백표체수평분별위2.20±0.48、1.23±0.14,명현고우RCS-rdy+대서적1.43±0.13화0.93±0.10,차이균유통계학의의(t=3.78、4.28,P＜0.05)；P25단백재17일령적RCS대서급RCS-rdy+대서적시망막조직중균미검측도,단재25일령화35일령적RCS대서시망막조직중표체수평(A340)위0.300±0.003、0.230±0.004,명현고우RCS-rdy+대서적0.040±0.004화0.070±0.004,차이균유통계학의의(t=121.81、77.51,P＜0.01)；Cdk5단백재각일령적RCS대서시망막조직중적표체수평여RCS-rdy+대서상비차이균무통계학의의(t=-0.60、0.19、1.62,P＞0.05)；17일령RCS대서화RCS-rdy+대서시망막조직중Cdk5/P25격매활성차이무통계학의의(t=0.19,P＞0.05),25일령화35일령적RCS대서시망막조직중Cdk5/P25격매활성분별위0.0058±0.0005、0.0056±0.0004,명현고우RCS-rdy+대서적0.0038±0.0003화0.0032±0.0007,차이균유통계학의의(t=8.07、5.97,P＜0.01)；25일령화35일령RCS대서시망막조직중tau단백린산화수평명현고우RCS-rdy+대서,차이균유통계학의의(t=4.71、3.17,P＜0.05). 결론 RCS대서시망막조직중CdkS/P25격매활성여P25단백표체수평화tau단백린산화수평적변화추세일치,제시P25표체증가가능유도Cdk5/P25격매활성승고,병통과린산화기작용저물인기시망막신경세포조망.
Background Retinitis pigmentosa (RP)is a common hereditary blinding eye disease in ophthalmology.Current researches documented that RP may have the common pathophysiologic basis to Alzheimer disease and chronic neurodegenerative disease.Understanding this mechanism will offer a new therapeutic target for RP.Objective The purpose of the present study was to investigate the roles of cyclin-dependent kinase 5 (Cdk5)/P25 activation in the apoptosis of retinal neural cells of RCS rats.Methods Eighteen SPF RCS rats and 18 RCS-rdy+ rats were randomized into 17-,25-and 35-day groups respectively and 6 rats for each.The rats were sacrificed at corresponding time points and retinal hemogenete was prepared.Expressions of CdkS,P35,P25 and tau phosphorylation in the retinas were detected by Western blot,and the kinase activity of Cdk5/P25 was analyzed by quantitative colorimetric assay.Results The expressing level of P35 protein(A340) in the retinas of 17-day-old RCS rats was near that of 17-day-old RCS-rdy+ rats(t =0.52,P＞0.05).In 25-and 35-day-old RCS rats,the expressing levels of P35 protein were 2.20±0.48 and 1.23±0.14,which were higher than those of RCS-rdy+ rats(1.43±0.13 and 0.93±0.10),showing significant differences between them(t =3.78,4.28,P＜0.05).The expression of P25 was undetectable at postnatal 17 days in RCS rats and RCS-rdy+ rats,but it showed significantly higher in RCS rats(0.300±0.003 and 0.230±0.004) than that in RCS-rdy+ rats(0.040±0.004 and 0.070±0.004) at postnatal 25 days and 35 days(t=121.81,77.51,P＜0.01).No significant difference was found in the expression of Cdk5 in RCS rats and RCS-rdy+ rats at different ages (t =-0.60,0.19,1.62,P＞ 0.05).The kinase activity of Cdk5/P25 did not show significantly different between RCS and RCS-rdy+ rats at postnatal 17 days(t =0.19,P＞0.05),but significantly higher kinase activity of Cdk5/P25 was seen in RCS rats (0.0058 ±0.0005 and 0.0056±0.0004) than that in RCS-rdy+ rats(0.0038±0.0003 and 0.0032 ±0.0007) at postnatal 25 days and 35 days (t =8.07,5.97,P＜ 0.01).No expression of tau phosphorylation was detected in RCS rats at postnatal 17 days,but significantly higher tau phosphorylation level was seen in RCS rats at postnatal 25 days and 35 days(1.80±0.22 and 1.23±0.17),which were significant different in comparison with RCS-rdy+ rats at postnatal 25 days and 35 days(1.60 ±0.20 and 1.04 ±0.12)(t=4.71,3.17,P＜0.05).Conclusions The Cdk5/P25 kinase activity shows a consistent trend with theexpressions of P25 and tau phosphorylation in the RCS rats,indicating that the upregulation of P25 induces the enhance of enzyme activity of Cdk5,which phosphorylate its substrates to result in more apoptosis of retinal neural cells.
