中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2015年
1期
5-9
,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
Cdk5/p25%抑制剂%Roscovitine%凋亡%RCS大鼠%视网膜变性/遗传性
Cdk5/p25%抑製劑%Roscovitine%凋亡%RCS大鼠%視網膜變性/遺傳性
Cdk5/p25%억제제%Roscovitine%조망%RCS대서%시망막변성/유전성
Cdk5/p25%Inhibitor%Roscovitine%Apoptosis%Rat,RCS%Retinal degeneration/genetics
背景 视网膜色素变性(RP)与阿尔茨海默病有着共同的发病机制,细胞周期依赖性蛋白激酶(Cdk5)活性及其激活剂参与中枢神经系统的退行性病变.Cdk5抑制剂roscovitine可以抑制Cdk5/p25通路的活性,从而抑制细胞凋亡,但其对RP的影响尚不清楚. 目的 探讨玻璃体腔注射roscovitine对RCS大鼠视网膜组织中p35、p25和tau蛋白表达的影响.方法 12只RCS大鼠于出生后17d右眼玻璃体腔注射4 μlroscovitine作为实验眼,左眼未进行任何干预作为对照眼.分别于注射后8d(出生后25 d)和18d(出生后35 d)用过量麻醉法各处死6只大鼠并分离视网膜,Western blot法检测RCS大鼠视网膜组织中Cdk5、p35、p25蛋白的相对表达水平和tau蛋白磷酸化水平,采用分光光度仪(光谱法)测定大鼠视网膜组织340 nm处的吸光度(A)值,定量分析大鼠视网膜中Cdk5/p25激酶活性,采用配对t检验比较实验眼和对照眼的检测结果.结果 玻璃体腔注射后8d和18d,大鼠实验眼视网膜组织中p35蛋白的相对表达水平分别为1.186±0.019和1.069±0.019,明显低于对照眼的1.364±0.016和1.214±0.008,差异均有统计学意义(t=-6.294、-6.477,均P＜0.05);大鼠实验眼视网膜组织中p25蛋白的相对表达水平分别为0.312±0.009和0.269±0.018,明显低于对照眼的0.595±0.013和0.473±0.011,差异均有统计学意义(t=-36.508、-11.879,均P＜0.05);实验眼与对照眼间大鼠视网膜中Cdk5蛋白的相对表达水平差异均无统计学意义(t=0.213、-0.540,均P＞0.05);实验眼大鼠视网膜组织中tau蛋白磷酸化水平均低于对照眼,差异均有统计学意义(t=-9.854、-6.744,均P＜0.05).玻璃体腔注射后8d和18d,比色定量测定法测得大鼠实验眼视网膜中Cdk5/p25活性分别为(0.003 83±0.000 14)mol/(s·mg)和(0.00201±0.000 11)mol/(s·mg),明显低于对照眼的(0.005 47 ±0.000 27) mol/(s·mg)和(0.003 35±0.000 15) mol/(s·mg),差异均有统计学意义(t=-9.152,P=0.000;t=-9.248,P=0.000). 结论 玻璃体腔注射roscovitine可以在一定程度上抑制RCS大鼠视网膜组织中Cdk5/p25激酶活性及tau蛋白磷酸化水平.
揹景 視網膜色素變性(RP)與阿爾茨海默病有著共同的髮病機製,細胞週期依賴性蛋白激酶(Cdk5)活性及其激活劑參與中樞神經繫統的退行性病變.Cdk5抑製劑roscovitine可以抑製Cdk5/p25通路的活性,從而抑製細胞凋亡,但其對RP的影響尚不清楚. 目的 探討玻璃體腔註射roscovitine對RCS大鼠視網膜組織中p35、p25和tau蛋白錶達的影響.方法 12隻RCS大鼠于齣生後17d右眼玻璃體腔註射4 μlroscovitine作為實驗眼,左眼未進行任何榦預作為對照眼.分彆于註射後8d(齣生後25 d)和18d(齣生後35 d)用過量痳醉法各處死6隻大鼠併分離視網膜,Western blot法檢測RCS大鼠視網膜組織中Cdk5、p35、p25蛋白的相對錶達水平和tau蛋白燐痠化水平,採用分光光度儀(光譜法)測定大鼠視網膜組織340 nm處的吸光度(A)值,定量分析大鼠視網膜中Cdk5/p25激酶活性,採用配對t檢驗比較實驗眼和對照眼的檢測結果.結果 玻璃體腔註射後8d和18d,大鼠實驗眼視網膜組織中p35蛋白的相對錶達水平分彆為1.186±0.019和1.069±0.019,明顯低于對照眼的1.364±0.016和1.214±0.008,差異均有統計學意義(t=-6.294、-6.477,均P＜0.05);大鼠實驗眼視網膜組織中p25蛋白的相對錶達水平分彆為0.312±0.009和0.269±0.018,明顯低于對照眼的0.595±0.013和0.473±0.011,差異均有統計學意義(t=-36.508、-11.879,均P＜0.05);實驗眼與對照眼間大鼠視網膜中Cdk5蛋白的相對錶達水平差異均無統計學意義(t=0.213、-0.540,均P＞0.05);實驗眼大鼠視網膜組織中tau蛋白燐痠化水平均低于對照眼,差異均有統計學意義(t=-9.854、-6.744,均P＜0.05).玻璃體腔註射後8d和18d,比色定量測定法測得大鼠實驗眼視網膜中Cdk5/p25活性分彆為(0.003 83±0.000 14)mol/(s·mg)和(0.00201±0.000 11)mol/(s·mg),明顯低于對照眼的(0.005 47 ±0.000 27) mol/(s·mg)和(0.003 35±0.000 15) mol/(s·mg),差異均有統計學意義(t=-9.152,P=0.000;t=-9.248,P=0.000). 結論 玻璃體腔註射roscovitine可以在一定程度上抑製RCS大鼠視網膜組織中Cdk5/p25激酶活性及tau蛋白燐痠化水平.
배경 시망막색소변성(RP)여아이자해묵병유착공동적발병궤제,세포주기의뢰성단백격매(Cdk5)활성급기격활제삼여중추신경계통적퇴행성병변.Cdk5억제제roscovitine가이억제Cdk5/p25통로적활성,종이억제세포조망,단기대RP적영향상불청초. 목적 탐토파리체강주사roscovitine대RCS대서시망막조직중p35、p25화tau단백표체적영향.방법 12지RCS대서우출생후17d우안파리체강주사4 μlroscovitine작위실험안,좌안미진행임하간예작위대조안.분별우주사후8d(출생후25 d)화18d(출생후35 d)용과량마취법각처사6지대서병분리시망막,Western blot법검측RCS대서시망막조직중Cdk5、p35、p25단백적상대표체수평화tau단백린산화수평,채용분광광도의(광보법)측정대서시망막조직340 nm처적흡광도(A)치,정량분석대서시망막중Cdk5/p25격매활성,채용배대t검험비교실험안화대조안적검측결과.결과 파리체강주사후8d화18d,대서실험안시망막조직중p35단백적상대표체수평분별위1.186±0.019화1.069±0.019,명현저우대조안적1.364±0.016화1.214±0.008,차이균유통계학의의(t=-6.294、-6.477,균P＜0.05);대서실험안시망막조직중p25단백적상대표체수평분별위0.312±0.009화0.269±0.018,명현저우대조안적0.595±0.013화0.473±0.011,차이균유통계학의의(t=-36.508、-11.879,균P＜0.05);실험안여대조안간대서시망막중Cdk5단백적상대표체수평차이균무통계학의의(t=0.213、-0.540,균P＞0.05);실험안대서시망막조직중tau단백린산화수평균저우대조안,차이균유통계학의의(t=-9.854、-6.744,균P＜0.05).파리체강주사후8d화18d,비색정량측정법측득대서실험안시망막중Cdk5/p25활성분별위(0.003 83±0.000 14)mol/(s·mg)화(0.00201±0.000 11)mol/(s·mg),명현저우대조안적(0.005 47 ±0.000 27) mol/(s·mg)화(0.003 35±0.000 15) mol/(s·mg),차이균유통계학의의(t=-9.152,P=0.000;t=-9.248,P=0.000). 결론 파리체강주사roscovitine가이재일정정도상억제RCS대서시망막조직중Cdk5/p25격매활성급tau단백린산화수평.
Background Study determined that retinitis pigmentosa has a similar pathogenesis mechanism to Alzheimer disease,and activity of cyclin-dependent kinase 5 (Cdk5) and its activators participates in the degeneration of central nervous system.Roscovitine,an inhibitor of Cdk5,can suppress activity of Cdk5/p25 pathway and therefore inhibit cell apoptosis.However,the influence of roscovitine on retinitis pigmentosa(RP) is unclear.Objective This study was to investigate the expressions of p35,p25 and tau in the retinas of RCS rats.Methods Roscovitine of 4 μl was intravitreally injected in the right eyes of 12 SPF 17-day-old RCS rats,and the fellow eyes were not intervened as the control eyes.The rats were sacrificed on eighth day (postnatal 25 days) and eighteenth day (postnatal 35 days),and whole retinas were isolated to evaluate the relative expressions of Cdk5,p35,p25 and tau phosphorylation by Western blot,and the activity of Cdk5/p25 was analyzed by quantitative colorimetric assay.The results were compared between the right eyes and fellow eyes by paired t test.The use and care of the rats complied with Ethic Statement of Experimental Animal of Ningxia Medical University.Results In the eighth and eighteenth day after injection,the relative expression values (A values) of p35 in rat retinas were 1.186±0.019 and 1.069± 0.019 in the injected eyes,showing significant decreases in comparison with 1.364±0.016 and 1.214±0.008 of the fellow eyes (t =-6.294,-6.477,both at P＜0.05);the relative expression values (A values) of p25 in rat retinas were 0.312±0.009 and 0.269±0.018 in the injected eyes,which was significantly lower than 0.595±0.013 and 0.473±0.011 of the fellow eyes (t=-36.508,-11.879,both at P＜0.05).No significant difference was found in the relative expression of Cdk5 protein between the injected eyes and the fellow eyes in various time points after injection (both at P＞0.05).The activities of Cdk5/p25 were (0.003 83 ±0.000 14) mol/(s · mg) and (0.002 01 ± 0.000 11) mol/(s · mg) in the injected eyes,with significant decreases in comparison with the (0.005 47±0.000 27)mol/(s · mg)and (0.003 35±0.000 15) mol/(s · mg) of the fellow eyes (t=-9.152,P=0.000;t=-9.248,P=0.000),and the tau phosphorylation levels followed the same pattern in the eighth and eighteenth day after injection (t =-9.854,-6.744,both at P＜0.05).Conclusions Intravitreal injection of roscovitine can inhibit the activity of Cdk5/p25 and tau phosphorylation level in retinas of RCS rats to certain extend.
