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反式白藜芦醇对Aβ25-35诱导阿尔茨海默病大鼠海马神经元的保护影响

Protectiveeffectoftrans-resveratrol

onhippocampalneuronsofratswithAp25.35一inducedAlzheimer'sdisease

Objective:Toinvestigatetheprotectiveeffectoftrans.resveratrol

onhippocampalneuronsofratswithA1325.35一inducedAlzheimer’sdisease,andexploreitspossiblemechanisms.

Methods:Ratsare

randomlydividedintoshamgroup,positivecontrolgmup(donepezil),modelgroup(AH25.35),low-andhigh-doseofTRgroups,respectiveIy(n:9).

AfterinjectionofA]825.35intohippocampus,ratswereadministratedwith

1.0mg.kg-1

donepezilandl0and40mg‘kg~TRoncedailybygavageforconsecutive15

daysrespectively,whileshamandmodelgroupswereadministrated谢thv01ume.matched

normalsalinebygavage.The

learningandmemoryabilityofratsweredetectedbvMorris

wat钉mazetest?neuronalinjuryinhippocampusWasobserved

byhematoty.eosinstaining’

andthemRNAexpressionlevelsof

caspasel2weredetectedbyrealtimePCRandthe

Protemexpresslonsofcaspase8andcaspase12wereexaminedby

immunohistOChemistryandWesternblotintherathippocampus.

Results:IntheratshippocampusafterinjectionofA]325.35,the

escapelatencies(s)of

sh锄group,positivecontrolgroup(donepezil),modelgroup(A陈35),low.and

high

-doseofTRgroups

onthefirstdayinthenavigationtestbyMorriswatermazetestwere

59?0士1

8?2,65?2士27.8,98.8士19.9,77.7士20.4,66.6士25.9respectively;theescape

latencies(s)ontheseconddaywere

25.0+14.5,32.4士21.2,72.8士25.0,52.4+14.8,

33?3士15?1respectively;theescapelatencies(s)onthethirddaywere20.3士12.0,

25?1士12?9,67?2士17.8,44.8士23.2,26.2士19.7respectively;theescapelatencies(s)onthe

founhdaywere

10.3士4.8,12.0士5.8,62.6士22.1,30.3士11.5,14.2士4.7;Theadjusted

escapelatenciesonthefifthdayinspatialprobetestwere32.8士8.4,25.3士5.5,

15.6+1.2,21.7+4.8,28.0+4.9respectively.ThemRNAexpressionsofcaspasel2of

shamgroup,positivecontrolgroup(donepezil),modelgroup(A1325.3s),low-andhigh

-doseofTRgroupsbyrealtimePCRwere55.70士10.12,91.11+7.06,181.49士38.68,

138.43.26士15.28,98.06士23.05respectively;Theprotein

expressionsofcaspase8by

westernblotwere0.18士0.03,0.29士0.02,0.64士0.04,0.57士0.05,0.31士0.03

respectively;Theproteinexpressionsofcaspase12byWesternblotwere0.19士0.05,0.29士0.03,0.68士0.03,0.60士0.05,0.32士0.04respectively.Comparedtothe

sham—treatedrats,A1325.35injectionsignificantly

meanescapelmencyinthe

prolongedthe

navigationtestandshortenedtheadjustedescapelatencyinspatialprobetest(P<0.o1),

NeumalinjuryinhippocampusandthemRNAexpressionofcaspasel2,aSwellasthe

proteinexpressionsofcaspase8andcaspase12inrathippocampuswere

increased(P<0.01).Comparedwiththemodelrats,Trans?resveratrol-treated

groups

significantlyshortenedthemeanescapelatencyinthenavigationtestandprolongedthe

adjustedescapelatencyinspatialprobetest∽<0.05),andattenuatedneuronalinjuryinrathippocampus,andsignificantlydecreasedthemRNAexpressionsofCaspasel2,aSwellaStheproteinexpressionsfcaspase8andCaspasel2inrathippocampus(P<O.05).

Conclusion:Thereisprotectiveeffectoftrans?resveratrolonhippocampalneuronsof

ratswithAp25.35‘inducedAlzheimer’Sdisease,itsmechanismsmaybeatleastpartlyduetodecreasingthemRNAandproteinsexpressionofcaspase8andcaspase12inrat

hippocampus.

KeyWords:trans-resveratrol;蝤ppoc举;caspase8;caspasel2;

既往研究TR对AB。¨j诱导AD大鼠有保护作用,其机理可能部分与抑SwJiNOS、caspase8、caspase9mRNA表达有关n0’川。目前关于TR对AD的保护作用研究,通过内质网应激凋亡通路caspasel2表达的影响,国内外研究较少。本实验以相关文献报道的白藜芦醇抗AD作用为依据,通过观察TR对AD大鼠海马神经元细胞形态影响,探讨TR对AD的保护作用及潜在的机制。进一步证实了TR对AD大鼠海马神经元的保护作用,可能与抑制caspasel2mRNA,caspasel2和caspase8蛋白表达有关,为TR临床应用打下基础,同时提供TR治疗AD的药理学依据及可能的发病机制。

反应体系的组成如下表:

试剂使用量SYBRsuperMix荧光混合物7.5ul

模板混合液(上游、下游引物)0.5ul

DEPC水

稀释6.5倍cDNA反应总体积4u13.0ul15ul

反应条件如下:

Stage1:预变性

95℃10min1Cycle

Stage

2:PCR反应40Cycles

95℃10s:57.9℃1min

Stage3:融解曲线分析

80Cycles95℃1min;55℃lmin;55℃10S。

③引物:

引物合成TaKaRa生物工程公司,见表1。

表1引物序列

Table1Thesequenceofprimers

Forwardprimer(5'-3’)ene

Lj7Reverseprimer(5'-3’)

Caspase12CAATTCCGACAAACAGCTGAGTTTACATGGGCCACTCCAACATTTAC3-actinGGAGATTACTGCCCTGGCTCCTAGACTCATCGTACTCCTGCTTGCTG

5)结果分析采用相对定量法

以Ct值为统计参数依次计算出如下数据:

a.Ctaverage=(Ctl+Ct2)/2(重复管)

b.dCt=Ctaverage-中间值

C.基因的表达=2^(-dCt)

d.相对定量=目的基因的表达/内参基因的表达

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