| Object：UCH-L1plays a key role in neurodegenerative diseases.It is necessary tomaintain the normal synaptic and cognitive function. In this study, We choseSAMP8mice as experimental objects, discuss the distribution of UCH-L1inCA1area of hippocampus of aging,, and the expression level of the protein,mRNA of UCH-L1in changes with aging, using Western Blot,Immuneohistochemistry, and PCR technology. It will be a preliminary studyon UCH-L1, UPP in the rapid aging and neurodegeneration in effect, enrichthe neural mechanism of learning and memory.Methods:1Animal grouping:3,6,9,12-month-old male SAMP8/SAMR1micewere selected each for10.2Tissue preparation and dyeing observation: Groups of mice (n=3pergroup)were taken out, perfused quickly with normal saline and subsequentlyfixed with4%paraform via left ventricle and broken right auricle. Thehippocampus was taken between superior collicμlus and optic chiasma, thebrain made the same two parts through midsagittal by razor blade. One wasusing for UCH-L1immunohistochemical staining; the other was made use forGolgi staining, and count each group of mice hippocampal CA1region of2to3apical dendrites dendritic spine density.3Protein extraction and detection of activity of deubiquitinating enzymein different organs of SAM mice: Take6-month-old SAMR1and SAMP8mice each one, with6%chloral hydrate anesthesia, quickly takes out heart,liver, testis, hippocampus, frontal cortex. After PBS washing, quickly placedin lysis buffer, fμlly homogenized on ice at rest30min;4℃,12000RPM centrifuge for20minutes,discard the pellet, the supernatant, small part of thesupernatant was used for the determination of protein concentration (measuredby Coomassie brilliant blue method), the rest was combined with activedeubiquitinating enzyme probe with HA markers for1hours, for detectiondistribution of active deubiquitinating enzyme in various organs.4Extraction of mRNA and PCR: Groups of mice (n=3per group) weretaken out, After6%chloral hydrate anesthesia rapidly decapitation, removedthe skμll, stripped of meninges, after exposed brain tissue, isolated from thehippocampus. After PBS cleaning, quickly set in Trizol, conventionallyextracted mRNA, quantified using NanoDrop2000C, reverse transcribedinto cDNA,-20℃preservation,for PCR.5Protein extraction and Western Blot Groups of mice (n=3per group)were taken out, after6%chloral hydrate anesthesia rapidly decapitation,removed the skμll, stripped of meninges, after exposed brain tissue, isolatedfrom the hippocampus. After PBS washing, quickly placed in lysis buffer,fμlly homogenized on ice at rest30min;4℃,12000rpm centrifuge for20minutes, discard the pellet, the supernatant, small part of the supernatant wasused for the determination of protein concentration (measured by Coomassiebrilliant blue method), put the rest of supernatant in-80℃refrigerator ofstorage, used for Western Blot detection.Results:1Detect to activity of ubiquitination enzymes in different organs ofSAM mice: active deubiquitinating enzyme expression in different organs ofSAM mice, in the hippocampus,36kD has obvious bands, suggesting thatUCH-L1in the hippocampus has a number of active expression.2The results of anti-UCH-L1immunohistochemistry.The hippocampusCA1region of6-mouth-old SAMR1group was dyed deep, the optical densityvalue was0.374±0.018, significantly higher than other groups (P<0.05); Thehippocampal CA1region of12-mouth-old SAMP8group were stained light,the optical density value was0.171±0.003, significantly lower than the othergroups (P<0.05);the optical density of9-mouth-old SAMR1group was 0.256±0.011,which is higer than it in9-mouth-old SAMP8group (P<0.05);Optical density of12-mouth-old SAMR1group was0.224±0.013,which ishigher than it in12-mouth-old SAMP8group (P<0.05);The optical density of3-mouth-old SAMR1group was0.251±0.006and the optical density of3-mouth-old SAMR1group was0.239±0.001compared with each other thereis no statistically significant difference (P>0.05)3Expression of UCH-L1mRNA PCR：the expression of UCH-L1mRNA of6-mouth-old SAMR1group is higher than other groups, theexpression of UCH-L1mRNA of12-mouth-old SAMR1group is lower thanother groups; the R1group each month expression level was0.391±0.087,0.786±0.072,0.541±0.062,0.348±0.009,more than any other in the samemonth old SAMP8rats were0.390±0.020,0.700±0.038,0.354±0.015,0.299±0.010(P<0.05).4The result of Western Blot: the expression of UCH-L1in thehippocampus of6-mouth-old SAMR1group, the IOD value was2.224±0.089,was significantly higher than other groups(P<0.05);Theexpression of UCH-L1in hippocampus of12-mouth-old SAMP8group, theIOD value was1.033±0.140,was significantly lower than the other groups(P<0.05);the expression of UCH-L1in the hippocampus of9-mouth-oldSAMR1group was1.486±0.101,which is higer than it in9-mouth-old SAMP8group(P<0.05);The expression of UCH-L1in the hippocampus of3-mouth-oldSAMR1group was1.280±0.095and the expression of UCH-L1in thehippocampus of3-mouth-old SAMR1group was1.235±0.038compared witheach other there is no statistically significant difference (P>0.05).5Gogli staining: dendritic spines in CA1area of hippocampus of6-mouth-old SAMR1group arranged densely, dendritic spine number is1.314±0.028/μm2, significantly higher than the other groups (P<0.05); dendriticspine number of12-mouth-old SAMP8group spine was0.887±0.022/μm2,significantly lower than that of other groups; the dendritic spine density of9-mouth-old SAMR1group was1.158±0.025/μm2,which is higer than it in9-mouth-old SAMP8group (P<0.05); Dendritic spine density of12-mouth-old SAMR1group was1.015±0.021/μm2,which is higher than it in12-mouth-oldSAMP8group (P<0.05). the dendritic spine density of3-mouth-old SAMR1group was1.127±0.024/μm2and the dendritic spine density of3-mouth-oldSAMR1group was1.107±0.024/μm2compared with each other there is nostatistically significant difference (P>0.05).Conclusion:1Deubiquitinating enzymes express in different organs of SAM mice,UCH-L1has a lot of expression in testis and the nervous system.2In gene and protein levels,6,9,12months of age, content of UCH-L1inSAMR1mice is more than UCH-L1in SAMP8mice with same age. TheUCH-L1of SAMP8mice and SAMR1mice in the age of March did not showdifferences.3In the gene level and protein level, the content of UCH-L1of SAMR1and SAMP8reduce with increasing of age.4In the different month, the number of dendritic spines in SAMR1mouse is more than SAMP8mice in the same month old. The UCH-L1ofSAMP8mice and SAMR1mice in the age of March did not show differences.5In different months, the number of dendritic spine of SAMR1andSAMP8reduce with increasing of age. |
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