Study On The Differential Gene Expression Of Human Traumatic Cerebral Cortex By Genechip

Objective To analyze the difference of gene expression profile between thetraumatic and normal human cerebral cortex,and look for new molecular biological strategies for preventing and treating brain trauma.Methods The traumatic cerebral cortex samples were taken from 5traumatic brain injury (TBI) patients pericontusional tissue during decompression operation within 2 days post injury.Total RNA was extracted respectively in Trozal reagent, purified with RNAeasy mini spin column,and quantified with spectrophotometer.The total RNA quality was estimate by formaldehyde gel electrophoresis. 1 normal total RNA of human cerebral cortex was supplied by Ambion company. The total RNA was retrotranscribed into double-chains cDNA with cDNA Synthesis Kit and T7—Oligo (dT) 15 primer ,then the double-chains cDNA was purified with QIAquick PCR Purification Kit and was transcribed into with T7 RiboMAX Express Large Scale RNA Production System. The cRNA was retrotranscribed into 1st cDNA with SuperscriptII and 9 Random Primer. The 2nd cDNA was synthesize and labeled with cy3- or cy5 fluorescence in the system including 1st cDNA, 9 Random Primer , dNTP, Cy5(3)-dCTP,and KLENOW.The traumatic cortex was labeled with cy5 and the normal control one was labeled with cy3. The labeled 2nd cDNA probes were mixed and hybridized with human genome Oligo genechip containing 21,329 genes ,12 housekeeper genes as positive control, 12 heterogenous genes as negative control, and 8 yeast genes as outer mark. The genechips were premade in 75mm×25 mm chip base .The probes and genechips were hybridized in hybridization reagent at 42 ℃ for a night ,thenwere wash and dried. LuxScan Scanner was used to scan the signals of hybridization and GenePix Pro 4.0 software was applied to export data . Those bad spots ,whose cy3 and cy5 signal were both less than 300 ,and weak spots,whose dispersion of background and foreground were less than 30% self-expression pels ,were excluded. The data was statistically analyzed in Lowess way and normalized and corrected. After normalization and correction ,the Cy-5/Cy-3 value was looked as standand ratio,and ratio >2 or <0.5 was the statistical standard to judge differentially expressed genes.Four differentially expressed genes were randomly selected to confirm the genechip data by quantitative real-time PCR (qRT-PCR) .At last,the expression of three differentially expressed genes were studied in 29 traumatic human brain by RT-PCR.Results We found that there were 8828 expression genes on TlvsN chipand 362 of them were differentially expressed genes(210 up-regulation and 152 down-regulation). Those index were respectively 9911 , 476( 249, 227) on T2vsN chip;9213 , 627( 381, 246) on T3vsN chip;9078 , 973( 573, 400) on T4vsN chip;and 8766 , 1757( 823, 934) on T5vsN chip. There were 87 common genes in those differentially expressed genes from trauma injuried brains compared to the normal brain,60 genes up-regulated and 27 down-regulated. In the up-regulated genes,44 genes were be categorised into 12 functional categories,and they were main metabolism(26), response to stress(12),apoptosis(9),signal(9);the other 16 genes' function were unknown or ESTs.In the down-regulated genes,7 genes were be categorised 4 functional categories, they were main metabolism(6);the other 20 genes' function were unknown or ESTs.We detected the mRNA level of 4 genes by qRT -PCR and found that the ratio of Cy-5/Cy-3 was almost consistent with the corresponding chip data.We detected the mRNA level of sppl - cyr61 and plscrl in 29 traumatic human brain by RT -PCR and found that they all expressed increasedly in all patients.Conclusions We investigated traumatic human brain injury by genechip techniche,and found that the commonly differentially expressed genes was 87 ones.In these 87 genes,some genes such as cd4^ ccl2> tiegN veg^ cdl^ idl^ xbpK egrK rgs2^ ft^ il-lb^ timpK gadd45aN cdkn^ cnn3 had been reported in literatures on brain trauma through Pubmed ,some genes such as clicl n plscrl > cyr6K bag3> gbeK tlr2 ^ sppl were not reported in brain trauma literature before.Those differentially expressed genes found lately may become new targets for the treatment on traumatic brain injury.