Effects Of SAO On Apoptosis Expression Of Bcl-2,Bax In Glioma

ObjectiveSurvivin, is a new member of apoptosis inhibiting protein (IAP) family, it' s N-end merely contain a single pole virus IAP repeat section sequence (BIR) while C-end without RING finger frame. Survivin expresses in most tumor tissues and do not in adult end differentiation tissues. Survivin has double functions of inhibiting apoptosis and regulating mitotic, induces tumor apoptosis and inhibit tumor proliferate through low-down Survivin expression. In recently, some researchers begin to study Survivin expression in glioma, which offering a new aspect to glioma diagnosis and therapy; Survivin and tumor suppress gene P53 express together in tumor, there is a significant correlation (r=0.037) . Expression of apoptosis related gene Bcl-2 Bax and maintaining functional stability play an important role in the process of glioma deteriorate and relapse. Survivin offers a new target on a lot of facet in the cause of it' s unusual biological characteristic such as apoptosis proliferate and angiogenesis. AntisenseSurvivin study aiming at Survivin abnormity expression: Ambrosini et al investigate that endogenesis lowing-down expression of Surviving apoptosis augment chasten the growth of transform cells induced by EPR-1. Apoptosis induced by Survivin antisense oligonucleartide(SAO) processes on the level of cell surface receptor or mitochondrion. Shangkar restrains oligodendroglioma (TC620) cell cleavage boost apoptosis dealing with Survivin antisense oligonucleartide (SAO). Mesri discovered Survivin antisense oligonucleartide(SAO) restraining endothelium cell safeguard function guided by VEGF, accelerated endothelium cell apoptosis during tumor duct shaping and blood vessel receding.Survivin play an important role in the control of apoptosis related gene Bcl-2 Bax cell cycle and maintaining inner stability . Study on Survivin as a marker of tumor diagnosis and therapy target showes a finer application foreground. We believe that the study on Survivin antisense oligonucleartide( SAO) will discover internality relationships of glioma apoptosis and contribute a new break way to neoplasm unite gene therapy.MethodClinical immunohistochemistry study :Material:Glioma is a most frequent nerve center system tumor, reaching about40%⁵0% in adult intracranial neoplasm[l].Glioma relapse is malignancy marked biology behaviors, still a ordinary death causation [2].Study on correlation between expression of p53 and Survivin in relapsed glioma has not been reported in national literature. We collected 96 cases relapsed post operation low grade (WHO I—II grade) glioma samples during 1979, 10-2003, 10 in our hospital.All formalin fixup and paraffin embeded filed samples including male 52 cases, female 44 cases, age range from 25 to 68 years, including typical hypocacoastrocytoma 45 casesn bristleastrocytoma 27 cases > pleomorphicxanthoastrocytoma 15 cases > amalgamoastrocytoma 9 cases.2. Methods:Using immunohistochemistry, mouse anti-p53 monoclonal antibody and streptomycete avidin peroxides immunohistochemistry dyeing super sensitivity reagent box bought from America Maxim Co, Survivin polyclonal antibody bought from Neo Markers Co..3. Statistics manage :EXCEL software recordes experiment datum , progressing statistics analysis using SAS software, x2 inspection T test and pearson correlation analysis. .₁n Transgenic study on Survivin antisense oligonucleartide( SAO) in C61. MaterialGlioma cell line C6 bought from Chinese Academy of the Sciencer of shanghai cell biology academe. Survivin adenovirus vectors PAdCMV-Survivin and empty vectors PAdCMV-LacZ situ apoptosis reagent box and related reagent bought from BOEHRINGR M Pr ANNHEIM Co., restriction enzyme buy from MBI Co; MTT\ PI bought from Sigma Co.; Survivin mice monoclonal antibody bought from Santa Cruz Biotechnology, sheep anti-mouse HRP marker second antibody, Survivin sheep poly-anti and rabbit anti-sheep HRP marker second antibody bought from BOSD Co.2. Primer sequence s designing and synthesizingPrimer designed by Primer5 software, Survivin gene sense: 5'-GGACCACCGCATCTCTACAT-3',antisense:5'-GCACTTTCTTCGCAG TTTCC-3', annealing temperature at 72°C, expected expand segment is 338 bp.3. Survivin vectors adenovirus transfectes C6 cell:Infecting respectively C6 cell with Survivin vectors adenovirus and recombined empty vectors PAdCMV-LacZ empoty reporting gene LacZ according to 100MOI, 37°C culture for 48h, 2% formalin +0.2% glutaral 4 °C fixed for 2h, X-gal dyeing overnight, observe blue cell under microscope, inoculate C6 cell, adding Survivin adenovirus vectors while anchorage-dependent growth cell fusion efficiency reach 40%—50%, countneeded virus according to 100MOI, 27°C adsorb 2h, swag one time per lOmin, PBS rinse 3 times, add media contain 2% calf serum culture on.4. Detecting of extrinsic Survivin expressionDistill general RNA of C6> C6-Survivin cell, synthesize the first chain of cDNA after disposed by DNase I, and regard it as model, also use antisense Survivin oligonucleartide gene primer and actin primer to process PCR amplification, reaction condition at: 94°C\ 3min, 94°C\ 45sec, 58 °C > 50sec, 72°C> 50sec, cycle numbers: 30 times, prolong: 72°C> 7min. Agarose gel electrophoresis analysis, gather C6 -, C6-aSurvivin cell , distilling general protein after Survivin adenovirus infected 48h , processing protein SDS-PAGE electrophoresis, analysis protein expression level with western blot.5. The situ apoptosis examine (TUNEL)Inoculate C6^ C6-aSurvivin cell respectively to cover slice, infect slice cells with vector PAdCMV-Survivin and empty vector PAdCMV-LacZ of 100 MOI next day, examine apoptosis signal after 48h infecting using TUNEL regent box, working-out average positive rate (LI) .6. Expression of Bcl-2> BaxUsing immunohistochemistry of SP to examine PAdCMV-Survivin C6 cell, observe Bcl-2 -, Bax immunal reaction substance under optics microscope .Positive cells exceed 5% be determined positive.7. Statistics manageSPSS 10.0 for windows software was used in data processing, X2, T test was used.Results ^ Clinical immunohistochemistry study : l.The expression of p53 and Survivinexpression of p53 and Survivin in relapsed glioma shows respectively 44.8% (43/96) and 21.9% (21/96); while 29.2% (28/96) and 14.9%(14/96) (P<0.05) in original glioma. Positive sample of p53 is partial nucleus coloration and plasma coloration, while Survivin is all plasma coloration. Statistics shows significant advance expression of p53 and Survivin in relapsed glioma (P<0.05) .2. The correlation between expression of p53 and Survivin and relapsed timeRelapsed time in expression of p53 and Survivin greatly bring ahead respectively to 3.9 years and 4.1 years (PO.05), relapsed time in together expression bring ahead to 2.7 years (PO.01) .3. The correlation between expression of p53 and Survivin and relapseAbout 56.3% cases of original glioma show positive expression of p53 and Survivin, while in relapsed glioma reach 70%, show great significant difference and great positive correlation (r=0.673, PO.01) .-^ Transgenic study on Survivin antisense oligonucleartide: l.The expressions of antisense Survivin oligonucleartide gene in C6RT-PCR shows PAdCMV-Survivin transfect caused the level of Survivin mRNA in C6 cell greatly decline. PAdCMV-Survivin virus drop degree at 7.0x108 pfu /ml, PAdCMV-LacZ at 5.0x108 pfu/ ml. WESTERN BLOT analysis why expression of Survivin protein in C6-aSurvivin cell is higher than in C6 cell.2. Result of situ apoptosis examination(TUNEL)Apoptosis signal in C6-aSurvivin cell is higher than in C6 -LacZ cell (p<0.05) .3. Expression of Bcl-2> BaxIn C6-aSurvivin cell, protein of Bcl-2 show some positive expression, but the number of apoptosis cells show no significant difference with negative expression (p>0.05), while protein of Bax shows great positive expression and significant difference to negative expression (p<0.05 ) .Conclusions1. Average relapsed time of the cases with expression of p53 and Survivin respectively bring ahead greatly to 3.9 years and 4.1 years (P<0.05) .While co-expression bring ahead to 2.7 years (P<0.0\), show significantlydifference.2. Expressions of Survivin suggests the advance of glioma relapse time .3. Signifficent correlation on expression of p53 and Survivin shows defmitude prognosis meaning of glioma relapse. Post operation life span of positive expression of Survivin is lower than negative.4. Paying great attention to the cases of expression of p53 and Survivin is absolutely necessary in clinical. Early callback should be emphasized to early detect and early deal with relapsed glioma.5. While simultaneously antisense Survivin increase number of apoptosis cell, number of apoptosis cell on Bcl-2 show no difference between positive and negative expression (/?>0.05), it explain that the apoptosis restraining function of Survivin equally not go down.6. Number of apoptosis cell in Bax positive expression is increased more than in negative (p<0.05), it explain that the apoptosis restraining function of Survivin distinctively go down.7. The signals of modulating apoptosis may be passed by a positive cycle composed of Survivin antisense oligonucleartideN Bax> Bcl-2, ultimately start-up and strengthen Caspase—3 inducement function of apoptosis domino offect.