| IntroductionC - myb is a kind of intranucleus cancer gene, it was found in mature cellular leukemia virus and leukamia virus E26 in poultry at first . At earlier, c -myb was only found in mature marrow cells and chest gland cells in experiment animals and human bodies. Then c - myb also was found in external germinal layer, digest channel and blood vessel muscular cells. Some experiments show that the expression of c - myb has a positive relation with the malignant degree of tumor,more malignant the tumor is, higher the expression of c - myb index is. It is discovered that some benign tumor with higher c - myb index show cell proliferating actively, growing invadly, changing malignantly, higher reappearing removed surgically. Some articals report that c - myb also has a positive relation with the radio sensitivity, more higher c — myb index is, more sensitive radiation is. With the progress of the reserch to c - myb ,c - myb has been used extensively. It may become a marker to determin the prognosis of tumor and radio sensitivity.Nowadays, antisense gene therapy may be a better developing direction. Because antisense oligodeoxyribonucleolide (ASODN) has the advantages of to use convenienly and to make easily.. At the same time its specificity is higher than traditional medicine. The antisense gene therapy means directly to carry artificial ASODN to target cells or tissues by specific carrier and to utilize " the geng seal" to close the expression of cancer gene. At last it can make cancer cells to split in normal orbit or bring cells to apoptosis.125I is a kind of radioactive nuclear element which release energy by the way of electronic capture and interchange.125I has no significant cellular toxicitywhen it lies in cell membrance and cell plasma, but when it entrance cellular DNA,125I is extrally poisonous,this is the DNA target treament of 125I. However, 125IUdR is a kind of better carrier to entrance cellular DNA and it can kill the cells of S period.x IUdR is a specificial radioactive medicine of cell period.Apoptosis is a form of cell death characterized by active cellular suicide during T - cell colonal deletion, embryogenesis, and DNA damage. Apoptotic cell death is often associated with distinctive characteristics, such as nuclear fragmentation, cytoplasma blebbing, and internucleosomal fragmentation of DNA. The Bel - 2 family plays a central role in the control of apoptosis. The familiy include a number of proteins which have amino acid sequence homology, including anti - apoptotic member such as Bel - 2 and Bel - xl, as well as pro - apoptotic members including Bax and Bad. Overexpression of Bax has been shown to accelerate the cell death. Conversely, overexpression of antiapoptotic proteins such as Bel - 2 represses the death function of Bax. Thus, the ratio of Bel - 2 to Bax appears to be a critical determinant of a cell s threshold for undergoing apoptosis.In this study, to adopt Wistart rats C6glioma cell is worthwhile to research the work of ASODN and 125IUdR - ASODN. In order to inveatigate whether ASODN and 125IUdR -ASODN could restrain proliferation and induce apoptosis in C6glioma cell and what the mechanism is. It could provide us with theory and test foundation that ASODN and 125IUdR - ASODN have an obvious action in resisting glioma cell growth.Materials and methods1. Materials: MTT, TUNEL, monocolonal anti - c - myb antibody mono-colonal anti - Bax, Bel - xL antibody were obtained from Boster Co. ( Wuhan, China). The RNA collects the TRIZOL reagent that the reagent is the American GIBCOBRL corporation. PCR kit act as Shanghai student workman's productions. The BcaBest kit that the Japan treasure living beings( Dalian) corporations were fastend to the system is writed down in the inversion invert. The Iipo, ASODN and 125IUdR - ASODN were shopped from Institude of atomic energy ofChinese Academy of Sciences, Shanghai, China. The samples of different grade glioma were from the pathology department of the first affiliated hospital of China Medical University. The Wistar rats were obtained from China Medical University experimental animal ministry.2. Cell line;The cell line of C6glioma was used in our study (Institute of Cytobiology of Chinese Academy of Science, Shanghai, China). Cells were grown in culture flasks containing PRMI —1640 culture medium suplemented wih 10% FCS at 37X. in a humidified atmosphere with 5% C023. Methods:(1) S - P immune histochemistry stainning;To check the expression of c -myb protein in different grade glioma.(2) MTT assay: 1 x 105 cells/well in a 96 - well plate after 24 hours incubation were treated with Lipo, ASODN and 125IUdR - ASODN for 24 hours. lOul of 5g/L of MTT was added to the cells in every well and incubated for 4 hours at 37^. Culture medium were discarded followed by addition of 0. 2ml of DMSO and vibrated for lOminutes. The absorbance ( OD) was measured at 570 nm u-sing a microplate reader. The cell growth inhibitory rate was calculateed as fol-'lows: ( OD of control group - OD of experimental group )/OD of control group 100%.(3) TUNEL: To examine the apoptosis rate of C6 glioma cells treated by ASODN and I25IUdR -ASODN for 24 hours.(4) Type Blue stain: To measure the quantity of living cells and to calculate the percent rate of living cells.(5) To monitor the change of the tumor in weight, volume and living period after C6 glioma cells were implanted into rates abdominal cavity and growing tumors were treated by ASODN and 125IUdR -ASODN.(6) Transmission electron microscope: The C6 glioma cells treated with ASODN and 125IUdR - ASODN for 24 hours were trypsinized and harvested after 24hours. Subsequently the cells were fixed in 4% glutaral, imbedded in capsules and converged for 72 hours at 60 T!, The cells were prepared into ultratin section (60nm) and stained with uranyl acetate and lead citrate. Cells morphology was examined by transmission electron microscope.(7) Assessing the expression of the Bax and Bel - xl mRNA: For RT -PCR analisis of Bax and Bel -xl expression in C6glioma cells,RNA was isolated using RNAase spin columns. RT reactions were carried out on RNA from approximately 1000 cells using sensiscript RT and an oligo dT primer according to the manufacturers instructions. Control reactions with no enzyme were carried out in parallel. PCR amplification was carried out on one - fifth of the RT or control reaction. The expression of Bax and Bel -xl mRNA expression was analysed;primer sequences for these were:The Bax antisense primer was 5'- GGG AAT TCT GGA GCT GCA GAG GAT GAT T - 3 ' , and the sense primer was: 5 ' - GCG GAT CCA AGT TGC CAT CAG CAA ACA T - 3';The Bel - xl antisense primer was :5' - AGG CTG GCG ATG AGT TG A A - 3' , and the sense primer was :5f- CGG CTC TCG GCT GCT GCA TT - 3';(3 - action antisense primer was :5' -CAT TTC CGG TGC ACG ATG GAG -3', and the sense primer was: 5'-GCC ATC CTG CGT CTG GAC CTG -3'.(8) Assessing the expression of c - myb protein;Cells were rinsed twice with PBS, and protein was performed according to the manufacturer^ instruction. Protein concentration in homogenates was determined using a BSA - Protein assay employing boving serum albumin as the standard. The extracted protein (20ug) were subjected to 15% polyacrylamide electrophoresis in the presence of sodium dodecyl sulfate under reducing conditions, and then transferred onto poly vinylidene difluoride {PVDF) membranes. Rat monoclonal anti - rabbit c -myb antibodies were used as the primary antibody, and horseradish peroxidasela-beled anti - rat immunoglobulin was used as the secondary reagent. Detection was performed using an ECL system.(9)Statistical analysis;Results are presented as means SEM. Experimental groups were compared by one - way ANOVA, followed by Scheffe's post - hoc test . Comparisons bewteen groups were performed by SPSS11.0 software.Results(1) C - myb can be expressed in various grade glioma and there are significant difference between various grade glioma. Higher the grade is, more the ex-pression is.(2) Compared with control group ASODN and 125IUdR - ASODN have obvious effect in restraining proliferation, promoting apoptosis and confining growth of C6glioma cells. At the same time, 125IUdR -ASODN is better than ASODN.(3) Transmission electron microscope;After 24 hours, the C6 glioma cells treated by ASODN and !25IUdR -ASODN were observed apoptosis characteristic morphological changes.(4) Analysis of Bax and Bel -xl mRNA expression in C6glioma cells;The test results indicate that Bax mRNA ascends and Bel - xl mRNA descends along with the time prolonged of ASODN and 125IUdR - ASODN and there were statistically significant difference. The action of 125IUdR - ASODN is more obvious than that of ASODN.(5) Analysis of c - myb protein expression in C6 glioma cells;c - myb protein descends along with the time prolonged of ASODN and 125IUdR -ASODN, there were statistically significant difference, between 6 hours and 12 hours, 12 hours and 24 hours, 24 hours and 48 hours. The effect of 125IUdR - ASODN is more obvious than that of ASODN.DiscussionC - myb is a kind of intranucleus cancer gene. At earlier, c — myb was on-ly found in mature marrow cells and chest gland cells in experiment animals and human bodies. Then c -myb also was found in external germinal layer, digest channel and blood vessel muscular cells. Some experiments show that the expression of c - myb has a positive relation with the malignant degree of tumor. It is discovered that some benign tumor with higher c - myb index show cell proliferating actively, growing invadly, changing malignantly, higher reappearing removed surgically. Some articals report that c - myb also has a positive relation with the radio sensitivity. Therefore it may become a marker to determin the prognosis of tumor and radio sensitivity. The antisense gene therapy means directly to carry artificial ASODN to target cells or tissues by specific carrier and to utilize " the geng seal" to close the expression of cancer gene. At last it canmake cancer cells to split in normal orbit or bring cells to apoptosis. 125I is a kind of radioactive nuclear element 125I has no significant cellular toxicity when it lies in cell membrance and cell plasma,but when it entrance cellular DNA,1251 is ex-trally poisonous, this is the DNA target treament of 125I. However, 125IudR is a kind of better carrier to entrance cellular DNA and it can kill the cells of S period. 125IudR is a speciflcial radioactive medicine of cell period.Apoptosis is a form of cell death characterized by active cellular suicide during T - cell colonal deletion, embryogenesis, and DNA damage. Apoptotic cell death is often associated with distinctive characteristics, such as nuclear fragmentation, cytoplasma blebbing, and internucleosomal fragmentation of DNA. The Bel - 2 family plays a central role in the control of apoptosis. The familiy include a number of proteins which have amino acid sequence homology, including anti - apoptotic member such as Bel - 2 and Bel - xL, as well as pro - apoptotic members including Bax and Bad. Overexpression of Bax has been shown to accelerate the cell death. Conversely, overexpression of antiapoptotic proteins such as Bel -2 represses the death function of Bax. Thus,the ratio of Bel -2 to Bax appears to be a critical determinant of a cells threshold for undergoing apoptosis.In the present study, MTT assay was used to observe the effect of ASODN and 125IUdR - ASODN on the grpwth of the cell line C6 indicating that the two drugs could inhibit the growth of the cell line C6. Our results show the effect of 125IUdR - ASODN is more stronger than that of ASODN.Transmission electron microscope showed apoptosis characteristic morphological changes after treatment of cell line C6 with ASODN and 125IUdR -ASODN for 24 hours. The test of RT - PCR results indicate that Bax mRNA ascends and Bel - xL mRNA descends along with the time prolonged of ASODN and 125IUdR -ASODN, and there were statistically significant difference.Western - blot was used to detect the expression of c - myb protein. The results showed after treatment of C6 cells with ASODN and 125IUdR - ASODN for 6,12,24,48 hours,c - myb protein descends along with the time prolonged of ASODN and 125IUdR - ASODN , there were statistically significant difference between 6h and 12h,12h and 24h,24h and 48h.The resrech in our laboratory demonstrated ASODN and 125IUdR - ASODN are able to induce the apoptosis in C6 cells. This apoptosis may be mediated by down - expression of apoptosis - regulated gene Bel - xL and up - expression of apoptosis - regulated gene Bax. The mechanism of ASODN and 125IUdR -ASODN as a chemotherapeutic drug in anti - glioma chemotherapy should be further studied. The results also prompts ASODN and 125IUdR — ASODN induce the apoptosis in C6 cells cultured by means of Bax 's/Bcl - xl's method. The ASODN and 125IUdR - ASODNadjust some segments that Bax and Bel - xl regulated to happen is living to transcript formerly the level. From all above,we think that IUdR - ASODN has more obvious effece than ASODN in promoting apoptosis, restraining growth of glioma cell.Conclusions(1) C — myb can be expression in various grade glioma and there are significant difference between various grade glioma. Higher thr grade is, more the expression is.(2) ASODN and 125IUdR - ASODN have the function of antiproliferation and inducing apoptosis on C6 cells. The function of IUdR — ASODN is more apparent than that of ASODN.(3 ) The regulation pathway of Bax/Bcl - xl maybe a mechanism ASODN and125 IUdR - ASODN induce apoptosis on C6 cells. The regulation of Bax and Bel - xl by ASODN and125 IUdR - ASODN occurs at a point before transduction.
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