Effection On The Biological Characteristics And Inhibitor Of Dna Binding 4 Of Raji Cell By Cdr

【Background and objective】Non-Hodgkin's lymphoma(NHL) which is about 80%-90%of the total number of lymphoma,is the most common type of lymphoma.In recent years the NHL epidemiological studies have shown that its threat to human health is becoming increasingly serious,and its world wide growth is wide spread.At present,the NHL study of the treatment strategy focuses on multidisciplinary treatment,which requires the patient to carry out accurate early prevention,diagnosis,staging,evaluation,to determine prognosis.The tumor markers play an important role in these areas,and is used widely in the clinical work.The cause of malignant lymphoma and pathogenesis is not clear so far,most of the current research that the cause of malignant lymphoma may be related to the following factors:bacteria and viruses,immune defects,and occupational exposure to environmental factors,diet,genetic factors,life habits,blood transfusion and iatrogenic outside of estrogen,and so on.In recent years,as some scholars put forward the pathogenesis of lymphoma of the human body is infected with some viruses(such as EBV),the virus penetrated the normal human cells can be caused by spontaneous mutation or gene fusion can also be caused by the body's antigen receptor gene variant and defective immune function in the right environment factors will be induced tumors.But it seems that the final results of all of these factors is that it is the gene lead to structural changes or changes in gene expression in order to promote tumor occurrence and development.The occurrence of tumors is a complex multi-step,in connection with cell proliferation,differentiation,integration of mutation of the genome of the proto-oncogenes,tumor suppressor genes and other important cellular genes.In recent years a large number of studies have shown that the promoter region of a number of DNA loci of abnormal CpG methylation and the occurrence and development of a variety of tumors are closely related.Inhibitor of DNA binding 4(Id4) is a kind of suppressor of DNA binding,it is also called differentiation inhibitory factor,and it widely exists in mammalian cells in the nuclear transcription factor negative regulatory protein,contains four Id family members:Id1,Id2,Id3 and Id4,it is a bHLH transcription factor protein of the dominant negative regulator,which could hinder the adoption of the latter with DNA binding,thereby inhibiting cell division, resulting in tumorigenesis.At present,some studies have shown them with tumor cell proliferation,differentiation,apoptosis and invasion is closely related to biological behavior.In these four genes,the functions of the former three more studies,and research on gene Id4 relatively small.Expression studies have shown that Id proteins play critical roles in early embryonic development.They are also involved in angiogenesis,lymphocyte development,cell cycle control,and cellular senescence .The involvement of Id proteins in neoplastic processes has been suggested. Increased Id1 and Id2 expression has been reported in various tumor types,including adenocarcinomas arising from the colon and pancreas.Transgene expression of Id1 and Id2 in mice has resulted in tumor formation in the intestinal epithelium and lymphoid organs,respectively.Expression of Id3 has been more variable;studies report both up-regulation and down-regulation in different tumor types.In force in the international community YU LI found for the first time in 2004:Id4 gene in many tumor cell lines were methylation and gene silencing occurs,resulting in the suppression of gene expression,thereby introducing the gene is likely to be a new tumor suppressor gene,and maybe is closely related to the development and the prognosis of lymphoma.Also there was reported at a foreign literature that Id4 gene methylation and its protein expression change and the occurrence and development of colon cancer was a certain relationship,In normal tissues gene methylation had the low incidence and its protein expression in the cytoplasm was higher;the outcome of cancer tissue was opposite,another study showed,compared with normal tissue,a variety of malignant lymphoma in a gene hypermethylation Id4 change.These studies showed that both at home and abroad Id4 expression of genes and their proteins in a variety of cell development,differentiation plays an important role,not only involved in cell cycle regulation,but also involved in the regulation of tumor cell proliferation, differentiation,invasion,several key processes of cell biology and may be associated with tumor progression and prognosis.Id4 protein and Id1,Id2,Id3 comparison, despite the similar structure,but recent studies have shown that there is an obvious expression differences,studies have shown that Id molecules in human tumor tissue and in vitro cultured tumor cells showed a trend of high expression,and Id4 in seminoma in abnormally high expression;However,recent studies also show that Id4 protein reduced the expression in colon cancer and the majority of gastric cancer cell lines.DNA methylation refers to the transfer of DNA methylation enzymes(DNA methyltransferases,DNMT) role,using S-adenosyl-L-methionine(S-adenosyl methionine,SAM) as a donor of activated DNA to introduce a subgrade mission chain, in the genomic CpG dinucleotide of cytosine 5' carbon-bit combination of a covalent methyl group to form 5 - methyl cytosine(5-methylcytosine,m5 C).Mammalian DNA in 70%～80%CG sequences in the cytosine resIdues were methylated 5, referred to as methylation sites of the CpG.Caused by methylation of cytosine 5' position of the modification is the only known naturally occurring genome of covalent modification.DNA methylation pattern and gene expression was negatively correlated.This gene covalent modification of bases in the regulation of gene transcription,X chromosome inactivation,gene imprinting,cell differentiation and tumorigenesis have played an important role,as well as some scientists call the "fifth base".DNA demethylation agents mainly methyl transferase inhibitors,the role of the mechanism and cytotoxicity induced by demethylation.Through integration into the DNA,inhibiting DNA methyltransferase,in dividing cells during DNA replication,so that can not be transferred to cytosine methylation,with the lowering of the ability of DNA to accept methyl,which carried out with the split,the degree of methylation of DNA in the progeny cells gradually get lower.So that the tumor suppressor gene whose transcripttion inactivate because of its CpG island methylation restore its function to reverse the biological activity of tumor cells.With immunocytochemical SP,flow cytometry,methylation-specific polymerase chain reaction(MS-PCR) technology our experiment mainly focused on the effection of demethylation drug 5-aza-2'- deoxy-cytosine(CdR)on Burkitt lymphoma cell line-Raji cell apoptosis,cell cycle,differentiation inhibitory factor 4 gene methylation and its protein expression.Then we will find the signifation of Id4 on Burkitt's lymphoma.【Materials and methods】1.The main medicine and reagent:5-aza-2'- deoxy-cytosine dry powder:Id4 rabbit polyclonal antibody:One step modified base kit;Takara Ex Taq enmyze.2.Cell line and culturing:human Burkitt's lymphoma cell line Raji cells purchased from the CAS Shanghai Institute of Cell Biology,2×10⁵/ml cells were inoculated with new RPMI 1640 culture medium(containing 10%fetal calf serum, 100 U/ml penicillin and 100 mg / ml streptomycin) in the culture(37℃,5%CO₂).3.According to related literature by 0.1μmol / L 0.5μmol / L,1.0μmol / L,2.5μmol / L,5.0μmol / L,respectively,after the daily check dosing treated group and control group cells we count the total number of living cells for seven days in light microscope and make a growth curve.4.According to the growth curve,in this experiment were chosen 0.5μmol / L, 5.0μmol / L concentrations of CdR deal with Burkitt lymphoma cells 24h,48h,72h. And we chose the culture medium control group as the control group.Then with immunocytochemical SP method,flow cytometry,methylation-specific polymerase chain reaction(MS-PCR) technology,we test the expression of Id4 protein,the cell apoptosis and cell cycle,the Id4 gene methylation status of Raji cells.5.Using SPSS 13.0 software for statistical analysis.Measurement data with mean±standard deviation((?)±s) that the analysis of variance of experimental data for statistical analysis,the test to a=0.05 for standard test of significance.【Results】1.Results of determination of growth curve:Different drug concentrations under a certain extent with the dose of and the role of increased time on the Raji cells,the more obvious.2.Immunocytochemical Results:Non-Hodgkin's lymphoma cells,Id4 protein expression was low,only expressed in the cytoplasm;to 0.5μmol / L,5.0μmol / L of two concentrations of drug demethylation CdR role 24h,48h,72h after the cytoplasm Id4 protein than the control group(0.0μmol / L) increased expression,the nucleus is also the expression of 5.0μmol/LCdR role and to express the highest levels 72h,three time periods as the concentration increased,ID4 protein expression increased,the time period the experimental group compared to each other,the differences were statistically significant(P＜0.05),3 concentration with time,Id4 protein expression increased the dose of the experimental group compared to each other,the differences were significance(P＜0.05),but there was no difference among the dealt group (P＞0.05).3.Flow cytometry results:(1) Detected by flow cytometry at different times with different concentrations in treatment group and normal control group showed apoptosis situation,with the role and the role of higher concentration of time,Raji cell apoptosis rate increased.CdR role 24h,48h,72h after the different concentrations between the apoptosis rate by ANOVA,there was a significant difference(P＜0.05),the concentration of different time periods compared(P＜0.05),the difference was significant.(2) Detected by flow cytometry at different times with different concentrations in treatment group and normal control group,cell cycle situation. Status display,with the role and the role of higher concentration of time,cells in S phase increased in proportion,G0 / G1 and G2 / M decline in the proportion(P＜0.05). 4.MS-PCR results:CdR role without Raji cells were Id4 gene methylation status, low drug concentration group 24h group was completely methylated,48h,72h group and the high drug concentration group 24h,48 h group was part of methylation,high drug concentrations group 72h totally non-methylation.【Conclutions】1.Id4 protein expression is low in Raji cells,CAR can increase Id4 protein expression.2.CdR can significantly inhibit Raji cell proliferation,increase its apoptosis induced by effective dose of a time-replied;CdR effectively induces cell cycle arrest.3.In Raji cells without CdR,Id4 gene methylation is completely changed.CdR effectively induces Id4 gene demethylation.4.The mechanism of demethylation drug CdR to Raji cell apoptosis may be raised to achieve Id4 gene expression.