| Objective:With the development of medicine, the morbidity and mortality of acute infectious diseases what threaten children seriously have decreased greatly. Malignant tumor has become one of the principal diseases which has threatened the children's lives.The majority of the malignant tumor are the neoplastic hematologic disorder, whose the attack rate has increased year by year and precisely etiopathogenisis is still unknown.So it is limited with early discovery and etiological treatment. At present, the neoplastic hematologic disorder has become the chief killer of the children. Traditional chemotherapy is still the main method of therapy. In recent years, due to the continuous improvement of chemotherapy, the long-term survival rates have also been increased, but there are still some children can not be relieved or recurrence. Therefore, it is significant that searching for etiological factor and exploring a more effective prevention and cure measure to improve the patient's prognosis and quality of life. DNA methylation is the important section of epigenetics and plays a substaintial part in neoplastic hematologic disorder's genesis and development. The abnormal DNA methylation of some gene can be taken as biomarker of neoplastic hematologic disorder to be used in clinical.ZO-1(zona occludens 1)is a tight junction protein,it belongs to membrane-associated guanylate kinase homologs(MAGUKs).ZO-1 is very essential for maintaining the integrity and polarity of cells and plays an important role in cell junction and signal conduction. Recently some scholars firstly found the promoter region of ZO-1 gene was methylated in mouse leukemia with RLGS,indicating it might be a leukemia correlative gene. There was still no similar report in the pediatric neoplastic hematologic disorder. This dissertation, by detecting methylated degree of ZO-1 gene in the neoplastic hematologic disorder patients's bone marrow, investigates the function of the methylated degree of ZO-1gene in the development mechanism of pediatric neoplastic hematologic disorder, then searchs a new pediatric neoplastic hematologic disorder, molecule maker, which provides effective theoretical and experimental basis for early diagnosis, curative effect evaluation,prognosis appraisal,minimal residual disease(MRD)monitoring and molecular therapy target.Methods:We adopted bone marrow samples of children whose were diagnosed with leukemia[Acute lymphoblastic leukemia(ALL),acute myeloid leukemia (AML),chronic myeloid leukemia (CML)],lymphoma [Hodgkin's disease(HD),non-Hodgkin's lymphoma(NHL)]and neuroblastoma, that were collected before treatment, early stage of chemotherapy, intermediate stage of chemotherapy, advanced stage of chemotherapy, clinical remission and relapse as a study group, and used Non-neoplastic hematologic disorder children's as a control group.Using the method of Methylation-Specific Polymerase Chain Reaction (MS-PCR) to detect the ZO-1 gene methylation status in study group and control group, Reverse Transcription-Polymerase Chain Reaction (RT-PCR) to detect the expression of ZO-1 gene mRNA in study group and control group.At the same time, the bands of ZO-1 methylation DNA were amplified by the MS-PCR, then semi-quantitatively analysed with gel imaging system to obtain integrated optical density (IOD values).Observed the dynamic changes of IOD values in research group, and analyzed the relation of IOD values with the number of white blood cells in peripheral blood and the number of malignant cells in bone marrow. Used SPSS 13.0 statistical software for statistical analysis.All measurement data showed by mean±standard deviation(X±S), analysed with t-est, the data which were continuously observed used repeated-measures analysis of variance, count data analysed with chi-square test or Fisher s exact propability, the correlation between two factors analysed with linear correlation analysis. There is a obvious statistically difference if P value less tan 0.05.Results: 1 Study group compared with control group,there was no significant difference in age and gender (P>0.05).2 MS-PCR showed:There were 52 examples in Study group before treatment,47 examples appeared ZO-1 gene methylation bands before treatment, the positive rate was 90.4% (47/52examples),while the Control group had no methylation bands(n=20).There was statistical significance(P <0.001).The positive rate of Lymphoma patients was 92.6%(25/27 examples).[NHL was 95.2%(20/21 examples),HD was 83.3%(5/6 examples)].The positive rate of leukemia was 86.7%(13/15 examples), [ALL was 100%(5/5 examples),AML was 75% (6/8 examples),CML was 100%(2/2 examples)].The positive rate of neuroblastoma was 90% (9/10 examples).It suggested that there was no difference among these different kinds of tumors or among those different types of the same tumor(P>0.05). Dynamicly Observing Study Group,(during chemotherapy, there were 11 patients gave up treatment,2 patients died, so there were 39 patients could be observed continuously)The Methylation-positive rate before treatment was 92.3% (36/39examples), early stage of chemotherapy was 84.6% (33/39examples), intermediate stage of chemotherapy was 76.9% (30/39examples), advanced stage of chemotherapy was 69.2% (27/39examples), clinical remission was 38.5% (5/13examples).There was no significant difference between the Pre-treatment and early stage of chemotherapy or intermediate stage of chemotherapy, while compared with advanced stage of chemotherapy, there was significant difference. With the progress of chemotherapy, the bone marrow of patients gradually released, and the positive rate of complete methylation reduced gradually, while positive rate of transitional partial methylation increased gradually until disappeared. In the course of Chemotherapy, two patients recurrented, the ZO-1 gene becomed complete methylation status from partial methylation. Study group (39 cases) included 21 Lymphoma, there were 20 Lymphoma examples occurred ZO-1 gene methylation before treatment, positive rate of methylation was 95.2% (20/21 examples), it was 80.95% (17/21 examples) in early stage of chemotherapy,76.2%(16/21 examples) in intermediate stage of chemotherapy,66.7% (14/21 examples) in advanced stage of chemotherapy, 22.2% (2/9examples) in clinical remission. There was no significant difference between the Pre-treatment and early stage of chemotherapy or intermediate stage of chemotherapy(P>0.05).But there was significant difference compared with advanced stage of chemotherapy(P<0.05).One NHL patient recurrented, the ZO-1 gene becomed complete methylation status from partial methylation.11 Leukemia,the positive rate of methylation was 90.9% (10/11 examples) before treatment, it was 90.9%(10/11 examples) in early stage of chemotherapy,81.8% (9/11 examples)in intermediate stage of chemotherapy,72.7%(8/11 examples)in advanced stage of chemotherapy, 66.7% (2/3examples) in clinical remission. There were no significant differences between before treatment and each period after chemotherapy. One M3 patient recurrented, the ZO-1 gene becomed complete methylation status from partial methylation.7 Neuroblastoma,the positive rate of methylation in neuroblastoma was 85.7% (6/7examples) before treatment, it was 85.7% (6/7examples) in early stage of chemotherapy,71.4% (5/7examples) in intermediate stage of chemotherapy,71.4% (5/7examples) in advanced stage of chemotherapy,100%(1/1 examples) in clinical remission. There were no significant differences between before treatment and each period after chemotherapy. The ZO-1 gene methylation of neuroblastoma and leukemia were higher than lymphoma in clinical remission.3 RT-PCR showed:52 examples of study group were extracted RNA before treatment, there were five examples expressed the ZO-1 gene, the positive rate was 9.6%(5/52 examples), the control group were all expressed. It suggested that the result had statistical significance, and showed that the ZO-1 gene unexpressed in Study group before treatment. Dynamicly observed the positive rate of ZO-1 genes expression, it was 7.7% before treatment(3/39examples),15.4%(6/39examples) in early stage of chemotherapy,23.1%(9/39 examples) in intermediate stage of chemotherapy, 30.8%(12/39examples)in advanced stage of chemotherapy,61.5% in clinical remission(8/13examples).There was no significant difference between the Pre-treatment and early stage of chemotherapy or intermediate stage of chemotherapy(P>0.05).But there was significant difference compared with advanced stage of chemotherapy(P<0.05).4 The IOD showed:The IOD mean value of before treatment is (20,715.87±10,854.81799),and that of the early stage of chemotherapy is (12,183.64±7,093.12756),and that of the intermediate stage of chemotherapy is(9,703.744±6,938.87355),and that of the advanced stage of chemotherapy is (5,669.667±4,761.52117).Compared with before treatment, it was found that all the results of the three periods had statistical significance (P<0.05). There is aslo a significant difference among the three periods after chemotherapy (P<0.001).It showed the degree of methylation had become decreased.5 The correlation analysis of IOD values in Study group:52 cases in Study group, there was no statistical significance of IOD between male and female. There was no correlation between age and IOD values (P= 0.620> 0.05).There was no correlation between the total number of peripheral white blood cells and IOD values(P=0.575>0.05).There was correlation between the number of malignant cells in bone marrow and the IOD values (P=0.000<0.05),and it was a positive correlation. It suggested that the degree of methylation was no correlation with gender, age and the total number of peripheral white blood cells, but has correlation with the malignant cells.Conclusions:1 ZO-1 gene shows specific hyper-methylation status in children hematological malignancy, which can be detected in lymphoma, leukemia and neuroblastoma. ZO-1 gene is closely related to the development and turnover of the children hematological malignancy, which is a factor of unfavourable prognosis.2 The hypermethylation status of ZO-1 gene leads to silent expression. ZO-1 gene may play a role as a tumor-suppressor gene.3 There is no correlation between methylation status of ZO-1 gene and gender, age or white blood cell.But it relates to the quantity of malignant cells in bone marrow.4 There is a big change of ZO-1 gene methylation status before and after treatment. The positive rate of methylation gradually reduces with the progress of chemotherapy. But ZO-1 gene methylation status are still detected in part of patients who are in clinical remission. So it may be a new molecular marker, which may provide an index for early clinical diagnosis, determining efficacy, prognosis analysis and ditection of minimal residual disease.
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