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Objective: With the development of medical science, a serious threat to the morbidity and mortality of children with acute infectious diseases has dropped significantly, cancer has become one of the major diseases that threaten children's lives, including hematologic malignancies majority, and the incidence The rate increased year by year trend. The cause is not clear, early detection is limited due to treatment has become a major killer of children. Chemotherapy is still the primary means of treatment, long-term survival rates have also been increased in recent years due to the continuous improvement of chemotherapy, but there are still some children can not ease or recurrence. Therefore explore the cause, has a very important significance to improve the prognosis and quality of life of children seeking a more effective prevention and treatment measures. DNA methylation is an important part of the epigenetic modifications, methylation abnormalities occur in the blood tumor development plays an important role. Hematologic malignancies abnormal methylation of specific genes as molecular markers for observation in the clinical efficacy and therapeutic targets. ZO-1 (zonula occludens) gene expression is a tight junction protein, membrane-associated guanylate kinase family members, an essential protein for maintaining cell structural integrity and normal polarity connection and signal transduction in cells play an important role, and participate in the regulation of cell proliferation and differentiation. In recent years, scholars applied the RLGS technology first found in murine leukemia cells showed high methylation status of ZO-1 gene promoter region, resulting in the inhibition of the expression of the gene, suggesting that ZO-1 may be a leukemia-associated genes. But no related research reports of the gene in the tumor of the blood of children. ZO-1 gene methylation in the experimental bone marrow by detecting blood tumor in children, in order to investigate its occurrence in children's blood tumor development, aimed at finding a new children's blood tumor molecular markers to provide early diagnosis for clinical determine the efficacy, prognostic analysis of the theoretical and experimental basis of minimal residual disease detection and targeted therapy. 's lymphoma (NHL) and neuroblastoma cell tumors in children before treatment, the pre-chemotherapy, chemotherapy medium-term chemotherapy late clinical remission and recurrence bone marrow samples as a research group, non-blood tumor bone marrow of children as a control group. By methylation-specific polymerase chain reaction (MS-PCR) methods were used to detect the bone marrow of children ZO-1 gene methylation status of the study group and the control group, reverse transcriptase polymerase chain reaction (RT-PCR) method integration value of the fluorescence intensity detected in the study group and the control group ZO-1 gene mRNA expression, with the semi-quantitative analysis of the fluorescence intensity of the gel imaging system using ZO-1 gene by the MS-PCR amplification after methylation article ( IOD values), the the observation study group during chemotherapy IOD of dynamic change, with the number of malignant cells in the peripheral blood leukocyte count and bone marrow correlation analysis. SPSS13.0 statistical software for statistical analysis. All measurement data were presented as mean ± standard deviation ((?) ± S) and t-test, and continuous observation data using repeated measures analysis of variance, count data using the chi-square test or Fisher s exact test, the correlation between the two factors Sexually linear correlation analysis. P lt; 0.05 considered statistically significant. Results: 1 study group compared with the control group: two groups of age, gender had no significant difference (P gt; 0.05). 2 MS-PCR test results: The study group before treatment of 52 cases of ZO-1 gene methylation bands in 47 cases, the positive rate was 90.4% (47/52 patients) in the control group of 20 cases, no case of The two groups, the difference was significant (P lt; 0.001). Lymphoma in children was 92.6% (25/27 cases) [NHL was 95.2% (20/21 cases), HD 83.3% (5/6)]; leukemia was 86.7% (13/15 cases), [ALL 100% (5/5 cases), 75% (6/8) in AML, CML is 100% (2/2)]; neuroblastoma was 90% (9/10). Positive rate between the different sub-type of the disease and the same disease was no significant difference (P gt; 0.05). Dynamic observational study group, 39 patients (11 patients abandon the treatment in the course of chemotherapy, 2 deaths, a total of 39 cases of chemotherapy continuous observation), before treatment Methylation was 92.3% (36/39 patients), chemotherapy pre was 84.6% (33/39 patients), chemotherapy mid-to 76.9% (30/39 patients), 69.2% (27/39 cases) chemotherapy late clinical remission in 13 cases, five cases may be detectable in 38.5% (5/13). Before treatment with chemotherapy early, mid-There was no significant difference (P gt; 0.05), the difference was significant (P lt; 0.05) compared with chemotherapy late. With the progress of chemotherapy, bone marrow of children gradually ease, and can be seen fully methylated strip tape reduce transitional partially methylated article brought to disappear. Course of chemotherapy in two cases of recurrence, ZO-1 gene methylation by partially methylated bar band into a fully methylated bands. The dynamic observation group, 39 patients, lymphoma and 21 cases, treatment before the ZO-1 gene methylation in 20 cases, the positive rate of 95.2% (20/21), the the chemotherapy early 17 cases, the positive rate of 80.95% (17/21 cases ) chemotherapy, the interim 16 cases, the positive rate was 76.2% (16/21 patients), the the chemotherapy late 14 cases, the positive rate of 66.7% (14/21 patients), clinical remission in 9 cases, 2 cases detected, the positive rate of 22.2% (2/9). Before treatment with chemotherapy before interim difference no statistical significant (P gt; 0.05), the difference was significant (P lt; 0.05) compared with chemotherapy late. One cases of NHL in children with relapsed methylation status by partial methylation into a fully methylated; the leukemia 11 cases before treatment ZO-1 gene methylation in 10 cases, the positive rate of 90.9% (10/11 cases) , 10 cases chemotherapy preliminary, positive rate of 90.9% (10/11 patients), chemotherapy in the mid-nine cases, the positive rate of 81.8% (9/11 patients), chemotherapy late eight cases, the positive rate of 72.7% (8/11 patients), clinical ease the three cases, two cases detected in 66.7% (2/3 cases). Before treatment with chemotherapy before, in the late difference there was no significant sex (P gt; 0.05). Patients with acute promyelocytic leukemia in children with recurrent methylation status of partially methylated into fully methylated; seven cases of neuroblastoma, treatment before the ZO-1 gene methylation six cases, the positive rate of 85.7% (6/7), chemotherapy pre-six cases, the positive rate of 85.7% (6/7 patients), chemotherapy the interim five cases, the positive rate of 71.4% (5/7 patients), chemotherapy the late five cases, the positive rate of 71.4% (5/7), clinical remission in 1 case, may be detectable in the positive rate of 100% (1/1 cases). Before treatment with chemotherapy before, in the late methylation difference was no significant sex (P gt; 0.05). Achieve clinical remission ZO-1 gene methylation detection higher neuroblastoma and leukemia. 3 RT-PCR test results: 52 cases of the study group before treatment to extract total RNA, five cases seen ZO-1 gene expression, the positive rate was 9.6% (5/52 cases); the control group were expressed. The two groups have a significant difference (P lt; 0.001), indicating that the study group therapy before ZO-1 gene silencing. Dynamic observation group, 39 patients ZO-1 gene expression positive rate before treatment was 7.7% (3/39 patients), chemotherapy pre-15.4% (6/39 cases), 23.1% (9/39) mid-chemotherapy, chemotherapy late was 30.8% (12/39 cases), clinical remission in 13 cases, 8 cases of ZO-1 gene expression, the positive rate was 61.5% (8/13 cases). Before treatment with chemotherapy preliminary, interim difference was no significant sex (P gt; 0.05), the difference was significant (P lt; 0.05) compared with chemotherapy late. 4 IOD value of test results: The study group dynamic observation of 39 cases before treatment mean IOD values ??20,715.87 ± 10,854.81799, the chemotherapy pre 12,183.64 ± 7,093.12756, chemotherapy mid 9,703.744 ± 6,938.87355 chemotherapy late to 5,669.667 ± 4,761.52117, before chemotherapy, post respectively, compared with before treatment, with a significant difference (P lt; 0.05), before chemotherapy, between late comparison, the difference was significant (P lt; 0.001). After shows that, after chemotherapy, ZO-1 gene methylation level decreased. 5 study groups before treatment IOD value analysis: 52 cases in men and women do not IOD values ??showed no significant difference (P gt; 0.05). Age IOD values ??linear relationship (P gt; 0.05). The total number of peripheral blood leukocytes IOD value of the non-linear relationship (P gt; 0.05). Malignant tumor cells and the IOD value of linear correlation (P lt; 0.001) in the bone marrow, and a positive correlation. Illustrate the degree of methylation with gender, age, independent of the WBC, is closely related to the number of malignant cells. Conclusion: 1 ZO-1 gene in children's blood tumor methylation status showed high specificity in terms of lymphoma, leukemia, neuroblastoma can be detected with the children's blood tumor occurrence is closely related to the development and prognosis is poor prognostic factor. 2 ZO-1 gene methylation status lead to gene silencing of ZO-1 gene may play a role in the tumor suppressor gene. 3 ZO-1 gene methylation status, gender, age, WBC regardless of the number, the number of malignant cells and bone marrow. 4 with the conduct of the chemotherapy, ZO-1 gene methylation status before and after treatment with significant changes in the degree of methylation decreased clinical remission in children is still part can be detected, it is likely to become the new molecular markers, provide early diagnosis for clinical, therapeutic efficacy, prognosis analysis, the detection of minimal residual disease indicators.
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