Study Of Experiment, Clinical And Evidence-based Medicine Of Childhood Acute Leukemia

Objective:Leukemia cell lines were usually used as research target in most childhood leukemia study, however, cell lines were lack of cell heterogeneity and easy to mutate, which affect of the experimental accuracy. To provide biologically and genetically similar cells as those in vivo, we explored the isolation and culture methods in childhood primary leukemia cells and proposed the optimal cultivation methods. Our study serves as important references in childhood leukemia study in vitro.Methods:The primary leukemia cells were collected from 25 children with acute leukemia. The primary leukemia cells were isolated by human lymphocyte separation medium. The cell morphology was observed by inverted microscope. The cell proliferation activities were analyzed by CCK-8 method under various cell densities and cell culture medium.Results:Cell morphology and CCK-8 shows that the primary leukemia cells have higher viability within 72 h. Cells at high density [(4-10)×106/ml]have higher cell proliferation than those at low density[(1-2)×106/ml]. The fetal bovine serum was better for primary cell proliferation than patients' own serum (P<0.05)Conclusions:The primary leukemic cell can be successfully isolated by human lymphocyte separation medium.6×106/ml planting density and 10% fetal bovine serum medium were the most optimal culture condition for primary leukemia cells. Objective:To investigate berberine-induced cell proliferation and apoptosis in childhood primary leukemia.Methods:The primary leukemic cells were isolated from bone marrow or peripheral blood by human lymphocyte separation medium in 27 children with newly diagnosed acute leukemia. These primary leukemic cells were divided into the following four groups:(1) the control group,(2) 25uM berberine group, (3)50uM berberine group,(4) 100 uM berberine group. Primary leukemic cell proliferation inhibition rate of berberine was obtained by cell counting kit-8 assay (CCK-8) at 24 h,48 h, and 72 h. The berberine-induced apoptosis of primary leukemia cells was analyzed by flow cytometry and optical microscopy at 24h.Results:After 24,48,72 h treatment of berberine, proliferation of primary leukemia cell were remarkably inhibited in a time-and dose-dependent manner, which was of significant differences (F=125.86,12.67 P<0.01). Cell morphology changes and typical apoptotsis were observed under optical microscope at 24h of berberine treatment. The berberine induced apoptotic rates at 25 uM,50 uM,100 uM were 28.51%,31.98%,48.60% at 24 h, which was detected by flow cytometry.Conclusion:Berberine induces apoptosis and inhibits the proliferation of primary leukemia cells in a time-and dose-dependent manner. Objective:To explore the relationship of MDM2, P53 gene expression and cell growth inhibition of berberine in childhood leukemic cell.Methods:Forty patients with newly diagnosed childhood leukemia were collected from Department of Pediatrics, Tongji Hospital as research objective, and leukemic cells were isolated by human lymphoid separation liquid, and leukemia cell growth inhibition rate of berberine was detected by CCK-8 methods. MDM2, P53 mRNA expression were detected by RT-PCR; MDM2, P53 protein expression were detected by immunohistochemistry in leukemia cells. The correlations between cell growth inhibition rate and the expression of MDM2mRNA, P53mRNA were investigated.Results:The higher expression of MDM2, P53 gene were observed in childhood leukemia. After 24 h of treatment, berberine induced cell growth inhibition in various concentration in MDM2 positive cells were significantly higher than that in MDM2-negative cells, while similar results was not found in P53 positive cells.Conclusion:Berberine may inhibit the growth of leukemia cells, which is correlated with the expression of MDM2, but not with the expression of P53.Objective:As the antigen expression in leukemic cells are not necessarily the same as normal hematopoietic cells. leukemic cells were often lost those normal antigens present those abnormal antigen. No specific antigen patterns were found in childhood leukemia cells. To solve the problem, we analyzed immunophenotype reaults which were detected by flow cytometry in childhood acute leukemia (AL) and compared with their clinical data.Methods:2-3 ml bone marrow or blood with heparin anticoagulation from children were used to test immunophenotype classification by flow cytometry. The first remissions were inducted by VDLP in ALL children, while in children with AML were DA or DAE or ARTA+DA schema.Results:1. According to characteristics of the immunophenotype in 195 AL children, the number of ALL was 139 cases (71.28%), AML were 42 cases (21.54%); AMLL 14 cases (7.18%). In 139 ALL children, B-ALL 103 cases (74.1%), T-ALL 24 cases (17.27%), T/B biphenotypic 12 cases (8.63%).In 42 children of AML, the incidence rate of M2 was the highest (40.78%), followed by M3(28.57%), M5 (16.67%), M4 (9.52%), M1 (4.76%). In this study, the consistent diagnosis rate with FAB classification was 92.82%.2. In 103 children of B-ALL, expression of the major antigen were CD19 (90.29%), CD10 (83.50%), CD20 (27.18%); In 24 children with T-ALL, expression of the major antigens were CD3 (79.17%), CD7 (66.67%), CD5 (33.33%). In 12 cases of B/T-ALL, the expression of T-lymphoid antigens were CD7 (50%), CD5 (41.67%), while the B lymphoid antigens were CD19 (50%), CD10 (33.33%). In 139 children of ALL, myeloid antigen expression (My+ALL) was 32 cases(23.02%), and the main expression antigen were CD 13, CD33, CD 14, MPO and CD 15 was not expressed; In 139 children of ALL, expression of CD34 in 31 cases with ALL, followed by T/B-ALL, B-ALL, T-ALL. CD34-positive expression (15.62%) in My+ALL was significantly lower than that of My-ALL (24.30%). In 82 cases of HLA-DR expression, the sequence was B-ALL, T/ B-ALL, T-ALL.3. In 42 children of AML, the major antigen were CD13 (64.29%), CD33 (59.52%), MPO (42.86%), CD15 (33.33%). For expression of CD13, CD33, MPO in all of ALL, whereas no expression of CD 15, the myeloid lineage specificity of CD 15 was superior to that of CD13, CD33, MPO. In M1, CD13, CD117, CD15 were mainly expressed; M2 was CD13, CD33, MPO. M3 was CD13, CD33, MPO. M4 was CD13, CD33, CD15. M5 was CD 14. In 13 children of AML were associated with lymphoid antigen expression of CD 19, CD7, CD3. In 42 cases of AML, expression of CD34 was 15 cases, which were the most common in M1, M2. Ly+ AML in the CD34-positive expression of Ly-AML was not different from that of Ly+ AML.And expression of HLA-DR was 25 cases, which were the most common in M1, M2, M4, M5.4. In 14 cases of AMLL, the main expression of B lineage antigens were CD 19, CD 10, CD22;the main expression of T antigens were CD7, CD3, CD5; myeloid antigen expression were CD13, CD14, CD33, CD15. Nine cases received chemotherapy in 14 cases of AMLL,6 patients achieved complete remission (CR) (66.67%) after first chemotherapy, which was lower than that of ALL and AML. In 14 cases of AML,2 cases expressed CD34; 7 cases expressed HLA-DR.5. The number of sex, bleeding in My+ALL group and My-ALL group were significantly different (x2=4.20,20.03 P<0.05), while the other indexes wasnot different significantly (P>0.05). CR rate (56.25%) of My+ALL group and My-ALL group (57.94%) have more differences (x2= 6.18 P<0.05). The number of cases of liver large in Ly+AML group compared with in the Ly-AML are different (x2= 6.48 P<0.05), while the other indexes did not differ significantly (P>0.05). CR rate (31.03%) in Ly+AML group and Ly-ALL group was not also different (x2= 0.15 P> 0.05).Conclusions:1. Immunetyping helps to differentiate the sources of leukemic cells, and helps to get the correct typing in most cases of childhood leukemia.2. To identify ALL and AML, the myeloid lineage specificity of CD 15 is superior to that of CD13, CD33 and MPO.3. HLA-DR negative could be of certain value in the diagnosis of AML M3. 4. In My+ALL, the major expression of myeloid antigens was CD13 and CD33. And in Ly+AML, the major expression of lymphoid expression are CD 19, CD3. And the myeloid expression and the lymphoid expression are not related with their prognosis.5. AMLL is a unique acute leukemia expresses both myeloid and lymphoid antigen, which has worse prognosis than either AML or ALL.Objective:To evaluate of the correlations of methylenetetrahydrofolate reductase gene (MTHFR) polymorphisms and childhood acute leukemia (AL).Methods:MTHFR gene C677T and A1298C genotype frequencies in children with AL and control group were used as statistical quantity. Relevant literatures were extensively searched and screened according to the type of leukemia and population subgroup. These results of research data were extracted and the gene loci were tested for heterogeneity. Various research datas consolidation, combined OR values and their 95% CI were statistically tested by RevMan 4.2; Funnel plots was used for the bias analysis of published literature; sample size alteration were used to analyze the results sensitivity in order to evaluate the stability of Meta analysis.Results:Seventeen literatures were searched for Meta analysis. Except for MTHFR gene 677 loci genotype TT had no heterogeneity (P>0.05), the other MTHFR gene 677 loci genotypes all had heterogeneity. the frequency distribution of MTHFR gene 677 loci genotype TT in AL group was significantly higher than that in control group, and it's combined OR values and 95% CI were 0.81(0.69-0.97)(P<0.05). MTHFR gene 677 loci genotype CT, TT+CT of the frequency in AL group compared with that in control group had no significant difference (P>0.05). Compared to control group, there were no significant difference of frequency distribution of MTHFR gene 1298 loci AC, CC, AC+ CC in pediatric AL group. Combined OR values and 95%CI were 1.11 (0.83-1.49),1.15 (0.93-1.42),1.18 (0.95-1.46) (P>0.05).Subgroup analysis:1.the results suggest that MTHFR 677 loci CT, CT+TT and 1298 loci AC, CC, AC+CC were not correlated with ALL; In ANLL group, analysis results indicated that MTHFR gene polymorphism in ANLL group has nothing to do with control group.2. The analysis results suggested that MTHFR1298 loci CC, AC+CC was related with childhood AL in Asian populations (Z=2.05,2.01,P<0.05) while in Europe population, MTHFR 677 loci CT, CT+TT was related with childhood AL (Z=2.91,2.08,P <0.05).In other population, MTHFR677,1298 was not related with childhood AL. The bias and sensitivity analysis confirmed that the Meta analysis of the publicated literatures in this article werestable and reliable.Conclusions:MTHFR gene 677 loci TT is closely related with pediatric AL; And MTHFR gene 677 CT, CT+TT are also contributed to the occurrence of AL in European children. MTHFR gene 1298 loci CC is the protective factors of AL in European children, and MTHFR gene 1298 loci AC+CC can led to the occurrence of AL in the Asian children. Objective:To investigate the relationship of cytochrome P4501A1 (CYP1 A1) MspⅠgene polymorphism and childhood acute leukemia (AL).Methods:The CYP1 A1 MspⅠgene frequencies were used as statistical quantity in AL and control group. Relevant literatures were extensively searched and screened according to the type of leukemia and the subgroup of population. These results of research data were extracted and the gene loci were tested for heterogeneity. Various research datas consolidation, combined OR values and their 95% CI were statistically tested by RevMan 4.2; Funnel plots was used for the bias analysis of published literature.Results:Six related literatures were found to meet the requirements of screen,including 837 cases in AL group and 1252 cases in control group. According to heterogeneity test result, there was no significant difference (x2=11.68, P>0.05)in homozygous types, but in two others types, there were significant difference (x2= 16.38,14.77, P all<0.05). For the wild CYP1A1 MspⅠhomozygous for the reference group, the combined OR of heterozygous mutation, homozygous, heterozygous + homozygous mutation in AL and control groups were 1.18(0.94-1.49),0.96(0.31-3.00),1.10(0.77-1.57). Subgroup analysis: combined analysis(Z value) of CYP1A1 MspⅠhomozygous, heterozygous + homozygous in acute lymphoblastic leukemia (ALL) and control group were 0.10,0.76 respectively, P all > 0.05; Z value of CYP1A1 MspⅠhomozygous, heterozygous + homozygous in non-acute lymphoblastic leukemia (ANLL) and control group were 0.74,0.75, P all> 0.05.Conclusion:There is no correlation between CYP1 A1 MspⅠgene polymorphism and the susceptibility of pediatric AL. Objective:To explore the relationship between maternal alcohol consumption during pregnancy and childhood acute leukemia (AL), which provides a basis for the prevention of childhood AL.Methods:These relevant literatures of maternal alcohol consumption during pregnancy were comprehensively searched and screened in AL group and control group, and according to the type of leukemic subgroup, Meta analysis was used. These results of research data of maternal alcohol consumption during pregnancy were tested for heterogeneity. The combined OR values and their 95%CI were statistically tested by Rev Man 4.2; and the funnel plots was used for the bias analysis of all the published literatures.Results:Ten pieces of related literatures meeted the requirements of data screen. The number of AL cases and control group were 4593 and 6157 respectively. According to heterogeneity test result, there was difference (x2= 16.26, P<0.05), and combined OR values and 95% CI in AL and control group by random effects model were 1.00 (0.90-1.11), and the total effect Z value was 0.02, P>0.05. Subgroup analysis:combined OR values and 95% CI in childhood acute lymphoblastic leukemia (ALL) and control group were 0.92 (0.84-1.00), and the total effect (Z value) of the results was 1.92, P= 0.05. Combined OR values and 95% CI in childhood acute nonlymphoblastic leukemia (ANLL) and control group were 0.82(0.61-1.11), and the total effect (Z value) of the results was 1.30, P> 0.05.Conclusion:Maternal alcohol consumption during pregnancy is a risk factor in childhood ALL, but not in ANLL.