BACKGROUND:Studies concerning bone marrow mesenchymal stem cells (BMSCs) cultured by umbilical cord serum in vitro were hot topic to avoid the heterogenous serum rejection during BMSC culture and improve culture efficiency of BMSCs.OBJECTIVE:To compare biological feature of BMSCs by the umbilical cord serum and adult autoserum in vitro.DESIGN,TIME AND SETTING:The opening experiment was performed at the Laboratory of Cell Biology,Xiangya Second Hospital,Central South University,China from October 2006 to June 2008.MATERIALS:Sixteen bone marrow samples were provided by Xiangya Second Hospital,Central South University and Xiangtan Central Hospital of Hunan Province.METHODS:BMSCs were obtained from 16 fresh adult bone marrow by gradient centrifugation with Ficoll Paque,cultured with DMEM/F12 with 10% umbilical cord blood serum and 10% adult autoserum.The fourth passage was used in this study.The surface antigens of BMSCs were detected by flow cytometry.BMSCs could differentiate into ostenblasts and adipocytes under culture in conditioned medium for osteogeneais and adipogenesis and the differentiated BMSCs were identified by morphological observation,immunophenotype and immunochemical staining.MAIN OUTCOME MEASURES:The following parameters were measured:morphological observation;cell cycle and cell count;identification of surface antigen;observation of osteoblasts and adipocytea induced from BMSCs by immunochemical staining.RESULTS:The quantity and velocity of BMSCs in umbilical curd blood serum was better than those in adult autoserum (P ＜ 0.05)Only few cells were proliferating in both medium,BMSCs in S phase in umbilical cord blood serum was more than those in adult autoserum (P ＜ 0.05).The positive rate of CD29,CD73 and CD105 on BMSCs in umbilical cord serum (above 90%) was higher than those in adult autoserum (P ＜ 0.05),and the positive rates of CD31,CD34 and CD45 were lower than 2%,and the positive rate of CD31 was lower than those in adult autoserum (P ＜ 0.05).The positive rate of BMSCs differentiated into osteoblasts and adipocytes in umbilical cord blood serum was also higher than these in adult autoserum (P ＜ 0.05).CONCLUSION:Purity and differentiated potency of BMSCs in umbilical cord blood serum are better than those in the adult autoserum in vitro.The umbilical cord serum is more adapt to clinical application.

Objective To investigate the changes of T cells and dendritic cells (DCs) and their related factors in children with immune thrombocytopenia (ITP) before and after therapy,and to analyze their clinical significance.Methods T-cells and DCs subsets were determined by flow cytometry both in 64 children with ITP (ITP group) before and after therapy and the control group.The serum levels of interleukin (IL)-4,interferon-γ (IFN-γ),transforming growth factor beta 1 (TGF-β1),and IL-27 were detected by enzyme linked immunosorbent assay (ELISA).Results Treatments of glucocorticoid or IVIg were effective in 41 cases of 64 ITP children.Compared to the control group,helper T cells (Th),Th/suppressor T cells (Ts),T regulatory cells (Treg),plasmacytoid DC (pDC),pDC/myeloid DC (mDC),and TGF-β1 in ITP patient group before treatment were significantly lower,while IFN-γ and IFN-γ/IL-4 were significantly higher (P ＜0.05).In ITP group,Th,Th/Ts,Treg,pDC,pDC/mDC,TGF-β1,and IL-27 were significantly increased,while IFN-γ and IFN-γ/IL-4 were decreased in children with ITP after therapy and achieved response (P ＜ 0.05).However,there was no significant difference between before and after therapy in ITP children without treatment response (P ＞ 0.05).Conclusions T cells and DCs subsets disorder and abnormal cytokine levels are observed in children with ITP,which can be corrected by immunosuppressive therapy,indicating that Th1 overactivity and the decrease of Treg and pDC both in quantity and function may be related to the pathogenesis of ITP in children.

OBJECTIVETo study the effects of minimal residual disease (MRD) level on day 33 of remission induction and IKZF1 genotype on the survival of children with B-lineage acute lymphoblastic leukemia (B-ALL).

METHODSA total of 152 children with newly-diagnosed B-ALL who had complete remission after the first cycle of the chemotherapy and had complete follow-up information were enrolled in this study. According to the MRD detection by flow cytometry on day 33 of remission induction, they were divided into three groups: standard-risk (SR) group (MRD <10; n=60), intermediate-risk (IR) group (10≤ MRD <10; n=55), and high-risk (HR) group (MRD ≥10; n=37). Nested RT-PCR was used to determine the IKZF1 genotype of all children before chemotherapy. The effects of MRD level on day 33 of remission induction and IKZF1 genotype on the recurrence-free survival (RFS) of children with B-ALL were analyzed.

RESULTSThere were 7 common IKZF1 subtypes in all the 152 children with B-ALL: IK1, IK2/3, IK4, IK6, IK8, IK9, and IK10. Of the 152 children, 130 had functional subtypes of IKZF1 and 22 had non-functional subtypes of IKZF1. During the follow-up period, relapse occurred in 26 (17%) children, and the recurrence rate was highest in the HR group (P<0.05). However, there was no significant difference in the recurrence rate between the SR group and the IR group (P>0.05). The cumulative recurrence rate of the children with non-functional subtypes of IKZF1 was significantly higher than that of those with functional types of IKZF1 (P<0.01). The predicted 5-year RFS rates in the SR, IR, and HR groups were (94.2±2.9)%, (86.7±3.8)%, and (56.2±4.5)% respectively (P<0.05). The 5-year RFS rate of the children with functional subtypes of IKZF1 was significantly higher than that of those with non-functional subtypes of IKZF1 (P<0.01). There was no significant difference in the predicted 5-year RFS rate between the children with functional subtypes of IKZF1 and those with non-functional subtypes of IKZF1 in the SR group (P>0.05). However, the predicted 5-year RFS rate of the children with functional subtypes of IKZF1 was significantly higher than that of those with non-functional subtypes of IKZF1 in the IR group and the HR group (P<0.05).

CONCLUSIONSB-ALL children with non-functional subtypes of IKZF1 have a high recurrence rate, and the recurrence rate will be even higher in B-ALL children with non-functional subtypes of IKZF1 and MRD ≥10 on day 33 of chemotherapy.

Antineoplastic Combined Chemotherapy Protocols ; Child ; Child, Preschool ; Female ; Genotype ; Humans ; Ikaros Transcription Factor ; genetics ; Male ; Neoplasm, Residual ; genetics ; mortality ; therapy ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; mortality ; therapy ; Prognosis ; Recurrence ; Remission Induction ; Survival

Objective:To investigate the anti-leukemia effect of icaritin on 697 cells of children relapsed acute lymphoblastic leukemia in vitro, and to analyze its possible mechanism. Methods:697 cells were cultured in vitro and divided into control group and icariin groups (5, 10, 20 μmol/L). After treated with icaritin, the cell proliferation was detected with cell counting kit-8 (CCK-8) method, and apoptosis was measured with Hoechst 33258 staining and Flow Cytometry Assay. Expression of Bcl-2, Cyt-C, Bax and Caspase3 proteins in 697 cell were detected by Western blot. Results:After treatment with various concentrations (5-20 μmol/L) of icaritin for different time-points (24, 48, 72 h), the growth inhibition rate exhibited a dose and time-dependent manner ( r=0.76, r=0.89); the apoptotic rate exhibited significant increase for 48 h ( P<0.05). Bcl-2 protein decreased significantly ( P<0.05), while Bax, Cyt-C and Caspase3 cleavage protein increased significantly in icaritin treated group for 48 h compared with control group ( P<0.05). Conclusions:Icaritin shows the effects of anti-leukemia by inducing apoptosis and inhibiting proliferation of 697 cell. Icaritin maybe induce apoptosis of 697 cell by down-regulation of Bcl-2 expression and up-regulation of the Bax, Cyt-C and cleaved Caspase 3 expression.

Objective:To investigate the changes of regulatory B cells (Bregs) and lymphocyte subsets in children with primary immune thrombocytopenia (ITP), and to analyze their relationship with ITP duration and remission time.Methods:Proportion of different immune cell subsets were detected in the peripheral blood of 88 ITP children before treatmen, 84 ITP children after treatmen and 45 normal controls by flow cytometry. The treatments included glucocorticoids, intravenous IgG as first-line treatment. The changes of lymphocyte subsets in ITP children with different remission time after treatment were compared.Results:The Th, natural killer (NK), Breg cell counts and Th/Tc ratio in all children with ITP were significantly lower than those in the control group ( P<0.05), while the Tc and CD19 + B cells were significantly higher than those in the control group ( P<0.05). The Breg cell count in the persistent and chronic ITP groups was significantly lower than that in the newly diagnosed ITP ( P<0.05). A total of 84 children with ITP were relieved after treatment: 58 cases (69.1%) achieved long-term remission, and 26 cases (30.9%) had short-term remission. The Breg and lymphocyte subsets in the short-term remission ITP group after treatment were not significantly different from those before treatment, but there were still significant differences from the control group ( P<0.05); The Breg and lymphocyte subpopulations in the long-term remission ITP group after treatment were not significantly different from those in the control group, but compared with those before treatment, other cell subpopulations except Tc were significantly increased ( P<0.05); The NK cells and Breg cells in the long-term remission ITP group were significantly higher than those in the short-term remission ITP group ( P<0.05). Conclusions:The Bregs in peripheral blood of long-duration ITP children significantly decrease. After treatment, the Bregs in ITP children with long-term remission increase significantly, indicating that the restoration of the immune mechanism of Bregs may be related to the long-term remission of children with ITP.