Histidine re-sensitizes pediatric acute lymphoblastic leukemia to 6-mercaptopurine through tetrahydrofolate consumption and SIRT5-mediated desuccinylation.

Despite progressive improvements in the survival rate of pediatric B-cell lineage acute lymphoblastic leukemia (B-ALL), chemoresistance-induced disease progression and recurrence still occur with poor prognosis, thus highlighting the urgent need to eradicate drug resistance in B-ALL. The 6-mercaptopurine (6-MP) is the backbone of ALL combination chemotherapy, and resistance to it is crucially related to relapse. The present study couples chemoresistance in pediatric B-ALL with histidine metabolism deficiency. Evidence was provided that histidine supplementation significantly shifts the 6-MP dose-response in 6-MP-resistant B-ALL. It is revealed that increased tetrahydrofolate consumption via histidine catabolism partially explains the re-sensitization ability of histidine. More importantly, this work provides fresh insights into that desuccinylation mediated by SIRT5 is an indispensable and synergistic requirement for histidine combination therapy against 6-MP resistance, which is undisclosed previously and demonstrates a rational strategy to ameliorate chemoresistance and protect pediatric patients with B-ALL from disease progression or relapse.

Individualized Use of 6-Mercaptopurine in Chinese Children with ALL: A Multicenter Randomized Controlled Trial.

Continuous 6-mercaptopurine (6-MP) dose titration is necessary because of its narrow therapeutic index and frequently encountered dose-limiting hematopoietic toxicity. However, evidence-based guidelines for gene-based 6-MP dosing have not been established for Chinese children with acute lymphoblastic leukemia (ALL). This multicenter, randomized, open-label, active-controlled clinical trial randomly assigned Chinese children with low- or intermediate-risk ALL in a 1:1 ratio to receive TPMT-NUDT15 gene-based dosing of 6-MP (N = 44, 10 to 50 mg/m2 /day) or standard dosing (N = 44, 50 mg/m2 /day) during maintenance therapy. The primary end point was the incidence of 6-MP myelosuppression in both groups. Secondary end points included frequencies of 6-MP hepatotoxicity, duration of myelosuppression and leukopenia, event-free survival, and steady-state concentrations of active metabolites (6-thioguaninenucleotides and 6-methylmercaptopurine nucleotides) in erythrocytes. A 2.2-fold decrease in myelosuppression, the primary end point, was observed in the gene-based-dose group using ~ 50% of the standard initial 6-MP dose (odds ratio, 0.26, 95% confidence interval, 0.11 to 0.64, P = 0.003). Patients in the gene-based-dose group had a significantly lower risk of developing thiopurine-induced myelosuppression and leukopenia (P = 0.015 and P = 0.022, respectively). No significant differences were observed in the secondary end points of the incidence of hepatotoxicity and steady-state concentrations of active metabolites in erythrocytes between the two groups. TPMT- and NUDT15-based dosing of 6-MP will significantly contribute toward further reducing the incidence of leukopenia in Chinese children with ALL. This trial is registered at www.clinicaltrial.gov as #NCT04228393.

Intratracheal administration of umbilical cord-derived mesenchymal stem cells attenuates hyperoxia-induced multi-organ injury via heme oxygenase-1 and JAK/STAT pathways.

BACKGROUND: Bronchopulmonary dysplasia (BPD) is not merely a chronic lung disease, but a systemic condition with multiple organs implications predominantly associated with hyperoxia exposure. Despite advances in current management strategies, limited progress has been made in reducing the BPD-related systemic damage. Meanwhile, although the protective effects of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) or their exosomes on hyperoxia-induced lung injury have been explored by many researchers, the underlying mechanism has not been addressed in detail, and few studies have focused on the therapeutic effect on systemic multiple organ injury. AIM: To investigate whether hUC-MSC intratracheal administration could attenuate hyperoxia-induced lung, heart, and kidney injuries and the underlying regulatory mechanisms. METHODS: Neonatal rats were exposed to hyperoxia (80% O2), treated with hUC-MSCs intratracheal (iT) or intraperitoneal (iP) on postnatal day 7, and harvested on postnatal day 21. The tissue sections of the lung, heart, and kidney were analyzed morphometrically. Protein contents of the bronchoalveolar lavage fluid (BALF), myeloperoxidase (MPO) expression, and malondialdehyde (MDA) levels were examined. Pulmonary inflammatory cytokines were measured via enzyme-linked immunosorbent assay. A comparative transcriptomic analysis of differentially expressed genes (DEGs) in lung tissue was conducted via RNA-sequencing. Subsequently, we performed reverse transcription-quantitative polymerase chain reaction and western blot analysis to explore the expression of target mRNA and proteins related to inflammatory and oxidative responses. RESULTS: iT hUC-MSCs administration improved pulmonary alveolarization and angiogenesis (P < 0.01, P < 0.01, P < 0.001, and P < 0.05 for mean linear intercept, septal counts, vascular medial thickness index, and microvessel density respectively). Meanwhile, treatment with hUC-MSCs iT ame liorated right ventricular hypertrophy (for Fulton's index, P < 0.01), and relieved reduced nephrogenic zone width (P < 0.01) and glomerular diameter (P < 0.001) in kidneys. Among the beneficial effects, a reduction of BALF protein, MPO, and MDA was observed in hUC-MSCs groups (P < 0.01, P < 0.001, and P < 0.05 respectively). Increased pro-inflammatory cytokines tumor necrosis factor-alpha, interleukin (IL)-1ß, and IL-6 expression observed in the hyperoxia group were significantly attenuated by hUC-MSCs administration (P < 0.01, P < 0.001, and P < 0.05 respectively). In addition, we observed an increase in anti-inflammatory cytokine IL-10 expression in rats that received hUC-MSCs iT compared with rats reared in hyperoxia (P < 0.05). Tran scriptomic analysis showed that the DEGs in lung tissues induced by hyperoxia were enriched in pathways related to inflammatory responses, epithelial cell proliferation, and vasculature development. hUC-MSCs administration blunted these hyperoxia-induced dysregulated genes and resulted in a shift in the gene expression pattern toward the normoxia group. hUC-MSCs increased heme oxygenase-1 (HO-1), JAK2, and STAT3 expression, and their phosphorylation in the lung, heart, and kidney (P < 0.05). Remarkably, no significant difference was observed between the iT and iP administration. CONCLUSION: iT hUC-MSCs administration ameliorates hyperoxia-induced lung, heart, and kidney injuries by activating HO-1 expression and JAK/STAT signaling. The therapeutic benefits of local iT and iP administration are equivalent.

A prospective multicenter study investigating rituximab combined with intensive chemotherapy in newly diagnosed pediatric patients with aggressive mature B cell non-Hodgkin lymphoma (CCCG-BNHL-2015): a report from the Chinese Children's Cancer Group.

The evidence for the safety and efficacy of adding rituximab to intensive chemotherapy in pediatric patients with aggressive mature B cell non-Hodgkin lymphoma/leukemia (B-NHL/B-AL) is not yet robust. In this prospective multi-institutional trial, 419 evaluable patients ≤ 16 years of age with newly diagnosed B-NHL/B-AL were enrolled. Patients were stratified into 4 risk groups according to stage, resection status, and serum lactate dehydrogenase. Patients in group R1 received 3 therapy courses in the treatment order A-B-A. Patients in group R2 received 5 courses A-B-A-B-A. Patients in group R3 received 6 courses A-BB-AA-BB-AA-BB. For patients in group R4, rituximab was added to the chemotherapy backbone for patients in R3 (A-RBB-RAA-RBB-RAA-BB). At a median follow-up of 54 months, the 4-year event-free survival (EFS) for the entire group was 88.3 ± 1.6% (76.0 ± 4.3% in the historical study). The EFS rates according to the intention-to-treat principle were 100%, 98.6 ± 1.2%, 94.2 ± 1.8%, and 73.5 ± 3.7% for patients in treatment groups R1, R2, R3, and R4, respectively (P < 0.001). There were 9 (2.1%) toxic deaths due to infection during treatment. Regarding the toxicities of rituximab, grade 3/4 thrombocytopenia, mucositis, and infection occurred in 44.0%, 33.3%, and 64.0% after courses R-BB and grade 3/4 neutropenia, thrombocytopenia, and infection occurred in 96.3%, 77.8%, and 54.1% after courses RAA. The addition of rituximab to intensive chemotherapy is feasible even in a developing country. EFS was significantly improved when compared with the historical data. clinicals.gov identifier: NCT02405676.

[Effect of miltirone on proliferation and apoptosis of leukemia THP-1 cells].

To investigate the toxicity and related mechanism of miltirone to human acute myeloid leukemia THP-1 cells. To be specific, the active components and targets of miltirone were retrieved from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP), and the target proteins were converted into standard gene names with UniProt. Acute leukemia-rela-ted target genes were screened from GeneCards and DisGeNET. Venn diagram was constructed with Venny 2.1 to yield the common targets of the disease and the drug. The protein-protein interaction(PPI) network was constructed by STRING and Cytoscape 3.8.2. THP-1 cells in the logarithmic growth phase were treated with dimethyl sulfoxide(DMSO), and 2.5, 5, 10, 15, and 20 µmol·L~(-1) miltirone for 24 h, respectively. The proliferation rate of cells was analyzed by carboxyfluorescein diacetate succinimidyl ester(CFSE), apoptosis rate by flow cytometry with Annexin V-PE/7 AAD staining, and cell morphology by acridine orange staining. Real-time quantitative PCR(qPCR) was employed to detect the mRNA levels of nuclear receptor coactivator 2(NCOA2), poly(ADP-ribose) polymerase-1(PARP1), B-cell lymphoma-2(Bcl-2)-associated X protein(Bax), Bcl-2, and cysteine aspartyl protease-3(caspase-3). The effect of miltirone on apoptosis was detected in presence of caspase inhibitor Z-VAD-FMK. A total of 26 targets of miltirone, 1 046 genes related to acute leukemia, and 6 common targets of the two were screened out. Flow cytometry result showed miltirone at 10 µmol·L~(-1) can inhibit proliferation and promote apoptosis of THP-1 cells. The typical manifestations of apoptosis, such as cell shrinkage, nuclear rupture, and chromatin agglomerate were displayed by acridine orange staining. The decreased mRNA levels of NCOA2 and PARP1 and increased Bax/Bcl-2 ratio and the activity of pro-apoptotic protein caspase-3 were observed. Z-VAD-FMK can attenuate the apoptosis-inducing effect of miltirone. This study indicates that miltirone can inhibit the proliferation and promote the apoptosis of THP-1 cells, by down-regulating NCOA2 and PARP1, raising Bax/Bcl-2 ratio, and activating caspase-3.

Prior transfusion of umbilical cord mesenchymal stem cells can effectively alleviate symptoms of motion sickness in mice through interleukin 10 secretion.

BACKGROUND: Motion sickness (MS) is a disease that occurs during unbalanced movement, characterized by gastrointestinal symptoms and autonomic nervous system activation. Current clinical treatments for MS are limited. Recent evidence indicates that the levels of pro-inflammatory cytokines increase during MS and are associated with an inner ear immune imbalance. In the present study, mesenchymal stem cells (MSCs) have been shown to exert strong immuno-suppressive effects. AIM: To explore whether umbilical cord-derived mesenchymal stem cells (UC-MSCs) can prevent the occurrence of MS, and the underlying mechanism regulated by MSCs in a mouse model of MS. METHODS: A total of 144 (equal numbers of males and females) 5wkold BALB/c mice were randomly divided into five groups: Normal group (n = 16), MS group (n = 32), MSCs group (n = 32), MS + MSCs group (n = 32), and MS + AS101/MSCs group (n = 32). The MSCs group (n = 32), MS + MSCs group (n = 32), and MS + AS101/MSCs group (n = 32) were preventively transplanted with UC-MSCs or AS101-treated UC-MSCs (1 × 106 cells/mouse). Mice in the MS (n = 32), MS + MSCs, and MS + AS101/MSCs groups were subjected to rotation on a centrifuge for 10 min at 8 × g/min for MS model establishment on days 3, 5, 8, and 10 after UC-MSCs injection. The Morris water maze (MWM) test was used to observe the symptom of dizziness. Enzyme-linked immunosorbent assay (ELISA) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to detect the levels of inflammatory cytokines in mice peripheral blood and the petrous part of the temporal bone samples. Western blot analysis was performed to analyze the JAK2/STAT3 signaling pathway in the cochlear tissues. Histological examination was performed by hematoxylin and eosin (HE) staining for conventional morphological evaluation in the petrous part of temporal bone samples. RESULTS: The MWM test demonstrated that UC-MSCs improved the symptoms of MS. The MS + MSCs group was faster than the MS group on days 3 and 5 (P = 0.036 and P = 0.002, respectively). ELISA and RT-qPCR showed that the serum and mRNA levels of interleukin-10 (IL-10) in the cochlear tissues were increased after transplantation with UC-MSCs (MS + MSCs group vs MS group at 3 and 5 d, P = 0.002 and c P < 0.001, respectively). RT-qPCR results confirmed a significant increase in IL-10 levels at four time points (MS + MSCs group vs MS group, P = 0.009, P = 0.009, P = 0.048, and P = 0.049, respectively). This suggested that UC-MSCs reduced the sensitivity of the vestibular microenvironment by secreting IL-10. Moreover, Western blot analysis showed that the MSCs activated the JAK2/STAT3 signaling pathway in the cochlear tissues. The levels of IL-10, IL-10RA, JAK2, STAT3, and phosphorylated JAK2 and STAT3 in the MS + MSCs group were increased compared to those of the MS group (P < 0.05). The morphological changes in the four groups showed no significant differences. The role of IL-10 secretion on the ability of UC-MSCs to successfully improve the symptoms of MS was confirmed by the diminished therapeutic effects associated with treatment with the IL-10 inhibitor ammonium trichloro (dioxoethylene-o,o') tellurate (AS101). CONCLUSION: Prophylactic transplantation of UC-MSCs can alleviate the clinical symptoms of MS in mice, particularly at 3-5 d after preventive transplantation. The mechanism for UC-MSCs to reduce the sensitivity of vestibular cortex imbalance may be the secretion of IL-10. The next step is to demonstrate the possibility of curing MS in the vestibular environment by intermittent transplantation of MSCs. Above all, MSCs are expected to become a new method for the clinical prevention and treatment of MS.

[Duration of automated auditory brainstem response test for the initial hearing screening and influencing factors for the duration in neonates].

OBJECTIVE: To study the duration of automated auditory brainstem response (AABR) test for initial hearing screening and the factors influencing the duration in neonates. METHODS: A total of 472 neonates who were admitted to the neonatal intensive care unit (NICU) were enrolled as the study group and 182 healthy neonates were enrolled as the healthy control group. The influence of the duration of AABR test on the initial screening results was observed in the two groups. The influencing factors for the AABR test duration were analyzed. RESULTS: In the AABR screening of 180, 360, and 540 seconds, the study group had a failure rate of 41.5%, 28.4%, and 24.4% respectively, while the healthy control group had a failure rate of 31.3%, 19.8%, and 15.4% respectively, showing a decreasing trend with the extension of test time in both groups (P<0.05). In the two groups, the screening results of 180-second testing were moderately consistent with those of 360- or 540-second testing (Kappa<0.75, P<0.05), and the screening results of 360-second testing were highly consistent with those of 540-second testing (Kappa>0.75, P<0.05). In the study group, the median duration of AABR test was 108 seconds (95%CI: 97-120 seconds), which was significantly longer than the duration of 75 seconds (95%CI: 65-85 seconds) in the healthy control group (P<0.05). The Cox regression analysis showed that maternal age ≥35 years, anemia, and electrolyte disturbance (RR<1, P<0.05) were independent risk factors for prolonged AABR test duration, while the prolonged continuous positive airway pressure-assisted ventilation was a protective factor (RR>1, P<0.05). CONCLUSIONS: The AABR test time of 360-540 seconds for initial hearing screening helps to reduce false positive results due to environmental and risk factors in neonates. It may be useful to reduce the false positive results of AABR screening before discharge by taking corresponding intervention measures for NICU neonates with high risk factors.

Clinical presentation and outcome of pediatric patients with hemophagocytic lymphohistiocytosis in China: A retrospective multicenter study.

BACKGROUND: Hemophagocytic lymphohistiocytosis (HLH) is a heterogeneous disease with major diagnostic and therapeutic difficulties. A large-scale multicenter study of pediatric HLH is still lacking in China. PROCEDURE: The Histiocytosis Study Group of the Chinese Pediatric Society conducted this retrospective study in 2014. A total of 323 patients diagnosed with HLH between 2011 and 2013 from 12 hospitals were registered. RESULTS: The median age at diagnosis was 2.2 years (range, 0-14.6 years), with a peak age of HLH onset at 0 to 3 years (63%). Mutations in HLH-related genes were found in 27.9% (24/86) patients who underwent genetic testing. PRF1, UNC13D, STXBP2 and LYST were the predominant genes involved. Sixteen patients (66.7%) presented with only monoallelic mutations in one gene. Epstein-Barr virus (EBV) infection was the major condition related to HLH, which was documented in 74.4% (201/270) of the patients who underwent EBV detection. Of 252 evaluable patients, 64.7% (163) achieved non-active disease at the eighth week and patients treated with a protocol containing etoposide presented higher remission rates (75.6% vs. 46.8%, P < 0.001). In multivariate analysis, a younger age at diagnosis (<12 months), platelet count less than 80×109 /L, central nervous system involvement, and initial treatment using a protocol without etoposide (not HLH-94/04) were independent prognostic factors indicating resistant disease. DISCUSSION: This study first multicenter assessment of HLH in China shows some different features in Chinese children with HLH compared with those in western countries, including older age, vulnerability to EBV infection, and a high proportion of patients with single monoallelic genetic mutations.

Angiopoietin-1 Modified Human Umbilical Cord Mesenchymal Stem Cell Therapy for Endotoxin-Induced Acute Lung Injury in Rats.

PURPOSE: Angiopoietin-1 (Ang1) is a critical factor for vascular stabilization and endothelial survival via inhibition of endothelial permeability and leukocyte- endothelium interactions. Hence, we hypothesized that treatment with umbilical cord mesenchymal stem cells (UCMSCs) carrying the Ang1 gene (UCMSCs-Ang1) might be a potential approach for acute lung injury (ALI) induced by lipopolysaccharide (LPS). MATERIALS AND METHODS: UCMSCs with or without transfection with the human Ang1 gene were delivered intravenously into rats one hour after intra-abdominal instillation of LPS to induce ALI. After the rats were sacrificed at 6 hours, 24 hours, 48 hours, 8 days, and 15 days post-injection of LPS, the serum, the lung tissues, and bronchoalveolar lavage fluid (BALF) were harvested for analysis, respectively. RESULTS: Administration of fluorescence microscope confirmed the increased presence of UCMSCs in the injured lungs. The evaluation of UCMSCs and UCMSCs-Ang1 actions revealed that Ang1 overexpression further decreased the levels of the pro-inflammatory cytokines TNF-α, TGF-ß1, and IL-6 and increased the expression of the anti-inflammatory cytokine IL-10 in the injured lungs. This synergy caused a substantial decrease in lung airspace inflammation and vascular leakage, characterized by significant reductions in wet/dry ratio, differential neutrophil counts, myeloperoxidase activity, and BALF. The rats treated by UCMSCs-Ang1 showed improved survival and lower ALI scores. CONCLUSION: UCMSCs-Ang1 could improve both systemic inflammation and alveolar permeability in ALI. UC-derived MSCs-based Ang1 gene therapy may be developed as a potential novel strategy for the treatment of ALI.

[Effect of Human Umbilical Cord Mesenchymal Stem Cells on Etoposide-induced Nalm-6 Cell Apoptosis].

OBJECTIVE: To investigate the effect of human umbilical cord mesenchymal stem cells (hUC-MSC) on VP16-induced apoptosis of Nalm-6 cells. METHODS: hUC-MSC were isolated and identified using morphological observation and flow cytometry, then Nalm-6 cells were treated with hUC-MSC with or without VP16. Apoptosis and cell cycle were assayed by FACS. The mRNA levels of apoptosis-related genes BCL-2, BAX and caspase-3 were detected by quantitative RT-PCR, and the protein levels of BCL-2, BAX and caspase-3 were examined by Western blot. RESULTS: FACS showed that hUC-MSC inhibited the proliferation and decreased apoptosis of Nalm-6 cells resulted from VP16. The quantitative RT-PCR showed that hUC-MSC increased the mRNA expression level of BCL-2 and decreased the expression level of BAX and caspase-3 (P < 0.05). Western blot showed that the protein expression level of BCL-2 increased, and expression level of BAX and caspase-3 decreased in Nalm-6 cells after co-culture with hUC-MSC (P < 0.05). CONCLUSION: hUC-MSC may protect Nalm-6 cells from apoptosis induced by VP16 through regulation of BCL-2, BAX and caspase-3.

CD4+ T cells from patients with acute myeloid leukemia inhibit the proliferation of bone marrow-derived mesenchymal stem cells by secretion of miR-10a.

BACKGROUND: The abnormality of bone marrow-derived mesenchymal stem cells (BM-MSCs) has been reported to contribute to the pathogenesis of acute myeloid leukemia (AML). T cell immunodeficiencies play important roles in the progression of leukemia. This study investigated the effect of CD4+ T cells from AML patients on the proliferation of BM-MSCs. METHODS: The growth rate of BM-MSCs from AML patients and healthy donor was compared. CD4+ T cells were separated and identified from AML patients. Through co-culturing CD4+ T cells from AML patients and BM-MSCs from healthy, we detected the proliferation of BM-MSCs from healthy by MTT assay. qRT-PCR was performed to examine the expression of miR-10a. Luciferase reporter assay was used to analyze the regulation of miR-10a on the expression of BCL6. RESULTS: Here, we observed that BM-MSC from AML patients grew slower than that from healthy. CD4+ T cells from AML patients inhibited the proliferation of BM-MSCs through secreting miR-10a. In addition, miR-10a was found to target BCL6 and regulated its expression in transcription and translation levels. Correlation analysis revealed that the level of miR-10a in serum of AML patients was negatively correlated with BCL6 in BM-MSC. CONCLUSION: This study provides evidence that CD4+ T cells from AML patients suppress the proliferation of BM-MSCs via secreting miR-10a.

Therapeutic effect of placenta-derived mesenchymal stem cells on hypoxic-ischemic brain damage in rats.

BACKGROUND: Oxidative stress is involved in the development of hypoxic-ischemic brain damage (HIBD). In this study, we investigated the therapeutic effects of placenta-derived mesenchymal stem cells (PD-MSCs) and explored the NF-E2-related factor-2/heme oxygenase-1 (Nrf2/HO-1) signaling pathway in treating HIBD. METHODS: P7 rats were subjected to hypoxic-ischemic brain injury and randomly divided into four groups (control, HIBD, HIBD+PD-MSCs, and HIBD+fibroblasts). Forty-eight hours after the induction of HIBD, 5×10(5) of PD-MSCs were injected into cerebral tissue in the HIBD+PD-MSCs group, while the same dose of fibroblasts were injected in the HIBD+fibroblasts group. Morris Water Maze, gross and pathological changes were tested at P28. The level of malondialdehyde (MDA) was detected in rats' hippocampus. RT-PCR and western blot analysis were used to evaluate the changes of Nrf2/HO-1. RESULTS: The HIBD group showed significantly longer escape latency and a lower frequency of original platform crossing in the Morris Water Maze compared with the control group. Rats receiving PD-MSCs showed significant improvement of HIBD. The pathological changes were evident after HIBD, but ameliorated in the PD-MSCs group. Compared with the control group, HO-1 and Nrf2 were up-regulated at gene and protein levels in the HI brain, beginning at 6 hours and peaking at 48 hours (P<0.05). The expression of HO-1 and Nrf2 in the PD-MSCs treatment group was more pronounced than in the HIBD group (P<0.01). PD-MSCs also decreased MDA production in the brain tissue. CONCLUSION: These results demonstrate that PD-MSCs have neuroprotective effect during the treatment of HIBD and that the mechanism may be partly due to alleviating oxidative stress.

[Immunomodulatory ability of senile mesenchymal stem cells].

This study was aimed to investigate the immunomodulatory ability of human umbilical cord mesenchymal stem cells (UB-MSC) along with prolonging of culture time and increasing of passages in vitro. Mesenchymal stem cells (MSC) were isolated from human umbilical cord and cultured in vitro. The morphological changes and nucleocytoplasmic ratio of MSC were observed using Giemsa staining. MSC of the 5th passage were selected as control group, and MSC of the 13th passage were taken as senile group. The degree of cell senescence was detected by aging cells in situ test kit. Cell Counting Kit-WST-8 was used to determine the proliferation of lymphocytes in mixed lymphocytes coculture system with different passages of MSC. The expression of immunomodulation-related genes was detected by RT-PCR. The results showed that the length-breadth ratio of MSC increased and nucleocytoplasmic ratio decreased along with the increasing of passages. The senium degree of cells of the 13th passage was higher than that of the 5th passage cells. The capacity of suppressing lymphocyte proliferation of the 13th passage MSC was enhanced, compared with the 5th passage. Moreover, the expression of immunosuppression-related genes of senile MSC increased and the expression of most anti-inflammation associated genes declined as compared with young MSC by RT-PCR. It is concluded that the degree of MSC senescence gradually develops with increasing of culture passage, but the immunosuppressive ability of MSC strengthens with continuous culture.

[The expression and regulatory mechanism of microRNA-708 in pediatric common B-cell acute lymphoblastic leukemia].

OBJECTIVE: To evaluate the expression of microRNAs and reveal the regulatory mechanism of miRNA-708 in pediatric common acute lymphoblastic leukemia (ALL) (common-ALL). METHODS: The expressions of microRNAs in common-ALL patients were detected by microarrays in 3 pediatric common-ALL samples, and then verified by stem-loop quantitative RT-PCR in 34 common-ALL samples. The target genes of miR-708 were found by bioinformatics software, and verified by dual-luciferases reporter assay, RT-PCR and Western blot. RESULTS: Compared to normal bone marrow samples, of all the 2006 detected miRNAs, the expression of miR-708, miR-181b and miR-210 were 16.886 ± 16.854, 5.710 ± 4.652, and 9.789 ± 1.178, retrospectively, being significantly up-regulated expressed than those in normal control (1.872 ± 0.339, 1.276 ± 0.531 and 1.005 ± 0.080, retrospectively) (P < 0.05), while miR-27b and miR-345 were the two most down-regulated ones (0.524 ± 0.085 and 0.675 ± 0.086, retrospectively) (normal control: 1.123 ± 0.066 and 1.204 ± 0.140, retrospectively) (P < 0.05). And the expression of miR-708 and miR-181b were significantly correlated with the clinical types in common-ALL. In high risk common-ALL, miR-708 and miR-181b were much higher than in standard and middle risk common-ALL (P < 0.05). The further verification research in 293 cell line showed that miR-708 decreased the expression level of its target genes CNTFR, NNAT and GNG12 by combining with 3'-UTR of the 3 genes, moreover, miR-708 combined with CNTFR 3'-UTR in 394 ∼ 400 bp sequence region. CONCLUSION: MicroRNAs plays an important regulatory role during the occurrence and development of the pediatric common-ALL and miR-708 is an important factor for high risk common-ALL.

[Influence of vascular endothelial growth factor on endothelial components in human bone marrow and umbilical cord mesenchymal stem cells].

This study was aimed to compare the proportion of endothelial cells (EC) in human bone marrow mesenchymal stem cell (BM-MSC) and human umbilical cord mesenchymal stem cells (UC-MSC), and to investigate the influence of vascular endothelial growth factor (VEGF) on proportion of EC in MSC. Flow cytometry was used to detect the proportion of CD34(+)CD133(+) and vWF(+)CD31(+) double positive cells in MSC. Wright's staining was employed to observe the influence of VEGF on morphology of MSC. The expressions of CD34, CD133, CD31, vWF were detected by immunofluorescence. qRT-PCR was performed to detect the influence of VEGF on EC marker genes' expression of MSC. The results showed that there were a small amount of EC and endothelial progenitor cells (EPC) in obtained BM-MSC and UC-MSC. After exposed to VEGF 10 ng/ml for 24 h, aspect ratio of MSC and the proportion of EC increased, while proportion of EPC decreased. Expression of EC related marker genes such as Tie-2 and ecNOS up-regulated, especially in UC-MSC. It is concluded that small amount of EC and EPC exists in cultured BM-MSC and UC-MSC, VEGF can enhance the proportion and function of EC in MSC.

[Changes of biological characteristics and gene expression profile of umbilical cord mesenchymal stem cells during senescence in culture].

This study was purposed to investigate the changes of biological properties and expression patterns of the aging related genes in umbilical cord mesenchymal stem cells (UC-MSC) during in vitro culture. UC-MSC at passage 3 were served as the control cells and those at passage 15 were considered as the aged cells. The biological features of those two kinds of cells including morphology, proliferation activity and phenotypic profile were observed, and the differences of gene expression were analysed by the whole human genome oligo microarray. Several differential genes were selected for further confirmation by quantitative reverse transcription-polymerase chain reaction. The results showed that UC-MSC at passage 15 were larger in size and their proliferation rate was slower compared with those of cells at passage 3, while the positivity of CD44 and CD105 remained unchanged. Compared with UC-MSC at passage 3, relatively aged cells expressed higher levels of genes that are associated with small subunit of ribosome. Further analysis with Gene Ontology functional categories showed that the up-regulated genes were concentrated in those related to steroid biosynthesis, galactose metabolism and the development of autoimmune diseases and degenerative diseases and the down-regulated genes in UC-MSC at passage 15 were concentrated in cytoskeleton molecules, DNA structure binding, mRNA binding and protein function. Functional analysis with Kyoto Encyclopedia of Genes and Genomes functional pathway revealed that the expression of some genes responsible for ribosome composition was elevated while those of associated with extracellular matrix, focal adhesion and cell cycle progression were down-regulated. It is concluded that UC-MSC become senescent due to the declines in metabolism and proliferation activities.

[Effect of TGF-ß1 on proliferation and extracellular matrix and gene expressions of hUC-MSC].

This study was purposed to investigate the effects of transforming growth factor-ß1 (TGF-ß1) on proliferation and extracellular matrix (ECM) and gene expressions of human umbilical cord derived mesenchymal stem cells (hUC-MSC). The hUC-MSCs were isolated and cultured by transplantation technique from umbilical cords. Surface antigens of the fifth passage cultured hUC-MSCs were detected by flow cytometry. The effects of TGF-ß1 0.1 - 20.0 ng/ml on hUC-MSC proliferation were determined by Cell Counting Kit-8. The mRNA expression of ECM components and the components involved in the mechanism of ECM production regulation were detected by qRT-PCR. Expressions of collagen I (Col-I), collagen IV (Col-IV), fibronectin (FN) and laminin (LN) were observed by immunocytochemistry. The gene expression profiles of hUC-MSC in response to TGF-ß1 were examined by microarray. The results showed that no significant changes of the OD values were observed with the increasing doses of TGF-ß1, while all OD values of 0.1 ng/ml group showed relative peaks at 24, 48 and 72 hour time points. The mRNA expression level of Col-I, MMP-2, MMP-9 and TIMP-2 increased to maximum in 0.5 ng/ml group, and the mRNA expression of Col-IV, FN, LN, Integrin, Tenascin-C, MMP-1 and TIMP-1 reached the peak level in 0.1 ng/ml group. Immunocytochemical analysis for Col-I and Col-IV revealed that their production increased slightly in 0.5 and 0.1 ng/ml groups, respectively, while immunostaining for FN showed diffuse cell membrane and cytoplasmic staining randomly distributed, but LN staining was absent in both groups. Microarray analysis showed that 9 genes closely related to cell movement and migration were up-regulated. Analysis by Pathway and Go indicated that differentially expressed genes were involved in transcription, translation, biosynthesis, metabolism, signal transduction, migration and adhering junction etc. It is concluded that TGF-ß1 0.1 ng/ml promotes the proliferation of hUC-MSC. TGF-ß1 of different concentrations upregulates the expression of different ECM components. TGF-ß1 may play important roles in various aspects of hUC-MSC including transcription, translation, biosynthesis, metabolism, signal transduction, migration and adherens junction and so on.

MicroRNA miR-24 inhibits erythropoiesis by targeting activin type I receptor ALK4.

MicroRNAs have been suggested to modulate a variety of cellular events. Here we report that miR-24 regulates erythroid differentiation by influencing the expression of human activin type I receptor ALK4 (hALK4). Ectopic expression of miR-24 reduces the mRNA and protein levels of hALK4 by targeting the 3'-untranslated region of hALK4 mRNA and interferes with activin-induced Smad2 phosphorylation and reporter expression. Furthermore, miR-24 represses the activin-mediated accumulation of hemoglobin, an erythroid differentiation marker, in erythroleukemic K562 cells and decreases erythroid colony-forming and burst-forming units of CD34+ hematopoietic progenitor cells. ALK4 expression is inversely correlated with miR-24 expression during the early stages of erythroid differentiation, and the forced expression of miR-24 leads to a delay of activin-induced maturation of hematopoietic progenitor cells in liquid culture. Thus, our findings define a regulation mode of miR-24 on erythropoiesis by impeding ALK4 expression.

[Study of single cell PCR for HLA typing].

OBJECTIVE: To apply the single cell nested multiplex polymerase chain reaction (PCR) to HLA typing, and analyze the influence factors on the amplification results. METHODS: Single cell DNA templates were prepared with different methods. The exon 2, 3 and intron 2 of HLA-A, B, and exon 2 of DRBI were amplified using multiplex PCR. The second round of SSP-PCR HLA typing was carried out according to the large scale routine HLA typing results. RESULTS: Enzyme lysis method was the most efficient procedure for preparing the single cell DNA template, with a success rate (SR) of 93.3%, while the SRs of alkali lysis and freezing-thaw lysis methods were 83.3% and 73.3%, respectively. The second round amplification using enzyme lysis and SSP-PCR in 20 samples obtained a 95% success rate and a 15% allele drop out rate. The time for performing the whole procedure was less than 6 hours. CONCLUSION: The modified nested multiplex PCR technique is efficient for single cell HLA typing and might be applied to clinical preimplantation genetic diagnosis.

[In vitro expansion and function of cord blood megakaryocyte].

OBJECTIVE: Cord blood (CB) provides a rich source of stem cells for transplantation. CB transplantation has been used widely after myeloablative therapy. One major disadvantage of CB transplantation is delayed platelet engraftment. The aim of this study was to hasten platelet engraftment by investigating the ability of different hematopoietic growth factor combinations to generate large numbers of megakaryocyte (Mk) from CB and by evaluating the biologic characteristics and function of the expanded Mk. METHODS: CB samples were obtained at the end of normal full-term deliveries with informed consent. Mononuclear cells (MNCs) were isolated from CB using Ficoll density centrifugation. MNC population was positively selected for CD(34) expression by magnetic cell sorting (MACS). CD(34)(+) cells were cultured in serum-free and stroma-free medium containing the following two different cytokine combinations: thrombopoietin (TPO) + stem cell factor (SCF) + interleukin (IL) -3 + IL-6 and TPO + SCF. Cultures were characterized after 3, 7, 10 and 14 days by flow cytometry, colony forming unit-megakaryocyte (CFU-Mk) and maturation evaluation (Mk ploidy). The expanded Mk function was examined by the platelet activation in vitro and severe combined immunodiffiency (SCID) mice transplantation in vivo. RESULTS: Different results were observed with different culture conditions. With the first cytokine combination optimal expansion of CD(41)(+) cells was observed on day 10, but the optimal expansion of Mk progenitors (CD(34)(+)CD(41)(+)) was observed on day 7, with a median 121 and 44-fold increase at the starting cell dose. This result was also proven by CFU-Mk. The largest numbers of CFU-Mk were also observed on day 7. The degree of maturation of Mk cells also increased as suggested by DNA content of CD(41)(+) cells, which means that CD(34)(+) cells cultured for 3 - 7 days were richer in primitive Mks, while those cultured for 10 - 14 days had greater numbers of more differentiated Mks. For the second cytokine combination, CD(41)(+) and CD(34)(+)CD(41)(+) cells were fewer than the first one, but it produced 36 and 85-fold CD(34)(+)CD(41)(+) and CD(41)(+) respectively on day 7. Platelet activation test confirmed that the expanded Mks had normal function. Therefore, the expanded Mks could be transplanted into the SCID mice bone marrow and produce human platelet in the peripheral blood of the mice. CONCLUSION: Ex vivo expanded Mk might facilitate CB transplantation and help shorten the period of post-transplant thrombocytopenia.