Marrow Mononuclear Cells Of Patients Research Articles

Role of Vascular Endothelial Growth Factor-C during Stem Cell Therapy Using Autologous Bone Marrow Mononuclear Cells in Patients with Lower Limb Lymphedema

Introduction: Vascular endothelial growth factor-C (VEGF-C) is the primary lymphangiogenic factor that stimulates lymphangiogenesis by signaling via specific receptor, vascular endothelial growth factor receptor 3 (VEGFR3). This study was conducted to evaluate the change in the level of VEGF-C before and after autologous bone marrow mononuclear cell transplantation for treatment of Lower limb lymphedema. Patient and methods: Forty patients with lower limb lymphedema were divided into two groups. Group I included 20 patients with chronic lower limb lymphedema who underwent autologous bone marrow mononuclear cell transplantation. Group II included 20 patients with chronic lower limb lymphedema who were exposed only to compression therapy as a control group. VEGF-C level in the diseased limbs was measured in both groups at the beginning of the study then 3 and 6 months respectively. Results: Group I included 20 patients, 8 patients were male (40%) and 12 patients were females (60%) with mean age 29.5 ± 12.15 while group II included 20, 10 patients were male (50%) and 10 patients were females (50%) with mean age 39.5 ± 11.5. In group I, the specimens were taken at 3 and 6 months after transplantation showed a marked decrease in the VEGF-C level with statistically significant p value, 0.02 and 0.001 respectively. In group II the level of VEGF-C after compression therapy alone at 3 and 6 months interval showed fluctuation with statistically non-significant p value, 0.64 and 0.55 respectively. Conclusion: VEGF-C is essential for regulation of lymphangiogenesis. The level of VEGF-C was found elevated in patients with lymphedema and decrease after autologous mononuclear bone marrow cells, however these results were statically non-significant.

Expression and Clinical Significance of MMSA-8 and MMSA-1 in Bone Marrow of Patients with M2 Acute Myeloid Leukemia

To detect the expression of multiple myeloma-associated antigen (MMSA)-8 and MMSA-1 in bone marrow mononuclear cells of patients with acute myeloid leukemia, and explore their roles in acute myeloid leukemia. A total of 83 patients with M2 acute myeloid leukemia in our hospital from January 2019 to January 2020 were selected as research group, during the same period, 15 patients diagnosed iron deficiency anemia were selected as control group. Real-time fluorescence quantitative PCR was used to detect the levels of MMSA-8 and MMSA-1 in bone marrow mononuclear cells. Patients in the research group were divided into remission and non remission according to the clinical curative effect, and were divided into good prognosis, medium prognosis, and poor prognosis according to the prognosis. The relationship between MMSA-8, MMSA-1 and clinical efficacy, prognosis was analyzed. In addition, the general data of patients in the research group were collected, including white blood cell count (WBC), hemoglobin (Hb), platelet count (PLT), and percentage of bone marrow progenitor cells at admission. Pearson method was used to analyze the correlation between MMSA-8, MMSA-1 and clinical data, and MMSA-8 and MMSA-1. The analysis results about mRNA levels of MMSA-8 and MMSA-1 in bone marrow mononuclear cells of patients showed that patients in the research group were significantly higher than those in the control group (P<0.05); In the research group, patients without remission were also significantly higher than those with remission, as well as those with medium and poor prognosis than with good prognosis, while only mRNA level of MMSA-1 in patients with poor prognosis was significantly higher than those with medium prognosis (P<0.05). Pearson analysis showed that MMSA-8, MMSA-1 were positively correlated with WBC (r=0.468, r=0.516), and MMSA-8 was positively correlated with MMSA-1 (r=0.318). The levels of MMSA-8 and MMSA-1 in bone marrow mononuclear cells of patients with M2 acute myeloid leukemia are increased, which are closely related to the occurrence and development of the disease, and have certain value for the prognosis.

Immunosuppressive role of CD11b+ CD33+ HLA-DR- myeloid-derived suppressor cells-like blast subpopulation in acute myeloid leukemia.

ObjectiveMyeloid‐derived suppressor cells (MDSCs) facilitate tumor growth and development by suppressing T cell function; however, their role in acute myeloid leukemia (AML) remains unclear. Here, we investigated the immunosuppressive role and prognostic value of blasts with an MDSC‐like phenotype.MethodsCD11b+CD33+HLA‐DR− MDSC‐like blasts from bone marrow mononuclear cells of patients with AML were analyzed. To investigate their T cell‐suppressing function, MDSC‐like blasts were isolated using flow cytometry and co‐cultured with CD8+ cytotoxic T cells and NB4 leukemic cells. Treatment outcomes were then compared between the MDSC‐like blasts low (≤9.76%) and high (>9.76%) groups to identify clinical significance.ResultsMDSC‐like blasts showed higher expression of arginase‐1 and inducible nitric oxide synthase. Isolated MDSC‐like blasts significantly suppressed CD8+ T cell proliferation induced by phytohemagglutinin A. NB4 cell proliferation was significantly suppressed upon co‐culture with CD8+ cytotoxic T cells and partially restored upon co‐culture with MDSC‐like blasts. Patients with high MDSC‐like blasts at diagnosis showed substantially shorter overall survival and leukemia‐free survival relative to low MDSC‐like blasts patients, with subgroup analysis showing statistically significant differences in patients not receiving allogeneic hematopoietic stem cell transplantation.ConclusionWe demonstrated that MDSC‐like blasts drive AML‐specific immune‐escape mechanisms by suppressing T cell proliferation and restoring T cell‐suppressed NB4 cell proliferation, with clinically higher fractions of MDSC‐like blasts at diagnosis resulting in poor prognosis.

Strengthening effects of bone marrow mononuclear cells with intensive atorvastatin in acute myocardial infarction

ObjectiveTo test whether intensive atorvastatin (ATV) increases the efficacy of transplantation with autologous bone marrow mononuclear cells (MNCs) in patients suffering from anterior ST-elevated myocardial infarction (STEMI).MethodsThis clinical trial was...

Diagnostic Value of miR-34a in Bone Marrow Mononuclear Cells of Acute Myeloid Leukemia Patients.

The current study aims to analyze the expression of miR-34a in bone marrow mononuclear cells (BMMCs) of acute myeloid leukemia (AML) patients and its possible role in the development of leukemia. Real-time fluorescent quantitative PCR was used to detect the expression of miR-34a in bone marrow of 40 patients with initial treatment of AML, 22 patients with complete remission AML, 13 patients with refractory recurrence, 20 patients with non-hematological tumors and healthy persons. Pearson's correlation was performed to analyze the correlation between the expression level of miR-34a and clinical indicators in patients. The expression of miR-34a in the initial treatment group, complete remission group and refractory relapse group of AML patients was higher than that in the control group. In addition, the expression of miR-34a in the complete remission group was higher than that in the initial treatment group. Moreover, the expression of miR-34a in the refractory relapse group was lower than that in the initial treatment group. The expression level of miR-34a in initial treatment AML group was negatively correlated with the number of bone marrow primary immature cells, but not with the number of peripheral blood leukocytes, platelet count, hemoglobin content, age, gender, and chromosomal abnormalities. The expression of miR-34a in bone marrow mononuclear cells of patients with AML was down-regulated and related to tumor load. It may play an important role in the occurrence and development of leukemia and may become a new molecular marker for the diagnosis of AML.

Expression Analysis and Epigenetics of MicroRNA-28-5p in Multiple Myeloma

To investigate the expression, mechanism and methylation level of miR-28-5p in multiple myeloma (MM), so as to provide the expirement basis for searching new targeted therapy. RT-PCR was used to detect the expression levels of miR-28-5p and potential target CCND1 in CD138+ cells of the patients with MM and bone marrow mononuclear cells of patients with iron defficiency anemia(IDA) as control, Methylation-specific PCR(MSP) was used to detect methylation levels of CpG island in LPP/miR-28-5p promoter region and the correlation with other clinical indicators was analyzed. The 5-aza-2'-deoxycytidine (5-Aza-dC，DAC) was used to treat MM cell line U266; after drug treatment，MSP was used to analyze the methylation status of the CpG islands in LPP/miR-28-5p promoter; the qPCR was used to detect the expression levels of miR-28-5p，and the regulatory mechanism of miR-28-5p expression was explored furtherly. The methylation level of CpG island in LPP/miR-28-5p promoter region of MM patients was significantly higher than that of IDA patients. The relative expression level of miR-28-5p in MM patients was significantly lower than that of IDA patients. The relative expression level of miR-28-5p in newly diagnosed MM patients was higher than that in relapsed/progressive patients. The miR-28-5p target CCND1 was expressed at high levels in MM patients with LPP / miR-28-5p methylation, the expression level of miR-28-5p in MM patients correlated with β2-MG concentration. 5-aza-dc could significantly inhibit the growth of U266 cell line, arrest the cell cycle in G1 phase, inhibit the biosynthesis of cellular RNA and protein and promote cell apoptosis. At the same time, up-regulation of miR-28-5p expression was found. The expression of miR-28-5p in MM patients is regulated by methylation of CpG islands in the promoter region of the genome.miR-28-5p may act as a tumor suppressor gene, and its low expression may be involved in the occurrence and development of MM, suggesting that miR-28-5p may become a new target for the treatment of MM.

Expression of WT1 gene in bone marrow mononuclear cells of patients with myelodysplastic syndromes

Objective To analyze the relationship between the expression of WT1 gene in bone marrow mononuclear cells and the cytogenetic characteristics, curative effect and prognosis in patients with myelodysplastic syndromes (MDS). Methods The quantitative expression of WT1 gene was detected by real-time quantitative polymerase chain reaction (RQ-PCR) in bone marrow mononuclear cells of 115 MDS patients who were admitted to Taixing People's Hospital from August 2010 to March 2018, and the relationship between the expression of WT1 gene and the cytogenetic characteristics, curative effect and prognosis were evaluated. Results WT1 gene was highly expressed in 80 cases (69.6%), lowly expressed in 15 cases (13.0%), and unexpressed in 20 cases (17.4%). Of the 80 patients with high WT1 expression, 56 cases(70.0%) were refractory anemia with excess blasts (RAEB), and 22 cases (27.7%) were refractory cytopenia with multilineage dysplasia (RCMD). In 55 patients with very poor cytogenetic prognosis, 44 patients had high expression of WT1 gene, and in 28 patients with poor prognosis, 19 patients had high expression of WT1 gene. The complete remission rate of WT1 gene high expression group [12.5% (10/80)] was lower than that of low expression group [26.7% (4/15)] and unexpressed group [40.0% (8/20)], and the difference of complete remission rate between the three groups was statistically significant (χ2= 8.96, P < 0.05). Conclusion MDS patients with high expression of WT1 gene have low remission rate and poor prognosis. Key words: Myelodysplastic syndromes; WT1 gene; Cytogenetics; Prognosis; Treatment outcome

Intraportal therapy of autologous bone marrow mononuclear cells in patients on the waiting list for liver transplantation

This study was carried out to explore the methodology of intraportal infusion of autologous bone marrow mononuclear cells in patients in the waiting list for liver transplantation. The study included eight patients. The Model for End-Stage Liver Disease (MELD) score as a predictor of hepatic failure exceeds 10 points, i.e., 12-22 points. According to Child-Turcotte-Pugh (CTP) classification, the severity of cirrhosis ranged from class B to C. The bone marrow volume of 224-320 ml was harvested by bilateral aspiration from the posterior iliac crest. The total volume of the mononuclear cells (MNC) was 47.1-58.3 ml (median, 47 ml). The absolute viable count of isolated MNC was 1.24 · 109-5.84 · 109 and CD34+ cells 2.54 · 106-48.20 · 106. After portal vein catheterization, direct transhepatic portography was performed, and portosystemic collaterals were found in all cases. Portal flowmetry was performed to determine the optimal selective perfusion rate of the segmental branches of the portal vein. Portal vein infusion was performed at 0.3-1.5 ml/sec. Serious side effects were not detected during bone marrow harvesting and MNC infusion. All patients had stable condition (no deterioration was initially noticed) after MNC infusion according to the MELD score and CTP criteria during a 3-month observation. Moreover, improvement in physical health status was observed and obtained using the SF-36 questionnaire. This study confirmed the safety of using intraportal therapy of autologous bone marrow MNC in patients on the waiting list for liver transplantation. Herein, we presented the technique of target delivery of autologous bone marrow MNC to the liver to minimize their loss during intraportal infusion. Prolonged follow-up time and improvement of the present technique are needed for further evaluation of the efficiency of autologous bone marrow MNC.

Maternal folate genes and aberrant DNA hypermethylation in pediatric acute lymphoblastic leukemia.

BackgroundThere is evidence that maternal genotypes in folate-related genes are associated with pediatric acute lymphoblastic leukemia (ALL) independent of offspring genotype. We evaluated the relationship between maternal genotypes in methionine synthase (MTR) and DNA methylation status in ALL to better characterize the molecular mechanism underlying this association.ProcedureWe obtained bone marrow samples from 51 patients with ALL at diagnosis and from 6 healthy donors. Mothers of patients provided a saliva sample and were genotyped at 11 tagSNPs in MTR. DNA methylation was measured in bone marrow mononuclear cells of patients and six healthy marrow donors. We used hierarchical clustering to identify patients with a hypermethylator phenotype based on 281 differentially methylated promoter CpGs. We used logistic regression to estimate the effects of maternal genotype on the likelihood of DNA hypermethylation in ALL and Ingenuity Pathway Analysis to identify networks enriched for differentially methylated genes.ResultsTwenty-two cases (43%) demonstrated promoter hypermethylation, which was more frequent among those with ETV6-RUNX1 fusion and initial white blood cell count < 50 x 109/L. Maternal rs12759827 was associated with aberrant DNA methylation (odds ratio [OR] 4.67, 95% confidence interval 1.46–16.31); non-significantly elevated ORs were observed for all other SNPs. Aberrantly methylated promoter CpGs aligned to genes with known cancer-related functions.DiscussionMaternal folate metabolic genotype may be associated with DNA methylation patterns in ALL in their offspring. Therefore, the effect of maternal genotypes on ALL susceptibility may act through aberrant promoter methylation, which may contribute to the in utero origins of ALL.

In Vitro Effect of Pomalidomide on Bone Marrow Mononuclear Cells From Multiple Myeloma Patients

Background Many features of anticancer drugs, including cytotoxicity and/or cytokine induction, are studied using cell lines or human blood leukocytes. However, in a disease such as multiple myeloma, most cancerous cells are resided within bone marrow mononuclear cells. In the present study, we investigated the effect of pomalidomide on apoptosis and IL-2 production of bone marrow mononuclear cells in patients with multiple myeloma and controls.Materials and Methods The Bone Marrow Mononuclear Cells from 10 multiple myeloma patients and 10 controls were cultured using pomalidomide for 48-hour. Apoptosis induction rate, viability, cytotoxicity, and IL-2 production were evaluated. Results The results showed that apoptosis and cytotoxicity significantly increased in bone marrow mononuclear cells of multiple myeloma patients cultured in the presence of pomalidomide (PConclusion The results of the current study confirmed the toxic effect of pomalidomide on bone marrow mononuclear cells of patients, with no toxic effect on controls. On the other hand, pomalidomide has the potential to increase IL2 production within bone marrow mononuclear cells and consequently make changes in the immune response, which requires further studies for more clarifications.

Effects of Decitabine on the proliferation of K562 cells and the expression of DR4 gene.

ObjectiveTo investigate the role of DR4 gene in the occurrence, development and prognosis of acute myeloid leukemia (AML), find a new regulatory gene of Decitabine for the treatment of AML, namely DR4 gene, and explore the molecular mechanism of AML in the treatment of AML.MethodsThe methylation level and the mRNA expression level of DR4 gene promoters of bone marrow mononuclear cells in 122 patients with newly diagnosed AML and 24 patients with iron deficiency anemia (IDA) were detected using Methylation specific PCR (MS-PCR) and Q-RT-PCR, respectively, and a correlation analysis of them was conducted. The effects of Decitabine on the proliferation of K562 cells were detected using CCK-8 assay. Then, the effects of Decitabine on the methylation level and the mRNA expression level of DR4 genes of K562 cells treated with Decitabine were detected using MS-PCR and Q-RT-PCR, respectively. The effects of Decitabine on the cell cycle and apoptosis of K562 cells were detected using flow cytometry.ResultsCompared with the control group, the methylation level (P = .002) of DR4 genes of bone marrow mononuclear cells in patients with newly diagnosed AML was high. The methylation level (P = .01) of DR4 genes of bone marrow mononuclear cells in patients of the positive group of enlargement of liver, spleen and lymph node was lower than that of the negative group, and the methylation level (P = .006) of DR4 genes in patients of the high risk group of clinical stage was lower than that of the low risk group, and the methylation level (P = .03) of DR4 genes in patients of the group where patients did not achieve complete remission (CR1) after a course of induction chemotherapy was lower than that of the group where patients achieved complete remission (CR1) after a course of induction chemotherapy. There was a significant negative correlation (P < .01) between the methylation level and the mRNA expression level of DR4 genes of bone marrow mononuclear cells in 122 patients with newly diagnosed AML. After the K562 cells were treated with Decitabine for 48 h, the methylation level of DR4 gene promoters gradually decreased, while the mRNA expression level of DR4 genes gradually increased, both of which showed a concentration-dependent relationship. After the K562 cells were treated with 5 µmol/L Decitabine for 48 h, the K562 cells in G0/G1 phase and G2/M phase increased significantly, and the K562 cells in S phase decreased significantly.ConclusionDR4 gene played an important role in the occurrence and development of AML. Decitabine can effectively inhibit the proliferation of K562 cells, which probably partly because it can terminate the methylation effect of DR4 gene promoters and restore the mRNA expression of DR4 genes.

Expression of lymphoid enhance factor 1 in chronic myeloid leukemia and its clinical significance

Objective To analyze the mRNA expression level of lymphoid enhance factor 1 (LEF-1), and to investigate its clinical significance in bone marrow mononuclear cells of patients with chronic myeloid leukemia chronic-phase (CML-CP) after initial diagnosis and chemotherapy, and to analyze its clinical significance. Methods The real-time fluorescence quantitative polymerase chain reaction was used to measure the expression level of LEF-1 gene in 38 CML-CP patients after initial diagnosis and chemotherapy and 20 persons without blood system diseases and neoplastic diseases as normal control. The difference of LEF-1 expression level between the patients and healthy control was compared, and the effect of imatinib on the main molecular response (MMR) was analyzed. Results The expression of LEF-1 mRNA in 38 newly diagnosed patients [0.002 14 (0.000 20-0.021 20)] was significantly higher than that in normal controls [0.001 01 (0.000 09-0.002 33)] (U= 163.0, P 0.05). The level of LEF-1 mRNA expression of non-MMR group was also higher than that of the normal control group (U= 14.0, P < 0.01). The rate of acquiring MMR was significantly higher in high LEF-1 mRNA expression group [84.2 % (16/19)] than that in low expression group [47.4 % (9/19)] (χ 2 = 4.209, P < 0.01). The time of acquiring MMR was significantly shorter in the high LEF-1 mRNA expression group [(10.0 ± 4.5) months] than that in the low expression group [(14.6 ± 3.8) months] (t = 2.705, P < 0.01). Conclusions LEF-1 may be involved in the occurrence and development of CML, and reflects the tumor burden. It may be one of the indicators to predict the efficacy of imatinib. Key words: Leukemia, myeloid, chronic; Polymerase chain reaction; Lymphoid enhance factor 1; Prognosis

Expression of lymphoid enhance factor 1 in acute myelogenous leukemia patients with intermediate-risk and its clinical significance

Objective To quantitatively analyze the mRNA expression level of lymphoid enhance factor 1 (LEF-1) in bone marrow mononuclear cells of patients with acute myeloid leukemia (AML) at intermediate-risk after initial diagnosis and chemotherapy, and to analyze its clinical significance. Methods The real-time fluorescence quantitative polymerase chain reaction (RT-PCR) was used to measure the expression level of LEF-1 gene in AML patients at intermediate-risk after initial diagnosis and chemotherapy, and its relationship with effectiveness and survival were analyzed. Results The LEF-1 mRNA level in preliminarily diagnosed patients with AML was significantly higher than that in control arm [0.005 19 (0.000 15-0.092 07) vs. 0.001 01 (0.000 09-0.002 33)], and the difference was statistically significant (u= 134.50, P < 0.01). The LEF-1 mRNA level in patients after chemotherapy was significantly declines as compared to that in patients before chemotherapy [0.001 07 (0.000 08-0.007 44) vs. 0.005 19 (0.000 15-0.092 07)], and the difference was statistically significant (u= 317.00, P < 0.01) and LEF-1 mRNA expression level before chemotherapy in complete remission (CR) patients was significantly higher than that in non-CR patients [(0.011 08 (0.001 64-0.092 07) vs. 0.001 10 (0.000 15-0.009 16)], and the difference was statistically significant (u= 19.00, P < 0.01). High LEF-1 expression predicted a significantly better overall survival in AML patients with intermediate-risk cytogenetics (χ2= 4.549, P= 0.033). Conclusions LEF-1 may be involved in the development and progression of AML at intermediate-risk patients and is closely related to tumor burden and treatment efficacy. LEF-1 may be a good predictor of better prognosis and a novel target for therapeutic effect. Key words: Leukemia, myeloid, acute; Polymerase chain reaction; Lymphoid enhance factor 1; Prognosis

IL-32 mRNA Expression of Bone Marrow Stromal Cells and Its Correlation with Cell Apoptosis in Patients with Myelodysplastic Syndrome

To investigate the IL-32 mRNA expression of bone marrow stromal cells and its correlation with apoptosis of bone marrow mononuclear cells in patients with myelodysplastic syndrome (MDS). Bone marrow samples from 26 MDS patients and 10 iron deficiency anemia (IDA, as control) patients were collected, RT-PCR was used to detect the IL-32 mRNA expression of bone marrow stromal cells, and the apoptosis of bone marrow mononuclear cells was detected by flow cytometry with Annexin V-FITC/PI dowble staining. The born marrow lymphocytes and NK cells were detected by means of direct immunofluorescence labeling whole blood hemolysis and flow cytometry. IL-32 mRNA expression of bone marrow stromal cells in the MDS patients was significantly higher than that of control group, the IL-32 mRNA expression of bone marrow stromal cells in patients with RA, RAS and RCMD was significantly higher than that in patients with RAEB. There was no obvious difference between RAEB and the control groups. The apoptosis of bone marrow mononuclear cells in MDS group was significantly higher than that in the control group, the apoptosis of bone marrow mononuclear cells in patients with RA, RAS and RCMD was significantly higher than that in RAEB. There was no significant difference between RAEB group and control group. The IL-32 mRNA expression in bone marrow stromal cells significantly correlated with the apoptosis of bone marrow mononuclear cells in MDS patients. The NK cell number in born marrow of MDS patients and the control group had no significant difference. The expression of IL-32 mRNA in bone marrow stromal cells significantly relates with the apoptosis of MDS cells, and the secretion of IL-32 by bone marrow stromal cells may be one of the reasons for the apoptosis of MDS bone marrow cells. It is speculated that the abnormal MDS bone marrow microenvironment is involved in the apoptosis of bone marrow cells.

Aberrant Expression Notch1 and Asb2 mRNA in Bone Marrow from Patients with P210(+) Chronic Myeloid Leukemia

To explore the mRNA expression level of Notch1 and ASB2 in bone marrow mononuclear cells of patients with P210(+) chronic myeloid leukemia and the correlation between Notch signaling pathway and ubiquitination in chronic myeloid leukemia, so as to provide the valuable information for investigating the pathogenesis of chronic myeloid leukemia and clinical treatment. Bone marrow was collected from 32 patients with newly diagnosed chronic myeloid leukemia and 34 non-hematopathic and healthy individuals (control group), respectively. The mRNA expression levels of Notch1 and ASB2 were detected by real-time quantitative PCR. The expression of Notch1 mRNA in CML patients were significantly different from that of healthy individuals group (t=36.3, P<0.01), which was 337.8 times of the healthy individuals. Moreover, the expression level of ASB2 mRNA in CML group was significantly higher than that in healthy individuals (t=19.4, P<0.01). The mRNA expression of Notch1 and ASB2 gene was positive correlation (r=0.504, P<0.01). The aberrant expression of Notch1 and Asb2 exists in patients with P210 positive CML, which may be involved in incidence and development of CML.

Autophagy level of bone marrow mononuclear cells in patients with myelodysplastic syndromes

目的研究骨髓增生异常综合征（MDS）患者骨髓单个核细胞（BMMNC）自噬水平的变化，探讨细胞自噬在MDS中的作用。方法以38例MDS患者为实验组，以26例巨幼细胞贫血患者为对照组，抽取骨髓并分离BMMNC。应用透射电镜观察自噬情况；采用单丹（磺）酰戊二胺（MDC）染色法检测自噬泡水平；采用RT-PCR、免疫荧光技术及蛋白免疫印迹法检测微管相关蛋白轻链3（LC3）、Beclin1 mRNA及蛋白的表达水平。结果MDS患者BMMNC内易见自噬泡；MDC染色显示MDS患者BMMNC含自噬泡细胞数量显著高于对照组[（9.75±2.63）%对（2.90±0.89）%，P<0.05]；MDS患者BMMNC LC3阳性细胞比例显著高于对照组[（6.13±1.03）%对（1.50±0.58）%，P<0.05]。低危/中危-1 MDS患者BMMNC Beclin1及LC3A mRNA表达水平显著高于对照组（3.61±3.02对1.55±1.03，P<0.05；6.56±3.97对1.21±0.95，P<0.05）；低危/中危-1 MDS患者BMMNC自噬负性调控基因-哺乳动物雷帕霉素靶点（mTOR）mRNA水平显著低于对照组（0.39±0.37对1.50±1.03，P<0.05）；中危-2/高危MDS患者BMMNC Beclin1、LC3和mTOR mRNA表达水平与对照组比较差异均无统计学意义（P值均>0.05）。蛋白免疫印迹法显示低危/中危-1患者BMMNC Beclin1蛋白表达（1.257±0.197）高于对照组（0.528±0.086）及中危-2/高危（0.622±0.118），差异均有统计学意义（P<0.05）。中危-2/高危组和对照组比较差异无统计学意义（P>0.05）。结论低危/中危-1 MDS患者BMMNC自噬水平升高，中危-2/高危MDS组患者BMMNC自噬水平无明显改变，自噬对MDS患者可能起保护性作用，自噬水平相对不足可能与MDS的进展有关。

Breathing pattern in a phase I clinical trial of intraspinal injection of autologous bone marrow mononuclear cells in patients with amyotrophic lateral sclerosis

The safety of autologous bone marrow mononuclear cells (ABMNC) intraspinal infusion in amyotrophic lateral sclerosis (ALS) patients was evaluated considering breathing and sleep patterns. Patients between 20 and 65 years old were eligible if they had definite ALS, spinal onset, a disease duration between 6 and 36 months, FVC>50%, and a below 90% oxygen saturation (T90) <2% of sleep time. The transplant was performed 6 months after enrollment. ABMNC were infused at thoracic 3–4 level. Eleven patients were included. The REM sleep decreased slightly one year after the cell transplant but not significantly. There were no differences in apnea–hipopnea index, mean oxygen saturation and nadir desaturation evolution. An increase of T90 was observed 180 and 360 days after injection (2.95±1.51% and 4.30±4.10% respectively), although it was not statistically significant. The central drive determined by occlusion pressure (P01) and inspiratory flow showed non-significant differences after one year. Intramedullary injection of ABMNC did not worsen the cortico medullar diaphragmatic pathways.

TWIST-1 promotes cell growth, drug resistance and progenitor clonogenic capacities in myeloid leukemia and is a novel poor prognostic factor in acute myeloid leukemia.

Alterations of TWIST-1 expression are often seen in solid tumors and contribute to tumorigenesis and cancer progression. However, studies concerning its pathogenic role in leukemia are scarce. Our study shows that TWIST-1 is overexpressed in bone marrow mononuclear cells of patients with acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). Gain-of-function and loss-of-function analyses demonstrate that TWIST-1 promotes cell growth, colony formation and drug resistance of AML and CML cell lines. Furthermore, TWIST-1 is aberrantly highly expressed in CD34+CD38- leukemia stem cell candidates and its expression declines with differentiation. Down-modulation of TWIST-1 in myeloid leukemia CD34+ cells impairs their colony-forming capacity. Mechanistically, c-MPL, which is highly expressed in myeloid leukemia cells and associated with poor prognosis, is identified as a TWIST-1 coexpressed gene in myeloid leukemia patients and partially contributes to TWIST-1-mediated leukemogenic effects. Moreover, patients with higher TWIST-1 expression have shorter overall and event-free survival (OS and EFS) in AML. Multivariate analysis further demonstrates that TWIST-1 overexpression is a novel independent unfavourable predictor for both OS and EFS in AML. These data highlight TWIST-1 as a new candidate gene contributing to leukemogenesis of myeloid leukemia, and propose possible new avenues for improving risk and treatment stratification in AML.

Impact of BMMSCs from different sources on proliferation of CD34⁺ cells.

There are significant differences on the biological characteristics of bone marrow mesenchymal stem cells (BMMSCs), immunological response, and antigen-presenting functions between patients with psoriasis and normal subjects, but there are no significant differences in aborted fetuses. We examined the differences in BMMSCs between aborted fetuses and patients with psoriasis in this study. Bone marrow from normal subjects, aborted fetuses, and patients with psoriasis were obtained using a MidiMACS machine. Density gradient centrifugation method was used to isolate the bone marrow mononuclear cells of patients with psoriasis and aborted fetus and the cells were cultivated. Bone marrow CD34(+) cells from normal subjects were isolated. MTT colorimetric detection was used to test the proliferation activity of bone marrow CD34(+) cells. The purity of bone marrow CD34(+) cells and BMMSCs was determined by flow cytometry. The BMMSC culture supernatant fluid of patients with psoriasis and aborted fetuses showed no statistically significant difference with bone marrow CD34(+) cell proliferation in normal subjects (P > 0.05).

Direct intramyocardial transthoracic transplantation of bone marrow mononuclear cells for non-ischemic dilated cardiomyopathy: INTRACELL, a prospective randomized controlled trial.

ObjectiveWe tested the hypothesis that direct intramyocardial injection of bone marrow mononuclear cells in patients with non-ischemic dilated cardiomyopathy can improve left ventricular function and physical capacity.MethodsThirty non-ischemic dilated cardiomyopathy patients with left ventricular ejection fraction <35% were randomized at a 1:2 ratio into two groups, control and treated. The bone marrow mononuclear cells group received 1.06±108 bone marrow mononuclear cells through mini-thoracotomy. There was no intervention in the control group. Assessment was carried out through clinical evaluations as well as a 6-min walk test, nuclear magnectic resonance imaging and echocardiogram.ResultsThe bone marrow mononuclear cells group showed a trend toward left ventricular ejection fraction improvement, with magnectic resonance imaging - at 3 months, showing an increase from 27.80±6.86% to 30.13±9.06% (P=0.08) and returning to baseline at 9 months (28.78%, P=0.77). Magnectic resonance imaging showed no changes in left ventricular ejection fraction during follow-up of the control group (28.00±4.32%, 27.42±7.41%, and 29.57±4.50%). Echocardiogram showed left ventricular ejection fraction improved in the bone marrow mononuclear cells group at 3 months, 25.09±3.98 to 30.94±9.16 (P=0.01), and one year, 30.07±7.25% (P=0.001). The control group showed no change (26.1±4.4 vs 26.5±4.7 and 30.2±7.39%, P=0.25 and 0.10, respectively). Bone marrow mononuclear cells group showed improvement in New York Heart Association functional class, from 3.40±0.50 to 2.41±0.79 (P=0.002); patients in the control group showed no change (3.37±0.51 to 2.71±0.95; P=0.17). Six-minute walk test improved in the bone marrow mononuclear cells group (348.00±93.51m at baseline to 370.41±91.56m at 12 months, P=0.66) and there was a non-significant decline in the control group (361.25±90.78m to 330.00±123.42m after 12 months, P=0.66). Group comparisons were non-significant.ConclusionThe trend of intragroup functional and subjective improvement was not confirmed when compared to the control group. Direct intramyocardial application of bone marrow mononuclear cells in non-ischemic dilated cardiomyopathy was not associated with significant changes in left ventricular function. Differences observed within the bone marrow mononuclear cells group could be due to placebo effect or low statistical power.