Screening of aplastic anaemia-related genes in bone marrow CD4+ T cells by suppressive subtractive hybridization / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>CD4(+) T cells play a crucial role in the pathogenesis of aplastic anaemia. However, the mechanisms of over-proliferation, activation, infiltration of bone marrow and damage to haematopoietic cells of CD4(+) T cells in aplastic anaemia are unclear. Therefore, we screened differentially expressed genes of bone marrow CD4(+) T cells of aplastic anaemia patients and normal donors by suppressive subtractive hybridization to investigate the pathogenesis of aplastic anaemia.</p><p><b>METHODS</b>The bone marrow mononuclear cells of a first visit aplastic anaemia patient and a healthy donor of the same age and sex were isolated using lymphocyte separating medium by density gradient centrifugation. With the patients as "tester" and donor as "driver", their CD4(+) T cells were separated with magnetic bead sorting and a cDNA library established by suppressive subtractive hybridization. Then 15 of the resulting subtracted cDNA clones were randomly selected for DNA sequencing and homological analysis. With semiquantitative RT-PCR, bone marrow samples from 20 patients with aplastic anaemia and 20 healthy donors assessed the expression levels of differentially expressed genes from SSH library.</p><p><b>RESULTS</b>PCR detected 89 clones in the library containing an inserted fragment of 100 bp to 700 bp. Among 15 sequenced clones, 12 were known genes including 3 repeated genes. Compared with normal donors, there were 9/12 genes over-expressed in bone marrow CD4(+) T cells of patients with aplastic anaemia. The effects of these genes included protein synthesis, biology oxidation, signal transduction, proliferative regulation and cell migration. Not all these genes had been reported in the mechanisms of haematopoietic damage mediated by CD4(+) T cells in aplastic anaemia.</p><p><b>CONCLUSIONS</b>Screening and cloning genes, which regulate functions of CD4(+) T cells, are helpful in elucidating the mechanisms of over proliferation, activation, infiltrating bone marrow and damaging haematopoietic cells of CD4(+) T cells in aplastic anaemia.</p>

Analysis of expression of mitosis checkpoint gene chfr in bone marrow cells of acute leukemia patients / 中国实验血液学杂志

This study was purposed to investigate the significance of mitosis checkpoint gene chfr expression in acute leukemia (AL). 2 ml of bone marrow were extracted from each of 46 AL patients and 10 normal donors as control and their mononuclear cells were isolated. Then, their chfr expression was detected by using RT-PCR and immunohistochemistry. Normal control blood samples were also analyzed. The results showed that in 15 out of 28 cases of acute non-lymphocytic leukemia and 13 out of 18 cases of acute lymphocytic leukemia expression of chfr gene mRNA and protein significantly decreased as compared with control. The cytogenetic analysis of patients with a decreased Chfr expression revealed abnormal chromosome. In conclusion, Chfr gene is a leukemia-related gene and may play an important role in leukemia pathogenesis.

Expression of P120ctn in non-Hodgkin's lymphoma and its significance / 中国实验血液学杂志

To evaluate the expression of P120ctn in non-Hodgkin's lymphoma (NHL) and to explore its clinical significance, immunohistochemistry stain method was applied to comparatively investigate the protein expression of P120ctn in paraffin-embedded lymph node tissue slices from 40 cases of NHL and 10 cases of reactive hyperplasia of lymph node. The results showed that P120ctn was not detected in reactive hyperplasia of lymph node, but was detected in 55% (22/40) cases of NHL. P120ctn expression increased with the tumor malignancy of NHL, there was a significant difference between the expression rates of P120ctn in low grade (16.7%, 2/12) and intermediate to high grade malignant (71.4%, 20/28) NHL (P < 0.001). Moreover, P120ctn was also detected in vascular endothelial cells of NHL. It is concluded that the level of P120ctn expression is closely related to the malignant grade of NHL, it suggests that P120ctn possibly plays an important role in the malignant proliferation of lymphoma with a certain significance in diagnosis and therapy of lymphoma.

Effects of ligustrazine on expression of VCAM-1/VLA-4 in syngenic bone marrow transplantation of mice / 中国实验血液学杂志

To explore the effect of ligustrazine on the expression of adherent molecule VCAM-1/VLA-4 of bone marrow cells in syngenic bone marrow transplantation (BMT) mice, the mice were divided into 3 groups: normal group (which received no treatment), BMT control group and ligustrazine-treated groups. BMT mouse models were established. The BMT control group and the ligustrazine-treated group were orally administered 0.2 ml saline per mouse and 2 mg ligustrazine per mouse, respectively, twice a day. On the day 7, 14, 21, 28 after BMT, mice were respectively killed. Bone marrow nucleated cells were detected, and then the expression of VCAM-1/VLA-4 was assayed by immunohistochemistry, RT-PCR and flow cytometry analysis, respectively. The results showed that in ligustrazine-treated group, the accounts of bone marrow nucleated cells on the day 7, 14, 21, 28 after BMT were all higher than that in BMT control group. The expression level in the ligustrazine-treated group was significantly higher than that in the BMT control group (P < 0.05 or P < 0.01). It is concluded that ligustrazine can enhance VCAM-1/VLA-4 expression in bone marrow after syngenic bone marrow transplantation in mice, which may be related to the mechanisms underlying the ligustrazine accelerating hematopoietic reconstitution in allogenic bone marrow transplantation.

Expansive effects of aorta-gonad-mesonephros-derived stromal cells on hematopoietic stem cells from embryonic stem cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Hematopoietic stem cells (HSCs) give rise to all blood and immune cells and are used in clinical transplantation protocols to treat a wide variety of refractory diseases, but the amplification of HSCs has been difficult to achieve in vitro. In the present study, the expansive effects of aorta-gonad-mesonephros (AGM) region derived stromal cells on HSCs were explored, attempting to improve the efficiency of HSC transplantation in clinical practice.</p><p><b>METHODS</b>The murine stromal cells were isolated from the AGM region of 12 days postcoitum (dpc) murine embryos and bone marrow (BM) of 6 weeks old mice, respectively. After identification with flow cytometry and immunocytochemistry, the stromal cells were co-cultured with ESCs-derived, cytokines-induced HSCs. The maintenance and expansion of ESCs-derived HSCs were evaluated by detecting the population of CD34+ and CD34+Sca-1+ cells with flow cytometry and the blast colony-forming cells (BL-CFCs), high proliferative potential colony-forming cells (HPP-CFCs) by using semi-solid medium colonial culture. Finally, the homing and hematopoietic reconstruction abilities of HSCs were evaluated using a murine model of HSC transplantation in vivo.</p><p><b>RESULTS</b>AGM and BM-derived stromal cells were morphologically and phenotypically similar, and had the features of stromal cells. When co-cultured with AGM or BM stromal cells, more primitive progenitor cells (HPP-CFCs) could be detected in ESCs derived hematopoietic precursor cells, but BL-CFC's expansion could be detected only when co-cultured with AGM-derived stromal cells. The population of CD34+ hematopoietic stem/progenitor cells were expanded 3 times, but no significant expansion in the population of CD34+Sca-1+ cells was noted when co-cultured with BM stromal cells. While both CD34+ hematopoietic stem/progenitor cells and CD34+Sca-1+ cells were expanded 4 to 5 times respectively when co-cultured with AGM stromal cells. AGM region-derived stromal cells, like BM-derived stromal cells, could promote hematopoietic reconstruction and HSCs' homing to BM in vivo.</p><p><b>CONCLUSIONS</b>AGM-derived stromal cells in comparison with the BM-derived stromal cells could not only support the expansion of HSCs but also maintain the self-renewal and multi-lineage differentiation more effectively. They are promising in HSC transplantation.</p>

Effect of Ligustrazine on the expression of vascular endothelial growth factor in bone marrow stromal cells of radiation injured mice / 中国实验血液学杂志

To evaluate the effect of Ligustrazine on the expression of VEGF in bone marrow stromal cells (BMSCs) of radiation injured mice and to explore the effect of VEGF on the recovery of hematopoiesis and the mechanism of signal transduction, the protein expression of VEGF, focal adhesion kinase (FAK) and mitogen-activated protein kinase (MAPK) in BMSCs were assayed by Western blot, the cell cycle and apoptosis rate of BMSCs were tested by flow cytometry. The effect of Ligustrazine on the hematopoiesis was evaluated at the same time. The results showed that the protein expression of VEGF in BMSCs was decreased significantly after irradiation and increased slowly with the time. The value in Ligustrazine-treated group almost reached normal level, but it remained lower than that in control group on day 14. The changes of phosphorylated FAK and MAPK protein expression had the same tendency. After (60)Co gamma-irradiation, the BMSCs were arrested in G0-G1 phase and apoptosis rate increased; these values recovered slowly with the time and remained higher than that in normal control group on day 14. The recovery of these values in Ligustrazine-treated group was sooner than that in irradiated control group, and they almost reached to the normal levels on day 14. It is concluded that irradiation could inhibit the expression of VEGF in BMSCs and induce apoptosis. The Ligustrazine promotes the recovery of bone marrow microenvironment probably by increasing the expression of phosphorylated FAK and MAPK in BMSCs.

Effect of ligustrazine on the expression of LFA-1 and ICAM-1 in bone marrow transplantation recipient mice / 中国实验血液学杂志

The objective of study was to investigate the effect of ligustrazine on the expression of LFA-1 and ICAM-1 in bone marrow and the mechanism of hematopoietic reconstitution after bone marrow transplantation (BMT). The 150 mice were randomly divided into 3 groups: normal group, saline group and ligustrazine group. The normal group was not treated. The saline group was given normal saline (0.2 ml/mouse, twice a day) through gastric tube, while the ligustrazine group was given ligustrazine (0.2 ml/mouse, twice a day) through gastric tube. At days 7, 14, 21 and 28 after BMT, the survival rate, colony forming unit of spleen (CFU-S), peripheral blood cells, bone marrow mononuclear cells (BMMNC), histologic changes of bone marrow, as well as the expression level of LFA-1 and ICAM-1 were observed. The results showed that in ligustrazine group CFU-S counts on day 10 and survival rate, WBC and Plt amount in peripheral blood, BMMNC counts, hematopoietic tissue volume as well as the expression level of LFA-1 at 7, 14, 21, 28 days after BMT were higher than in saline group (P < 0.01 or P < 0.05). However, mature RBC volume and expression level of ICAM-1 were lower than in the saline group (P < 0.01 or P < 0.05). Furthermore, fat tissue volume was higher at 7 and 14 days (P < 0.01) and was lower at 21 and 28 days (P < 0.01) after BMT than in the saline group. It is concluded that ligustrazine could improve bone marrow microenvironment and promote hematopoietic reconstitution.

The effect of ligustrazine on the expression of CD44 in bone marrow of bone marrow transplantation mice / 中国实验血液学杂志

The aim of this study was to investigate the effects of ligustrazine on the expression of CD44 in bone marrow tissue at the early phase of hematopoietic reconstruction after bone marrow transplantation (BMT) in mice. The mice were divided into 3 groups: normal mice without BMT treatment, BMT only and BMT added with ligustrazine groups. On the days 7, 14, 21, 28 after BMT, peripheral blood cells and bone marrow nucleated cells (BMNC) were counted, while the expression of CD44 was assayed by flow cytometry. CFU-S was determined on the day 10 after BMT. The results showed that in ligustrazine group the WBC, Plt and BMNC counts and hematopoietic tissue area estimation in bone marrow on the days 7, 14, 21, 28 after BMT were all higher than in BMT controlled group, CFU-S counts were higher than in BMT only group too. On the contrary, mature RBC volume was lower than in the BMT only group. The expression of CD44 in ligustrazine group on days 7, 14, 21, 28 after BMT was significantly higher than in BMT only group. It is concluded that ligustrazine can enhance CD44 expression in bone marrow of syngenic BMT mice, which may be one of mechanisms of ligustrazine accelerating hematopoietic reconstitution in bone marrow.