Gene mutation and myelodysplastic syndromes with ring sideroblast excess / 中国实验血液学杂志

Myelodysplastic syndromes (MDS) are heterogeneous clonal hematopoietic stem cell disorders with different mechanisms and diverse prognosis. The excess of ring sideroblasts (RS) is an important presentation MDS, but the mechanisms of RS appearance are obscure and the treatment of MDS-RS is intractable. Splicing factors play a very important role in the maturation process of eucaryon mRNA, recent studies indicate that there is a significant causal relationship between splicing factor 3B subunit 1 (SF3B1) mutation and the presence of ring sideroblasts. Lucubrating the downstream molecular of the mutated SF3B1 can facilitate exploring the mechanisms and new therapeutic strategies of MDS-RS.

Effect of Gli1 gene silencing on proliferation of K562 cells and its mechanisms / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of Gli1 gene silencing by RNA interference (RNAi) on proliferation of K562 cells and its mechanisms.</p><p><b>METHODS</b>The small interference RNA (siRNA) was synthesized in vitro. K562 cells were transfected with Gli1 siRNA by the way of lipofection (lipofectamine 2000). Non-specific siRNA transfected cells were used as control. Transfection efficiencies of different siRNA concentrations were detected by flow cytometry and the best siRNA concentration was selected. The silencing effect of siRNA was demonstrated by real time PCR and Westem blot analysis. Cell proliferation was measured by MTT method, cell cycle by PI assay, c-myc and p21 mRNA level was detected by real time PCR analysis.</p><p><b>RESULTS</b>Transfection efficiency of siRNA was increased in a dose-dependent manner when siRNA concentration was below 200 pmol, and the highest transfection efficiency reached (80.11 ± 5.63)%. Both the mRNA and protein level of Gli1 was down-regulated in Gli1 specific siRNA group, the mRNA level was (52.60 ± 3.57)% of that of control group after 24 h (t = 20.33, P < 0.01) and the protein level was (79.31 ± 5.58)% of that of control group after 48 h (t = 6.54, P < 0.01). The cell proliferation rate in Gli1 siRNA group was (94.41 ± 3.58)% (t = 2.40, P = 0.05) and (90.22 ± 3.34)% (t = 4.37, P < 0.01) of that of control group after 24 h and 48 h, respectively. G(2)/M cell cycle arrest was observed, the mRNA level of c-myc was down-regulated while p21 was up-regulated in Gli1 siRNA group after 24 h and 48 h (P < 0.05).</p><p><b>CONCLUSIONS</b>Targeted silencing of Gli1 gene by RNAi inhibits the proliferation of K562 cells, which acts through the down-regulation of c-myc and up-regulation of p21 expression.</p>

Relation of notch pathway to senescence of murine bone marrow stromal cells / 中国实验血液学杂志

This study was purposed to investigate the relation of the Notch signaling pathway to senescence of murine bone marrow stromal cells in vitro. Intracellular domain of Notch 1 (ICN) was transfected into cultured murine bone marrow stromal cells by lipofectamine transfection. After transfection for three days the proliferation of transfected cells was measured by MTT, cell cycle distribution was analyzed by flow cytometry. The percentage of senescence associated beta-galactosidase (SA-beta-Gal) positive cells were measured by cytochemical method, and the expression rates of P53 and p21Cip1/Waf1 at gene and protein levels were analyzed by RT-PCR and Western blot respectively. The results showed that after transfection for 3 days the proliferation of murine bone marrow stromal cells was inhibited with induction of G1 arrest, the percentage of SA-beta-gal positive cells increased and the p53 and p21Cip1/Waf1 mRNA and protein expression levels were upregulated. It is concluded that the activated Notch signaling can induce premature senescence of bone marrow stromal cells through the p53-p21Cip1/Waf1 pathway.

Diagnostic role of BIOMED-2 multiplex polymerase chain reaction for antigen receptor genes rearrangement in lymphoproliferative disorders / 中华血液学杂志

<p><b>OBJECTIVE</b>To establish a sensitive and effective method for detection of immunoglobulin and T-cell receptor (Ig/TCR) gene rearrangement,and to explore its role in diagnosis and differential diagnosis of lymphoproliferative disorders.</p><p><b>METHODS</b>Fifty-eight lymphoid tissue samples from 54 patients with lymphoproliferations were evaluated by the novel BIOMED-2 multiplex polymerase chain reaction (PCR) for antigen receptor genes rearrangement.</p><p><b>RESULTS</b>Multiplex PCR demonstrated monoclonal Ig/TCR gene rearrangements in 22 of 25 (88.0%) B-cell malignancies and 8 of 15 (53.3%) T-cell malignancies. Among 17 benign lymphoproliferations confirmed histopathologically, polyclonal rearrangements were detected in 14 cases (82.4%). In total, the clonality analysis and the final clinico-histopathological diagnosis were concordant in 77.2%. Combination detection of Iglambda and TCR delta gene rearrangements did not increase the detection rate of monoclonal rearrangement of Ig/TCR, but might help to the detection of Iglambda+ or TCR delta+ lymphomas.</p><p><b>CONCLUSION</b>The novel BIOMED-2 multiplex PCR strategy is a rapid, reliable and sensitive approach to detecting clonality in suspected lymphoproliferations, especially in atypical cases.</p>

Aberrant methylation at promoter region of HOX A gene cluster in leukemia cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the characteristics of CpG islands methylation at promoter region of HOX A gene cluster in leukemia cells before and after all-trans retinoic acid (ATRA) treatment.</p><p><b>METHODS</b>Eleven human leukemia cell lines, bone marrow cells from leukemia patients before and after therapy and white blood cells from normal subjects were collected. HL-60 and K562 cells were treated by 2-deoxy-5-azacytidine (DAC) or ATRA respectively. Bisulfite modified DNA of these cells were amplified with PCR and quantitatively analyzed by pyrosequencing for methylation of CpG islands.</p><p><b>RESULTS</b>In normal cells, CpGs at all loci of HOX A cluster were unmethylated. In HOX A4, A6, A7, A9, A10 and A11, many CpG sites were methylated (>20%) or hypermethylated (>50%) in leukemia cell lines. Percentages of methylated CpGs were higher in T-cell leukemia (71.4%) and B-cell leukemia (85.7%) than in others. For individual CpGs methylations there were HOX A4 in all leukemia cells, HOX A6 and HOX A7 in most of the leukemia samples and HOX A10 and HOX A11 in K562 and HL-60 cells (38%-86%). HOX A9 CpGs showed hypomethylation in most of myeloid leukemia cells, whereas HOX A11 CpGs were hypermethylated in B-cell leukemia (>50%). Methylation levels of HOX A4 and A6 in AML and ALL patients after complete remission were decreased obviously, and so did HOX A6 and A9 in CML patients. Methylation levels of HOX A4, A6 and A10 in HL-60 cells and of HOX A6 in K562 cells were reduced by ATRA treatment.</p><p><b>CONCLUSIONS</b>In all leukemia cell lines, aberrant methylation of CpGs was observed at promoter regions of 6 HOX A cluster genes, and some of these genes showed leukemia-type-specific hypermethylation. CpGs methylation of some HOX A genes in leukemia cell lines, especially in HL-60 cells, were down-regulated by ATRA.</p>

Establishment of a bone marrow failure mouse model by immune injury / 中华血液学杂志

<p><b>OBJECTIVE</b>To establish a mouse model for the study of pathophysiologic mechanism and treatment of bone marrow failure (BMF).</p><p><b>METHODS</b>Balb/c mice (recipient) were irradiated 5.0 Gy by gamma rays of (60)Co, and then infused 5 x 10(6) lymph node (LN) cells from DBA/2 mice (donor) in 4 hours. Pancytopenia was monitored by cell counting, bone marrow damage was assessed by histological staining and mononuclear cell counting. Serum IFN-gamma concentration was measured by ELISA. The proportion of Treg in spleen was detected by flow cytometry.</p><p><b>RESULTS</b>Irradiation and infusion of LN cells led to rapid development of severe pancytopenia and BM hypoplasia, which reached the most severity at d14. The pancytopenia remained at d28 and displayed no signs of recovery. The bone marrow was full of adipose cells with scarcity of hematopoietic cells at d14 and persisted at least for 28 days, being similar to the feature of aplastic anemia. Serum IFN-gamma concentration was 6.3 fold increased \[(170.0 +/- 17.0) vs (27.7 +/- 7.1) pg/ml\] at d6. Tregs were decreased after infusion, and then increased \[(3.38 +/- 0.52)%\] and recovered to normal \[(4.04 +/- 0.44)%\] at d21. The expression level of the specific transcription factor Foxp3 was similar to normal.</p><p><b>CONCLUSION</b>The MHC antigen of Balb/c mice is identical to that of DBA/2 mice, but their minor antigen differs. 5.0 Gy irradiation and then 5 x 10(6) lymphocyte infusion can induce BMF similar to the features of aplastic anemia.</p>

Study on Wnt and Notch signalling involves in regulation of hematopoietic microenvironment / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the mechanism of Wnt and Notch pathway involved modulating time and spatial restricted hematopoiesis.</p><p><b>METHODS</b>Murine hematopoietic stem and progenitor cells (HSPCs) were isolated from bone marrow (BM) by using c-kit microbeads. E10.5 aorta-gonad-mesonephros (AGM), E12.5, E14.5, E16.5 fetal liver (FL) and adult BM derived stromal cells (StroCs) were isolated and co-cultured with c-kit(+)HSPCs. The floating cells in co-culture system were sorted and counted by FACS. Gene expressions of Wnt and Notch pathway were detected by quantitative PCR and protein expressions by immunostaining.</p><p><b>RESULTS</b>Co-culturing HSPCs with AGM and FL-derived StroCs resulted in an expansion of c-kit(+)population from 0.4 x 10(5)/well to (19.2 +/- 3.2) x 10(5)/well and (26.8 +/- 5.4) x 10(5)/well, respectively, being greater than that with BM-derived StroCs (P < 0.05). The percentage of c-kit(+)cells detected in AGM- and BM- derived StroCs culture system was (75.2 +/- 7.1)%, (74.1 +/- 6.2)% respectively, being higher than FL- derived StroCs culture system (63.4 +/- 5.3)% (P < 0.05). Wnt and Notch pathway genes expression varied at different phases of hematopoiesis. Wnt was highly expressed in AGM and FL derived StroCs, and, Notch did in AGM and BM derived StroCs.</p><p><b>CONCLUSION</b>Wnt and Notch pathway are important modulators in regulating time and spatial restricted hematopoiesis.</p>

Biological characteristics of hematopoietic progenitor cells at different stages of hematopoietic development / 生理学报

The aim of the present paper is to better understand the mechanism of hematopoietic development through studying the biological characteristics of hematopoietic progenitor cells at different stages of development. Firstly, the c-kit expression levels of the mononuclear cells from murine embryonic aorta-gonad-mesonephros (AGM) region at embryonic day (E)10.5 and E11.5, fetal liver (FL) at E12.5, E14.5, E16.5, E18 and bone marrow (BM) were assayed with fluorescence activated cell sorting (FACS). Secondly, hematopoietic progenitor cells derived from AGM at E10.5, FL at E14.5 and BM were isolated by using c-kit microbeads. Isolated c-kit(+) population cells from AGM, FL and BM were then co-cultured with E14.5 FL-derived stromal cells in transwell co-culture system in vitro. After 3, 7, 10 days of co-culture, numerous floating cells were generated. The floating cells generated in transwell inserts were collected for FACS cell count, migration activity detection and colony forming unit (CFU) formation assay. The results showed that the c-kit was highly expressed in E10.5 AGM, with the percentage of c-kit(+) cells declining during AGM development. c-kit expression was highly expressed again in E12.5 FL, declining along with the progressive development of the FL region. Co-cultured with FL-derived stromal cells, E10.5 AGM-derived c-kit(+) cells produced the highest number of hematopoietic cells, while BM-derived c-kit(+) cells produced the lowest number of hematopoietic cells. Compared with E10.5 AGM-derived c-kit(+) cells, E14.5 FL- and BM- derived c-kit(+) cells inclined to differentiate after 7 to 10 days of culture in vitro. E10.5 AGM and E14.5 FL-derived c-kit(+) cells exhibited a higher migration activity than BM-derived c-kit(+) cells. Moreover, E10.5 AGM-derived c-kit(+) cells showed a higher ability to form mixed colony-forming unit (CFU-Mix) colony. In conclusion, compared with FL- and BM-derived c-kit(+) cells, E10.5 AGM-derived c-kit(+) hematopoietic progenitor cells exhibit better proliferation, migration potential, and have a higher ability to maintain the undifferentiation state in vitro, providing an insight into their clinical manipulation.

Clinical analysis of amphotericin B in the treatment of invasive fungal infections in 121 patients with hematologic diseases / 中华血液学杂志

<p><b>OBJECTIVE</b>To observe the efficacy and safety of amphotericin B for treatment of invasive fungal infections (IFI) in patients with hematologic diseases.</p><p><b>METHODS</b>121 patients were given amphotericin B 5 -50 mg/d for 5 - 101 d with a median of 19 d.</p><p><b>RESULTS</b>The clinical efficacy rate was 67.3%, and fungal elimination rate 66.7%. The adverse events included rigor and fever, hypokalaemia, hepatic damage, nephrotoxicity, nausea and vomiting, phlebitis and teeter.</p><p><b>CONCLUSION</b>Amphotericin B is still a high-efficiency drug in the treatment of IFI, although it has many side effects. With monitoring of hepatic and renal function, it is still a relatively safe and effective drug.</p>

Mechanism of ligustrazine promoting hematopoietic reconstitution in syngenic bone marrow transplanted mice / 中国实验血液学杂志

The objective of this study was to investigate the effect of ligustrazine on the expression of stem cell factor mRNA (SCF) in bone marrow tissue and explore the mechanism of hematopoietic reconstitution after bone marrow transplantation (BMT). The colony forming unit of spleen (CFU-S) were counted, the survival rate at days 7, 14 and 21 after BMT were measured, as well as the expression level of SCF mRNA was detected by RT-PCR. The results showed that in ligustrazine group CFU-S counts on day 10 and survival rate, expression level of SCF mRNA on day 7, 14 and 21 after BMT were higher than that in the control group (p < 0.01 or p < 0.05). In conclusion, ligustrazine promotes the recovery of hematopoietic cells in bone marrow, enhances the repair of bone marrow microvessels, and then improves bone marrow microenvironment and promotes hematopoietic reconstitution.

Effect of adriamycin on Gfi-1 expression in K562 cells and its relationship with the relevant apoptotic genes / 中国实验血液学杂志

The study was purposed to explore the effect of adriamycin (ADM) on K562 cells in vitro and the mechanism of expression changes of relevant apoptotic genes and oncogene Gfi-1. The apoptosis was assayed by flow cytometry (FCM) and the DNA electrophoresis; the expression changes of Gfi-1, Bcl-2, bax mRNA and protein were detected by RT-PCR and FCM after K562 cells were treated with different concentrations of ADM for 24 hours. The results showed that when K562 cells were treated with 0 - 2.0 mg/L ADM for 24 hours, the typical apoptotic DNA electrophoresis band of K562 cells were observed with the dose increasing. When concentration of ADM was 0.5 and 2.0 mg/L, the expression of Gfi-1 decreased and the expression of bax increased; when concentration of ADM was 0.5 - 2.0 mg/L, the expression of Bcl-2 was not found to be significantly changed, the levels of Bcl-2 mRNA and protein were of no statistical difference. When dose of ADM was higher than 2.0 mg/L, the percentage of apoptotic K562 cells decreased with cell necrosis. It is concluded that at certain range of concentration, apoptosis or necrosis of K562 cells can be induced by ADM, the percentage of apoptosis, the changes of expression of Bcl-2, bax and Gfi-1 depend on the dose of ADM. The mechanism of apoptosis in K562 cells induced by ADM may be related to suppression of Gfi-1 oncogene and activation of expression of bax gene.

Role of the sonic hedgehog pathway in regulation of the proliferation, migration and differentiation of hemangioblast derived from AGM / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the role of sonic hedgehog (Shh) pathway in regulating the proliferation, migration and differentiation of hemangioblasts derived from aorta-gonad-mesonephros (AGM).</p><p><b>METHODS</b>The hemangioblasts were isolated from AGM region of 11-day postcoitum (dpc) murine embryos by using the immuno-magnetic with CD34 and Flk1 monoclonal antibodies. The phenotypic analysis of hemangioblasts and AGM-derived stromal cells were detected by flow cytometry. The secretion of Shh was examined by immunohistochemical staining. The roles of Shh in regulating the proliferation, migration and differentiation of hemangioblasts in the transwell non-contact coculture system with AGM-derived stromal cells were observed by adding exogenous Shh N-Terminus and its antibody.</p><p><b>RESULTS</b>The protein of Shh was highly expressed on AGM-derived stromal cells. The proliferation of hemangioblasts was promoted when co-cultured with AGM-derived stromal cells, and the effects of the latter could be blocked by antibody of Shh. The proliferation of hemangioblasts was strengthened further and kept for a long time without differentiation and apoptosis when exogenous Shh N-Terminus was added into the transwell non-contact co-culture system with AGM-derived stromal cells. When exogenous Shh N-Terminus was added into the cultural supernatant of hemangioblasts without AGM-derived stromal cells, the hemangioblasts were observed to be induced to apoptosis or differentiation after a short time of proliferation. Furthermore, the ability of migration could be promoted in the co-cultured hemangioblasts by adding exogenous Shh N-Terminus.</p><p><b>CONCLUSION</b>Shh pathway probably involves in the regulation of the proliferation, differentiation, apoptosis and migration of hemangioblasts, and is regulated by the AGM microenvironment.</p>

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>

ERK pathway change in the differentiation of human MDS cell lines SKM-1 induced by sodium butyrate / 中国实验血液学杂志

The study was purposed to investigate the role of extracellular signal-regulated kinase (ERK) pathway in the differentiation of human MDS cell lines SKM-1 induced by sodium butyrate (NaB), and to elucidate the molecular mechanism of differentiation in SKM-1 cells induced by NaB. The expression levels of total ERK and phosphorylated-ERK were determined by Western blot. The effect of NaB in combination with the ERK inhibitor PD98059 on the proliferation/differentiation of SKM-1 cells was studied, and then the expression levels of the P21 and HDAC protein were detected by Western blot. The results showed that the expression level of phosphorylated ERK was down-regulated by the 1 mmol/L NaB, and the level of total ERK had not changed. NaB or combination of the MEK inhibitor PD98059 with NaB could increase the differentiation of the SKM-1 cells and up-regulated the levels of the P21 and HDAC protein, but the effect of combination of NaB with PD98059 was higher than that of NaB alone. It is concluded that the inhibition of ERK may be involved in sodium butyrate inducing differentiation in SKM-1 cells.

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.

Ph+ acute lymphoblastic leukemia combined with lung and brain invasive aspergillosis / 中国实验血液学杂志

This study was aimed to investigate the clinical features and therapy of Ph(+) acute lymphoblastic leukemia (Ph(+)ALL) combined with invasive aspergillosis. A series of examination, including routine blood and bone marrow picture analysis, chest roentgenography, cranial computerized tomography and detection of cell genetics etc were carried out for a Ph(+)ALL patient combined with invasive aspergillosis. This patient received chemotherapy with DVCP, idarubicin and imatinib mesylate and was treated with sporanox and amphotericin B (Amb; including Amb-L) and cerebrotomy for drainage because the invasive aspergillosis occurred during myelosuppression. The results showed that patient gained complete remission and the invasive aspergillosis was controlled successfully. It is concluded that patient with Ph(+)ALL has poor prognosis despite intensive conventional chemotherapy, imatinib mesylate may prove to be an effective treatment for Ph(+)ALL. Because detection rate of the fungus is very low, itraconazole in combination with surgical excision of focus is the best treatment of lung and brain invasive aspergillosis.

Relationship between endostatin and vascular cell adhesion molecule-1 expressions on bone marrow stromal cells in BMT-mice / 中国实验血液学杂志

This study was aimed to investigate the relationship between endostatin and vascular cell adhesion molecule-1 (VCAM-1) expressions on bone marrow stromal cells (BMSC) in mice after bone marrow transplantation (BMT) and effect of ligustrazine on their expressions. The mice were randomly divided into 3 groups: normal group (without treatment), saline group (control of BMT) and ligustrazine group (BMT + ligustrazine). BMT mouse models were established. 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) also through gastric tube. On day 7, 14, 21 and 28 after BMT, mice were killed by euthanasia. The expression levels of endostatin and VCAM-1 in bone marrow stromal cells were detected by immunohistochemistry and RT-PCR analysis respectively. The results showed that the endostatin protein mainly expressed in nuclei of BMSCs, the VCAM-1 protein mainly expressed in plasma of BMSCs. On day 7, 14, 21 after BMT the expression levels of endostatin mRNA and protein in ligustrazine and saline groups were significantly lower than that in normal group (P < 0.01 or P < 0.05), while their expression levels in ligustrazine group were lower than that in saline group. On day 28 the expression levels in saline group returned to normal, while the expression levels in ligustrazine group not were normalized. On day 7, 14, 21 after BMT the expression levels of VCAM-1 mRNA and protein in ligustrazine and saline groups were significantly lower than that in normal group (P < 0.01 or P < 0.05), but their expression levels in ligustrazine group were significantly lighter than that in saline group (P < 0.01 or P < 0.05). On day 28 the VCAM-1 expression level in ligustrazine group returned to normal, while its expression level in saline group not were normalized. The difference between these two groups was significant (P < 0.01). Correlation analysis revealed that there was a negative correlation between endostatin and VCAM-1 expression in saline group, there was a positive correlation between endostatin and VCAM-1 expression in ligustrazine group. It is concluded that the endostatin can influence hematopoiesis in bone marrow by affecting VCAM-1 expression on BMSC and hindering connection between stromal cells and hematopoietic cells as well as extracellular stroma and hematopoietic cells, while ligustrazine can enhance the adhesion molecule expression on stromal cell surface of bone marrow in BMT-mice, accelerate the homing and proliferation of HSPC in bone marrow after BMT, meanwhile can promote the repair of bone marrow microenvironment, accelerate hematopoietic reconstitution of bone marrow after BMT through feedback regulation of endostatin expression of BMSC in BMT-mice.

Antisense oligonucleotide targeting endostatin enhances hematopoiesis reconstitution in BMT mice / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the effect of antisense oligonucleotide targeting endostatin (endostatin-ASON) transfecting bone marrow stromal cells ( BMSC) on hematopoiesis reconstitution in BMT mice.</p><p><b>METHODS</b>Inhibition of endostatin / VCAM-1 protein and mRNA expression was investigated by transfection of antisense oligonucleotide targeting endostatin with confocal microscopy, Western blot and RT-PCR. Bone marrow stromal cells were cultured and divided into 4 groups: group (1) without any treatment; group (2) BMT only; group (3) BMT + endostatin-ASON transfection; group (4) BMT + endostatin scrambled sequence transfection.</p><p><b>RESULTS</b>(1) Endostatin-ASON was successfully introduced into BMSC in vitro, and the transfecting rate was 86% ;(2) After Endostatin-ASON transfected into BMSC, the expression of Endostatin mRNA and its protein on the BMSC was signficantly inhibited at different time point after BMT [the grey value of Endostatin was (0.09 +/- 0.03) - (1.44 +/- 1.19) and (0.02 + 0.02) - (0.14 +/- 0.05), respectively] (P < 0.01 and P < 0.05); (3) Transfecting with Endostatin-ASON effectively promoted the expression of VCAM-1 mRNA and its protein on the BMSC [the gray value of VCAM-1 was (1.60 +/- 0. 92) - (8.05 +/- 0.87) and (0.07 +/- 0.02) - (0.67 +/- 0.09) , respectively] (P <0.01 and P <0.05) ; (4) There was no effects of transfecting Endostatin scrambled sequence on the expression of Endostatin and VCAM-1 on the BMSC (P > 0.05).</p><p><b>CONCLUSION</b>Endostatin-ASON could inhibit Endostatin expression and enhance VCAM-1 expression in BMSC after syngeneic-BMT in mice, which might be one of the mechanisms underlying the endostatin-ASON accelerating hematopoiesis reconstitution after allogeneic-BMT.</p>

Expression of plk-1 gene in acute leukemia patients and its significance / 中国实验血液学杂志

To investigate expression of plk-1 gene and PLK-1 protein in acute leukemia patients and its clinical significance, and to observe the distribution of PLk-1 protein in acute leukemin cells, the mononuclear cells were separated from the bone marrow or peripheral blood of acute leukemia patients, bone marrow benign proliferation individuals and normal individuals. The expression of plk-1 gene and PLK-1 protein in those cells were detected with RT-PCR and flow cytometry respectively, the distribution pattern of PLK-1 was observed by fluorescent inverted microscope. The result showed that the expressions of plk-1 gene and PLK-1 protein in mononuclear cells of acute leukemia patients were much higher than that of bone marrow benign proliferation individuals and normal individuals. Fluorescent inverted microscopy revealed that PLK-1 was highly concentrated in cytoplasm of acute leukemia cells during interphase of mitosis, and it was found that PLK-1 was mainly distributed between sister chromatid during the mitosis in mononuclear cells of acute leukemia patients, but the expressions of plk-1 gene and PLK-1 protein almost were not observed in cells of benign proliferative bone marrow and normal bone marrow. It is concluded that increased plk-1 gene and protein perhaps play an important role in abnormal proliferation of acute leukemia cells and correlate with the malignamcy of leukemia. plk-1 gene or PLK-1 protein may be considered as a new target of therapy, and one of useful indicators in evaluation of curative efficiency and prognosis.

Effect of ligustrazine on the expression of bFGF in bone marrow stromal cells of mice after BMT / 中国实验血液学杂志

This study was purposed to investigate the effect of ligustrazine on the expression of bFGF in bone marrow stromal cells (BMSC) and to explore the mechanism of hematopoietic reconstitution after bone marrow transplantation (BMT). The mice were randomly divided into 3 groups: normal group, saline group and ligustrazine group. BMT mouse models were established. The mice of normal group were not treated, the mice of saline group were given normal saline (0.2 ml/mouse, twice a day) through gastric tube, while the mice of ligustrazine group were given ligustrazine (0.2 ml/mouse, twice a day) through gastric tube. On day 7, 14, 21 and 28 after BMT, the femora were taken and the bone marrow mononuclear cell (BMMNC) suspensions were used for the cultivation of bone marrow stromal cells according to Dexter's culture method. The mRNA and protein expressions of bFGF in BMSC were assayed by RT-PCR and Western blot respectively. The results showed that the expression of bFGF in BMSC on the level of mRNA and protein were all reduced significantly after BMT, and increased slowly with the time. On day 7, 14 and 21 after BMT, the expressions of bFGF mRNA and protein in bone marrow stromal cells of ligustrazine group and saline group were lower than that in bone marrow stromal cells of normal group, but the expressions of bFGF mRNA and protein in ligustrazine group were obviously higher than that in saline group (P < 0.01 or P < 0.05). On day 28 after BMT, the expressions of bFGF mRNA and protein in ligustrazine group returned to normal level, while the expressions of bFGF mRNA and protein in saline group not returned to normal level, there was significant difference between these two groups. It is concluded that ligustrazine can enhance bFGF expression level in bone marrow stromal cells after syngeneic bone marrow transplantation in mice, which confirms that ligustrazine can enhance the repair of bone marrow microvessels, improve bone marrow microenvironment and promote hematopoietic reconstitution.