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>

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 and cloning the genes differentially expressed in human embryonic AGM-derived stromal cells / 中国实验血液学杂志

To screen and separate the genes differentially expressed in human embryonic aorta-gonad-mesonephros (AGM)-derived stromal cells, a subtracted library was generated through the suppression subtractive hybridization using the cDNA of human embryonic AGM-derived stromal cells as target and human fetal liver (FL)-derived stromal cells as drivers. Then a high though screening technique, gene chip, was used to screen the differentially expressed genes in the established subtractive library. Approximately 18 of the resulting subtracted cDNA clones were partially sequenced and analyzed by blastn in the GenBank database. The results showed that 211 Clones were selected and identified from the established subtractive library, the positive ratio was amount to 76.4%. 18 over-expressed genes were screened by gene chip with more than a 5-fold difference expression levels between AGM and FL-derived stromal cells, and were selected to sequence, results of sequencing indicated that the 18 sequences was compared to known sequences in the GenBank database, and among the sequenced clones, 14 sequences were considered as part of the known genes, and 4 sequences representing previously unknown genes. The known genes were reported to involve the regulation of cell migration, cell differentiation, cell proliferation, cell cycle, signal transduction, and angiogenesis. Most of these genes have not been reported to relate to the haematogenesis in ontogeny. It is concluded that many genes both known and unknown are differentially expressed in human embryonic aorta-gonad-mesonephros-derived stromal cells. Discovery of these genes provides a solid foundation to elucidate the mechanism of haematogenesis in ontogeny.

Screening for Causative Genes Involved in Children with Minimal Change Nephritic Syndrome / 实用儿科临床杂志

Objective To screen for the causative genes involved in the occurrence and development of minimal changes nephritic syndrome(MCNS) and to furtherly assist the genetic diagnosis and treatment of MCNS.Methods Human genome U133 Array Set from Affymetrix Inc was used to evaluate gene expression patterns in peripheral blood mononuclear cells(PBMC) isolated from 7 children with primary MCNS and 7 age-matched health volunteers.Reverse transcription-polymerase chain reaction(RT-PCR) and real-time PCR were performed to identify the findings of gene chip.Results Of 33 000 genes detected,969 genes showed significant difference between children with(MCNS) and healthy volunteers;552 genes were up-regulated,while 417 genes down-regulated significantly.Findings from RT-PCR and real-time PCR were consistent with those of gene chip.Conclusions Gene chip of expression patterns is a powerful method to detect expression difference of genes correlated with MCNS.Occurrence and development of MCNS can be a complicated process that many correlative genes may participate in.

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>

An experimental study on astrocytes promoting production of neural stem cells derived from mouse embryonic stem cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The production of neural stem cells (NSCs) derived from embryonic stem (ES) cells was usually very low according to previous studies, which was a major obstacle for meeting the needs of clinical application. This study aimed at investigating whether astrocytes could promote production of NSCs derived from ES cells in vitro.</p><p><b>METHODS</b>Mouse ES cells line-D3 was used to differentiate into NSCs with astrocytes as inducing stromal cells by means of three-stage differentiation procedure. Another group without astrocytes served as control. The totipotency of ES cells was identified by observation of cells' morphology and formation of teratoma in severe combined immunodeficiency disease (SCID) mice. The quantity and purity of NSCs derived from ES cells were analyzed using clonogenic assay, immunohistochemical staining and flow cytometry assay. The plasticity of NSCs was detected by differentiating test. Octamer-binding transcription factor 4 (Oct-4) and nestin, the specific marker genes of ES cells and NSCs respectively, were detected continuously using reverse transcription-polymerase chain reaction (RT-PCR) method to monitor the process of cell differentiation.</p><p><b>RESULTS</b>The ES cells of D3 line could maintain the ability of differentiating into cellular derivations of all three primary germ layers after continuous passage culture. At the end of two-stage of inducing process, 23.2 +/- 3.5 neurospheres per plate formed in astrocyte-induced group and only 0.8 +/- 0.3 per plate in the control group (clonogenic assay, P < 0.01), and the ratio of nestin positive cells was (50.2 +/- 2.8)% in astrocyte-induced group and only (1.4 +/- 0.5)% in the control group (flow cytometry, P < 0.01). With the induction undergoing, the expression of Oct-4 gradually decreased and then disappeared, while the expression of nestin was increased step by step, and the ratio of nestin positive cells was up to 91.4% by the three-stage differentiation. The nestin positive cells could be further induced into neurons, astrocytes, and oligodendrocytes in differentiating medium supplemented with fetal calf serum. The results of differentiating test showed that the ratio of NF-200 and NSE positive cells was (42.7 +/- 2.6)% in astrocyte-induced group and only (11.2 +/- 1.8)% in the control group (P < 0.01).</p><p><b>CONCLUSIONS</b>Astrocytes can not only increase the production of NSCs derived from ES cells but also promote the differentiation of NSCs toward neuronal lineage.</p>

The effects of Mcl-1 gene on ATRA-resistant HL-60 cell / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the role of Mcl-1 gene in resistance of all-trans retinoic acid (ATRA) of leukemia cells.</p><p><b>METHODS</b>Long-term, intermittent and repetitive exposure of HL-60 cells to ATRA was used to establish a multidrug-resistance cell line (HL-60/ATRA). HL-60/ATRA cells were transfected with Mcl-1 small interference RNA (siRNA) by Lipofectamine 2000. Western blot was used to detect the expression of Mcl-1. The proliferation, apoptosis and differentiation were evaluated by MTT assay, in situ nick end-labeling (TUNEL) and NBT assay, respectively.</p><p><b>RESULTS</b>The HL-60/ATRA could keep its undifferentiated and proliferative status to a high concentration of ATRA (100 nmol/L) with highly expressed Mcl-1 protein (relative grey scale 0.624 +/- 0.127). Mcl-1 gene knockdown by siRNA (relative grey scale 0.267 +/- 0.086) could reverse the resistance of ATRA of HL-60/ATRA by inhibiting proliferation, and inducing differentiation and apoptosis [apoptosis rate (18.5 +/- 4.5)%].</p><p><b>CONCLUSION</b>Mcl-1 gene might be involved in ATRA resistance in HL-60 cells and inhibiting its expression could be a new approach to ATRA resistance reversion.</p>