Research Progress in the Role of Tim3/galectin-9 in Hematological Malignancy--Review / 中国实验血液学杂志

The incidence of hematological malignant tumor is increasing year by year, and seriously affecting the human health. In addition to the traditional radiation and chemotherapy, immunotherapy has achieved a certain effect in the treatment of blood tumor, but it is limited by exhaustion of CD8

Effect of U2AF1 Mutation to Inflammatory Cytokine Expression in SKM-1 Cells through FOXO3a-Bim Signaling Pathway / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of U2AF1 gene mutation to inflammatory cytokine in SKM-1 cell of human myelodysplastic syndromes (MDS), and whether the above effects were mediated by FOXO3a-Bim signaling pathway.@*METHODS@#Wide-type U2AF1 and mutant U2AF1 (the serine residue 34 was replaced by phenylalanine, and named as S34F) recombinant expression plasmids were constructed. Lentiviruses were packaged and transfected into SKM-1 cells. The expression of FOXO3a was up-regulated by lentiviruses, and its transfection rate was investigated. The cell proliferation was detected by CCK-8 method. Flow cytometry was used to detect the apoptosis and cycle of the cells. The expression pro-inflammatory cytokine IL-1β, IL-6, TNF-α and anti-inflammatory cytokine IL-4 were detected by qRT-PCR. FOXO3a, Bim, Bcl-2 and Bax protein expression levels were detected by Western blot.@*RESULTS@#Compared with the control group, the cell apoptosis rate, pro-inflammatory cytokine IL-1β and TNF-α transcription levels were significantly increased in the S34F group (P<0.05); cell cycle was blocked at the G@*CONCLUSION@#U2AF1 S34F mutation can regulate inflammatory phenotype in SKM-1 cells, which may be mediated through FOXO3a-Bim signaling pathway.

Role of Macrophages in Erythropoiesis -Review / 中国实验血液学杂志

Terminal erythroid differentiation and its regulation occurs in the bone marrow specialized structure termed erythroblastic islands. The role of macrophages in erythropoiesis is closely related with the integrity of erythroblastic island, which is reliant on adhesive interactions between erythroid and macrophage cells. In regulating red blood cell homeostasis of adult, macrophages have 2 major roles, retaining erythroblast in bone marrow, controlling the final maturation of erythroblasts into reticulocytes and clearing old erythrocytes. Recent research demonstrated that body can affect erythropoiesis by regulating the function of macrophages in physiological or pathological conditions. This paper review focuses on the new advances of this aspect in recent years, including adhesion factor and regulatory factor between macrophage and red blood cells, role of macrophage in development of red blood cells, effect of macrophage on erythropoiesis in different conditiones and so on.

Anti-Tumor Effect of Rigosertib on HEL and K562 Cells and Its Related Mechanism / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects of rigosertib on the apoptosis, proliferation and cell cycle of HEL and K562 cells.</p><p><b>METHODS</b>The HEL and K562 cells were treated with different concentration of rigosertib at different time points, the cell apoptosis, proliferation and cycle were determined by using flow cytometry with Annexin V/PI double staining, WST-1 method and 7-AAD assay, respectively. Intracellular signaling proteins were detected by flow cytometry (FCM).</p><p><b>RESULTS</b>Rigosertib induced obvious apoptosis in HEL and K562 cells, and the apoptotic effect was both time-dependent and dose-dependent manner (P<0.05). The low dose of rigosertib inhibited obviously the proliferation of HEL and K562 cells after treatment from 6 to 54 h, Rigosertib arrested HEL and K562 cells into G/M phase. In addition, Rigosertib obviously increased the expression of apoptosis-related proteins such as cleaved caspase 3 and PARP, and reduced the proliferation-related proteins such as BCL-2 and Cyclin D1. Rigosetib inhibited the activation of AKT-GSK signaling through decreasing the expression of AKT, pAKT(Ser473) and GSK-3α/β (S21/9).</p><p><b>CONCLUSION</b>Rigosertib inhibites proliferation, induces apoptosis and cell cycle arrest in G/M phase of HEL and K562 cells. This agent may have potential application prospect in leukemia therapy.</p>

Disorders of Ribosomal Proteins and Related Hematologic Diseases -Review / 中国实验血液学杂志

Ribosomal proteins are key elements of protein synthesis machinery, ribosome. Besides the essential role in ribosome assembly and protein synthesis, there are lots of extraribosomal functions for ribosomal proteins, ranging from regulating cell proliferation and apoptosis to mediating DNA repair, cellular development and differentiation. Based on the typical features of hematologic system, the ribosomal proteins are related with many hematologic diseases, such as Diamond-Blackfan anemia, 5q- syndrome. In this review, the recent research progress on extraribosomal functions and related hematologic diseases are summarised.

Study on phenotypic and cytogenetic characteristics of bone marrow mesenchymal stem cells in myelodysplastic syndromes / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate phenotype, cell differentiation and cytogenetic properties of bone marrow (BM) mesenchymal stem cells (MSC) separated from the myelodysplastic syndrome (MDS) patients. And to analyze cytogenetic aberration of MSC derived from MDS (MDS-MSC) and its mechanism in pathogenesis of MDS.</p><p><b>METHODS</b>Adherent MSC from both myelodysplastic (n = 22) and normal (n = 7) marrow were obtained by a stromal culture procedure. Morphological features were observed by optical microscope. The cell-surface antigens were performed by flow cytometer(FCM). Adipogenic and osteogenic differentiation potential of MSC were identified under specific induction conditions. Standard cytogenetic analysis of both hematopoietic cells and MSC were performed by trypsin-Giemsa (GTG) banding. The karyotype analysis DNA content was determined by FCM to verify the results.</p><p><b>RESULTS</b>The morphology of MDS-MSC was typical slender spindle-shaped cells, MSC obtained from MDS patients had a MSC immunophenotype, lacked the expression of hematopoietic antigens-CD34, CD45 and expressed MSC markers, such as CD73, CD90, and CD105. MDS-MSC layers showed the capability to differentiate towards adipocytes, chondrocytes and osteoblasts. Cytogenetic aberrations were observed in MSC from 14 (64%) MDS patients, usually involve the loss of chromosomal material (92%), and the clonal loss (7 cases, 50%). Two cases of structural aberrations were also detected. Abnormal karyotypes in MSC were still more frequently identified in abnormal hematopoietic cells group (12 out of 13, 92% vs 3 out of 9, 33%, P < 0.05). There were not exactly the same type of chromosomal aberrations between hematopoietic cells and MSC, but different type of the aberrations in the same chromosome were involved.</p><p><b>CONCLUSION</b>MDS-MSC retains the phenotyping characteristics and differentiated function of normal MSC, but has different type of chromosomal abnormalities. A high proportion of loss of chromosomal may be a marker of chromosomal instability of MDS-MSC. Detection of abnormalities in MDS-MSC suggests enhanced genetic susceptibility of these cells in MDS. This may indicate potential involvement of MSC in the pathophysiology of MDS.</p>

GOMA: functional enrichment analysis tool based on GO modules / 癌症

Analyzing the function of gene sets is a critical step in interpreting the results of high-throughput experiments in systems biology. A variety of enrichment analysis tools have been developed in recent years, but most output a long list of significantly enriched terms that are often redundant, making it difficult to extract the most meaningful functions. In this paper, we present GOMA, a novel enrichment analysis method based on the new concept of enriched functional Gene Ontology (GO) modules. With this method, we systematically revealed functional GO modules, i.e., groups of functionally similar GO terms, via an optimization model and then ranked them by enrichment scores. Our new method simplifies enrichment analysis results by reducing redundancy, thereby preventing inconsistent enrichment results among functionally similar terms and providing more biologically meaningful results.

p15(INK4B) methylation on prognosis and response to decitabine in patients with MDS / 中华血液学杂志

<p><b>OBJECTIVE</b>To detect p15(INK4B) methylation levels and the kinetics of the methylation status before and after decitabine to explore its influences on prognosis and response to decitabine in myelodysplastic syndromes (MDS) patients.</p><p><b>METHODS</b>We examined 261 MDS patients (143 male and 118 female) with the median age of 52 years (32-78). Of them, 172 cases were low-risk group (low-risk 104 cases, intermediate-1 68 cases), 89 cases high-risk group (intermediate-2 52 cases, high risk 37 cases). Collections of bone marrow mononuclear cells of MDS patients and extracted the genomic DNA, the methylation status of p15(INK4B) was detected by methylation-specific PCR (MSP) method. Survival analysis was conducted according to the level of p15(INK4B) methylation in the cohort of patients. The kinetics of the methylation levels of p15(INK4B) in 58 MDS patients before and after 2 courses of decitabine have been assessed with the method of MSP.</p><p><b>RESULTS</b>The methylation level of p15(INK4B) in low-risk group patients was significantly lower than that in high-risk group (117.22 vs 157.63, P<0.05 ). The expected 2-year survival rate of p15(INK4B) methylation positive patients was lower than that of negative ones (91.8% vs 69.8%, P<0.05); the expected 2-year survival rate of p15(INK4B) methylation positive patients was shorter than that of negative ones in low-risk group（78.2% vs 92.0%, P<0.05）, meanwhile there was no significant difference in terms of expected 2-year survival rate and median expected survival between p15(INK4B) methylation positive and negative patients in high-risk group [35.6% vs 38.5%, (17.0±9.3) month vs (18.0±5.7) month, P>0.05]. Multivariate analysis showed p15(INK4B) methylation degree was an independent prognostic factor for overall survival. No statistical difference of overall response (OR) rates were found between p15(INK4B) methylation positive patients and negative patients before decitabine（65.9% vs 76.5%, P>0.05）, and complete remission (CR) rates between these two groups also showed no statistical difference（22.0% vs 29.4%, P>0.05）. p15(INK4B) methylation levels had no obvious change before and after treatment in decitabine responders（P>0.05）.</p><p><b>CONCLUSION</b>The survival of newly diagnosed MDS patients with positive p15(INK4B) methylation was comparatively shorter, but p15(INK4B) methylation had no influence on response to decitabine.</p>

Cytogenetic study of autosomal monosomies among myelodysplastic syndrome patients / 中国实验血液学杂志

Monosomic karyotype (MK) has recently been associated with poor prognosis of myelodysplastic syndromes (MDS). The objective of the current study was to investigate the prevalence and spectrum of autosomal monosomies in an unselected cohort of patients with known or suspected MDS by using retrospective analysis. The results showed that bone marrow cytogenetic studies (1532 cases) were performed between 2004 and 2012, and an abnormal karyotype was found in 538 cases (35.1%). In the 538 cases, 202 (37.5%) cases had autosomal monosomies including sole (n = 47, 23.3%), part of two (n = 33, 21.3%) or more (n = 122, 78.7%) anomalies. Almost all 22 autosomes were involved, but monosomy 7 was by far the most frequent, constituting 66.1% of all isolated monosomies and the highest fraction of those with two or more abnormalities. Other recurrent sole monosomies included chromosomes 20 (15.0%) and 13 (8.5%). Monosomy 13 (12.5%), 18 (8.3%), 20 (6.3%), 17 (7.3%), 21 (5.2%), 5 (5.2%) and 12 (5.2%) were also recurrent in the setting of 3 abnormalities. Bone marrow histology and clinical information were reviewed in all cases with isolated monosomy; associated clinical phenotypes were found in RCMD (n = 20, 13 were -7), RAEB (n = 12, 11 were -7), RA (n = 9, 3 were -7) and chronic myelomonocytic leukemia (CMML, n = 6, 4 were -7) cases. Sole monosomy 20 (n = 7, RA 3 case and RCMD 4 cases) was not detected in RAEB or CMML cases. It is concluded that the presence of at least 1 autosomal monosomy was documented in approximately 37.5% of all abnormal cases, which has potential impact on a more than trivial fraction of patients with MDS. The preponderance of monosomy 7 implicates a pathogenetic role for haploinsufficiency of genes associated with chromosome 7. The rarity of sole monosomy involving other chromosomes other than 7, 20, and 13 suggests that haploinsufficiency involving entire chromosomes is detrimental to cell survival, unless their effect is overcome by the presence of other genetic changes that are often associated with additional chromosomal abnormalities. The observation is consistent with the usually favorable prognostic profile associated with sole monosomy 20.

Study of aberrant p73 promoter methylation in patients with myelodysplastic syndrome / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the methylation status of p73 gene promoter in patients with myelodysplastic syndrome (MDS) and explore its significance with clinical prognosis.</p><p><b>METHODS</b>Methylation of p73 promoter was detected in bone marrow cells from 135 MDS patients and 13 healthy controls by methylation-specific PCR (MSP). The results of MSP were confirmed by bisulfite sequencing. The expression of p73 mRNA was detected by real-time quantitative PCR. Primary bone marrow cells from MDS patients were treated with decitabine, the changes of p73 methylation status and p73 mRNA expression were measured. The role of p73 methylation in the prognosis of MDS and the correlated clinical data were explored.</p><p><b>RESULTS</b>p73 hypermethylation was present in 37.04% of MDS cases and patients with high risk MDS (RAEB-1 and RAEB-2) exhibited a significantly higher frequency of p73 methylation than that of low risk MDS (58.8% vs 29.7%, P = 0.002). The expression of p73 mRNA in the methylated group was decreased compared to that of the unmethylated group (P = 0.032). Decitabine treatment decreased the level of p73 methylation and increased the level of p73 transcripts. Patients with p73 methylation progressed rapidly to AML (P < 0.001) and had shorter survival (P = 0.002) than those who did not have p73 methylation. In the multivariate Cox regression model, BM blast and p73 methylation status emerged as independent prognostic factor for overall survival and leukemia free survival.</p><p><b>CONCLUSION</b>p73 gene methylation is common in patients with MDS and may indicate poor prognosis. p73 may be a therapeutic target in MDS.</p>

Primary study on origination of bone marrow abnormal clones in patients with myelodysplastic syndrome / 中国实验血液学杂志

The study was aimed to investigate the origination of abnormal clones in hematopoietic cells of MDS patients. That is to say if there are abnormal clones in CD34(+)CD38(-) and CD34(+)CD38(+) cells and their proportions in MDS patients. Immuno-magnetic bead technique was used to sort CD34(+)CD38(-) and CD34(+)CD38(+) in bone marrow mononuclear cells of 9 MDS patients with chromosome abnormalities (four cases with trisomy 8, 1 case with trisomy 8 complex karyotype, 2 cases with 5q(-), 1 case with 5q(-)complex karyotype, 1 case with 5q(-) accompanying trisomy 8) and smears were made respectively. Then the percentage of abnormal clones in CD34(+)CD38(-) and CD34(+)CD38(+) cells were compared by using FISH. The results indicated that abnormal clones were involved in the two population cells in 9 patients. The percentage of abnormal clones in CD34(+)CD38(-) cells (41.8 ± 8.4%)was obviously lower than that in CD34(+)CD38(+) cells(72.4 ± 7.7%) (p < 0.001), and the percentage of abnormal clones in karyocytes was 70.8 ± 9.2%. It is concluded the abnormal clones of bone marrow hematopoietic cells may originate from stem cell stage in MDS patients with 5q(-) and +8, and the abnormal clones are predominant at stage of progenitors.

The affection of bisphosphonates combined with chemotherapy on bone metabolism index in multiple myeloma / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the clinical value of urine N-telopeptides of type I collagen (uNTX) and serum bone specific alkaline phosphatase (sBAP) in myeloma bone disease, and to understand the role of bisphosphonates therapy for multiple myeloma(MM) osteolytic bone lesion.</p><p><b>METHODS</b>Thirty-three MM cases were treated with bisphosphonates combined with chemotherapy (considered as treatment group), and 20 untreated MM cases with chemotherapy alone considered as control group. uNTX was detected by ELISA, and sBAP by chemiluminescence analysis.</p><p><b>RESULTS</b>(1) There was no significant differences in uNTX between treatment \[(173.74 ± 14.55) µg/L\] and control groups \[(129.79 ± 12.13) µg/L\] before bisphosphonates treatment (P > 0.05). After six-month treatment, there was significant differences between two groups \[(85.71 ± 8.23) µg/L and (121.59 ± 12.43) µg/L, respectively\] (P < 0.05); Meanwhile, there were significant differences in uNTX between before and after three-month treatment (P = 0.045) and between before and after six-month treatment (P < 0.01) in treatment group. (2) There was no significant differences in sBAP concentration between treatment and control groups \[(4.78 ± 0.55) µg/L and (8.42 ± 1.32) µg/L, respectively\] before treatment (P > 0.05). After six-month treatment, there were significant differences between them \[(16.01 ± 0.52) µg/L and (9.62 ± 1.29) µg/L, respectively\] (P < 0.01). Meanwhile, in treatment group, there was no significant differences between before and after three-month treatment (P > 0.05), but being significant difference between before and after six-month treatment (P < 0.01).</p><p><b>CONCLUSION</b>uNTX, sBAP are important early sensitive index to measure the osteolytic bone lesion in MM patients. Bisphosphonates can significantly improve the osteopathy in MM cases.</p>

Biological behavior of stromal cell-derived factor-1 on migration, adhesion and apoptosis in different kinds of AML cell lines / 中国实验血液学杂志

The study was aimed to investigate the biological behavior of stromal cell-derived factor-1 (SDF-1) in migration, adhesion and apoptosis as well as the related signaling transduction pathways in different kinds of acute myeloid leukemia (AML) cell lines. The expression of surface molecules on AML (KG1a, ML1 and U937) cells were analyzed by flow cytometry. The cell adhesion was detected by MTT assay. The cell migration was checked by transwell assay. Bcl-xl was checked by immunoblotting after activation of phosphionositide-3 kinase (PI3K) in AML cells treated with SDF-1. The results indicated that the expressions of the surface molecules on AML (KG1a, ML1 and U937) cells were different. The list of the expression showed CD34 (KG1a = 95.6%, ML1 = 4.6%, U937 = 4.8%), CD45 (KG1a = 98.3%, U937 = 97.5%, ML1 = 17.8%), CXCR4 (ML1 = 85.4%, U937 = 43.6%, KG1a = 3.8%), ICAM (KG1a = 75.8%, U937 = 41.8% and ML1 = 46.3%). SDF-1 could not upregulate their expression, but could trigger the establishment of polarized morphology of the cells which expressed CXCR4 high. SDF-1 promoted ML1 and U937 cell adhesion to the stroma cells (HS5, HS27), stimulated PI3K in the cells. It was also confirmed that SDF-1 could increase the leukemic cell survival by stimulate this pathway. After addition of wortmaninn or PTX, the cell death increased. It is concluded that the SDF-1 increases the leukemic cell adhesion, migration and survival by stimulating the PI3K pathway. These functions can be depressed by the PI3K inhibitor and also the inhibitor of G protein as well.

Preliminary study of biological characteristics of myelodysplastic syndromes clonal cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the biological difference of clonal cells between myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML).</p><p><b>METHOD</b>Bone marrow (BM) clonal cells (which had cytogenetic markers detected by FISH assay) and blasts were quantitatively analysed in 51 MDS and 11 AML patients. The clonal cell percentage in orthochromatic normoblasts, granulocytes and megakaryocytes were assayed. The biological functions for phagocytosis and oxidation of MDS peripheral blood (PB) neutrophils were compared with that of normal controls.</p><p><b>RESULTS</b>Almost all MDS patients BM had a higher clonal cell percentage (mean 48.2%) than blasts percentage (mean 6.7%) (P < 0.01), but with the subtype of MDS advancing this percentage gap was closing up, and in 11 AML patients no such gap was observed. This gap in MDS patients with + 8 abnormality was smaller than in those with 5q -. In MDS BM, clonal cells were detected in segmented granulocytes (mean 45.9%), orthochromatic normoblasts (mean 46.0%) and mature megakaryocytes (mean 38.0%). In Addition, an approximate amount of clonal cells with the same karyotype abnormality in BM were detected in MDS PB (mean 37.3% in blood vs 48.6% in marrow). Functional analysis showed that the neutrophils in MDS PB could exert nearly normal physiological functions (P > 0.05), but those from AML could not as compared to healthy donors (P < 0.01).</p><p><b>CONCLUSION</b>There is a significant difference in the biological features between MDS and AML clonal cells.</p>

Abnormal polarization of T lymphocyte induces negative hematopoietic regulation in myelodysplastic syndromes / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore polarization of T lymphocyte and its relationship with apoptosis of marrow cells in patients with myelodysplastic syndromes (MDS).</p><p><b>METHODS</b>Measurements of Th1, Th2, Tc1, Tc2 subsets in bone marrows from 34 patients with MDS and 13 normal controls were performed by flow cytometry. INF-gamma and TNF-alpha in marrow serum were determined by ELISA (Enzyme-linked immunosorbent assay). Apoptosis index of marrow cells was detected by TUNEL (TdT-mediated dUTP nick end labeling). Correlations between Th1, Th2, Tc1, Tc2 subsets and INF-gamma, TNF-alpha levels as well as apoptosis index were analyzed, and relationship between TNF-alpha, INF-gamma levels and apoptosis index was also investigated.</p><p><b>RESULTS</b>(1) The percentage of Th1 cells [(10.1 +/- 1.6)%], Tc1 cells [(24.0 +/- 3.6)%] and Tc1/Tc2 ratio (50.0 +/- 11.1) was significantly increased in patients with MDS than in normal controls [(4.0 +/- 0.5)%, (5.8 +/- 0.6)% and 13.4 +/- 2.7, respectively]. Levels of INF-gamma [(58.6 +/- 21.7) microg/L] and TNF-alpha [(15.7 +/- 3.8) microg/L] in marrow serum of MDS patients was markedly elevated compared to normal controls [0 and (0.3 +/- 0.2) microg/L, respectively]. An increased apoptosis index of nucleated cells was observed in MDS patients [(7.8 +/- 1.5)%] as compared to controls [(2.1 +/- 0.3)%, P < 0.05]. The Th1 cell percentage showed a positive correlation with the levels of INF-gamma and TNF-alpha (r = 0.38, P < 0.05 and r = 0.39, P < 0.05, respectively), and with apoptotic index of nucleated marrow cells in MDS patients (r = 0.33, P < 0.05). Furthermore, a positive correlation was observed between INF-gamma, TNF-alpha levels and apoptotic index of marrow cells (r = 0.74, P < 0.01 and r = 0.73, P < 0.01, respectively). (2) Th1, Tc1 cells and Tc1/Tc2 ratio in MDS-RCMD patients was markedly elevated (P < 0.01) but did not in RCMD-RS, RAEB-1 and RAEB-2 patients as compared to normal controls. (3) An elevation in the percentages of Th1, Tcl and Tc1/ Tc2 ratio was detected in patients with IPSS lower-risk but did not in higher-risk group as compared to controls. (4) Increased Th1 and Tc1 percentages and Th1/Th2 and Tc1/Tc2 ratios were observed in RCMD patients with normal karyotype, but did not in those with abnormal karyotype. Conclusions Th1/Th2 and Tc1/Tc2 in bone marrow of MDS patients were unbalanced, polarizing to type I reaction especially in patients with RCMD subtype, IPSS lower-risk and normal karyotype. The increased Th1 cells in bone marrow may account for the increased apoptosis of nucleated marrow cells in MDS, through proapoptotic cytokines such as INF-gamma and TNF-alpha.</p>

Gene profile for differentiation of vascular adventitial myofibroblasts / 生理学报

Our previous study demonstrated that TGF-beta1 could induce the differentiation of vascular adventitial fibroblasts (AFs) to myofibroblasts (MFs). The aim of this study was to identify the genes which might be responsible for the cell phenotypic change using genechips. Cultured rat AFs were treated with TGF-beta1 (10 ng/ml) for 0 min, 5 min, 15 min, 2 h, 12 h and 24 h, respectively. Then the cells were gathered to prepare total RNA. We examined TGF-beta1-induced gene expression profiling using Affymetrix oligonucleotide microarrays and analyzed data by GCOS1.2 software. Moreover, expressional similarity was measured by hierarchical clustering. Some of genechip results were confirmed by real-time quantitative RT-PCR. Microarray analysis identified 2121 genes with a 2-fold change or above after TGF-beta1 stimulation. 1318 genes showed a greater than 2-fold increase and 761 genes were reduced 2 folds or more at mRNA levels, whereas a small portion of the total regulated genes (42 genes) displayed dynamically up- and down-regulated pattern. Genes were further segregated for early (peak at 5 min, 15 min and/or 2 h), late (peak at 12 h and/or 24 h), and sustained (2-fold change or above at five time points) temporal response groups according to the time of their peak expression level. Among 1318 up-regulated genes, 333 genes (25.3%) responded rapidly to TGF-beta1 and 159 genes (12.1%) responded in a sustained manner. Most genes (826, 62.6%) were regulated at 12 h or later. For the 761 down-regulated genes, numbers of early and late responsive genes were 335 (44%) and 267 (36.1%), respectively. There were also 159 genes, 19.9% of total down-regulated genes, decreased at five time points treated by TGF-beta1. The results suggested that the gene expressions of secreted phosphoprotein 1 (APP1) and Rho-associated coiled-coil forming kinase 2 (ROCK2) had the same trends as alpha-smooth muscle-actin, a marker of MF differentiation. In addition, the gene expression of potassium voltage-gated channel, Shal-related family and member 2 (KCND2) was up-regulated. Furthermore, it was found that endothelin 1 (EDN1), some complement components, NADPH oxidase 4 (NOX4) and NAD(P)H dehydrogenase, quinone 1 (NQO1) might be involved in MF differentiation. Using microarrary technique, we confirmed some genes that have been identified by other techniques were implicated in MF differentiation and observed new genes involved in this process. Our results suggest that gene expression profiling study is helpful in identifying genes and pathways potentially involved in cell differentiation.

Gene expression profiling of microvascular endothelial cells during capillary morphogenesis in an in vitro model of angiogenesis / 中华外科杂志

<p><b>OBJECTIVE</b>To globally compare the gene expression profiles during the capillary morphogenesis of human microvascular endothelial cells (HMVECs) in an in vitro angiogenesis system with Affymetrix oligonucleotide array.</p><p><b>METHODS</b>A microcarrier-based in vitro angiogenesis system was developed, in which endothelial cells (ECs) migrated into the matrix, proliferated, and formed capillary sprouts. The sprouts elongated, branched and formed network. The total RNA samples from the HMVECs at the selected time points (0.5 h, 24 h, and 72 h) during the capillary morphogenesis were used for microarray analyses, and the data were processed with the software provided by the manufactory. The expression patterns of some genes were validated and confirmed by Semi-quantitative RT-PCR. The regulated genes were grouped based on their molecular functions and expression patterns, and among them the expression of chemokines/chemokine receptors were specially examined and their functional implications were analyzed.</p><p><b>RESULTS</b>About 1500 genes were found up- or down- regulated 2-folds or above detected by the arrays, and among them, about 400 genes regulated 3-folds or above. The regulated genes could be grouped into categories based on their molecular functions such as growth factor and receptor, cell proliferation, extracellular matrix, cell cycle and apoptosis, signaling molecule and transcription factor, and so on, using the Gene Ontology Mining Tool in The NetAffx Analysis Center. The regulated genes were also clustered into six groups based on their patterns of expression. As for chemokines, the CCL2/MCP-1, CCL5/RANTES and CX3CL1 were identified to be specially upregulated at 24 h time point when the sprouting characterized the morphological change. It was thus suggested that they might exert crucial roles at the early stage of angiogenesis.</p><p><b>CONCLUSIONS</b>Based on our angiogenesis model, and by oligonucleotide arrays, the present study demonstrates global profiles of the gene expression during endothelial capillary morphogenesis, and the results provide us much information about the molecular mechanisms of angiogenesis, with which further evaluation of individual genes can be encouraged.</p>