UNLABELLEDIn this study, the roles of hematopoietic inhibitors elaborated by bone marrow endothelial cells in the proliferation and differentiation of hematopoietic progenitors were investigated. Murine bone marrow endothelial cell conditioned medium (BMEC-CM) was collected and the components with > 10 kD and < 10 kD were obtained by centrifugal ultrafiltration. The effect of BMEC-CM and its components on proliferation of hematopoietic progenitors was evaluated by CFU-GM and HPP-CFC assay and antibody neutralization test. The expression of the inhibitors in BMEC and BMEC-CM was detected by RT-PCR and Western blot, and change of proliferation and differentiation-related genes during expansion of hematopoietic progenitors was examined by membrane hybridization technique.

THE RESULTS(1) When BME C-CM and its components directly were added to CFU-GM and HPP-CFC culture system, BMEC-CM had no effect on colony formation, > 10 kD component enhanced and < 10 kD component inhibited the formation of CFU-GM and HPP-CFC. (2) When BMEC-C M and its components were added to liquid culture system of marrow cells, after 24 hours incubation, CFU-GM decreased and HPP-CFC increased significantly in B MEC-CM group, CFU-GM increased and HPP-CFC had no significant change in > 10 kD component group; and both CFU-GM and HPP-CFC reduced in < 10 kD group. (3) MIP-2, MIP-1 alpha, MSP, TGF-beta, TNF-alpha, IFN-gamma and T beta 4 were expressed in murine marrow endothelial cells, and MIP-2, MIP-1 alpha, MSP, TGF-beta, TNF-alpha and T beta 4 were existed in BMEC-CM. (4) Antibody neutralization test results demonstrated that TGF-beta, MSP, MIP-1 alpha, IFN-gamma and T beta 4 existed in BMEC-CM had significant suppressive effects on CFU-GM and HPP-CFC. (5) T beta 4 combined with 5 hematopoietic cytokines (SCF, IL-3, IL-6, GM-CSF and EPO) added to CD34(+) cells expansion culture system, HPP-CFC significantly increased compared with 5 cytokines group. T beta 4 could downregulated the expression of proliferation and differentiation-related genes and signal transduction-related genes. It is concluded that BM EC-CM promotes the proliferation of early hematopoietic progenitor cells, and this effect is related with the inhibitors existed in BMEC-CM and it could be executed via influencing cell proliferation and differentiation-related genes and signal-related genes.

Animals ; Bone Marrow Cells ; metabolism ; Cell Division ; drug effects ; Cytokines ; genetics ; pharmacology ; Endothelium ; cytology ; metabolism ; Hematopoietic Stem Cells ; drug effects ; physiology ; Mice ; RNA, Messenger ; analysis ; Thymosin ; pharmacology

BACKGROUNDMacrophage stimulating protein (MSP) is produced by human bone marrow endothelial cells. In this study, we sought to observe its effects on inducing the expansion of early hematopoietic progenitor cells which were cultured in a liquid culture system in the presence of the combination of stem cell factor (SCF), interleukin 3 (IL-3), interleukin 6 (IL-6), granulocyte macrophage-colony stimulating factor (GM-CSF), erythropoietin (EPO) (Cys) and MSP or of Cys and bone marrow endothelial cell conditioned medium (EC-CM).

METHODSHuman bone marrow CD34(+) cells were separated and cultured in a liquid culture system for 6 days. Granulocyte-macrophage colony forming unit (CFU-GM) and colony forming unit-granulocyte, erythrocyte, macrophage, megakaryocyte (CFU-GEMM) were employed to assay the effects of different treatment on the proliferation of hematopoeitic stem/progenitor cells. The nitroblue tetrazolium (NBT) reductive test and hoechest 33258 staining were employed to reflect the differentiation and apoptosis of the cells respectively.

RESULTSMSP inhibited the proliferation of CFU-GM and CFU-GEMM in semi-solid culture and the inhibitory effect on CFU-GEMM was stronger than on CFU-GM. MSP inhibited the differentiation of early hematopoietic progenitor cells induced by hematopoietic stimulators. Bone marrow (BM) CFU-GEMM was 2.3-fold or 1.7-fold increase or significantly decreased in either Cys + EC-CM, Cys + MSP or Cys compared with 0 hour control in liquid culture system after 6 days.

CONCLUSIONMSP, a hematopoietic inhibitor, inhibits the differentiation of early hematopoietic progenitor cells induced by hematopoietic stimulators and makes the early hematopoietic progenitor cells expand in a liquid culture system.

Antigens, CD34 ; analysis ; Apoptosis ; drug effects ; Cell Differentiation ; drug effects ; Cells, Cultured ; Chemokine CCL3 ; Chemokines, CC ; pharmacology ; Hematopoietic Stem Cells ; drug effects ; Hepatocyte Growth Factor ; pharmacology ; Humans ; Proto-Oncogene Proteins ; pharmacology

To investigate whether lymphocyte function-associated antigen 1 (LFA-1) and very late antigen 4 (VLA-4) are involved in vasoendothelial adhesion and transendothelial migration of high proliferative potential endothelial progenitor cells (HPP-EPCs), flow cytometry was used to analyze the expression of integrin beta1 and beta2, the expression of intercellular adhesion molecule (ICAM-1, 2) and vascular cell adhesion molecule (VCAM-1) in mouse bone marrow endothelial cells (mBMECs). The adhesion and transmigration through endothelial cells of the HPP-EPCs blocked by functional grade neutralizing antibodies of VLA-4 and LFA-1 were studied in vitro. The results revealed that HPP-EPCs were positive for CD11a and CD49d in HPP-EPCs. The expression of ICAM-1and VCAM-1 of mBMECs increased after activated by IL-1beta and TNF-alpha. The results of adhesion in vitro revealed that the numbers of the adhered and migrated cells in the CD11a antibody group, in the CD49d antibody group and in the combinational antibody group were less than those in the isotype control antibody group. Furthermore, the number of adhered and migrated cells in the combinational antibody group was less than that in the CD11a or the CD49d antibody group (p < 0.05). It is concluded that both LFA-1 and VLA-4 are involved in vasoendothelial adhesion and transendothelial migration of HPP-EPCs.
Animals ; Antigens, CD ; metabolism ; Bone Marrow Cells ; cytology ; Cell Adhesion ; Cell Adhesion Molecules ; metabolism ; Cell Movement ; Cells, Cultured ; Endothelial Cells ; cytology ; Humans ; Integrin alpha4beta1 ; physiology ; Intercellular Adhesion Molecule-1 ; metabolism ; Lymphocyte Function-Associated Antigen-1 ; physiology ; Mice ; Stem Cells ; cytology ; Vascular Cell Adhesion Molecule-1 ; metabolism

OBJECTIVE: To observe the effects of endothelial cells from umbilical cord blood (UCB) on the amplification of human early hematopoietic cells from UCB in vitro. METHODS: Endothelial cells from UCB were cultured by the optimized medium of endothelial cells. There were 2 experiment groups: cytokines group (SCF+IL-3+IL-6+GM-CSF, CKs group) and noncontact group (endothelial cell layer with CKs without contacting the CD34+ cells group). CD34+ cells from UCB were isolated by MiniMACS. After the cells in the CKs group and the noncontact group were cultured for 7 days, the amplifying folds of early hematopoietic cells were assayed. RESULTS: Early hematopoietic cells from UCB were expanded in the CKs group or the noncontact group. The amplifying folds of the noncontact group on early hematopoietic cells were significantly more than those of the CKs group. CONCLUSION The amplification effect of the noncontact group on early hematopoietic cells is superior to that of the CKs group.
Antigens, CD34 ; analysis ; Cell Proliferation ; drug effects ; Coculture Techniques ; Culture Media ; pharmacology ; Cytokines ; pharmacology ; Endothelial Cells ; cytology ; metabolism ; Fetal Blood ; cytology ; metabolism ; Flow Cytometry ; Hematopoietic Stem Cells ; cytology ; metabolism ; Humans ; Immunohistochemistry

To study the effects of serum-free murine bone marrow endothelial cell conditioned medium (mBMEC-CM) on the growth of bone marrow endothelial cells, mBMEC-CM was collected and ultrafiltrated by Centriprep-10. The retentate of mBMEC-CM [molecular weight (MW)>10 kDa] and the filtrate of mBMEC-CM (MW<10 kDa) were obtained. The effect of bone marrow conditioned media, their components and exogenous cytokines on the formation of endothelial cell colonies were observed. The effect of bone marrow conditioned media, their components and exogenous cytokines on the proliferation of murine bone marrow endothelial cells were determined by [(3)H]-thymidine incorporation. The method of hybridizing to the Atlas cDNA array was used to determine the expression of cytokine mRNAs in bone marrow endothelial cells. The results obtained are as follows: vWF was expressed in bone marrow endothelial cells. The original mBMEC-CM and MW>10 kDa component of mBMEC-CM promoted the proliferation of bone marrow endothelial cell colonies and increased [(3)H]-thymidine incorporation of bone marrow endothelial cells. The MW<10 kDa component did not affect the production of endothelial cell colonies and did not increase [(3)H]-thymidine incorporation of endothelial cells. Six cytokines (IL-6, IL-11, SCF, GM-CSF, VEGF, bFGF) promoted the proliferation of bone marrow endothelial cell colonies. VEGF, bFGF and SCF increased [(3)H]-thymidine incorporation of bone marrow endothelial cells. According to the results of the Atlas cDNA array, GM-CSF,TGF-beta,BMP-2, bFGF, SCF, endothelin-2, thymosin beta10, MSP-1, connective tissue GF, PDGF-A chain, MIP-2 alpha, PlGF, neutrophil activating protein ENA-78, INF-gamma, IL-1, IL-6, IL-13, IL-11, inhibin-alpha mRNAs were expressed in endothelial cells. These results suggest that murine bone marrow endothelial cell conditioned medium promotes the proliferation of bone marrow endothelial cells.
Animals ; Bone Marrow Cells ; cytology ; Cell Line ; Cell Proliferation ; drug effects ; Cells, Cultured ; Culture Media, Conditioned ; pharmacology ; Culture Media, Serum-Free ; pharmacology ; Endothelial Cells ; cytology ; Hematopoiesis ; physiology ; Mice

OBJECTIVETo investigate whether the insertion/deletion(I/D) polymorphism in the angiotensin converting enzyme(ACE) gene is associated with essential hypertension in Xinjiang Kazakh isolated population.

METHODSThe study covered 201 hypertensives and 151 normotensive controls in Xinjiang Barlikun Kazakh population. The I/D polymorphism of ACE gene was determined by polymerase chain reaction.

RESULTSThe frequencies of D and I in the hypertensive group (0.44 and 0.56, respectively) were not significantly different from the controls(0.39 and 0.61, respectively, P=0.16). The frequencies of ACE genotypes of DD, ID, and II were 0.18, 0.52, 0.30 in hypertensives respectively and 0.17, 0.43, 0.40 in control group respectively. There was no significant difference in genotypes between hypertensive group and normotensive group (P=0.14).

CONCLUSIONThe results suggested that the I/D polymorphism of ACE gene might not be associated with hypertension in the Kazakh population of Xinjiang Barlikun area.

Asian Continental Ancestry Group ; genetics ; Blood Pressure ; genetics ; China ; ethnology ; Female ; Gene Frequency ; Humans ; Hypertension ; genetics ; INDEL Mutation ; Male ; Middle Aged ; Peptidyl-Dipeptidase A ; genetics ; Polymorphism, Genetic ; Population Groups