ObjectiveTo investigate the gene expressions of TAZ and Wnt/β-catenin on the postosteogenic cells of mesenchymal stem cells(MSC)in multiple myeloma(MM)patients and to explore the potential therapeutic target of multiple myeloma bone disease (MBD).MethodsBone marrow mononuclear cells MNC from MM and controls were isolated,cultured,expanded and then induced to osteogenic differentiation.Realtime quantitative RT-PCR was employed to detect the osteogenic markers (TAZ,Wnt/β-catenin,OPN,OC,ALP and Cbf α1); and alizarin red staining for mineral deposition.The mRNA expressions of TAZ and Wnt/β-catenin in the two groups were analysed.ResultsAlizarin red staining was positive and the red calcium nodules were appeared on the post-osteogenic cells of MSC.The mRNA expressions of OC,ALP and Cbf α1 were 2.0958±0.5665,2.6670±0.3847,0.8463±0.3473,respectively,on the post-osteogenic cells of MSC in the experimental groups,which were significantly higher than those of pre-osteogenic cells(1.3487±0.9291,1.1452±0.6054,0.4439±0.2945) (t =2.171,6.709,2.795; all P ＜ 0.05).The mRNA expressions of OPN,OC,ALP and Cbf α1 were 2.1096±0.8267,2.8991±0.3531,4.3045±0.2844,1.3273±0.4075,respectively,on the post-osteogenic cells of MSC in the controls,which were significantly higher than those of pre-osteogenic cells (1.2200±0.9091,0.8780±0.3927,1.9161±0.2684,0.6736±0.2513) (t =2.289,12.103,25.134,4.411; all P ＜ 0.05).The mRNA expressions of OPN,OC,ALP,Cbf α1 were 1.2710±0.5636,2.0958±0.5665,2.6670± 0.3847,0.8463+0.3473,respectively,on the post-osteogenic cells of MSC in the experimental groups,which were significantly lower than those of control groups(2.1096 ±0.8267,2.8991 ±0.3531,4.3045±0.2844,1.3273±0.4075) (t =-2.650,-3.805,-10.822,-2.841; all P ＜ 0.05).The mRNA expression of TAZ and β-catenin were 2.2315±1.0723 and 0.5801±0.2159 on the post-osteogenic cells of MSC in MM patients,which were significantly lower than those of control groups (4.4140±0.8325,0.9516±0.2920) (t =±5.085,-3.235;both P ＜ 0.05).ConclusionThe gene expressions of OPN,OC,ALP and Cbf α1,the osteogenesis related genes,are increased in post-osteogenic cells of MSC,which showed the MSC have been successfully induced to osteoblasts.Comparing with control groups,the osteogenic potential of MSC in MM patients is lower.Based on the above research,TAZ and Wnt/β-catenin may present a novel target for the future therapy of MBD.

OBJECTIVETo investigate the morphologic and functional characteristics of the immortalized human liver sinusoidal endothelial cell line (LSEC line).

METHODSImmunofluorescence staining and fluorescence microscopy were used to detect the classic endothelial cell markers in LSEC line, and flow cytometry was used to analyze the purity of the human LSEC line. The morphology (including W-P bodies and surface fenestrations) and phagocytotic capacity of the human LSEC line were observed by transmission and scanning electron microscope. The proliferation curve of the human LSEC line was analyzed by MTT assay. The functional differences between the human LSEC line and human primary LSEC in expression of ELAM-1 and ICAM-1, activities of fibrinolysis (PAI-1, t-PA, u-PA), releasing of IL-6 and IL-8 were compared respectively by enzyme linked immunosorbent assay. Comparison of the susceptibility to hypoxia-reoxygenation induced apoptosis between the human LSEC line and human primary LSEC were investigated by TUNEL.

RESULTSThe established human LSEC line maintained a high proliferative ability and has been passaged for more than 80 times in the absence of any growth factors. Immunofluorescence staining showed that the human LSEC line could express classic endothelial cell marks including von Willebrand Factor (vWF), and could take up acetylated low-density lipoproteins (Ac-LDL). The purity of the human LSEC line was confirmed over 95% by flow cytometric analysis. The W-P bodies and the phagocytosis of Dynabeads was demonstrated by transmission electron microscope. And fenestrations could be found cellular surface with scanning electron microscopy. When compared with human primary LSEC, the human LSEC line has an equivalent responsiveness to tumor necrosis factor in up-regulation of ELAM-1 and ICAM-1. The human LSEC line can also release PAI-1, t-PA, u-PA but can not release IL-6 and IL-8 to TNF-alpha. In contrast, human primary LSEC could release IL-6. The human LSEC line showed higher susceptibility to hypoxia-reoxygenation-induced apoptosis, and the percentage of apoptotic cells was as high as (38.4 +/- 6.7)%, while (28.6 +/- 4.5)% and (7.8 +/- 1.2)% respectively in primary LSEC and in human umbilical vein endothelial cells.

CONCLUSIONSThe established human LSEC line maintains the special phenotypes and the major functional characteristics, and especially maintains the high susceptibility to hypoxia-reoxygenation-induced apoptosis. Therefore it is feasible to use this cell line for the study of liver ischemia-reperfusion injury.

Apoptosis ; Cell Line ; Cell Proliferation ; E-Selectin ; metabolism ; Endothelial Cells ; cytology ; metabolism ; Humans ; Intercellular Adhesion Molecule-1 ; metabolism ; Interleukin-6 ; metabolism ; Interleukin-8 ; metabolism ; Liver ; cytology

OBJECTIVEIn order to investigate the roles of metalloproteinase in inflammatory bone destruction in ankylosing spondylitis (AS), and analyze the mechanism of preventing inflammatory bone destruction of Bushen Qiangdu decoction (BSQDD) in AS cases. Comparisons were made on the expressions of matrix metalloproteinase 9 (MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1) by peripheral blood mononuclear cells (PBMC) between AS patients and healthy controls. The effect of BSQDD was investigated on the expression and of MMP-9 and TIMP-1 produced by PBMC in AS patients.

METHODSFrom March 2005 to March 2006, 30 active AS cases of Kidney-asthenia, Du-cold and blood-stasis syndrome were selected as patients group in the China-Japan Friendship Hospital. There are 27 male patients and 3 female patients. The age range is from 16 to 45, averaging (30.8 +/- 8.8) years. Disease duration is from 0.5 to 10 years. Cases received three-month BSQDD treatment were considered as the treatment group. Twenty healthy persons were included in the control group. Serum and PBMC were separated. The PBMC were stimulated by PHA and PMA, and the supernatant was collected. The mRNA expression of MMP-9 and TIMP-1 in PBMC was analyzed by RT-PCR. The content of MMP-9 and TIMP-1 in serum and culture supernatant of PBMC were detected by ELISA.

RESULTSCompared with health control group, the serum concentration of MMP-9 and TIMP-1 in patients group before treatment increased (P<0.01, P<0.05), but the level of MMP-9 and TIMP-1 in the serum of patients after treatment decreased compared with pre-treatment cases (P<0.05). Furthermore,compared with health control group, PBMC of patients group before treatment expressed higher levels of MMP-9 and TIMP-1 both on transcript level and at protein level (P<0.01, P<0.05), and the expression levels of MMP-9 and TIMP-1 in PBMC in patients after treatment both on transcript level and at protein level was down-regulated compared with pre-treatment (P<0.01, P<0.05).

CONCLUSIONPBMC of AS patients had a higher potential capacity for MMP-9 and TIMP-1. BSQDD possibly prevented inflammatory bone destruction of AS through inhibiting production of MMP-9 and TIMP-1 produced by PBMC.

Adolescent ; Adult ; Case-Control Studies ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Female ; Gene Expression Regulation ; drug effects ; Humans ; Leukocytes, Mononuclear ; drug effects ; metabolism ; Male ; Matrix Metalloproteinase 9 ; biosynthesis ; blood ; genetics ; Middle Aged ; RNA, Messenger ; genetics ; metabolism ; Retrospective Studies ; Spondylitis, Ankylosing ; blood ; drug therapy ; genetics ; metabolism ; Tissue Inhibitor of Metalloproteinase-1 ; biosynthesis ; blood ; genetics ; Young Adult

The study was aimed to explore clinical result of cyclosporin A (CsA) and androgens for treatment of myelodysplastic syndrome (MDS) with refractory anemia. Four cases of MDS-RA were treated with CsA and androgens, while the changes of blood counts, bone marrow and chromosome were observed. The results showed that substantial hematological response was observed in all four patients, that their anemia improved and all transfusion-dependent patients achieved transfusion independence. In conclusion, CsA and Adr therapy was well tolerated. CsA and Adr therapy offer an alternative treatment of MDS with RA. The mechanisms of the benifical effect from this therapy remain the subject of an ongoing study.
Adolescent ; Adult ; Aged ; Androgens ; therapeutic use ; Blood Cell Count ; Cyclosporine ; therapeutic use ; Drug Therapy, Combination ; Female ; Hemoglobins ; analysis ; Humans ; Immunosuppressive Agents ; therapeutic use ; Male ; Middle Aged ; Myelodysplastic Syndromes ; blood ; drug therapy ; Treatment Outcome

OBJECTIVETo evaluate the effect of early application of high-volume hemofiltration treatment (HVHF) on the levels of lactic acid, pro-inflammatory cytokines and C-reactive protein (CRP) in plasma, as well as APACHE II score in patients suffering from severe sepsis.

METHODSThirty patients meeting the diagnosis of severe sepsis were enrolled in the trial within 24 hours of insults. The level of lactic acid, interleukin-6 (IL-6) and CRP in plasma were measured before HVHF and at 24, 48 or 72 h following HVHF treatment.

RESULTThe plasma levels of lactic acid and IL-6 decreased significantly at 24 h, 48 h, 72 h after HVHF (P <0.05), while, IL-10 did not differ significantly following HVHF (P>0.05), when compared with that before HVHF.

CONCLUSIONThe early application of HVHF could clear the plasma lactic acid and pro-inflammatory cytokines, and improve the tissue oxygenation in severe sepsis.

APACHE ; Adult ; C-Reactive Protein ; analysis ; Female ; Hemofiltration ; methods ; Humans ; Interleukin-10 ; blood ; Interleukin-6 ; blood ; Lactic Acid ; blood ; Male ; Middle Aged ; Sepsis ; blood ; therapy ; Treatment Outcome ; Young Adult

OBJECTIVETo analyze the phenotypic and functional characteristics of endothelial (T3A) cells derived from human hepatocellular cell carcinoma.

METHODSEndothelial cells were isolated from human hepatocellular carcinoma specimens. The identification of T3A cells was performed by checking von Willebrand Factor (vWF), CD31, CD34 and Dil-Ac-LDL uptake. The cell surface fenestrations, a specific morphological feature of tumor derived EC, were investigated by scanning and transmission electron microscopy. The phenotypic characteristics of T3A cells were analyzed by fluorescence-activated cell sorter (FACS) and were further conformed by real-time PCR at transcription level. Furthermore, tumor necrosis factor alpha (TNFalpha)-induced cytotoxicity was evaluated by 3-(4, 5-dimethythiazolyl) -2, -diphenyl-2H-tetrazolium-bromide (MTT) assay; Matrix metalloproteinase secretion was detected by zymography; Angiogenic ability in vitro was analyzed by culturing T3A cells in three-dimensional Matrigel plug. Coagulant and fibrinolytic activities were detected by enzyme-linked immunosorbent assay (ELISA).

RESULTSThe isolated T3A cells exhibited classic "spindle-shape" morphology and monolayer growth and contact inhibition properties. Immunofluorescent staining showed that T3A cells expressed vWF, CD31, CD34, and uptake of Dil-Ac-LDL at a high level. The cell surface fenestrations were observed on T3A cells by scanning and transmission electron microscopy. By FACS and real-time PCR, T3A cells were found to express alphav3, alphavbeta5 and TNF receptor p75 at high levels, and TNF receptor p55 and ICAM-1 at low levels, as compared with those in human liver sinusoidal endothelial cells (LSEC). In response to TNFalpha, LSEC exhibited a dose-dependent cytotoxicity, while T3A cells were resistant. Gelatin zymography showed that MMP-2 activity was higher in T3A cells than that in LSEC. In a three-dimensional plug of Matrigel, T3A cells exhibited stronger angiogenic ability as compared with LSEC. In addition, T3A cells released more tissue factor (TF), tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor (PAI-1) and urine plasminogen activator (u-PA) than LSEC in response to TNFalpha.

CONCLUSIONTumor-derived endothelial cells are phenotypically and functionally different from those derived from normal liver tissue.

Antigens, CD34 ; metabolism ; Carcinoma, Hepatocellular ; genetics ; metabolism ; pathology ; Cell Proliferation ; drug effects ; Cell Shape ; Cells, Cultured ; Endothelial Cells ; metabolism ; pathology ; ultrastructure ; Gene Expression ; Humans ; Integrin alphaVbeta3 ; metabolism ; Integrins ; metabolism ; Intercellular Adhesion Molecule-1 ; metabolism ; Lipoproteins, LDL ; metabolism ; Liver Neoplasms ; genetics ; metabolism ; pathology ; Lung ; blood supply ; metabolism ; pathology ; Matrix Metalloproteinase 2 ; metabolism ; Microscopy, Electron, Scanning ; Neovascularization, Pathologic ; metabolism ; pathology ; Phenotype ; Plasminogen Activator Inhibitor 1 ; metabolism ; Platelet Endothelial Cell Adhesion Molecule-1 ; metabolism ; Receptors, Tumor Necrosis Factor, Type I ; metabolism ; Receptors, Vitronectin ; metabolism ; Tissue Plasminogen Activator ; metabolism ; Tumor Cells, Cultured ; Tumor Necrosis Factor Decoy Receptors ; metabolism ; Tumor Necrosis Factor-alpha ; pharmacology ; von Willebrand Factor ; metabolism

OBJECTIVETo investigate the role of adhesion molecules alphavbeta3 and alphavbeta5 and their ligands Del-1 and L1 in the tumor-endothelial cell adhesion in vitro.

METHODSThe expression of alphavbeta3, alphavbeta5 and ICAM-1 in liver sinusoidal endothelial cells (LSEC) and liver cancer endothelial cells (T3A) cultured under normoxia or hypoxia were analyzed by RT-PCR and fluorescent activated cell sorter (FACS). The expression of Del-1 and L1 in six tumor cell lines under normoxia or hypoxia were analyzed by RT-PCR and Western blot, respectively. The adhesion of dye-labeled tumor cells and endothelial LSEC and T3A cells was measured by a fluorescence plate reader after their culture.

RESULTSThe expression of alphavbeta3 and alphavbeta5 were higher in T3A cells than that in LSEC cells, and were upregulated under hypoxia, while the expression of ICAM-1 was lower in T3A cells than that in LSEC cells, and was upregulated under hypoxia only in LSEC. The expression of Del-1 and L1 molecules were obviously different in various tumor cell lines and were differentially regulated under hypoxia. The adhesion of tumor cells with Del-1 or L1 expression was higher in T3A cells than that in LSEC cells, and was significantly increased under hypoxia condition. Furthermore, the adhesion of tumor cells to T3A could be inhibited by antibodies against alphavbeta3 and alphavbeta5, or SiRNAs for beta3 and beta5.

CONCLUSIONalphavbeta3 and alphavbeta5 and their ligands Del-1 and L1 may play an important role in tumor cell migration.

Antibodies ; immunology ; Cell Adhesion ; Cell Hypoxia ; Cell Line, Tumor ; Endothelial Cells ; cytology ; metabolism ; Humans ; Integrin alphaVbeta3 ; genetics ; immunology ; metabolism ; Intercellular Adhesion Molecule-1 ; immunology ; metabolism ; Ligands ; Neoplasms ; metabolism ; pathology ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; pharmacology ; Receptors, Vitronectin ; genetics ; immunology ; metabolism

OBJECTIVETo investigate the phenotypic and functional characteristics of human adrenal microvascular endothelial cells (AdrEC).

METHODSAdrEC were isolated and purified from a sample of human adrenal tissue by sub-cell clone method. The cells identified by flow cytometry for classical endothelial markers von Willebrand factor (vWF) and CD31, uptake of Dil-labeled acetylated low density lipoprotein (Dil-Ac-LDL), as well as phenotypes. The cell fenestrations were checked by scanning electron microscopy. The expressions of endogenous vascular endothelial growth factor (VEGF) mRNA and protein were detected by reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry. The glucocorticoid-induced cytotoxicities in different organs-derived microvascular endothelial cells were compared.

RESULTSHuman AdrEC expressed those classical endothelial markers such as vWF, CD31, and uptake of Dil-Ac-LDL. The phenotypic analysis indicated that alpha-1 proteinase inhibitor, tumor necrosis factor receptor p55, and intercellular adhesion molecule-1 were expressed in human AdrEC. Scanning electron microscopy demonstrated that there were many microvilli and fenestrations on cellular surface. RT-PCR and immunocytochemistry showed that there was expression of endogenous VEGF in AdrEC. In response to glucocorticoid-induced cytotoxicity, microvascular endothelial cells (MVEC) derived from human brain were highly susceptible, MVEC derived from human lung and human liver sinusoidal endothelial cells were sub-sensitive, while AdrEC were highly resistant.

CONCLUSIONHuman AdrEC are specially differentiated and have characteristics that are different from other organ-derived MVEC in phenotypes and functions.

Adrenal Glands ; blood supply ; Cells, Cultured ; Endothelial Cells ; cytology ; physiology ; Humans ; Phenotype ; RNA, Messenger ; biosynthesis ; genetics ; Vascular Endothelial Growth Factor A ; biosynthesis ; genetics

BACKGROUNDEndothelial cell damage is an important pathophysiological step of restenosis after angioplasty and stenting. Cell transplantation has great therapeutic potential for endothelial recovery. We investigated the effect of transplanting endothelial progenitor cells (EPCs) derived from human early fetal aortas in rat injured arteries.

METHODSThe carotid arterial endothelium of Sprague-Dawley rats was damaged by dilatation with a 1.5 F balloon catheter, and then EPCs derived from human early fetal aortas (<14 weeks) were injected into the lumen of the injured artery in transplanted rats, with an equal volume of normal saline injected into control rats. Rats were sacrificed at 2 and 4 weeks after treatment and transplanted cells were identified by immunohistochemical staining with anti-human CD31 and anti-human mitochondria antibodies. Arterial cross-sections were analyzed by pathology, immunohistochemistry, and morphometry.

RESULTSGreen fluorescence-labeled EPCs could be seen in the endovascular surface of balloon-injured vessels after transplantation. The intimal area and intimal/medial area ratio were significantly smaller in the transplanted group than in the control (P < 0.05) and the residual lumen area was larger (P < 0.05). After EPC transplantation, a complete vascular endothelial layer was formed, which was positive for human von Willebrand factor after immunohistochemical staining, and immunohistochemical staining revealed many CD31- and mitochondria-positive cells in the re-endothelialized endothelium with EPC transplantation but not control treatment.

CONCLUSIONEPCs derived from human early fetal aorta were successfully transplanted into injured vessels and might inhibit neointimal hyperplasia after vascular injury.

Animals ; Carotid Arteries ; pathology ; Cell Adhesion ; physiology ; Cell Survival ; physiology ; Cell Transplantation ; Endothelial Progenitor Cells ; cytology ; physiology ; Humans ; Immunohistochemistry ; Microscopy, Fluorescence ; Neointima ; therapy ; Rats ; Rats, Sprague-Dawley

AIMTo study the permeability of nerve growth factor (NGF) liposomes (NGF-L, NGF-SSL, NGF-SSL-T) on the blood-brain barrier (BBB) model and the distribution in vivo, and analyze the correlation between the results in vitro and in vivo.

METHODSThe BBB model in vitro was established by using mouse brain microvascullar endothelial cell, and the model was applied to study the permeability of NGF liposomes. The distribution of NGF of each group was studied by 125I labeled and SDS-PAGE method.

RESULTSThe highest encapsulation proportion was 34%, and the mean size of NGF liposomes was below 100 nm. The permeability of NGF liposomes on in vitro BBB model showed that the liposome could promote NGF to transport across the BBB, the permeability of NGF-SSL-T was the highest. The distribution in the brain showed in an order of NGF concentration NGF-SSL-T > NGF-SSL + RMP-7 > NGF-SSL > NGF-L. There was a close relationship between P(e) (permeability coefficient on in vitro BBB model) and BUI (brain uptake constant in vivo).

CONCLUSIONLiposomes can promote NGF to transport across the BBB, and the transporting ability BBB of NGF-SSL-T which RMP-7 incorporated into the surface of NGF liposomes is the best.

Animals ; Biological Transport ; drug effects ; Blood-Brain Barrier ; Bradykinin ; analogs & derivatives ; pharmacology ; Brain ; metabolism ; Cell Membrane Permeability ; Drug Delivery Systems ; Endothelial Cells ; cytology ; Liposomes ; Male ; Mice ; Nerve Growth Factor ; administration & dosage ; pharmacokinetics ; Particle Size ; Rats ; Tissue Distribution