BACKGROUND:Mesenchymal stem cells (MSCs) exist in human tissues.Presently,cell source is single;culture method has great differences;obtained results are not consistent.Thus,it cannot verfy that isolated and cultured cells are identical calls,which is difficult to compare.OBJECTIVE:To compare the biological features of MSCs derived form bone marrow (BM),perpheral blood (PB) and cord blood (CB) under in vitro culture conditions.DESIGN,TIME AND SETTING:The cytological in vitro controlled study was performed at the Department of Hematology,Navy General Hospital of Chinese PLA from June 2007 to December 2008.MATERIALS:A total of 10 donors of hemopoietic stem cell transplantation at the Department of Hematology,Navy General Hospital of Chinese PLA were selected.MB and PB cells were obtained from the same donor,and cell volumes were respectively 20 mL and 2 mL.CB cells (30 mL) were obtained from healthy primipara at the Department of Obstetrics,Navy General Hospital of Chinese PLA.METHODS:MSCs were obtained from BM,PB and CB by Percoll density gradient + adherence method,and then incubated in DMEM/F12 medium containing 10% fetal bovine serum.When 80%-90% confluency,cells were digested in trypsin-EDTA and made into 5×10~8/L cell suspension as P_0.Above-described operation was performed as P_1,and the rest may be deduced by analogy as P_2-P_5.MAIN OUTCOME MEASURES:The following parameters were measured:cell growth morphology;results of Wright-Giemsa staining;results of cytochemistry;cell proliferation amount;cell surface markers using flow cytometry.RESULTS:Time of adherence,time to 50% confluency and time to 80% confluency of BMSCs were earlier comarped with the PBMSCs and UCMSCs.Adherent cells from BM grew in whirpool-like type,while CB and PB did not at 5-7 days.Majority of aderent cells from BM were fibroblast-like cells,and small parts were endothelioid cells.Aderent cells from PB and CB at the fifth generation contained more endothelioid cells and mononuclear and macrophage-like cells besides fibroblast-like cells.PAS stain,Sudan black B stein,alkaline phosphatase (AKP) staining of adherent cells from BM,PB and CB were negative from P_1 to P_5.Compared with P0 cells,number of BMMSCs till P5 was significantly more in PBMSCs and UCMSCs (P ＜ 0.05).Positive rates of CD29,CD44,CD90,CD71,CD105,CD166 and HLA-ABC were 55.9% 92.8% at P0 to P5,but ≤6% following BMMSCs were incubated;19.7%-33.4% at P0 to P5,but ≤10% following PBMSCs were incubated;35.4%-93.2% at P_0 to P_5,but ≤20% following CBMSCs were incubated.Positive rates of CD34,CD45 and HLA-DR were low in BM-,PB-and CB-MSCs.Positive rates of CD14 and CD31 were low in BMMSCs;12.1%-28.3% in PBMSCs,and 8.1%-21.3% in CBMSCs.CONCLUSION:MSCs can be attained from BM,PB and CB.Quantities of MSCs form BM are the highest,with single component,followed by CBMSCs and PBMSCs,with multiple components.

OBJECTIVETo analyze 68 pediatric cases with functional univentricle heart who underwent bidirectional Glenn procedure during from April 1998 to December 2005.

METHODSThere were 47 males and 21 females in this group, aged from 5 months to 14 years old and weighed from 6.7 to 30.0 kg. Among them, 39 cases were received bidirectional Glenn procedure on the right side, 13 cases on the left side and 16 cases on both sides. Three cases had the pulmonary artery banded; one case had the pulmonary artery ligated;one case had the original A-P shunt cut off; six cases had the PDA ligated; four cases had the MAPCAs cut off; one case had TAPVC corrected contemporarily; two cases of PAPVC were also corrected; four cases had the atrial-ventricular valve repaired.

RESULTSThree cases died. The mortality was 4.4%. The mean post-operative pressure of super vena cava was (15.9 +/- 2.4) mm Hg (1 mm Hg = 0.133 kPa), higher than the pre-operative one (8.3 +/- 1.8) mm Hg (P < 0.01). The mean post operative SpO(2) was (89.3 +/- 4.2)%, higher than the pre-operative one (78.4 +/- 6.0)% (P < 0.01).

CONCLUSIONSBidirectional Glenn procedure is of satisfied effect on surgical treatment for functional univentricle heart. The persistent forward flow from pulmonary artery should be reserved in bidirectional Glenn procedure.

Adolescent ; Child ; Child, Preschool ; Female ; Follow-Up Studies ; Fontan Procedure ; methods ; Heart Defects, Congenital ; surgery ; Humans ; Infant ; Male ; Retrospective Studies ; Tricuspid Atresia ; surgery

OBJECTIVETo investigate the prevalence of JAK2V617F and MPLW515L/K mutation in patients with slightly elevated platelets (BPC) or hemoglobin (Hb) not meeting the criteria of polycythemia vera (PV) or essential thrombocythemia (ET).

METHODSGenomic DNA from bone marrow or blood mononuclear cells was screened with allele specific polymerase chain reaction (AS-PCR) for JAK2V617F and MPLW515L/K mutation. The history of thrombosis was assessed retrospectively by patients files.

RESULTSOf 30 patients, 14 (46.7%) were positive for the JAK2V617F mutation, none of them had the MPLW515L/ K. Five of these 14 patients had a history of thrombosis. Follow-up results were available in 22 patients. Among them, 12 patients with JAK2V617F mutation turned out to be MPD in 6-24 months; only 2 out of 10 patients without this mutation evolved to MPD.

CONCLUSIONJAK2V617F mutation could be one of the diagnosis criteria of early MPD. No MPLW515L/K expression was found in early MPD.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Early Diagnosis ; Female ; Follow-Up Studies ; Humans ; Janus Kinase 2 ; genetics ; metabolism ; Male ; Middle Aged ; Mutation ; Myeloproliferative Disorders ; diagnosis ; genetics ; metabolism ; Receptors, Thrombopoietin ; genetics ; metabolism ; Young Adult

In order to study the potential of Venus, lentiviral vector, applied to acute myeloid leukemia, the recombinant vector Venus-C3aR was transfected into 293T packing cells by DNA-calcium phosphate coprecipitation. All virus stocks were collected and transfected into HL-60, the GFP expression in HL-60 cells was measured by flow cytometry. The expression level of C3aR1 in transfected HL-60 cells was identified by RT-PCR and flow cytometry. The lentiviral toxicity on HL-60 was measured by using CCK-8 method and the ability of cell differentiation was observed. The results indicated that the transfection efficacy of lentiviral vector on HL-60 cells was more than 95%, which meets the needs for further study. C3aR1 expression on HL-60 cells increased after being transfected with recombinant lentiviral vector. Before and after transfection, the proliferation and differentiation of cells were not changed much. It is concluded that the lentiviral vector showed a high efficacy to transfect AML cells and can be integrated in genome of HL-60 cells to realize the stable expression of interest gene. Meanwhile, lentiviral vector can not affect HL-60 cell ability to proliferate and differentiate.
Genetic Vectors ; HL-60 Cells ; Humans ; Lentivirus ; genetics ; Transfection

The objective of study was to investigate tissue-specific transcriptional activity of WT1 (Wilms' tumor gene) promoter and enhancer in cell lines with diverse tissue origin for leukemic gene therapy depending on WT1 transcriptional regulation elements. WT1 promoter and enhancer were ligated into pEGFP-1 to construct a recombinant vectors with EGFP gene as a reporter. By using electroporation or lipofectamine, the resultant constructs were transfected into 13 cell lines including WT1-expressing hematopoietic cell lines (K562, NB4, THP-1 and SHI-1), WT1-nonexpressing hematopoietic cell lines (U937 and Jurkat), WT1-expressing nonhematopoietic cell lines (MCF-7, T47D and 293) and WT1-nonexpressing nonhematopoietic cell lines (ECV304, SMMC7721, HT-29 and SHG44). The mean fluorescence intensity (MFI) of EGFP representing the transcriptional activities of promoter and/or enhancer was analyzed by using flow cytometry in the transfected cells which stably expressed EGFP. The results indicated that the vectors, pEWP containing WT1 promoter and pEWPA containing both WT1 enhancer and promoter, were constructed by recombinant DNA technique. Among nonhematopoietic cell lines, pEWP induced the highest EGFP expression in ECV304 (16.54 +/- 2.45 times as high as pEGFP-1), mildly higher in MCF-7 and SHG44 (9.46 +/- 1.10 and 7.29 +/- 0.73 times of pEGFP-1 level), and lowest in HT-29 (0.99 +/- 0.02 times as much as pEGFP-1) respectively. Among hematopoietic cell lines, EGFP expression was highest in K562 cell line (2.93 +/- 0.27 times of pEGFP-1), which was statistically higher than those in Jurkat and SHI-1 cell lines (0.74 +/- 0.03 and 0.84 +/- 0.09 times of pEGFP-1 level) respectively. pEWPA, with WT1 enhancer inserted at Afl II site near SV40 polyA, increased basal transcription levels of the WT1 promoter in HT-29, SHI-1 and K562 cells by 4.81, 3.06 and 1.01-fold respectively. It is concluded that the transcriptional activities of WT1 promoter in the recombinant vector seem unrelated to the constitutional expression level of endogenous WT1 gene. The WT1 enhancer promotes the transcriptional activities of WT1 promoter in some of the cell lines regardless of the hematopoietic tissue origin.
Enhancer Elements, Genetic ; genetics ; Humans ; Jurkat Cells ; K562 Cells ; Promoter Regions, Genetic ; genetics ; Transcription, Genetic ; U937 Cells ; WT1 Proteins ; genetics ; metabolism

In order to investigate the feasibility of using EGFP as a reporter gene in WT1 transcriptional regulation study. WT1 promoter and enhancer were ligated into the vector pEGFP-1 by recombinant DNA technique and confirmed by restriction enzymes digestion. The resultant constructs were transfected into K562 cell line by DMRIE-C reagent and the function of these WT1 gene elements was detected by using a fluorescent microscope after transfection for 48 hours. The results indicated that the recombinant vectors, pEWP containing WT1 promoter, and pEWPE, pEWPA and pEWPD harboring both WT1 enhancer and promoter, had been successfully constructed. Fluorescence was observed in K562 cells transfected by pEWP, pEWPE, pEWPA and pEWPD, while no fluorescence could be detected in cells transfected by pEGFP-1. It is concluded that EGFP gene as a reporter gene can be applied to the WT1 transcriptional regulation study, which provides the basis for gene therapy.
Enhancer Elements, Genetic ; genetics ; Genes, Reporter ; genetics ; Green Fluorescent Proteins ; genetics ; Humans ; K562 Cells ; Transfection ; WT1 Proteins ; genetics

OBJECTIVETo evaluate the clinical effects of treatment with oral sildenafil on severe pulmonary hypertension after cardiac surgery.

METHODSFrom September 2002 to January 2005, oral sildenafil was added to the treatment regime in 27 cases of severe pulmonary hypertension after cardiac surgery. All these cases were given general treatments including intravenous prostaglandin E1 and inhalation of nitric oxide before the use of sildenafil, which did not show obvious effects on decreasing pulmonary pressure. Then a combined treatment [general treatment plus oral sildenafil (1-2 mg/kg, q8h; Pfizer Ltd)] was instituted. Pulmonary artery pressure, systolic pulmonary artery pressure/systolic systemic blood pressure (Pp/Ps) were measured before and every hour after adding sildenafil.

RESULTSOne hour after adding sildenafil, the patients' pulmonary artery pressure decreased remarkably (P < 0.01) with no adverse effects on systematic artery pressure. SO(2) and PaO(2) of all cases improved respectively (P < 0.05). One or two days later, the patients' hemodynamics were stable and some patients stopped inhaling nitric oxide and the dosage of prostaglandin E1 decreased. 25 cases stopped use of ventilator and were discharged safely. 2 cases died of multiple organ dysfunction.

CONCLUSIONSildenafil is a highly selective and effective pulmonary hypertension vasodilator, which can be given for the treatment of pulmonary hypertension after cardiac surgery.

Adolescent ; Adult ; Cardiac Surgical Procedures ; adverse effects ; Child ; Child, Preschool ; Female ; Humans ; Hypertension, Pulmonary ; drug therapy ; etiology ; Infant ; Male ; Middle Aged ; Piperazines ; therapeutic use ; Purines ; therapeutic use ; Sildenafil Citrate ; Sulfones ; therapeutic use ; Vasodilator Agents ; therapeutic use ; Young Adult

This study was aimed to investigate whether the thrombopoietin (rhTPO) may facilitate myelofibrosis or not. The modified Dexter culture system with various concentrations of rhTPO was used to culture the stromal cells in vitro; the proliferative activity of cells was detected by MTT method; the morphologic changes were observed by light and scanning electron microscopy; the staining changes of ALP, PAS, AS-D NCE and IV type collagen were observed by cytochemistry method; the changes of fibronectin, laminin and IV type collagen were assayed by immunohistochemistry method; the cell surface antigens were assayed by flow cytometry. The results indicated that rhTPO could promote the proliferation of stromal cells which was related to the concentrations of rhTPO. Proliferative activity of stromal cells increased with increasing of rhTPO concentration, and was not related to the exposure time. On day 3 stromal cells adhered to the wall, and became oval. On day 7 stromal cells turned to fusiform and scattered dispersively. On day 12 to 14 these cells ranged cyclically and became long fusiform. Cells covered 70%-80% area of bottle bottom at that time. By day 16 to 18 these cells covered more than 90% area of bottom and ranged cyclically. They displayed the same shape as fibroblasts. By light microscopy with Wrights-Giemsa staining, fibroblasts predominated morphologically, few macrophages, endothelial cells and adipose cells were found. There were no significant differences between experimental group and control group. On day 14 to 42 the adherent cells were positive with PAS staining, poorly positive with ALP and naphthol AS-D chloroacetate esterase (AS-D NCE) staining, and the difference in cytochemistry was not significant between two groups. When these cells were dyed with Masson's trichrome and Gomori's staining, neither collagen fibers nor reticular fibers were positive, but fibronectin, laminin, and collagen type IV appeared positive stronger in experimental group than those in control. The expressions of these molecules were not dependent on culture time. By scanning electron microscopy microvilli and fibers on cell surface appeared more and more, monolayer cells evolved into multilayer cells, and newly-formed fibroblasts appeared gradually as culture time prolonged. These alterations were not different among various groups. The expressions of CD34, CD45, CD105, CD106, and CD166 were not affected obviously by rhTPO. It is concluded that rhTPO had no effects on histochemical properties of stromal cells. Fiber staining and scanning electron microscopic examinations revealed that rhTPO can not facilitate fiber formation of stromal cells. But rhTPO may be able to augment the expressions of fibronectin, laminin and collagen type IV of stromal cells. Therefore it is still necessary to follow up the patients for a long time, who have received rhTPO therapy clinically.
Bone Marrow Cells ; cytology ; drug effects ; Cell Proliferation ; drug effects ; Fibroblasts ; Humans ; Stromal Cells ; cytology ; drug effects ; Thrombopoietin ; pharmacology

OBJECTIVEIn order to investigate whether or not thrombopoietin (TPO) could promote the fibrogenesis of bone marrow stromal cells in absence of megakaryocytes (MKs).

METHODSImproved dexter culture system with various TPO concentrations was used for ex vivo culture of bone marrow stromal cells. Relative proliferation index, the expressions of fibronectin, laminin and type IV collagen, and the systhesis of type III procollagen were detected at different time points during culture process.

RESULTSTPO stimulated the proliferation of bone marrow stromal cells. Relative proliferation index of the stromal cells increased with the TPO concentration increasing, and was not related to the exposure time. The expressions of fibronectin, laminin, and type IV collagen appeared stronger in the TPO groups than those in the control group. But the expressions of these molecules were not dependent upon the culture time. TPO could accelerate the synthesis of type III procollagen in bone marrow stromal cells, and this acceleration was unrelated to the TPO concentration.

CONCLUSIONThese findings suggested that TPO could stimulate the stromal cells with a consequence of increased syntheses and secretions of the extracellular matrix and collagen in absence of MKs. In other words, TPO could promote the fibrogenesis of bone marrow stromal cells without the existence of MKs.

Cells, Cultured ; Collagen Type III ; metabolism ; Collagen Type IV ; metabolism ; Extracellular Matrix ; metabolism ; Fibronectins ; metabolism ; Fibrosis ; pathology ; Humans ; Laminin ; metabolism ; Megakaryocytes ; cytology ; Mesenchymal Stromal Cells ; cytology ; metabolism ; pathology ; Thrombopoietin ; pharmacology

AIMTo explore proper cryopreservative systems for hematopoietic stem cells.

METHODSPeripheral blood mononuclear cells from 20 persons were mixed with different cryopreservative agent, dimethyl suflfoxide (DMSO) or combination of DMSO and hydroxyethyl starch (HES), then cooled in -80 degrees C low temperature refrigerator (Refr) or autocontrolled programmed cryogenic system (PCS), preserved in Refr or in liquid nitrogen. GM-CFU, LTC-IC, CD34+ cells and typeran blue resistance (TBR) were assayed after different period of cryopreservation.

RESULTSThe recovery rates of CFU-GM, LTC-IC, CD34+ cells and TBR in peripheral blood mononuclear cells which were cooled and preserved in Refr with 5% DMSO-6% HES were 82.2% +/- 14.7%, 83.0% +/- 12.2%, 94.2% +/- 4.3% and 97.7% +/- 3.9% respectively, significantly higher than that in Refr with 10% DMSO (P < 0.05). When cells were cryopreservated with the same cryopreservatives, there was no significantly difference of recovery rate in group of Refr and group of Refr with PCS. Meanwhile, there was not significantly difference of recovery rate among all three groups, preserved in Refr ahead of liquid nitrogen, in Refr merely, in liquid nitrogen with PCS within one year (p > 0.05). However, the recovery rate of CFU-GM, LTC- IC, CD34+ cells and TBR decreased dramatically if cells were cooled and preserved in Refr for two years. After cells were thawed, the cell activity declined gradually at room temperature if the cryopreservatives were not removed or diluted. The cell activity of 10% DMSO group was affected more than that of 5% DMSO-6% HES group.

CONCLUSION5% DMSO-6% HES is better than 10% DMSO as cryopreservatives for hematopoietic stem cells. Refr cryopreservation is a simple and effective method if cells would be cryopreserved for less than one year. If cells would be cryopreserved for more than one year, liquid nitrogen cryopreservation should be recommended. The cryopreservatives should be diluted or removed immediately after cells were thawed.

Blood Preservation ; methods ; Cell Survival ; drug effects ; Cryopreservation ; methods ; Cryoprotective Agents ; pharmacology ; Hematopoietic Stem Cell Transplantation ; methods ; Hematopoietic Stem Cells ; cytology ; drug effects ; Humans