Cells, Cultured ; Chemokine CCL4 ; Chemotaxis, Leukocyte ; drug effects ; Endothelial Cells ; cytology ; metabolism ; Homocysteine ; pharmacology ; Humans ; Macrophage Inflammatory Proteins ; biosynthesis ; genetics ; Monocytes ; physiology ; RNA, Messenger ; biosynthesis ; genetics ; Umbilical Veins ; cytology

OBJECTIVETo construct recombinant plasmid pEGFP-BMP7 and determine its expression in rat bone marrow mesenchymal stem cells (MSCs) in vitro.

METHODScDNA of target gene was obtained from neonatal rat kidney by RT-PCR. After sequencing the target gene, the cDNA was subcloned into a eukaryote plasmid pEGFP-N1 by directed cloning and then digested with two restrictive endonucleases to verify the correctiveness of the recombinant plasmid pEGFP-BMP7. Rat bone marrow MSCs were transiently transfected with the pEGFP-BMP7 and transfection efficiency of the Green Fluorescent Protein (GFP) was determined. RT-PCR and immunocytochemical analysis were also performed to detect the expression of BMP7 in rat MSCs.

RESULTS1 311 bp cDNA fragment was obtained by RT-PCR and sequence analysis showed it matched perfectly with that of rat BMP7 gene except a single nucleotide change at 756 bp from T to A. Digestion of the recombinant plasmid showed two 1.3 kb and 4.7 kb fragments and their size were same as those of BMP7 and pEGFP. This indicated that BMP7 cDNA was successfully subcloned into pEGFP. Transient transfection showed an efficiency of 33% at day 2 in rat MSCs. After transfection, transcription of BMP7 was detected in MSCs and expression of BMP7 protein was also verified.

CONCLUSIONRecombinant eukaryote plasmid pEGFP-BMP7 was successfully constructed and expressed in rat bone marrow MSCs. This procedure may provide a unique method for stimulation of callus formation in distraction osteogenesis and reconstruction of craniofacial bone defects.

Animals ; Bone Marrow Cells ; Bone Morphogenetic Protein 7 ; Genetic Vectors ; Green Fluorescent Proteins ; In Vitro Techniques ; Mesenchymal Stromal Cells ; Plasmids ; Rats ; Transfection

OBJECTIVETo observe the effects of mesenchymal stem cells (MSCs)on cranial suture under mechanical strain in growing goats.

METHODS10 growing goats were used in this study. A customized distractor was used for distraction of the coronal suture at a rate of 0.4 mm/day for 8 days. The experimental group(5 goats) was injected with autologous MSCs into the distracted region, whereas the control group (5 goats) with injection of physiological saline. All animals were killed at 4 weeks after the end of distraction. Scanning electron microscopy and histological analysis were taken to observe the samples.

RESULTS4 weeks after the end of distraction, the cranial sutures in all animals were separated successfully. The new bone formation at the edge of suture in the experimental group was superior to that in the control group.

CONCLUSIONAutologous MSCs transplantation may promote the cranial suture distraction osteogenesis in the growing goats.

Animals ; Cranial Sutures ; Goats ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; Osteogenesis, Distraction ; Skull

AIMUnderstanding the response of mesenchymal stem cells (MSCs) to mechanical strain and their consequent gene expression patterns will broaden our knowledge of the mechanobiology of distraction osteogenesis.

METHODOLOGYIn this study, a single period of cyclic mechanical stretch (0.5 Hz, 2,000 microepsilon) was performed on rat bone marrow MSCs. Cellular proliferation and alkaline phosphatase (ALP) activity was examined. The mRNA expression of six bone-related genes (Ets-1, bFGF, IGF-II, TGF-beta, Cbfa1 and ALP) was detected using real-time quantitative RT-PCR.

RESULTSThe results showed that mechanical strain can promote MSCs proliferation, increase ALP activity, and up-regulate the expression of these genes. A significant increase in Ets-1 expression was detected immediately after mechanical stimulation, but Cbfa1 expression became elevated later. The temporal expression pattern of ALP coincided perfectly with Cbfa1.

CONCLUSIONThe results of this study suggest that mechanical strain may act as a stimulator to induce differentiation of MSCs into osteoblasts, and that these bone-related genes may play different roles in the response of MSCs to mechanical stimulation.

Alkaline Phosphatase ; analysis ; Animals ; Antigens, Surface ; analysis ; Biomechanical Phenomena ; Bone Marrow Cells ; physiology ; Cell Culture Techniques ; Cell Differentiation ; physiology ; Cell Proliferation ; Core Binding Factor Alpha 1 Subunit ; analysis ; Fibroblast Growth Factor 2 ; analysis ; Insulin-Like Growth Factor II ; analysis ; Mesenchymal Stromal Cells ; physiology ; Osteoblasts ; physiology ; Osteogenesis, Distraction ; Pluripotent Stem Cells ; physiology ; Proto-Oncogene Protein c-ets-1 ; analysis ; Rats ; Stress, Mechanical ; Transforming Growth Factor beta ; analysis ; Up-Regulation ; physiology

OBJECTIVETo study the effect of nerve growth factor (NGF) and Schwann cells on axon regeneration of the inferior alveolar nerve following mandibular lengthening with distraction osteogenesis.

METHODSUnilateral mandibular osteodistraction was performed in 9 healthy adult male goats with a distraction rate of 1 mm/d. Every 3 goats were killed on days 7, 14 and 28 after mandibular lengthening, respectively. The inferior alveolar nerves in the distraction callus were harvested and processed for ultrastructural and NGF immunohistochemical study. The inferior alveolar nerves from the contralateral side were used as controls.

RESULTSOn day 7 after distraction, axon degeneration and Schwann cell proliferation were observed, and very strong staining of NGF in the distracted nerve was detected. On day 14 after distraction, axon regeneration and remyelination were easily observed, and NGF expression started to decline. On day 28 after distraction, the gray scale of NGF immunoreactivity recovered to the normal value and the Schwann cells almost recovered to their normal state.

CONCLUSIONSGradual mandibular osteodistraction can result in mild or moderate axon degeneration of the inferior alveolar nerve. Nerve trauma may stimulate the proliferation of Schwann cells and promote the synthesis and secretion of NGF in the Schwann cells. Schwann cells and NGF might play important roles in axon regeneration of the injured inferior alveolar nerve following mandibular lengthening.

Animals ; Axons ; pathology ; physiology ; Goats ; Immunohistochemistry ; Male ; Mandible ; surgery ; Mandibular Nerve ; physiology ; Nerve Growth Factor ; physiology ; Nerve Regeneration ; physiology ; Osteogenesis, Distraction ; Schwann Cells ; physiology

OBJECTIVETo explore the response of rat bone marrow mesenchymal stem cells (MSCs and calvarial osteoblasts to mechanical strain and the consequent changes of cytoskeleton F-actin.

METHODSBone marrow MSCs and calvarial osteoblasts were isolated from SD rats and cultured in vitro. Mechanical stretch was performed on passage 3 cells at 2 000 microepsilon for 0, 2, 6 and 12 hours using four-point bending system. The response of cells and the distribution of F-actin were observed using fluorescent staining under laser scanning confocal microscope and the morphological parameters were quantified using image analysis software Laserpix.

RESULTSUnder mechanical stretch, the fluorescent staining decreased obviously at both MSCs and osteoblasts, and F-actin filaments were rearranged and became tenuous, thinner, and abnormally distributed. The outline of nucleus became unclear and apoptotic changes were observed at some of both cells. Cellular size decreased more significantly in MSCs than in osteoblasts. Quantity analysis showed that total area of cells, total fluorescent density and green fluorescent density (F-actin) were all significantly decreased in MSCs (P < 0.05 or P < 0.01), and total fluorescent density, green fluorescent density and red fluorescent density (nuclei) did also in osteoblasts (P < 0.05 or P < 0.01).

CONCLUSIONMechanical stretch caused extensive response on both MSCs and osteoblasts which led to the rearrangement of F-actin filament and apoptosis in some of these cells. MSCs were more sensitive to mechanical strain than osteoblasts.

Actin Cytoskeleton ; metabolism ; Actins ; metabolism ; Animals ; Bone Marrow Cells ; Cells, Cultured ; Cytoskeleton ; Mesenchymal Stromal Cells ; Microtubules ; Osteoblasts ; Rats ; Stress, Mechanical

OBJECTIVETo explore the mechanism of 'erythroblast island (EI)' formation in the bone marrow of patients with immun-related hemocytopenia (IRP).

METHODSThe category of BM-auto antibody (au Ab) in 48 patients with IRP was detected with FCM. The BM-au Ab in the 'EI' of these cases were explored with immuonhistofluorescence (IF). Clinical and laboratory characteristics of these cases were also analyzed retrospectively.

RESULTSIgG could be detected in the 'EI' on the BM smear of 14 cases (29.17%), BM-au Ab mainly deposited at the edge/membranes between macrophage and erythroblasts rather than cyto plasm. Positive reaction were seen in all the cases with GlycoAIgG. The red blood cell count [(1.8 ± 0.5) × 10(12)/L] and hemoglobin level [(59.6 ± 16.2)g/L] were significantly lower than that in the IF(-) group [(2.5 ± 0.9) × 10(12)/L and (83.4 ± 25.0) g/L] (P < 0.05). The percentage of reticulocyte [(2.0 ± 0.8)%], serum level of IBIL [(9.4 ± 4.7) µmol/L], percentage of erythroblats in sternum BM (0.441 ± 0.139) and response rate to therapy (85.7%) in IF(+) group were significantly higher than that in IF(-)group [(1.3 ± 1.0)%, (6.6 ± 6.7)µmol/L, 0.298 ± 0.082, 61.3%, respectively] (P < 0.05).

CONCLUSIONMacrophage was connected with erythroblasts through autologous IgG in the 'EI's of some patients with IRP. 'EI' were the places where macrophages devoured and destroyed erythroblasts rather than erythroid development and differentiation. The pathogenetic mechanism of IRP might be associated with macrophages phagocytosing and destroying BM hematopoietic cells.

Blood Cell Count ; Bone Marrow ; Bone Marrow Cells ; immunology ; Coombs Test ; Erythroblasts ; Humans

OBJECTIVETo study the response of rat bone marrow mesenchymal stem cells (MSCs) to a single period of mechanical strain and expression patterns of transforming growth factor-beta (TGF-beta) and insulin-like growth factor-II (IGF-II) after mechanical stretch.

METHODSBone marrow MSCs were isolated from SD rats and cultured in vitro. A four-point bending apparatus were used to perform a single period of mechanical strain (2000 microepsilon, 40 min) on MSCs. Cellular proliferation and alkaline phosphatase (ALP) activity of MSCs were examined and gene expression patterns of TGF-beta and IGF-II were detected by SYBR green quantitative real-time RT-PCR.

RESULTSCell proliferation, ALP activity and expression of TGF-beta and IGF-II were all significantly up-regulated in stretched MSCs when compared with their controls. The mRNA levels of TGF-beta and IGF-II got top increase immediately after mechanical loading and increased about 51.44 and 8.92 folds, respectively, when compared with control cells. Expression of TGF-beta and IGF-II decreased with time and returned to control level at 12 h after mechanical stimulus, despite of a small increase at 6 h.

CONCLUSIONThe mechanical stretch can promote MSCs proliferation, up-regulate its ALP activity and induce a time-dependent expression increase of TGF-beta and IGF-II which in turn result in osteogenic differentiation of MSCs. Mechanical stimulus is a key stimulator for osteogenic differentiation of MSCs and vital for bone formation in distraction osteogenesis.

Animals ; Bone Marrow Cells ; Cell Differentiation ; Cell Proliferation ; Insulin-Like Growth Factor II ; Mesenchymal Stromal Cells ; Osteogenesis ; Osteogenesis, Distraction ; RNA, Messenger ; Rats ; Rats, Sprague-Dawley ; Somatomedins ; Transforming Growth Factor beta

OBJECTIVETo analyse the clinical outcome and the prognostic factor of childhood acute myeloid leukemia (AML).

METHODSDisease-free survival (DFS), event-free survival (EFS) and overall survival (OS) rates were estimated by Kaplan-Meier method and prognostic factors were evaluated by Cox regression with SPSS in 141 childhood AML in our hospital from August 1995 to July 2004. The patients were divided into 2 groups: acute promyelocytic leukemia (APL) as group A and AML other than APL as group B.

RESULTSOf the 90 group B patients, 54.4% (49/90) achieved complete remission (CR) after one course chemotherapy , with a total CR rate of 76.7%. The cumulative 5 year DFS and OS rate for group B patients were (28.4 +/- 9.0)% and (35.5 +/- 6.3)%, the 51 group A patients were (94.3 +/- 4.0)% and (81.4 +/- 5.7)%, and for total 141 AML patients were (56.9 +/- 6.3)% and (53.3 +/- 4.8)% respectively. Multivariate analysis demonstrated that higher bone marrow blast cell percentage at diagnosis, CR after more than one course of chemotherapy and less than six courses of consolidation chemotherapy were risk prognostic factors in childhood AML other than APL (P < 0.05).

CONCLUSIONThe prognosis of childhood APL is better, while of childhood t(8;21) AML is no better than other FAB subtypes.

Adolescent ; Bone Marrow Cells ; cytology ; Child ; Child, Preschool ; Disease-Free Survival ; Female ; Humans ; Infant ; Leukemia, Myeloid, Acute ; mortality ; therapy ; Male ; Prognosis ; Regression Analysis ; Retrospective Studies ; Treatment Outcome

Aim To identify the potential biomarkers associated with carbon tetrachloride(CCl 4 )-induced a-cute hepatic injury in rats and explore the therapeutic effect of Hugan Tablets(HGT). Methods The model was established by intraperitoneal injection of CCl4 in oil(1 : 1,V/ V)with a dosage of 1 mL·kg - 1 body weight to rats once. The levels of aspartate aminotrans-ferase(AST),alanine aminotransferase(ALT),alka-line phosphatase (ALP ) and lactate dehydrogenase (LDH)in serum of rats were determined. Moreover,a proton nuclear magnetic resonance (1 H-NMR)based metabonomic approach in combination with multivariate data analysis was applied to demonstrate CCl4-induced acute hepatic injury metabolic perturbations in rat urine and feces and identify the corresponding metabolic bio-markers. The intervention effect of HGT was evaluated based on the changes of metabolic phenotype and po-tential biomarkers related to acute hepatic injury. Re-sults The levels of AST,ALT,ALP and LDH in ser-um of rats with acute hepatic injury were significantly reduced by administration of HGT,respectively. The disturbed metabolic state associated with CCl4-induced acute hepatic injury in rat urine and feces could be re-stored by HGT. Meanwhile,five potential biomarkers (2-oxoglutarate,citrate,creatinine,trimethylamine N-oxide,hippurate)in rat urine and three potential bio-markers(butyrate,glucose,uracil)in rat feces related to acute hepatic injury were reversed by administration of HGT,respectively. Conclusion HGT exerts pro-tective effects against CCl4-induced acute hepatic inju-ry in rats,which is probably mediated by regulation of tricarboxylic acid cycle and gut microbiota metabolism.