10.3969/j.issn.1007-3969.2013.04.010

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

The aim was to study the apoptotic induction effect of thapsigargin on leukemia cell line K562 and its possible mechanism. After the treatment of leukemia cell line K562 by thapsigargin, morphological change of apoptotic cells was investigated by AO/EB fluorescent staining under fluorescent microscope; apoptosis rate was determined with annexin V-FITC/PI double staining by flow cytometry; intracellular calcium concentrations ([Ca(2+)]i) were measured by fluorescence spectrophotometer with calcium sensitive fluorescence indicator Fura-2/AM; mitochondrial transmembrance potentials (Delta Psi m) was detected on flow cytometry through staining of Rhodamine (Rh123); the changes of caspase-3, -7, -9, -12, cytochrome C, GRP78 proteins were detected by Western blot. The results showed that K562 cells cultured in 4 micromol/L thapsigargin for 48 hours exhibited typical morphological changes of apoptotic cells under fluorescent microscope, including shrinkage of cell, condensation of chromatin, breakage of nuclear, formation of apoptotic bodies, fluorescence of yellow green and pellet observed in early apoptoyic cells and hyacinth fluorescence of chromatin showed in late apoptotic cells. 24 and 48 hours after exposure to 1, 2, 4, 8 micromol/L thapsigargin, the apoptotic rates of K562 were respectively 7.51%, 11.65%, 23.22%, 30.56% and 12.85%, 20.27%, 31.51%, 44.16%, in dose-dependent manner, and were statistically significant when compared with the controls (P < 0.05). The apoptotic rate of K562 was dose- and time-dependent in experiment range. The enhancement of [Ca(2+)]i and the decrease of the Delta Psi m in K562 cells were induced by thapsigargin and were dose-dependent in experiment range, compared with control, P < 0.05. Western blot results indicated that cleavage and activation of caspase-3, -7, -9, -12, releasing of cytochrome C from mitochondria, upregulation of GRP78 expression at the endoplasmic reticulum were induced in K562 cells after 24 hours exposure of 4 micromol/L thapsigargin. It is concluded that thapsigargin induces endoplasmic reticulum stress-induced apoptosis in K562 cells. Endoplasmic reticulum is a novel important initiatory site of apoptosis in cells; the cleavage and activation of caspase-3, -7, -9, -12 play very important role in endoplasmic reticulum stress-induced apoptosis of K562 cells and is one of the important mechanisms for thapsigargin-induced apoptosis. Thapsigargin-induced apoptosis in K562 cells is associated closely with the disruption of the Delta Psi m and the release of cytochrome C from mitochondria, mitochondria participates in endoplasmic reticulum stress-induced apoptosis in K562 cells.
Apoptosis ; drug effects ; Calcium-Transporting ATPases ; antagonists & inhibitors ; Caspase 3 ; metabolism ; Caspase 7 ; metabolism ; Cytochromes c ; metabolism ; Endoplasmic Reticulum ; drug effects ; enzymology ; Enzyme Inhibitors ; pharmacology ; Heat-Shock Proteins ; metabolism ; Humans ; K562 Cells ; Leukemia ; pathology ; Mitochondria ; drug effects ; Molecular Chaperones ; metabolism ; Thapsigargin ; pharmacology

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 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

BACKGROUNDMolecular hydrogen, as a novel antioxidant, has been proven effective in treating many diseases. This study aimed to evaluate the therapeutic effects of hydrogen saturated saline in treatment of a rat model of diabetes mellitus and a rat model of insulin resistant.

METHODSA rat diabetes mellitus model was established by feeding a high fat/high carbohydrate diet followed by injection of a small dose of streptozotocin, and an insulin resistant model was induced with a high glucose and high fat diet. Hydrogen saturated saline was administered to rats with both models conditions on a daily basis for eight weeks. A pioglitazone-treated group and normal saline-treated group served as positive and negative controls. The general condition, body weight, blood glucose, blood lipids, and serum insulin levels of rats were examined at the 8th week after treatment. The oxidative stress indices, including serum superoxide dismutase (SOD), glutathione (GSH) and malondialdehyde (MDA) were also evaluated after eight weeks of treatment using the commercial kits.

RESULTSHydrogen saturated saline showed great efficiency in improving the insulin sensitivity and lowering blood glucose and lipids. Meanwhile, the therapeutic effects of hydrogen saturated saline were superior to those of pioglitazone. Hydrogen saturated saline markedly attenuated the MDA level and elevated the levels of antioxidants SOD and GSH.

CONCLUSIONHydrogen saturated saline may improve the insulin resistance and alleviate the symptoms of diabetes mellitus by reducing the oxidative stress and enhancing the anti-oxidant system.

Animals ; Diabetes Mellitus, Experimental ; drug therapy ; Hydrogen ; therapeutic use ; Hypoglycemic Agents ; therapeutic use ; Insulin Resistance ; Oxidative Stress ; drug effects ; Rats ; Sodium Chloride ; chemistry ; Thiazolidinediones ; therapeutic use

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

The mutations of BLM gene may result in Bloom's syndrome which includes immunodeficiency, predisposition to malignant tumors and so on, and enhances sister chromati exchange (SCE), DNA replication failure, genome instability, and increases cancer susceptibility. This study was aimed to investigate the variability of mRNA expression level and cDNA structure of BLM gene in tumor cell strains so as to look for a new cancerogenic mechanism and to find a new therapeutic target. The expression level of mRNA and the structure of cDNA of BLM gene in six tumor cell strains and the normal human bone marrow mononuclear cells were detected with RT-PCR and DNA sequencing was performed. The results indicated that these tumors cells expressed BLM mRNA higher than the normal human bone marrow mononuclear cells (P < 0.01), but no cDNA sequence abnormality of BLM gene in these tumors cells was observed. It is concluded that the increase of expressing level of BLM mRNA may play an important role in the development of these tumors.
Base Sequence ; Cell Line, Tumor ; DNA Helicases ; genetics ; Gene Expression Regulation, Neoplastic ; HL-60 Cells ; Humans ; Jurkat Cells ; Molecular Sequence Data ; RNA, Messenger ; biosynthesis ; genetics ; RecQ Helicases ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA

This study was aimed to explore the expression and significance of melanoma antigen gene-3 (MAGE-3) in endoplasmic reticulum stress-induced apoptosis. After the treatment of leukemia cell line K562 and its multidrug-resistant cell line K562/A02 by thapsigargin, intracellular calcium concentrations ([Ca(2+)]i) in K562 and K562/A02 were measured by fluorescence spectrophotometer with calcium sensitive fluorescence indicator Fura-2/AM; expression changes of glucose-regulated protein 78 (GRP78) were detected by Western blot; morphological change of apoptotic cell was investigated by AO/EB fluorescent staining under fluorescent microscope; apoptosis rate was determined by terminal deoxyribonucleotidyl transferase (TdT)-mediated dUTP nick end labelling (TUNEL) staining; the expression of MAGE-3 gene mRNA was detected by RT-PCR. The results showed that (1) thapsigargin induced the enhancement of [Ca(2+)]i with different extent in K562 and K562/A02 cells, and the enhancement of [Ca(2+)]i was dose-dependent in experiment range. At the same time, thapsigargin induced upregulation of GRP78 protein expression and typical apoptotic changes of K562 and K562/A02 cells, apoptotic rate was also dose-dependent in experiment range. The [Ca(2+)]i in K562/A02 cells were higher than that in K562 cells. (2) in the course of endoplasmic reticulum stress-induced apoptosis by thapsigargin, the expression of MAGE-3 gene mRNA was remarkably downregulated. Moreover, the expression of MAGE-3 gene mRNA in K562/A02 cells was higher than that in K562 cells. It is concluded that (1) thapsigargin induces endoplasmic reticulum stress-induced apoptosis of K562 and K562/A02 cells in experiment range, and this may be associated with downregulation of MAGE-3 mRNA expression or MAGE-3 gene may participates in the regulation of endoplasmic reticulum stress-induced apoptosis. (2) MAGE-3 gene may possess anti-apoptotic activity, multidrug resistance in K562/A02 cells can be associated with [Ca(2+)]i increase and upregulation of MAGE-3 expression.
Antigens, Neoplasm ; genetics ; Apoptosis ; drug effects ; physiology ; Blotting, Western ; Calcium ; metabolism ; Endoplasmic Reticulum ; metabolism ; Heat-Shock Proteins ; metabolism ; Humans ; In Situ Nick-End Labeling ; K562 Cells ; Microscopy, Fluorescence ; Molecular Chaperones ; metabolism ; Neoplasm Proteins ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Thapsigargin ; pharmacology

OBJECTIVETo explore the biomarker of manganese exposure by analyzing the relationship between manganese exposure and concentration in some biomaterials.

METHODSThe air samples were collected through the individual air sample. According to the manganese levels in the air, workers were assigned to control group, low concentration group and high concentration group, and manganese in the hair, urine, serum, blood cell and saliva from different group were measured respectively. The correlations between concentration of external manganese exposure and manganese concentrations in biomaterials, and years of employment and concentrations in biomaterials were analyzed.

RESULTSIn the high concentration group, saliva manganese was 32.17 µg/L, hair manganese was 37.39 mg/kg, urine manganese was 2.50 µg/L, plasma manganese was 29.61 µg/L, blood manganese was 14.49 µg/L, were higher than those in the control group (10.40 µg/L, 1.60 mg/kg, 0.77 µg/L, 10.30 µg/L, 4.56 µg/L respectively) (P < 0.01). The manganese concentration in the saliva was significantly correlated with airborne manganese concentration (r = 0.649, P < 0.01), with the years of employment (r = 0.404, P < 0.01), with the total exposure of manganese (r = 0.342, P < 0.01), with the manganese concentration of plasma (r = 0.303, P < 0.01) and with the manganese concentration in blood cells (r = 0.359, P < 0.01), respectively.

CONCLUSIONSThe concentration of manganese in saliva could work as a biomarker of manganese internal exposure.

Adult ; Air Pollutants, Occupational ; analysis ; Biomarkers ; analysis ; Hair ; chemistry ; Humans ; Manganese ; analysis ; blood ; urine ; Manganese Poisoning ; prevention & control ; Middle Aged ; Occupational Exposure ; prevention & control ; Saliva ; chemistry ; Young Adult