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Objective To investigate the effect of hypertonic saline on the expressions of aquaporin 4 (AQP4) and caspase-3 in the brain edema area after traumatic brain injury (TBI) in rats Methods Seventy-two male SD rats weighing 220-250 g were selected and randomly divided into three groups (24 rats per group):sham operation group (Group A),traumatic brain injury + normal saline group (Group B) and traumatic brain injury + hypertonic saline group (Group C).Moderate TBI model was induced by Feeney's free falling method.Normal saline and hypertonic saline were delivered respectively.The neurological score was measured at 6,24,and 48 hours after operation.The brain water content was measured,and the blood brain barrier stability was detected by Evans blue staining.AQP4 positive cells was detected by immunohistochemistry.The expressions of AQP4 and caspase-3 protein in brain tissue were detected by Western blot,and the apoptosis of neurons in brain tissue by TUNEL method.Results Compared with Group A,the neurological score of Group B were obviously decreased,while the water content in the brain tissue,Evans blue staining,AQP4 positive cells,AQP4 (6 hours:1.73 ±0.31 vs.0.33 ±0.13;24 hours:2.47 ±0.27 vs.0.33 ±0.14;48 hours:2.18 ± 0.19 vs.0.33 ±0.12),caspase-3 protein expression(6 hours:0.53 ±0.18 vs.0.34 ±0.07;24 hours:0.58 ±0.16 vs.0.33 ± 0.08;48 hours:0.59 ± 0.11 vs.0.33 ± 0.07) and apoptosis index in brain tissue in Group B were significantly increased (all P ＜ 0.05).Compared with Group B,the neurological score of Group C were obviously increased,while the water content in the brain tissue,Evans blue staining,AQP4 positive cells,AQP4 (6 hours:1.51 ±0.27 vs.1.73 ±0.31;24 hours:2.13 ±0.13 vs.2.47±0.27;48 hours:1.84 ±0.22 vs.2.18 ±0.19) and Caspase-3 protein expression (6 hours:0.44±0.09vs.0.53±0.18;24 hours:0.46±0.10vs.0.58±0.16;48 hours:0.48±0.12 vs.0.59 ± 0.11) and apoptosis index in brain tissue of Group C were significantly decreased (all P ＜ 0.05).Conclusion Hypertonic saline can attenuate TBI-induced brain edema and have a significant neuroprotective effect,possibly by down-regulating the expressions of AQP4 and caspase-3.
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The aim of this study was to label rabbit bone derived mesenchymal stem cells (BMSCs) with superparamagnetic iron oxide particles (SPIO) and to study the effects of magnetic labeling on the multi-differentiation of BMSCs. Rabbit BMSCs were isolated, purified, expanded, then coincubated with SPIO(25 microg/ml) complexed to protamine sulfate (Pro) transfection agents overnight. Prussian blue staining and transmission electron microscopy were performed to show intracellular iron. Cell differentiation was evaluated. Both labeled and unlabeled BMSCs were subjected to osteogenic, adipogenic and chondrogenic differentiation to assess their differentiation capacity for 21 d. Osteogenic cells were stained with alizarin red to reveal calcium deposition, adipogenic cells were stained with oil redO' respectively. Chondrogenic cells stained with Safranin-O, glycosamino glycans, and type II collagen production was assessed by standard immunohistochemistry. Cell with immunohistochemistry staining were detected by polarized light microscopy and analysed by Image-Pro Plus software. The results showed that intracytoplasmic nanoparticles were stained with Prussian blue and observed by transmission electron microscopy clearly except the unlabeled control. As compared with the nonlabeled cells, it showed no statistically significant difference on the differentiation of the labeled BMSCs. And the differentiation of the labeled cells were unaffected by the endosomal incorporation of SPIO. In summary, BMSCs can be labeled with SPIO without significant change in cell multi-differentiation capacity.
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Animals , Rabbits , Adipocytes , Cell Biology , Bone Marrow Cells , Cell Biology , Cell Differentiation , Physiology , Cell Proliferation , Cell Tracking , Cells, Cultured , Chondrocytes , Cell Biology , Dextrans , Ferric Compounds , Magnetite Nanoparticles , Mesenchymal Stem Cells , Cell Biology , Osteoblasts , Cell Biology , Staining and LabelingABSTRACT
To assess a novel cell manipulation technique of tissue engineering with respect to its ability to augment superparamagnetic iron oxide particles (SPIO) labeled mesenchymal stem cells (MSCs) density at a localized cartilage defect site in an in vitro phantom by applying magnetic force. Meanwhile, non-invasive imaging techniques were use to track SPIO-labeled MSCs by magnetic resonance imaging (MRI). Human bone marrow MSCs were cultured and labeled with SPIO. Fresh degenerated human osteochondral fragments were obtained during total knee arthroplasty and a cartilage defect was created at the center. Then, the osteochondral fragments were attached to the sidewalls of culture flasks filled with phosphate-buffered saline (PBS) to mimic the human joint cavity. The SPIO-labeled MSCs were injected into the culture flasks in the presence of a 0.57 Tesla (T) magnetic force. Before and 90 min after cell targeting, the specimens underwent T2-weighted turbo spin-echo (SET2WI) sequence of 3.0 T MRI. MRI results were compared with histological findings. Macroscopic observation showed that SPIO-labeled MSCs were steered to the target region of cartilage defect. MRI revealed significant changes in signal intensity (P<0.01). HE staining exibited that a great number of MSCs formed a three-dimensional (3D) cell "sheet" structure at the chondral defect site. It was concluded that 0.57 T magnetic force permits spatial delivery of magnetically labeled MSCs to the target region in vitro. High-field MRI can serve as an very sensitive non-invasive technique for the visualization of SPIO-labeled MSCs.
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BACKGROUND: Chitosan-disodium β-glycerol phosphate (C/GP) gel has been shown to be compatible with the entrapment of viable chondrocytes, and bone marrow mesenchymal stem cells (BMSCs) are considered to be the potential cells used in tissue engineering. This experiment is aimed to observe the cytocompatibility of BMSCs with C/GP gel.OBJECTIVE: To study the effect of C/GP gel on the growth, proliferation and chondrogenic differentiation in vitro cultured BMSCs and explore a new carrier for the application of cartilage tissue engineering.DESIGN: Completely randomized controlled experiment.SETTING: Department of Bone and Joint Surgery, Affiliated Hospital of Luzhou Medical College; Center Laboratory of Southwest Hospital Affiliated to the Third Military Medical University of Chinese PLA.MATERIALS: The experiment was performed in Center Laboratory of Southwest Hospital Affiliated to the Third Military Medical University of Chinese PLA between October 2005 and April 2006, Six adult female mini-pigss were employed. C/GP gel is prepared by mixture the HCI solution of chitosan with salt solution of β-glycerol phosphate, allowed gel at 37 ℃ in incubator for about 5 to 10 minutes.METHODS: ① BMSCs culture: 4-6 mL of bone marrow harvested from the posterior superior lilac crest were plated at 20 ×10<'6>/100 mm dish and then grown for 14 days in complete media, consisting of DMEM/F-12 supplemented with 10% fetal ovine serum. Cells were harvested and re-seeded for subculture. ② BMSCs differentiation assays: Osteogenic differentiation was assessed by histologic detection of alkaline phosphatase activity and calcium in cultures under osteogenic conditions. Chondrogenic differentiation was evaluated by histology for toluidine blue and immunohistochemistry for type Ⅱ collagen in cultures under chondrogenic conditions. ③ In vitro assays, expanded BMSCs were suspended in C/GP solution and allowed gel at 37 ℃ in incubator for about 5 to 10 minutes, then cultured under chondrogenic conditions for 3 weeks. Cells attached to and viability in C/GP gel was monitored with the aid of an inverted light microscope. Chondrogenic differentiation of cell in C/GP gel were assessed by histological and immunohistocbemistry. The cell proliferated was monitored by MTT after 2, 5, 8 days seeding.MAIN OUTCOME MEASURES: ① Characterization of mini-pigs' BMSCs; ② BMSCs attached to and viability in C/GP gel; ③ Chondrogenic differentiation of BMSCs in C/GP gel; ④ BMSCs proliferated in C/GP gel.RESULTS: ① Characterization of mini-pigs' BMSCs: Cultured BMSCs showed fibroblastic morphology and were able to differentiate to chondrocytes or osteogenic cells under chondrgenic or osteogenic cultured condition respectively. ②BMSCs attached to and viability in C/GP gel: BMSCs attached to and remained > 90% viable in C/GP gels immediately post-casting, and throughout the 21 days, using MTT staining. ③ Chondrogenic differentiation of BMSCs in C/GP gel: During 21 days culture period in vitro, chondrogenic induced BMSCs produced amounts of de novo cartilage matrix in the chitosan, as assessed by histological and biochemical criteria. ④ BMSCs proliferated in C/GP gel: Chondrogenic induced BMSCs cultured in C/GP gels continued to proliferate. There was a significant difference among the values of optical density in the cells-gel constructs compared to the controls without cells after 2, 5, and 8 days of culture (P<0.05).CONCLUSION: It is confirmed that C/GP gel shows good cytocompatibility with BMSCs and contributes to the growth, proliferation and chondrogenic differentiation for BMSCs in vitro culture. C/GP gel can be a potential cell-carrier for tissue engineering of articular cartilage.
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Objective To study the effect of chemotherapeutic agents of different doses on sensitivity of glioma U251 cell line to chemotherapy.Methods The rates of cell growth of the U251 cell line under the effect of different chemotherapeutic agents(DOX、VCR、VP-16 and with verapamil)of a series of doses were detected by MTT assay.Results Using DOX or VCR or VP-16 singly,the rates of cell growth were detected at a lever with positively correlates with the concentrations of these chemotherapeutic agents.Moreover the rates of cell growth changed more obviously within some range of concentrations,while the change is limited with the high or low concentrations.The combination of pates of chemotherapeutic agents and verapamil show synergistic effect.Conclusions The relationship between the doses of chemotherapeutic agents and sensitivity of chemotherapy gas a basis for choosing appropriate clinical chemotherapy doses.
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Objective To investigate the dynamic changes of serum insulin-like growth factor-1(IGF-1)levels in patients with acute cerebralhemorrhage.Methods Serum levels were determined with RIA in 40 patients with cerebralhemorrhage within 2 days and at 14 days and 20 healthy individuals serve as control groups.Results The serum IGF-1 levels in patients with cerebralhemorrhage were significantly lower than those in controls(P
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BACKGROUD: It is not consistent on whether melatonin correlates with the adolescent idiopathic scoliosis(AIS) demonstrated by the domestic and foreign scholars.OBJECTIVE: To study the pathogenesis of AIS and its correlation with melatonin.DESIGN: A nonrandomized age-matched controlled study screened with two tests.SETTING: The Second Department of Orthopaedics, Haikou People' s Hospital.PARTICIPANTS: The experiment was completed in the Second Department of Orthopaedics, Haikou People' s Hospital. Totally 8 198 in-school students.from over 10 schools of towns in Haifu District, 4 423 males and 3 775 females, aged 7 to 16 years were surveyed.METHODS: Forty-two adolescents who have been diagnosed with AIS were selected and 50 healthy age-matched adolescents were selected as controls. Melatonin was assayed with the radioimmunology and the data were processed statistically.MAIN OUTCOME MEASURES: The levels of serum melatonin in the two groups.RESULTS: The serum melatonin of the preadolescents below 10 years old in AIS group, especially of females, was less than that of adolescents in the control group, with significant differencein statistics.CONCLUSION: The serum melatonin correlates with pathogenesis of AIS.
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This experiment was designed to explore the correlation between the mechanism of immobilization-induced skeletal muscle atrophy and the apoptosis of muscular cells. The models of skeletal muscle atrophy induced by immobilization for different length of time were established according to Sievanen II methods. 24 rabbits, each of them having one hind leg fixed by the tubal plaster and the other one free as control, were randomly divided into four groups depending on time of fixation (3, 7, 14, and 28 days respectively). The animals were sacrificed by the end of fixation. TdT-mediated d-UTP nick end labeling (TUNEL) was used to investigate the apoptotic muscle cells in the animal's bone. By comparing the apoptotic muscle cells with the morphology of the skeletal muscle, the correlation between cell apoptosis and skeletal muscle atrophy were analyzed. Apoptotic muscle cells did appear after immobilization in the atrophied skeletal muscle. In various groups, some cells with false positive stained TUNEL were found in the atrophic muscle, which could be distinguished from apoptotic cells by their characteristics. In conclusion, cell apoptosis participates in the process of skeletal muscle atrophy induced by immobilization; the amount of apoptotic cells is strongly associated with the time of immobilization, its peak appears on the 14th day of immobilization; the distribution of apoptotic skeletal muscle cell varies with the time of fixation. The severity of skeletal muscle atrophy is associated with the degree of the muscle cell apoptosis.