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Objective To investigate the neuroprotective effect of microRNA-124 on dopaminergic neurons in Parkinson's disease models and its inflammation-related regulation mechanism.Methods The inflammation cell models were prepared by microglial BV2 murine cells; real-time quantitative PCR (qRT-PCR) was performed to analyze the expressions of inflammation-related miRNAs,including miR-21,miR-124,miR-155,miR-146a,miR-181c and miR-221-3p.The C57BL/6 mouse models of Parkinson's disease were established by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MTPT)intraperitoneal injection,and then,administration of miR-124 via tail vein injection was performed;immunohistochemistry was performed to observe the apoptosis of dopaminergic neurons (TH-staining)and the activation ofmicroglial cells (Iba1-staining) in the substantia nigra of animal models before and after treatment; additionally,Western blotting and qRT-PCR were performed to analyze the expressions of the apoptosis-related proteins (caspase-3 and caspase-8).Results As compared with other 5 miRNAs,miR-124 showed significantly higher expression in BV2 cells (P＜0.05),and presented higher down-regulation after the induction of inflammatory.As compared with those in the Parkinson's disease models,significantly increased TH-positive cells,decreased Iba1-positive cells and down-regulated expressions of caspase-3 and caspase-8 in the substantia nigra of animal models after miR-124 ministration were observed (P＜0.05).Conclusion MiR-124 can slow down the apoptosis of dopaminergic neurons though inhibiting the activation of microglial cells,and maybe it is the key molecule ofpathogenesis of Parkinson's disease.
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Objective To investigate the role of C6 glioma cells mediated by rapid freezing and thawing ofAr-He cryoablation in the maturation of marrow-derived dendritic cells (BM-DCs) in Wistar rats,and the anti-tumor effect of these DCs on rat models of intracranial gliomas. Methods C6 glioma cells were routinely cultured in vitro; rapid freezing and thawing of Ar-He cryoablation was employed in C6 glioma cells of the experimental group, and C6 glioma cells of the negative control group were only performed insertion of the probe; blank control group (using rapid freezing and thawing of Ar-He cryoablation on the same amount of PBS) was also employed.Bone marrow-derived mononuclear cells (MNCs) were first prepared from tibia and femur bones of Wistar rats. These cells were cultured with such cytokines as recombinant granulocyte-macrophage colony-stimulating factor (rmGM-CSF),recombinant interleukin-4 (rmIL-4) and tumor necrosis factor-alpha (TNFα) to induce their maturation; BM-DCs were pulsed with or without tumor cell lysate obtained by rapid freezing and thawing of Ar-Hecryoablation at a ratio of (DC:tumor cells =1:3) 7 d after that.Morphological observation of BM-DCs was performed by light microscopy and the expression of DCs costimulatory molecules CD80 and CD86 were measured by flow cytometry 48 h after the addiction; the IL-12 level in the supematant of DCs was detected by ELISA. In order to determine whether or not vaccination with C6 TP DCs can induce the therapeutic potential in the established glioma-bearing models, the C6 cells cultured in vitro were stereotaxically implanted into the left caudate nucleus of Wistar rat brain; glioma-bearing rats were injected with vaccination with DCs,cells from the blank control group and negative control group on the 3rd and 10th d. Survival time was observed and determined using the method of Kaplan-Meier and Log-Rank analysis. Results DCs from rats' bone marrow cells cultured with cytokines and pulsed with tumor lysates showed the characters of typical mature DCs.Morphologically,these cells were large with oval or irregularly shaped nuclei and with many small dendrites. Phenotypically, they expressed high levels of CD80 and CD86 antigens (71.8 1％± 1.10％ and 74.66％± 1.48％ in experimental group,49.49％±1.08％ and 51.20％±2.06％ in negative control group, and 48.47％±1.09％ and 49.53％±1.89％ in blank control group); significant difference was noted between each 2 groups (P＜0.05).Functionally,the IL-12level in the supernatant of DCs showed obvious increment in the experimental group (245.99 ±3.20pg/mL) as compared with that in the negative control group (138.68±3.20 pg/mL) and blank control group (135.16±2.88 pg/mL,P＜0.05).These cells gained the capacity of mediating immunotherapy against intracranial gliomas in rats:the median survival in the experimental group (33 d) was significantly higher than that in the negative control and blank control groups (22 and 24 d, respectively, P＜0.05).Conclusion C6 glioma cells mediated by rapid freezing and thawing of Ar-He cryoablation can induce maturation of BM-DCs in Wistar rats; these BM-DCs pulsed with tumor lysates, as new therapeutic vaccines,can mediate immunotherapy against intracranial gliomas in rats.
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Objective To label adipose-derived stromal cells (ADSCs) with different concentrations of ultrasmall superparamagnetic particles of iron oxide (USPIO) to investigate the biological characteristics of ADSCs treated with these USPIO,and determine the optimal concentration of USPIO in labeling ADSCs in vitro. Methods USPIO with different concentrations were prepared,and the particles were endocytosed by ADSCs generated from rat adipose tissue. Eight groups (negative control group,blank control group,and 11.25,22.5,45,90,135 and 180 μg/mL treatment groups) were chosen. Labeling efficiency and cellular uptake were analyzed by Prussian blue staining. Meanwhile,proliferation capacity and viability of ADSCs were evaluated by Alamar blue assay and Cell Counting kit-8. Results ADSCs could be effectively labeled with USPIO: approximately 95％ ADSCs were labeled when they were incubated with USPIO for 24 h under the concentration of USPIO was 45 μg/mL;and approximately 100％ ADSCs were labeled when the concentration of USPIO was 90 μg/mL and above. The CCK-8 and Alamar blue tests showed that USPIO of different concentrations (11.25-90 μg/mL) had little influence on cell growth viability,and no significant difference was noted between each 2 concentration groups (P＞0.05). In a word, 45-90 μg/mL USPIO were the optimal choice for transplantion of ADSCs in vivo. Conclusion ADSCs from the adipose tissue can be effectively labeled with USPIO with minimal effect on cell proliferation and viability.
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<p><b>OBJECTIVE</b>To investigate the biological features of human decidua basalis-derived mesenchymal stem cells (PDB-MSCs) in vitro and identify their capacity of multilineage differentiation.</p><p><b>METHODS</b>PDB-MSCs were harvested from the decidua basalis of term placental by enzymatic digestion and density gradient centrifugation, and the growth characteristics and morphological changes of the MSCs were observed by inverted microscope. The proliferative ability of the cells was assessed by Cell Counting Kit-8. The cell cycle and expressions of the surface markers (CD29, CD44, CD73, CD90, CD34, CD45, and CD14) of the MSCs were identified by flow cytometry. Multilineage differentiation capacity of the cells was tested by inducing their differentiation toward osteoblasts, adipocytes and chondroblasts in vitro.</p><p><b>RESULTS</b>MSCs isolated from human decidua basalis of term placental exhibited a morphology similar to that of bone marrow-derived MSCs, and grew into colonies in in vitro culture, where the cells proliferated rapidly after passage with a cell doubling time of 2.21∓0.21 days. More than 70% of the cells stayed in the resting stage (G(0)/G(1)) and showed positivity for CD29, CD44, CD73 and CD90, but not for CD14, CD34 or CD45. After induction, the cells showed positive results of alizarin red staining, oil red O staining and Alcian blue staining.</p><p><b>CONCLUSION</b>Human decidua basalis contains a rich source of MSCs, which can be easily isolated and cultured without affecting their capacity of multilineage differentiation. The PDB-MSCs may have the potential as a novel source of stem cells.</p>
Subject(s)
Female , Humans , Pregnancy , Cell Differentiation , Physiology , Cell Separation , Cells, Cultured , Decidua , Cell Biology , Mesenchymal Stem Cells , Cell Biology , Multipotent Stem Cells , Cell Biology , Placenta , Cell BiologyABSTRACT
Objective To investigate the methods of isolation of human umbilical cord mesenchymal stem cells (MSCs) in Wharton' s jelly and the differentiation of MSCs into dopaminergic neurons. Methods The umbilical cord mesenchymal tissue was scraped off from the Wharton's jelly,and then, collagenase Ⅳ was employed to isolate the MSCs. The isolated cells were primarily cultured in DMEM/F12 medium containing 10% FBS. Inverted microscopy was used to observe the cytomorphology, and flow cytometry was employed to detect the cell surface antigens and the cell cycle.We evaluated the cell viability using CCK8 kit. Two-step method was employed to induce the MSCs of the P3 generation to differentiate into dopaminergic neurons, and immunocytochemistry and Western blotting were used to detect the expressions of neuron specific enolase (NSE) and tyrosine hydroxylase (TH) 3, 6 and 9 d after the induction. Results The isolated MSCs showed fibroblast-like shape, with parallel arrangement and vortex-like growth. MSCs of the P3 generation expressed CD73, CD29, CD44and CD105, but did not express CD34, CD45, CD106 and HLA-DR. The doubling time in the exponential phase was at the 48th h of culture, and 91.13% cells were under G0-G1. These cells had similar morphology of the neurons. The immunocytochemical assay showed that the NSE and TH positive rates were 19.5% and 8.9% on the 9th d of induction; and Western blotting showed that MSCs obviously expressed NSE and weakly expressed TH.Conclusion MSCs can be isolated from the umbilical cord mesenchymal tissue, and be induced to differentiate into dopaminergic neurons in vitro.
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Objective To explore the influencing factors of bleeding in patients with cerebral arteriovenous malformation (AVM) through observing the angioarchitecture of AVM with cerebral DSA.Methods The clinical and imaging data of 199 patients with AVM, including 107 with bleeding and 92without bleeding, were retrospectively analyzed. The relationships between bleeding and such factors as the site and size of masses, the type and site of supplying arteries, the number and pathway of venous drainage, having vascular tumor-like changes or not, the relation between AVM and aneurysm in terms of location, and the number and size of concurrent aneurysms were determined by univariate analyses and Binary logistic regression with SPSS13.0. Results Univariate analysis indicated that such factors as micro-sized (0.5-1.0 cra) and small-sized (1.0-2.0 cra) masses ofAVM, AVM underdeep location,having exclusively deep draining veins, fewer draining veins and deep feeding arteries, concurrent small aneurysms (＜5 mm), aneurysms at the end of the blood-supply artery were positively correlated to hemorrhagic presentation. The number of feeding arteries and the number of concurrent aneurysms were not correlated with hemorrhagic presentation. Having vascular tumor-like changes can decreased the risk of bleeding. When stepwise multiple logistic regression analysis was employed, only micro-sized and small-sized masses of AVM, having deep feeding arteries, and aneurysms at the end of the blood-supply artery were dependent predictors of hemorrhagic presentation. Conclusion Micro-sized and small-sized masses of AVM, having deep draining veins and aneurysms at the end of the blood-supply artery are the most powerful risk predictors for hemorrhagic AVM presentation.
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<p><b>OBJECTIVE</b>To construct the eukaryotic expression vector pDsRed2-N1-SDF-1alpha and observe its expression in the mouse bone marrow mesenchymal stem cells.</p><p><b>METHOD</b>SDF-1alpha gene sequence with XhoI, EcoRI restriction enzyme cutting site was amplified from the total RNA of mouse smooth muscle cells by reverse transcription-polymerase chain reaction (RT-PCR) and inserted into the eukaryotic expression vector pDsRed2-N1 encoding red fluorescent protein gene, and the insertion was verified by endonuclease digestion and DNA sequencing. Mouse bone marrow mesenchymal stem cells identified with immunofluorescence assay for vimentin expression were transfected with the constructed plasmid pDsRed2-N1-SDF-1alpha, and the expression of sdf-1alpha was detected using immunofluorescence assay.</p><p><b>RESULTS</b>The DNA fragment amplified by PCR from the total RNA was identical to SDF-1alpha from the gene library, and an identical DNA fragment was also amplified from the recombinants. Sequence analysis confirmed the successful insertion of SDF-1alpha into the pDsRed2-N1 vector and the eukaryotic expression vector pDsRed2-N1-SDF-1alpha was successfully constructed. The cultured mouse bone marrow mesenchymal stem cells positive for vimentin protein showed SDF-1alpha expression 24 h after transfection with the recombinant vector.</p><p><b>CONCLUSION</b>The pDsRed2-N1-SDF-1alpha eukaryotic expression vector constructed is capable of expression of SDF-1alpha fusion protein in the mouse bone marrow mesenchymal stem cells.</p>