The study aimed to investigate the effect of the CD200R1 gene deletion on microglia activation and nigrostriatal dopamine neuron loss in the Parkinson's disease (PD) process. The CRISPR-Cas9 technology was applied to construct the CD200R1^-/- mice. The primary microglia cells of wild-type and CD200R1^-/- mice were cultured and treated with bacterial lipopolysaccharide (LPS). Microglia phagocytosis level was assessed by a fluorescent microsphere phagocytosis assay. PD mouse model was prepared by nigral stereotaxic injection of recombinant adeno-associated virus vector carrying human α-synuclein (α-syn). The changes in the motor behavior of the mice with both genotypes were evaluated by cylinder test, open field test, and rotarod test. Immunohistochemical staining was used to assess the loss of dopamine neurons in substantia nigra. Immunofluorescence staining was used to detect the expression level of CD68 (a key molecule involved in phagocytosis) in microglia. The results showed that CD200R1 deletion markedly enhanced LPS-induced phagocytosis in vitro by the microglial cells. In the mouse model of PD, CD200R1 deletion exacerbated motor behavior impairment and dopamine neuron loss in substantia nigra. Fluorescence intensity analysis results revealed a significant increase in CD68 expression in microglia located in the substantia nigra of CD200R1^-/- mice. The above results suggest that CD200R1 deletion may further activates microglia by promoting microglial phagocytosis, leading to increased loss of the nigrostriatal dopamine neurons in the PD model mice. Therefore, targeting CD200R1 could potentially serve as a novel therapeutic target for the treatment of early-stage PD.
Animals ; Microglia/physiology* ; Mice ; Phagocytosis ; Parkinson Disease/genetics* ; Disease Models, Animal ; Receptors, Cell Surface/physiology* ; Dopaminergic Neurons/pathology* ; Antigens, CD/metabolism* ; Gene Deletion ; Substantia Nigra ; Mice, Inbred C57BL ; Mice, Knockout ; Cells, Cultured ; Male ; alpha-Synuclein ; CD68 Molecule ; Orexin Receptors

PURPOSEMacrophages are known to be important for healing numerous injured tissues depending on their functional phenotypes in response to different stimuli. The objective of this study was to reveal macrophage phenotypic changes involved in exercise-induced skeletal muscle injury and regeneration.

METHODSAdult male Sprague-Dawley rats experienced one session of downhill running (16° decline, 16 m/min) for 90 min. After exercise the blood and soleus muscles were collected at 0 h, 6 h, 12 h, 1 d, 2 d, 3 d, 1 w and 2 w after exercise, separately.

RESULTSIt was showed that CD68 M1 macrophages mainly infiltrated into muscle necrotic sites at 1-3 d, while CD163 M2 macrophages were present in muscles from 0 h to 2 weeks after exercise. Using transmission electron microscopy, we observed activated satellite cells 1 d after exercise. Th1-associated transcripts of iNOS and Ccl2 were inhibited post exercise, while COX-2 mRNA was dramatically increased 12 h after running (p < 0.01). M2 phenotype marker Arg-1 increased 12 h and 3 d (p < 0.05, p < 0.01) after exercise, and Clec10a and Mrc2 were up-regulated in muscles 12 h following exercise (p < 0.05, p < 0.05).

CONCLUSIONThe data demonstrate the dynamic patterns of macrophage phenotype in skeletal muscle upon eccentric exercise stimuli, and M1 and M2 phenotypes perform different functions during exercise-induced skeletal muscle injury and recovery.

Animals ; Antigens, CD ; analysis ; Antigens, Differentiation, Myelomonocytic ; analysis ; Macrophages ; physiology ; Male ; Muscle, Skeletal ; injuries ; pathology ; Myoglobin ; blood ; Phenotype ; Physical Conditioning, Animal ; Rats ; Rats, Sprague-Dawley ; Receptors, Cell Surface ; analysis

To investigate the effect of apigenin on self-renewal for sphere-forming cells in human small cell lung cancer cell line NCI-H446 and the underlying mechanisms.
 Methods: Sphere-forming cells from NCI-H446 cell line were cultured in stem cell-conditioned culture medium with ultra-low attachment surface plates. The rate of sphere-forming cells in the second passage sphere-forming cells was used to evaluate the inhibitory effects of apigenin on the self-renewal for sphere-forming cells. The protein level of urokinase-type plasminogen activator receptor (uPAR) in spheroids was analyzed by Western blot.
 Results: Apigenin signifcantly inhibited the self-renewal of the second passage sphere-forming cells [0, 5.0, 10.0, 20.0 μmol/L apigenin: (18.2±1.9)%, (13.6±1.7)%, (10.6±1.6)%, (6.9±1.3)%, respectively] and down-regulated uPAR expression in a concentration-dependent manner (P<0.05).
 Conclusion: Apigenin inhibits the self-renewal capacity of sphere-forming cells in NCI-H446 cells, which may be associated with down-regulation of uPAR expression.
Apigenin ; pharmacology ; Cell Line, Tumor ; Down-Regulation ; drug effects ; genetics ; Humans ; Lung Neoplasms ; Neoplastic Stem Cells ; drug effects ; pathology ; physiology ; Receptors, Cell Surface ; Receptors, Urokinase Plasminogen Activator ; drug effects ; genetics ; metabolism ; Signal Transduction ; Small Cell Lung Carcinoma ; drug therapy ; pathology ; Spheroids, Cellular ; drug effects ; physiology ; Stem Cells

Mesenchymal stem cell (MSC)-mediated therapy has been shown to be clinically effective in regenerating tissue defects. For improved regenerative therapy, it is critical to isolate homogenous populations of MSCs with high capacity to differentiate into appropriate tissues. The utilization of stem cell surface antigens provides a means to identify MSCs from various tissues. However, few surface markers that consistently isolate highly regenerative MSCs have been validated, making it challenging for routine clinical applications and making it all the more imperative to identify reliable surface markers. In this study, we used three surface marker combinations: CD51/CD140α, CD271, and STRO-1/CD146 for the isolation of homogenous populations of dental mesenchymal stem cells (DMSCs) from heterogeneous periodontal ligament cells (PDLCs). Fluorescence-activated cell sorting analysis revealed that 24% of PDLCs were CD51(+)/CD140α(+), 0.8% were CD271(+), and 2.4% were STRO-1(+)/CD146(+). Sorted cell populations were further assessed for their multipotent properties by inducing osteogenic and chondrogenic differentiation. All three subsets of isolated DMSCs exhibited differentiation capacity into osteogenic and chondrogenic lineages but with varying degrees. CD271(+) DMSCs demonstrated the greatest osteogenic potential with strong induction of osteogenic markers such as DLX5, RUNX2, and BGLAP. Our study provides evidence that surface marker combinations used in this study are sufficient markers for the isolation of DMSCs from PDLCs. These results provide important insight into using specific surface markers for identifying homogenous populations of DMSCs for their improved utilization in regenerative medicine.
Adaptor Proteins, Signal Transducing ; analysis ; Adult ; Aggrecans ; analysis ; Antigens, CD ; analysis ; Antigens, Surface ; analysis ; CD146 Antigen ; analysis ; Cell Differentiation ; physiology ; Cell Lineage ; Cell Separation ; methods ; Cells, Cultured ; Chondrogenesis ; physiology ; Collagen Type II ; analysis ; Core Binding Factor Alpha 1 Subunit ; analysis ; Flow Cytometry ; methods ; Homeodomain Proteins ; analysis ; Humans ; Integrin alphaV ; analysis ; Mesenchymal Stromal Cells ; cytology ; physiology ; Multipotent Stem Cells ; cytology ; physiology ; Nerve Tissue Proteins ; analysis ; Osteogenesis ; physiology ; Periodontal Ligament ; cytology ; Receptor, Platelet-Derived Growth Factor alpha ; analysis ; Receptors, Nerve Growth Factor ; analysis ; SOX9 Transcription Factor ; analysis ; Time Factors ; Transcription Factors ; analysis

Mesenchymal stem cells (MSCs) are a promising tool in regenerative medicine due to their capacity to differentiate into multiple lineages. In addition to MSCs isolated from bone marrow (BMSCs), adult MSCs are isolated from craniofacial tissues including dental pulp tissues (DPs) using various stem cell surface markers. However, there has been a lack of consensus on a set of surface makers that are reproducibly effective at isolating putative multipotent dental mesenchymal stem cells (DMSCs). In this study, we used different combinations of surface markers (CD51/CD140α, CD271, and STRO-1/CD146) to isolate homogeneous populations of DMSCs from heterogeneous dental pulp cells (DPCs) obtained from DP and compared their capacity to undergo multilineage differentiation. Fluorescence-activated cell sorting revealed that 27.3% of DPCs were CD51(+)/CD140α(+), 10.6% were CD271(+), and 0.3% were STRO-1(+)/CD146(+). Under odontogenic conditions, all three subsets of isolated DMSCs exhibited differentiation capacity into odontogenic lineages. Among these isolated subsets of DMSCs, CD271(+) DMSCs demonstrated the greatest odontogenic potential. While all three combinations of surface markers in this study successfully isolated DMSCs from DPCs, the single CD271 marker presents the most effective stem cell surface marker for identification of DMSCs with high odontogenic potential. Isolated CD271(+) DMSCs could potentially be utilized for future clinical applications in dentistry and regenerative medicine.
Adult ; Adult Stem Cells ; cytology ; Antigens, CD ; analysis ; Antigens, Surface ; analysis ; Biomarkers ; analysis ; CD146 Antigen ; analysis ; Cell Culture Techniques ; Cell Differentiation ; physiology ; Cell Lineage ; Cell Separation ; methods ; Cells, Cultured ; Chondrogenesis ; physiology ; Dental Pulp ; cytology ; Flow Cytometry ; methods ; Humans ; Integrin alphaV ; analysis ; Mesenchymal Stromal Cells ; cytology ; Multipotent Stem Cells ; cytology ; Nerve Tissue Proteins ; analysis ; Odontogenesis ; physiology ; Receptor, Platelet-Derived Growth Factor alpha ; analysis ; Receptors, Nerve Growth Factor ; analysis

OBJECTIVETo study the effects of Jisuikang (Chinese characters) on Nogo-NgR gene expression, and to explore the protective effects and mechanism of Jisuikang (Chinese characters) on spinal cord injury in rats.

METHODSOne hundred eighty female rats were randomly assigned to 6 groups(30 rats per group). Sham group: T10 lamina was resected only and spinal cord was untreated. Model group: spine cord injury (SCI) was created with a modified impinger of Allen's by impacting on the T10 spinal cord. Prednisolone group: Prednisolone (0.06 g/kg) was given by intragastric administration at a time interval of 24 hours after operation. The Jisuikang (Chinese characters) high, moderate and low dose groups: Jisuikang (Chinese characters) was supplied with different dose (50 g/kg, 25 g/kg, 12.5 g/kg) by intragastric administration in rats after operation,for the first time at 30 min after surgery. Animals were killed 3, 7, 14 days after surgery. The expression levels of Nogo-A and NgR were observed by Western Blot and Real-time PCR.

RESULTSThe expression of Nogo-A and NgR was at the basic level at all time points in sham group. Compared with model group, the protein expression levels of Nogo-A and NgR in sham, prednisolone, Jisuikang (Chinese characters) moderate dose groups were statistically significant at all time points (P < 0.05). No difference was found in Jisuikang (Chinese characters) high and low dose groups (P > 0.05). Three days after surgery, the mRNA levels of Nogo-A and NgR in treatment group were significantly lower than that in model group (P < 0.01); 7 days after surgery,Nogo-A and NgR mRNA expression were dramatically upregulated and peaked; 14 days after operation, the expression was decreased, but still significantly higher than that in other treatment groups (P < 0.01). Prednisolone and Jisuikang (Chinese characters) moderate dose groups showed the most significant effects among all groups,but there was no statistically significant difference between two groups (P > 0.05).

CONCLUSIONThe decoction Jisuikang (Chinese characters) can promote the nerve cell regeneration by regulating Nogo-A and NgR gene expression, activating Nogo- NgR signaling pathways after acute spinal cord injury.

Animals ; Female ; GPI-Linked Proteins ; analysis ; genetics ; physiology ; Medicine, Chinese Traditional ; Myelin Proteins ; analysis ; genetics ; physiology ; Nerve Regeneration ; drug effects ; Nogo Proteins ; Nogo Receptor 1 ; Rats ; Rats, Sprague-Dawley ; Receptors, Cell Surface ; analysis ; genetics ; physiology ; Signal Transduction ; drug effects ; Spinal Cord Injuries ; drug therapy ; metabolism

Perineuronal nets (PNNs) are reticular structures resulting from the aggregation of extracellular matrix (ECM) molecules around the cell body and proximal neurite of specific population of neurons in the central nervous system (CNS). Since the first description of PNNs by Camillo Golgi in 1883, the molecular composition, developmental formation and potential functions of these specialized extracellular matrix structures have only been intensively studied over the last few decades. The main components of PNNs are hyaluronan (HA), chondroitin sulfate proteoglycans (CSPGs) of the lectican family, link proteins and tenascin-R. PNNs appear late in neural development, inversely correlating with the level of neural plasticity. PNNs have long been hypothesized to play a role in stabilizing the extracellular milieu, which secures the characteristic features of enveloped neurons and protects them from the influence of malicious agents. Aberrant PNN signaling can lead to CNS dysfunctions like epilepsy, stroke and Alzheimer's disease. On the other hand, PNNs create a barrier which constrains the neural plasticity and counteracts the regeneration after nerve injury. Digestion of PNNs with chondroitinase ABC accelerates functional recovery from the spinal cord injury and restores activity-dependent mechanisms for modifying neuronal connections in the adult animals, indicating that PNN is an important regulator of neural plasticity. Here, we review recent progress in the studies on the formation of PNNs during early development and the identification of CSPG receptor - an essential molecular component of PNN signaling, along with a discussion on their unique regulatory roles in neural plasticity.
Animals ; Central Nervous System ; physiology ; physiopathology ; Chondroitin Sulfate Proteoglycans ; Extracellular Matrix ; physiology ; Humans ; Neuronal Plasticity ; Neurons ; Receptors, Cell Surface ; physiology

The capability of the central vestibular system in utilizing cues arising from the inner ear determines the ability of animals to acquire the sense of head orientations in the three-dimensional space and to shape postural movements. During development, neurons in the vestibular nucleus (VN) show significant changes in their electrophysiological properties. An age-dependent enhancement of membrane excitability is accompanied by a progressive increase in firing rate and discharge regularity. The coding of horizontal and vertical linear motions also exhibits developmental refinement in VN neurons. Further, modification of cell surface receptors, such as glutamate receptors, of developing VN neurons are well-orchestrated in the course of maturation, thereby regulating synaptic efficacy and spatial coding capacity of these neurons in local circuits. Taken together, these characteristic features of VN neurons contribute to developmental establishment of space-centered coordinates within the brain.
Animals ; Ear, Inner ; physiology ; Electrophysiological Phenomena ; Movement ; Neurons ; physiology ; Rats ; Receptors, Cell Surface ; physiology ; Vestibular Nuclei ; physiology

BACKGROUNDRecent studies have indicated that human nucleus pulposus contain mesenchymal stem cells (NP-MSCs). However, the immunophenotypic variation of NP-MSCs in vitro was unclear. The present study was conducted to address the immunophenotypic variation of mesenchymal stem cells in nucleus pulposus under continuous proliferation in vitro and show the difference between mesenchymal stem cells and nucleus pulposus cell.

METHODSTissue samples were obtained from thoracolumbar burst fracture patients and degenerative disc disease patients who underwent discectomy and fusion procedures. Flow cytometric and laser scanning confocal microscope (LSCM) were used to detect the variation of mesenchymal stem cells in nucleus pulposus which were expressing CD105 and CD24 in condition with or without transforming growth factor β1 (TGF-β1).

RESULTSMore than 90% of the analyzed primary cells of mesenchymal stem cells in nucleus pulposus fulfilled the general immunophenotyping criteria for MSCs, such as CD44, CD105 and CD29, but the marker of mature NP cells characterized as CD24 was negative. In continuous cultures, the proportion of mesenchymal stem cells which were expressing CD44, CD105 and CD29 in nucleus pulposus gradually decreased. The mesenchymal stem cells in nucleus pulposus cells were positive for CD105 and CD29, with slight positivity for CD44. The CD24 expression gradually increased in proliferation. Biparametric flow cytometry and laser scanning confocal microscopy confirmed the presence of cells which were expressing CD105 and CD24 independently, and only a small part of cells expressed both CD105 and CD24 simultaneously. TGF-β1 could stimulate mesenchymal stem cells in nucleus pulposus to express CD24.

CONCLUSIONSNon-degenerative and degenerative NP contains mesechymal stem cells. The variation of CD24 can be used as a marker to identify the NP-MSCs differentiation into NP-like cells.

Adult ; Antigens, CD ; metabolism ; CD24 Antigen ; metabolism ; Cell Differentiation ; physiology ; Cells, Cultured ; Endoglin ; Female ; Flow Cytometry ; Humans ; Hyaluronan Receptors ; metabolism ; Integrin beta1 ; metabolism ; Intervertebral Disc ; cytology ; metabolism ; Male ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Receptors, Cell Surface ; metabolism ; Young Adult

OBJECTIVE: To investigate the self-renewal mechanism of CD133+ cancer stem cells from Hep-2 cell line. METHOD: The CD133+ cells were sorted by flow cytometry from Hep-2 cell line. Then the sorted CD133+ cells were cultured in RPMI1640. The ability of self-renewal of CD133+ cells were tested by MTT assay. mRNA and protein expression of self-renewal related genes were detected by western blot and RT- PCR. RESULT: (3.10 ± 0.21)% of Hep-2 cells expressed the membrane antigen CD133. CD133+ fraction was raised to (90.20 ± 5.51)% by flow cytometry. In vitro culture and growth curve showed CD133+ cells had more active proliferation ability than CD133- cells, which showed statistically significant difference between these two group (P < 0.01). RT- PCR and western blot results showed upregulated mRNA and protein expression of Fas, c-myc, survivin in CD133+ group (P < 0.01). In the same time, the ratio of Bcl-2/Bax gene expression was obviously increased in CD133+ group. Self-renewal related gene such as β-catenin, SHH, SMOH and Bmi-1,Gli-1 were all up-regulated in CD133+ group both in mRNA and protein. On the contrary, PTCH gene was down-regulated. CONCLUSION CD133 positive cells are a small proportion of a Hep-2 cell line. The results of this experiment verified that CD133 positive cells owned the properties of cancer stem cells. Upregulated anti-apoptotic gene is the foundatiom of self-renewal mechanism of CD133+ cells. Cancer stem cells related signal pathways such as Hedgehog, Wnt and Bmi-1 pathway are in state of activation. The identification of self-renewal mechanism about cancer stem cell provides a powerful tool to investigate the tumorigenic process in the larynx and to develop therapies targeting to these signal pathways.
AC133 Antigen ; Antigens, CD ; Apoptosis ; Cell Physiological Phenomena ; physiology ; Down-Regulation ; Flow Cytometry ; Glycoproteins ; Humans ; Laryngeal Neoplasms ; Neoplastic Stem Cells ; physiology ; Patched Receptors ; Patched-1 Receptor ; Peptides ; Receptors, Cell Surface ; genetics ; metabolism ; Signal Transduction ; beta Catenin ; genetics