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

OBJECTIVETo investigate the expression of nesfatin-1/NUCB2 and ghrelin in the gastric mucosa of rats with intrauterine growth retardation (IUGR) and its significance.

METHODSThe IUGR animal model was established by feeding rats low-protein diets during their pregnancy. Newborn rats were divided into catch-up growth, non-catch-up growth and control groups. Protein and mRNA levels of nesfatin-1/NUCB2 and ghrelin in the gastric mucosa of rats were determined by RT-PCR and Western blot, respectively.

RESULTSNesfatin-1/NUCB2 mRNA and protein were expressed in the gastric mucosa of rats immediately after birth, and their expression increased in an age-dependent manner in all three groups. Furthermore, the level of nesfatin-1/NUCB2 in the catch-up growth group was higher than that in the control group before weaning, whereas there was no significant difference in nesfatin-1/NUCB2 expression between the two groups after weaning. The level of nesfatin-1/NUCB2 in the non-catch-up growth group was lower than that in the catch-up growth group during the whole observation period. The level of ghrelin in the catch-up growth group was higher than that in the control group starting from day 12 after birth, whereas there was no significant difference in ghrelin expression between the two groups after weaning. The level of ghrelin in the non-catch-up growth group was lower compared with those in the catch-up growth and control groups from days 12 to 28 after birth.

CONCLUSIONSNesfatin-1 and ghrelin are co-expressed in the gastric mucosa of rats with IUGR after birth and interact with each other to produce long-term nutritional regulation.

Age Factors ; Animals ; Calcium-Binding Proteins ; analysis ; genetics ; DNA-Binding Proteins ; analysis ; genetics ; Female ; Fetal Growth Retardation ; metabolism ; Gastric Mucosa ; chemistry ; Ghrelin ; analysis ; genetics ; Male ; Nerve Tissue Proteins ; analysis ; genetics ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley

BACKGROUNDThe mechanism of retinal neovascularization is not understood completely. Many growth factors are involved in the process of retinal neovascularization, such as vascular endothelial growth factor (VEGF) and pigment epithelium-deprived factor (PEDF), which are the representatives of angiogenic and antiangiogenic molecules respectively. Oxygen induced retinopathy (OIR) is a useful model to investigate retinal neovascularization. The present study was conducted to investigate the feasibility of small interference RNA (siRNA) targeting VEGF gene in attenuating oxygen induced retinopathy (OIR) by regulating VEGF to PEDF ratio (VEGF/PEDF).

METHODSIn vitro, cultured EOMA cells were transfected with VEGF-siRNA (psi-HI(TM)/EGFP/VEGF siRNA) and Lipofectamine(TM) 2000 for 24, 48, and 72 hours, respectively. Expression of VEGF mRNA was evaluated by real time polymerase chain reaction (PCR) and the level of VEGF protein was analyzed by Western blotting. In vivo, OIR model mice were established, the mice (C57BL/6J) received an intra-vitreal injection of 1 µl of mixture of psi-HI(TM)/EGFP/VEGF siRNA and Lipofectamine 2000. Expressions of retinal VEGF and PEDF protein were measured by Western blotting, retinal neovascularization was observed by fluorescein angiography, and quantified.

RESULTSIn vitro psi-HI(TM)/EGFP/VEGF siRNA treatment significantly reduced VEGF mRNA and protein expression. In vivo, with decreased VEGF and VEGF-PEDF ratio, significant attenuation of neovascular tufts, avascular regions, tortuous, and dilated blood vessels were observed in the interfered animals.

CONCLUSIONSVEGF plays an important role in OIR, and the transfection of VEGF-siRNA can effectively downregulate VEGF expression in vivo, accompanied by the downregulation of VEGF-PEDF ratio, and simultaneous attenuation of retinal neovascularization was also observed. These findings suggest that VEGF/PEDF may serve as a potential target in the treatment of retinal neovascularization and RNA interference targeting VEGF expression, which represents a possible therapeutic strategy.

Animals ; Eye Proteins ; analysis ; Mice ; Mice, Inbred C57BL ; Nerve Growth Factors ; analysis ; RNA, Small Interfering ; genetics ; Retinal Neovascularization ; therapy ; Serpins ; analysis ; Vascular Endothelial Growth Factor A ; analysis ; genetics ; physiology

OBJECTIVETo study the effects of ganglioside 1 (GM1) and nerve growth factor (NGF) on neural stem cells (NSCs) proliferation in vitro.

METHODSNSCs were isolated and cultured in vitro. NSCs were cultured in the medium containing epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) or without the two agents. Different concentrations of GM1 and NGF were added into the two different medium. MTT and cell ball counting methods were used to ascertain the proliferation of NSCs. Immunohistochemical technology was used to observe the effect of GM1 and NGF on the proliferation of NSCs.

RESULTSHigh concentrations of GM1 (100 ng/L and 200 ng/L) promoted significantly the proliferation of NSCs in the medium containing EGF and bFGF (p<0.05). In the differentiation medium containing serum but no EGF and bFGF, NSCs proliferation increased with increasing concentration of GM1; the proportion of neurons and gliacytes increased with increasing concentration of NGF.

CONCLUSIONSHigh concentration of GM1 can promote NSCs proliferation and NGF can promote NSCs differentiation.

Animals ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Female ; G(M1) Ganglioside ; pharmacology ; Intermediate Filament Proteins ; analysis ; Male ; Nerve Growth Factors ; pharmacology ; Nerve Tissue Proteins ; analysis ; Nestin ; Neurons ; cytology ; drug effects ; Rats ; Rats, Sprague-Dawley ; Stem Cells ; drug effects ; physiology

BACKGROUNDMany studies have suggested that the imbalance of angiogenic factor and anti-angiogenic factor expression contributes significantly to the development of choroidal neovascularization (CNV), and ultrasound microbubble combination system can increase the gene transfection efficiency successfully. This study was designed to investigate whether ultrasound-mediated microbubble destruction could effectively deliver therapeutic plasmid into the retina of rat, and whether gene transfer of pigment epithelium-derived factor (PEDF) could inhibit CNV.

METHODSHuman retinal pigment epithelial cells were isolated and treated either with ultrasound or plasmid alone, or with a combination of plasmid, ultrasound and microbubbles to approach feasibility of microbubble-enhanced ultrasound enhance PEDF gene expression; For in vivo animal studies, CNV was induced by argon lasgon laser in rats. These rats were randomly assigned to five groups and were treated by infusing microbubbles attached with the naked plasmid DNA of PEDF into the vitreous of rats followed by immediate ultrasound exposure (intravitreal injection); infusing liposomes with the naked plasmid DNA of PEDF into the vitreous (lipofectamine + PEDF); infusing microbubbles attached with PEDF into the orbit of rats with ultrasound irradiation immediately (retrobular injection); infusing microbubbles attached with PEDF into the femoral vein of rats with exposed to ultrasound immediately (vein injection). The CNV rats without any treatment served as control. Rats were sacrificed and eyes were enucleated at 7, 14, and 28 days after treatment. Gene and protein expression of PEDF was detected by quantitative real-time RT-PCR, Western blotting and immunofluorescence staining, respectively. The effect of PEDF gene transfer on CNV was examined by fluorescein fundus angiography.

RESULTSIn vitro cell experiments showed that microbubbles with ultrasound irradiation could significantly enhance PEDF delivery as compared with microbubbles or ultrasound alone. In the rat CNV model, transfection efficiency mediated by ultrasound/microbubbles was significantly higher than that by lipofectamine-mediated gene transfer at 28 days after treatment. The study also showed that with the administration of ultrasound-mediated microbubbles destruction, the CNV of rats was inhibited effectively.

CONCLUSIONSUltrasound-microbubble technique could increase PEDF gene transfer into rats' retina and chorioid, in association with a significant inhibition of the development of CNV, suggesting that this noninvasive gene transfer method may provide a useful tool for clinical gene therapy.

Animals ; Cells, Cultured ; Choroidal Neovascularization ; prevention & control ; Eye Proteins ; genetics ; Female ; Genetic Therapy ; Humans ; Microbubbles ; Nerve Growth Factors ; genetics ; RNA, Messenger ; analysis ; Rats ; Rats, Long-Evans ; Retina ; metabolism ; Serpins ; genetics ; Transfection ; Ultrasonics

OBJECTIVETo observe the expression and distribution of adult rat axon guidance cues Netrin-1 and Slit2 at different time points after spinal cord injury and to investigate the guidance mechanism of regenerated axons.

METHODSTwenty adult Sprague Dawley (SD) rats were divided randomly into five groups with 4 in each. Four groups of them were used to make Allen's spinal cord punch models and we took materials randomly from one of them on the 2nd, 4th, 7th and 14th day respectively after operation. The left one group was taken as the control group. Immunofluorescence laser confocal scan was used to examine the co-expression and localization of Netrin-1 and Slit2 proteins in the injured site of the spinal cord.

RESULTSWithin two weeks after SCI, the expression of Netrin-1 and Slit2 proteins increased temporarily and there was co-expression of them on the neuron plasma membrane.

CONCLUSIONSSynchronous high expression and co-expression of axon attractant Netrin-1 and repellent Slit2 are found in the adult rat injured spinal cord in the damaged local and vicinity parts, and probably, they act as the key regulators of axon guidance regeneration.

Animals ; Female ; Intercellular Signaling Peptides and Proteins ; analysis ; Male ; Microscopy, Confocal ; Nerve Growth Factors ; analysis ; Nerve Regeneration ; physiology ; Nerve Tissue Proteins ; analysis ; Netrin-1 ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Spinal Cord Injuries ; metabolism ; Tumor Suppressor Proteins ; analysis

OBJECTIVETo detect the levels of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) in aqueous humor of patients with active choroidal neovascularization (CNV).

METHODSAqueous humor samples were obtained from 32 patients with active CNV. The concentrations of VEGF and PEDF in aqueous humor were measured by enzyme linked immunosorbent assay (ELISA) for quantitative analysis. VEGF and PEDF in 10 samples of aqueous humor from patients with cataract were also detected by the same methods as control.

RESULTThe mean VEGF and PEDF concentrations in aqueous humor of active CNV patients were higher than those in the control group (P=0.000).

CONCLUSIONThe patients with active CNV exhibit significantly higher VEGF and PEDF levels than those in control, indicating that VEGF along with PEDF may modulate the formation of CNV.

Adult ; Aged ; Aged, 80 and over ; Aqueous Humor ; chemistry ; Choroidal Neovascularization ; metabolism ; Eye Proteins ; analysis ; Female ; Humans ; Male ; Middle Aged ; Nerve Growth Factors ; analysis ; Serpins ; analysis ; Vascular Endothelial Growth Factor A ; analysis

OBJECTIVETo observe the effect of Kangxin Capsule (KXC) on the expression of nerve growth factor (NGF) as well as the morphology and amount of nerve synapse in the cortical parietal lobe and hippocampus CA, area of vascular dementia ( VD) model rats.

METHODSThe model rats of VD made by photochemical reaction technique were randomly divided into five groups: the model group (MG), the high-dose, middle-dose and low-dose KXC groups (HDG, MDG and LDG), and the Western medicine hydergin control group (WMG). They were treated respectively with distilled water, high, middle and low dosage of KXC suspended liquid, and hydergin for a month. Besides, a blank group consisting of normal (non-model) rats was set up for control (CG). The ultrastructure of nerve synapse in the cortical parietal lobe and hippocampus CA1 area of the rats were observed and its density estimated. The condition of NGF positive neurons in the above-mentioned two regions were also observed by immunohistochemical stain.

RESULTSAll the KXC or hydergin treated groups demonstrated a normal amount of nerve synapse with integral structure in the cortical parietal lobe and hippocampus CA, area, which approached that in the CG and was superior to that in the MG. Also, the NGF positive neuron in all the treated groups was much more than that in MG with significant difference ( P<0.01), approaching to that in the CG.

CONCLUSIONKXC could elevate the expression of NGF in the cortical parietal lobe and hippocampus CA, area, preserve the number and morphology of synapse, thus to protect the function of nerve system from ischemic injury.

Animals ; Capsules ; Dementia, Vascular ; drug therapy ; metabolism ; pathology ; Disease Models, Animal ; Female ; Hippocampus ; chemistry ; Immunohistochemistry ; Male ; Medicine, Chinese Traditional ; Microscopy, Electron ; Nerve Growth Factors ; analysis ; Neurons ; ultrastructure ; Parietal Lobe ; chemistry ; Rats ; Rats, Wistar ; Synapses ; ultrastructure

OBJECTIVE: To investigate the dynamics of the induction of S100beta in different parts of rat brain following the diffuse brain injury. METHODS: Immunohistochemistry and auto-image analysis were to determine the expression of astroglial S100beta after diffuse brain injury in rats. Forty rats were distributed into groups according to injury time of 30min, and2,4,12,24h, and 3,6 d after diffuse brain injury, and normal rats as control. RESULTS: The number of S100beta positive cells in the four areas increased significantly followed by a decrease, and then a further increase. The expression of S100beta could be detected increasing in 2h, and increased significantly in 4h, and it reached apex 12h after DBI, and decreased gradually to the level less than normal 3d, and returned to normal 7d following injury. In the postmortem injury groups, there were no significant changes in anti-S100beta immunoreactivities in four areas of brain compared to the control group. CONCLUSION The present study showed the time-dependent expression of S100beta is obvious following diffuse brain injury, and suggested S100beta be suitable as a marker for brain injury age determination.
Animals ; Brain/pathology* ; Brain Edema/pathology* ; Brain Injuries/pathology* ; Female ; Immunohistochemistry ; Male ; Nerve Growth Factors/analysis* ; Neuroglia/metabolism* ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; S100 Calcium Binding Protein beta Subunit ; S100 Proteins/analysis* ; Staining and Labeling ; Time Factors

OBJECTIVETo investigate the expression and pattern of vascular endothelial growth factor (VEGF) and its fetal liver kinase-1 (Flk-1) receptor in spinal cord and dorsal root ganglia after neurotomy of sciatic nerve in rats.

METHODSForty-five adult male Wistar rats were divided randomly into a control group (n=5) and an experimental group (n=40). The bilateral sciatic nerves of the rats in the experimental group underwent neurotomy and the L4-L6 spinal cord and the corresponding dorsal root ganglia were harvested respectively at 8 hours, and 1, 3, 5, 7, 10, 14 and 21 days (8 subgroups with 5 rats each) after operation. The rats in the control group only underwent an exposure of sciatic nerve without neurotomy. Immunohistochemistry and image analysis were used to study the expression of VEGF and its Flk-1 receptor.

RESULTSBoth VEGF and Flk-1 receptor expressed in the normal rat spinal cord and dorsal root ganglia. In response to neurotomy, their expression reached a higher level and persisted for a short time then declined to the normal level rapidly. Besides, positive staining of Flk-1 was observed in both glial cells and nerve fibers, which located in the white matter of the spinal cord.

CONCLUSIONSVEGF can promote the regeneration of peripheral nerves from the angle of central neurons, which establishes the experimental and theoretical foundation for VEGF treating peripheral nerve injuries.

Analysis of Variance ; Animals ; Ganglia, Spinal ; metabolism ; Immunoenzyme Techniques ; Male ; Random Allocation ; Rats ; Rats, Wistar ; Sciatic Nerve ; metabolism ; surgery ; Vascular Endothelial Growth Factor Receptor-2 ; biosynthesis ; Vascular Endothelial Growth Factors ; biosynthesis