Objective To establish a method for the cultivation of hain follicle outer root sheath (ORS) cells of murine vibrissa and to study their expression of uPA/uPAR. Methods The ORS cells of murine vibrissa were cultured by combination of enzyme digestion and tissue culture with the feeder layer of dermal sheath cells of the hair follicle. The cells and their uPA/uPAR expression were identified by immunocytochemistry (ICC). Results The feeder layer of DS was suitable for the culture of ORS cells. ORS cells expressed uPA/uPAR protein. Conclusion The DS of hair follicle is a better feeder layer for the growth of ORS cells. ORS cells have the ability to migrate and proliferate.

Objective To determine the protective effects of endotoxin preconditioning on the brain ischemic injury and explore its protective mechanism. Methods Global ischemia for 20 min by four-vessel occlusion model was used in 140 Wistar rats, in which 70 rats were pretreated with MPL at dose of 20 ?g/kg 24 h before global ischemia. Another 10 Wistar rats served as normal controls. Ischemic neurons and neuron density of the hippocampal CA1 region, MPO activity, TNF-? activity, IL-1? activity were assayed at 4, 12, 24, 48, 72, 120, 168 h after reperfusion. Results Pretreatment with endotoxin produced significant reductions in ischemic neuron number, MPO activity, TNF-? activity and IL-1? activity. Conclusion Endotoxin preconditioning protects brain from subsequent ischemic injury, in which the suppression of cellular inflammation may be the protective mechanism.

OBJECTIVEThis study aims to investigate the regulatory effects of calcitonin gene-related peptide (CGRP) on Nod-like receptor protein 3 (NLRP3) and interleukin-1β (IL-1β) to promote osteoblast differentiation.

METHODSDifferent concentrations of CGRP (0, 10, 30, 100 ng · mL⁻¹) were added to mouse osteoblasts in vitro. The mRNA and protein expression levels of both NLRP3 and IL-1β were examined using Real-time polymerase chain reaction and Western blot, respectively. Moreover, the concentrations of IL-1β protein and intracellular reactive oxygen species (ROS) were detected using enzyme-linked immunosorbent assay and flow cytometry, respectively. The osteogenic differentiation of mouse osteoblasts was identified through alizarin red staining.

RESULTSThe protein and mRNA expression levels of both NLRP3 and IL-1β significantly decreased (P < 0.05) with increasing CGRP concentration. Moreover, the contents of intracellular ROS gradually decreased (P<0.05). The osteogenic differentiation of the osteoblasts was more enhanced in the group treated with 100 ng · mL⁻¹ CGRP than in the empty group (0 ng · mL⁻¹ CGRP).

CONCLUSIONCGRP promotes osteoblast differentiation by inhibiting the expression of inflammatory factors.

Animals ; Blotting, Western ; Calcitonin ; Calcitonin Gene-Related Peptide ; Cell Differentiation ; Enzyme-Linked Immunosorbent Assay ; Interleukin-1beta ; Mice ; NLR Family, Pyrin Domain-Containing 3 Protein ; NLR Proteins ; Osteoblasts ; Osteogenesis ; RNA, Messenger ; Reactive Oxygen Species ; Real-Time Polymerase Chain Reaction

OBJECTIVETo investigate the nerve growth factor (NGF) regulating the expression of calcitonin gene-related peptide (CGRP) in promoting the proliferation of osteoblast-like cell (MG-63) and thus illustrate the mechanism of the NGF in wound healing.

METHODSDifferent concentrations of NGF were used to stimulate MG-63. The expression of CGRP was detected by real-time quantitative polymerase chain reaction (RT-QPCR) and enzyme-linked immunosorbent assay after 1, 2, 3, and 4 days. The proliferation of MG-63 was detected by cell counting kit-8 (CCK-8). The expression of CGRP mRNA and the proliferation of MG-63 were then detected by RT-QPCR and CCK-8 after adding the NGF receptor blocker.

RESULTSCompared with the blank control group, the expression of CGRP significantly increased by stimulating the NGF. The expression of CGRP was positively related to the concentration of NGF (P<0.05). Moreover, the expression of CGRP increased by prolonging the NGF stimulation time. The proliferation of MG-63 increased after stimulating the NGF (P<0.05). After adding the NGF receptor blocker, the expression of CGRP and the proliferation of MG-63 correspondingly decreased (P<0.05).

CONCLUSIONNGF can up-regulate the expression of CGRP and increase the proliferation of MG-63. Therefore, NGF plays a significant role in wound healing.

Animals ; Calcitonin ; Calcitonin Gene-Related Peptide ; metabolism ; Cell Line ; Ganglia, Spinal ; Humans ; Nerve Growth Factor ; metabolism ; Rats, Sprague-Dawley ; Receptor, Nerve Growth Factor ; Signal Transduction ; Up-Regulation

Objective To explore the roles of urokinase type PA (uPA) and uPA receptor (uPA R) in the course of repair after human embryo corneal alkali burn in vitro . Methods After the alkali burn model in vitro was established successfully, some techniques such as the observation of cell culture and morphology, ICC and image analysis were used. Results No significant difference was found between the cells from corneal limbus and central cornea and almost all of them expressed AE1/AE3. The expressions of uPA and uPA R increased to the maximum at about 24 h after alkali burn. uPA expression distributed mainly in the cytoplasm but uPA R expression distributed mainly in the cell membrane. Conclusion The corneal epithelial cells of comparatively high purity can be acquired by tissue culture. There probably exists difference in development and vitality between embryo and adult cornea. There might be commonness of the roles of uPA and uPA R in epithelial tissue.

Objective To determine the effects and significance of endotoxin preconditioning on iNOS activity,nNOS activity in rat cerebral cortex,hippocampus and cerebella region after global brain ischemia/reperfusion.Methods Global ischemia for 20 min was made by four-vessel occlusion model(4-vo) in 60 Wistar rats,among which 30 were injected of 20 ?g/kg MPL through caudal vein 24 h before model establishment.Another 8 rats undergoing sham operation served as controls.The dynamic change of iNOS activity,nNOS activity and neuron density of the cerebral cortex,hippocampus and cerebella region were observed at 1,4,8 h after reperfusion. Results The activity of iNOS and nNOS decreased significantly after endotoxin preconditioning in the regions mentioned above,as compared with that of rats only undergoing the ischemia/reperfusion.The neuron number in rat hippocampus decreased after ischemia/reperfusion,but no significant difference was found between control and endotoxin preconditioning groups.Conclusion The activity of iNOS and nNOS changed significantly after global brain ischemia/reperfusion.That endotoxin preconditioning decreased iNOS and nNOS activities may be the protective mechanism.

ObjectiveTo?investigate?the?nerve?growth?factor?(NGF)?regulating?the?expression?of?calcitonin?gene-related?peptide?(CGRP)?in?promoting?the?proliferation?of?osteoblast-like?cell?(MG-63)?and?thus?illustrate?the?mechanism?of?the?NGF?in?wound?healing.?Methods???Different?concentrations?of?NGF?were?used?to?stimulate?MG-63.?The?expression?of?CGRP?was?detected?by?real-time?quantitative?polymerase?chain?reaction?(RT-QPCR)?and?enzyme-linked?immunosorbent?assay?after?1,?2,?3,?and?4?days.?The?proliferation?of?MG-63?was?detected?by?cell?counting?kit-8?(CCK-8).?The?expression?of?CGRP?mRNA?and?the?proliferation?of?MG-63?were?then?detected?by?RT-QPCR?and?CCK-8?after?adding?the?NGF?receptor?blocker.?Results???Com-pared?with?the?blank?control?group,?the?expression?of?CGRP?significantly?increased?by?stimulating?the?NGF.?The?expression?of?CGRP?was?positively?related?to?the?concentration?of?NGF?(P<0.05).?Moreover,?the?expression?of?CGRP?increased?by?pro-longing?the?NGF?stimulation?time.?The?proliferation?of?MG-63?increased?after?stimulating?the?NGF?(P<0.05).?After?adding?the?NGF?receptor?blocker,?the?expression?of?CGRP?and?the?proliferation?of?MG-63?correspondingly?decreased?(P<0.05).?Conclusion???NGF?can?up-regulate?the?expression?of?CGRP?and?increase?the?proliferation?of?MG-63.?Therefore,?NGF?plays?a?significant?role?in?wound?healing.

Objective This study aims to investigate the regulatory effects of calcitonin gene-related peptide (CGRP) on Nod-like receptor protein 3 (NLRP3) and interleukin-1β (IL-1β) to promote osteoblast differentiation. Methods Different concentrations of CGRP (0, 10, 30, 100 ng·mL-1) were added to mouse osteoblasts in vitro. The mRNA and protein expression levels of both NLRP3 and IL-1β were examined using Real-time polymerase chain reaction and Western blot, respectively. Moreover, the concentrations of IL-1β protein and intracellular reactive oxygen species (ROS) were detected using enzyme-linked immunosorbent assay and flow cytometry, respectively. The osteogenic differentiation of mouse osteoblasts was identified through alizarin red staining. Results The protein and mRNA expression levels of both NLRP3 and IL-1β significantly decreased (P<0.05) with increasing CGRP concentration. Moreover, the contents of intracellular ROS gradually decreased (P<0.05). The osteogenic differentiation of the osteoblasts was more enhanced in the group treated with 100 ng·mL-1 CGRP than in the empty group (0 ng·mL-1CGRP). Conclusion CGRP promotes osteoblast differentiation by inhibiting the expression of inflammatory factors.

The cultivation of innovation ability of medical students is requirement of the Times.Considering the shortcomings of the existing teaching methods,many specialized designs for teaching were implemented in the extracurricular activities of cell biology such as critical literature study,independent experiment and participation in the research of department project.It is proved that this teaching activity did well in expanding the contents of the formal class,training the practicing skills,inspiring independent think ing and improving the ability of research and innovation.

Objective: Chronic graft-versus-host disease (cGVHD) is a major long-term complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT) . It is important to study the changes of serum biomarkers expression in patients for early diagnosis and treatment. Methods: The expression levels of five serum protein markers (IL-1b, IL-16, CXCL9, CCL19, CCL17) in patients with or without cGVHD after allo-HSCT were detected by liquid suspension microarray. Results: Compared with the control group without cGVHD, the expression levels of CXCL9 and CCL17 in serum of patients with cGVHD were significantly increased (P<0.05) . CCL17 was correlated with the severity of cGVHD (P<0.001) . CXCL9 was significantly increased in the serum of patients with skin lesion (P<0.01) , and CCL17 was significantly expressed in cGVHD patients with liver as the target organ (P<0.01) . Conclusion The combination of CXCL9 and CCL17 can be used as serum biomarkers of cGVHD, which has certain reference value in assisting the diagnosis and evaluation of cGVHD severity.