BACKGROUND:A series of studies indicate that autophagy is closely linked with differentiation. Bone morphogenetic protein 2 (BMP-2) is the classical pathway for C2C12 and MC3T3-E1 osteoblast differentiation, and the ideal model to study osteogenic differentiation process.
OBJECTIVE:To observe the relationship between autophagy and BMP-2-induced celllines C2C12, MC3T3-E1 osteoblast differentiation.
METHODS:Real-Time PCR was applied to detect osteogenic differentiation and autophagy related index after C2C12 and MC3T3-E1 were induced with BMP-2 (100μg/L) for 72 hours. The osteogenic index alkaline phosphatase in BMP-2-induced MC3T3-E1 and C2C12 cultured with different concentrations of 3-methyladenine (0, 1, 5, 10 mmol/L) was determined with alkaline phosphatase staining. Western blot analysis was applied to detect LC3-I/II expression levels in C2C12 and MC3T3-E1 induced with BMP-2 for different time points (0, 12, 24, 48, 72, 96 hours).
RESULTS AND CONCLUSION:The autophagy-related mRNA and protein expression showed an increasing tendency and autophagy-related protein LC3 levels was increased, which was associated with the time, during the BMP-2-induced celllines C2C12, MC3T3-E1 osteoblast differentiation. Meanwhile, alkaline phosphatase expression levels were inhibited by autophagy in the process of osteogenic differentiation. Therefore, there is a close relationship between autophagy and the BMP-2-induced celllines C2C12, MC3T3-E1 osteoblast differentiation.

Objective To evaluate the relationship between vascular endothelial growth factor (VEGF) and extracellular signal-regulated kinase (ERK) signaling pathway in spinal dorsal horns of rats with neuropathic pain.Methods Twenty male Sprague-Dawley rats,aged 2 months,weighing 160-320 g,were randomly divided into 4 groups (n =5 each):sham operation group (group S); chronic constrictive injury (CCI) group; normal saline group (NS group); VEGF antibody group.Neuropathic pain was induced by CCI.The animals were anesthetized with intraperitoneal 10％ chloral hydrate 350 mg/kg.The left sciatic nerve was exposed and 4 ligatures were placed on the sciatic nerve at 1 mm intervals.In group S,the left sciatic nerve was only exposed but not ligated.In VEGF antibody group,VEGF antibody 0.3μg/15 μl was injected intrathecally every 2 days for 4 times starting from 2 h after CCI,while the equal volume of normal saline was injected in NS group.Paw withdrawal threshold to von Frey filament stimulation (PWMT) and paw withdrawal latency to nociceptive thermal stimulation (PWTL) were measured at 1 day before CCI (T1),and 1,3,5 and 7 days after CCI (T2-5).The rats were sacrificed after PWMT and PWTL were measured and the L4-6 segments of the spinal cord were removed for determination of phosphorylated ERK (p-ERK) expression in spinal dorsal horns by immunohistochemistry and Western blot.Results Compared with group S,PWMT and PWTL were significantly decreased at T2-5,and the p-ERK expression in spinal dorsal horns was up-regulated in CCI and VEGF antibody groups (P ＜ 0.05).Compared with CCI group,PWMT and PWTL were significantly increased at T3-5,and the p-ERK expression in spinal dorsal horns was down-regualted in VEGF antibody group (P ＜ 0.05).Conclusion VEGF in the spinal dorsal horn is involved in the development and maintenance of neuropathic pain in rats through activating ERK signaling pathway.

Fourteen compounds were isolated from 95% ethanol extract by silica gel, MCI, and ODS column chromatography. These compounds were respectively identified as quercetin (1), kaempferol (2), (+)-catechin (3), fraxin (4), protocatechuic acid (5), gallic acid (6), methyl gallate (7), ethyl gallate (8), apocynol A (9), baccatin (10), cerevisterol (11), ellagic acid (12), 3, 3',4'-tri-0-methylellagic acid(13) and N-benzoyl-L-phenylalaninyl-N-benzoyl-L-phenylalaninate(14) by analyzing their spectral data and comparing with the previously reported literatures. Except for gallic acid (6), all other compounds were isolated from this plant for the first time. Compounds 1, 2 and 6 showed moderate anti-proliferation activities on tumor cells.
Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Drugs, Chinese Herbal ; chemistry ; toxicity ; Euphorbiaceae ; chemistry ; Humans ; Plants, Medicinal ; chemistry ; Spectrometry, Mass, Electrospray Ionization

Laboratory animals and animal experiments are foundations and important support conditions for life sciences, especially for medical research. The animal experiments have drawn extensive attention from the society because of the ethical issue. This paper takes Wenzhou Medical University as an example to give a brief introduction to the ethical review about laboratory animals in the university so as to further draw attention and concerns from the public about the ethical issue of laboratory animals. We successively introduce its scientific projects, nurturing environment and ethical review of laboratory animals.
Animal Experimentation ; ethics ; Animals ; Animals, Laboratory ; Universities

BACKGROUND:Long non-coding RNA (lncRNA) regulates a series of physiological processes and it is considered to play important roles in the gene regulation of development, differentiation and metabolism. MC3T3-E1, C2C12 and C3H10T1/2 cells are able to differentiate into different celllineages, such as bone cells and muscle cells, and they can be used in the study of musculoskeletal diseases.
OBJECTIVE:To study the role of lncRNA in osteogenic differentiation induced by bone morphogenetic protein 2.
METHODS:Osteogenic differentiation of MC3T3-E1, C2C12 and C3H10T1/2 cells was induced by bone morphogenetic protein 2, and microarray expression profiling of lncRNA was undertaken in osteogenic differentiation. LncRNA simultaneous changes in three cells were found out. The siRNA interference of the lncRNA was used to study its effects on the osteogenic differentiation induced by bone morphogenetic protein 2. Real-time PCR and alkaline phosphatase staining were applied to detect osteogenesis related indicators.
RESULTS AND CONCLUSION:In the process of osteogenic differentiation induced by bone morphogenetic protein 2, osteogenic differentiation indicators were increased, while myogenic differentiation indicator myogenin was reduced. LncRNA AK007000 was screened out to play a role in osteogenic differentiation induced by bone morphogenetic protein 2. Knockdown of lncRNA AK007000 decreased the expression of osteogenic differentiation indicators, while increased the expression of myogenin. Therefore, AK007000 may play a role in promoting osteogenic differentiation and inhibiting myogenic differentiation.

Background Fungal corneal ulcer is a visual-threatening eye disease,and drug therapy has a limiting efficacy.Corneal transplantation or eye enucleation sometimes is necessary to the severe patients.Corneal collagen cross-linking (CXL) is an effective method for some corneal diseases,but the study on CXL for fungal corneal ulcer is lack.Objective This study was to evaluate the clinical effectiveness and safety CXL for fungal corneal ulcer.Methods Fifteen 8-week-old healthy New Zealand white rabbits were used in this study and other 5 rabbits served as normal controls.Fungal corneal ulcer models were established in the right eyes of other 10 rabbits by infecting sickle bacteria liquid after corneal scratching and removing corneal epithelium,then decellularized ostrich corneal patch covered the defected cornea.The models were randomly divided into the non-treatment group and the CXL treatment group.Corneal lesions were examined under the slit lamp microscope every day,and cornea was pictured by laser scanning confocal microscope on the 3rd,7th,14th,21st and 28th day individually after CXL.All rabbits were sacrificed and corneal tissues were obtained 4 weeks after treatment,and the collagen fiber diameter and fibrocytes were observed under the scanning electron microscope.Results Fungal corneal ulcer models were successfully established by corneal scratching and decellularized ostrich cornea covering.The gray ulcer lesions and hypbae like bean pod were seen by slit lamp microscope and laser scanning confocal microscope 3 days after modeling.Corneal ulcer deepened and expanded 1 week later,and there were a large number of spore and hyphae criss-crossing as short rod in shallow stroma.Inflammatory cells were observed in corneal endothelial cells and ocular anterior chamber.In the CXL treatment group,the range of corneal epithelial deficiency was less than that in the nontreatment group on the 3rd,7th,14th,and 21st (all at P＜ 0.05).The diameters of collagen fibers were (24.6± 1.8) nm,(24.9 ± 1.9) nm and (43.0 ± 7.4) nm in the normal control group,non-treatment group and CXL treatment group,showing a significant difference among the 3 groups (F =27.05,P =0.00),and the collagen diameters were thicker in the CXL treatment group than those in the normal control group and non-treatment group (t =5.40,-5.30,both at P＜0.05),and fibrocytes were seen among the collagen fibers.No significant difference was found in the collagen diameters between the non-treatment group and normal control group,and the fibrocytes were less in the non-treatment group.Conclusions CXL therapy can treat fungal corneal ulcer by enhancing collagen,promoting fibrocytes proliferation,suppressing fungus and inflammatory response and accelerating tissue repair.

AlM:To study the refractive status characteristics aftser cataract surgery and the correlation between preoperative anterior chamber depth ( ACD) and refractive status.METHODS: Ninety-six cases of patients with cataract were randomly divided into two groups. The patients in phacoemulsification group were treated with phacoemulsification combined intraocular lens ( lOL ) implantation while the patients in small incision group were treated by small incision extracapsular cataract extraction combined with lOL implantation. Changes in ACD and postoperative refractive status and refractive fully corrected value were counted and the correlation of them were analyzed .RESULTS: ACD of the phacoemulsification group s deepened 0. 74mm while that of the small incision group deepened 0. 78mm after treatment and there was no significant difference (P>0. 05). After operation, the ACD of two groups significantly deepened ( P<0. 05 ). The postoperative visual acuity of two groups were significantly better than the uncorrected visual acuity of two groups (P<0. 05). The postoperative refraction of two groups patients were mainly 0 ~ +1. 0D ( 41. 67% and 54. 16%) and+1. 25~+2. 0D (43. 75% and 33. 33%) (P>0. 05). CONCLUSlON: ACD is significant deepened after operation. Surgeon needs full consideration of changes to improve the refractive lOL calculation accuracy.

Objective This paper was to study the absorption and metabolism differences of intestine and liver for multicomponent licorice.Methods The components were identified with the UPLC-MS/MS. In situ closed-loop method was used to carry out the comparative experiments of absorption and metabolism differences between intestine and liver.Results 13 components were identified by UPLC-MS/MS. The absorption and metabolism results indicated some components in licorice water extract could be absorbed into blood and metabolism happened during this process. 14 metabolites were detected in the plasma sample. The hepatic metabolism results indicated many components could experience complex metabolism and more metabolites could be generated.Conclusions Liver was the major metabolism organ for licorice water extract and some components could be metabolized along with the absorption process in intestine. The absorption and metabolism differences between intestine and liver were significant.

OBJECTIVETo analyze the genetic polymorphism, distribution of haplotypes, common and well-documented (CWD) and rare alleles of high-resolution HLA-A, B and DRB1 alleles by analysis from hematopoietic stem cell (HSC) donors in Jiangsu Han Chinese.

METHODSPCR-sequence-based typing and PCR-sequence specific oligonucleotide probes methods were applied for HLA-A, B and DRB1 high-resolution genotyping of 3238 unrelated healthy donors of hematopoietic stem cells in Jiangsu branch of Chinese National Marrow Donor Program registry.

RESULTS46 alleles of HLA-A,85 HLA-B and 51 HLA-DRB1 locus were found. The frequencies of the most common alleles were A * 11:01 (16.52%), B * 13:02 (11.60%) and DRB1 *07:01 (15.78%). That of the most common haplotype was A * 30: 01-B * 13: 02-DRB1 * 07: 01 (8.87%). 40 alleles of HLA-A,77 alleles of HLA-B, and 47 HLA-DRB1 alleles of HLA-DRB1 were CWD, which account for 99. 8% of total number of samples, and a few rare alleles not reported in Chinese population were found.

CONCLUSIONThe results of high-resolution, CWD and rare alleles showed the characteristics of HLA distribution in Jiangsu Han population, which may be useful for finding HLA matched unrelated donors, as well as for HLA correlation with population genetics and disease association studies.

Alleles ; Asian Continental Ancestry Group ; genetics ; Genotype ; HLA-A Antigens ; genetics ; HLA-B Antigens ; genetics ; HLA-DRB1 Chains ; genetics ; Haplotypes ; Hematopoietic Stem Cells ; Humans ; Tissue Donors

OBJECTIVETo explore the effect of clinical application of stand-alone MC+PEEK cage in anterior cervical fusion.

METHODSFrom January 2011 to January 2014,50 patients were treated with the MC+PEEK cage filled with autogenous cancellous illic-bone graft after anterior cervical discectomy. There were 22 patients with cervical spondylosis,26 patients with traumatic cervical disc herniation, 2 patients with cervical instability in these patients. There were 32 males and 18 females, aged from 30 to 79 years old with an average of 53.30 years old. There were 32 patients with single segment, 15 patients with double segments and 3 patients with three segments. Cervical AP and lateral and the flexion-extension X-rays were regularly taken in order to assess the cervical physiological curvature, the graft fusion and internal fixation related complications. Nerve function, clinical effect and bone fusion were respectively evaluated according to Japan Orthopedic Association (JOA), Otani grade and Suk method.

RESULTSAll patients were followed up from 6 to 36 months with an average of 20 months. No correlated surgical complications were found and all patients obtained bony fusion with an average time of 4.30 months. JOA score had significantly improvement after surgery (P < 0.05). The JOA score was 10.60 ± 3.00 before surgery and 16.10 ± 2.20, 16.40 ± 2.35 at one week and six months after surgery respectively. According to Otani grade,40 cases got excellent results, 9 good, 1 fair. No significant dysphagia and internal fixation related complications such as displacement of cages were found during the follow-up period.

CONCLUSIONUsing this cage in anterior cervical fusion can obtain satisfactory clinical effect with less operation injury and reduce the complications. It is a better fusion method in anterior cervical fusion.

Adult ; Aged ; Cervical Vertebrae ; surgery ; Female ; Humans ; Male ; Middle Aged ; Spinal Fusion ; adverse effects ; instrumentation ; methods