OBJECTIVETo study the protective effect of puerarin on MPP(+) -induced SH-SY5Y cells by chaperone-mediated autophagy (CMA).

METHODThe Parkinson's disease cell model was established by injuring SH-SY5Y cells with 1 mmol x L(-1) MPP+. The CCK-8 staining was adopted to detect the effect the puerarin of different concentrations on the survival rate of MPP(+)-induced SH-SYSY cells. The autophagosome formation was observed under transmission electron microscope. The AO staining showed the changes in the lysosome activity. RT-PCR was used to detect the changes in Lamp2a and Hsc70 mRNA expressions. The western blotting was adopted to test the expressions of Lamp2a, Hsc70 and alpha-synuclein protein in cells.

RESULTWithin the concentration range of 12. 5-50.0 micromol x L(-1), the pretreatment with puerain for 30 minutes could protect the injury of MPP+ in SH-SY5Y cells, and showed a certain dose-effect relationship. The AO staining and electron microscope showed the effect of puerain within the concentration range of 12.5-50.0 micromol x L(-1) on 1 mmol x L(-1) MPP(+)-induced SH-SY5Y cells; autophagosomes emerged in cells, and increased along with the rise in the puerarin dose. The results of the flow cytometry revealed that 50.0 micromol x L(-1) of puerarin could protect against the increase of the ROS level in 1 mmol x L(-1) MPP(+) -induced SH-SY5Y cells and prevent the oxidative injury. The results of RT-PCR and western blotting indicated that puerain within the concentration range of 12.5-50.0 micromol x L(-1) alleviated the MPP(+)-induced SH-SY5Y cell injury, and inhibited the accumulation of alpha-synuclein proteins in MPP(+) -induced SH-SY5Y cells by up-regulating Hsc70, Lamp2a mRNA and protein level.

CONCLUSIONPuerarin could protect against the MPP(+) -induced cell injury, whose protective mechanism may be related to the chaperone-mediated autophagy pathway of interventional molecules.

Autophagy ; drug effects ; genetics ; HSC70 Heat-Shock Proteins ; genetics ; Humans ; Isoflavones ; pharmacology ; Lysosomal-Associated Membrane Protein 2 ; genetics ; Molecular Chaperones ; genetics ; Parkinson Disease ; drug therapy ; genetics ; Phagosomes ; drug effects ; genetics ; Piperidines ; pharmacology ; Pyrazoles ; pharmacology ; Tumor Cells, Cultured ; Up-Regulation ; drug effects ; genetics

Master of biomedical engineering degree is necessary for students to adapt to the trend of social and scientific development.Based on the social demands,staff working at academy of biomedical engineering department in Wuhan University create the master course and modify teaching methods,in order to integrate positive factors into our master of biomedical engineering course.These new teaching methods are implemented through entire course,which aims to improve students' abilities of critical thinking,creativity and operation.We also explore the new teaching methods to enabte students to be qualified with biomedical engineering work in the future.

Background Antagonists against vascular endothelial growth factor (VECF) play key roles in treating and preventing neovascular ophthalmopathy. As a novel anti-angiogenic factor, insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) might be an antagonist against VEGF in eye. Objective This study was to explore the inhibitory effect of IGFBP-rP1, a novel anti-angiogenic factor, on VEGF-induced retinal angiogenesis in vitro. Methods The retina-choroid endothelial cell line ( RF/6A ) was cultured in DMEM containing 10% fetal bovine serum. Culture cells were divided into control group(free-serum culture group) ,10mg/L VEGF culture group and different concentrations of IGFBP-rP1 (50,100,200 mg/L) +10 mg/L VEGF group. The expression of IGFBP-rP1 in the cells was detected by immunofluorescence assay. The proliferation and migration of RF/6A cells were evaluated using MTS colorimetric assay and the chemotactic motility assay, respectively. Flow cytometry was used to detect the apoptosis of RF/6A cells. Results The immunofluorescence assay RF/6A cells showed the green fluorescence in cytoplasm and red fluorescence in nuclei after cells were exposed to any concentration of IGFBP-rP1 ,but only red fluorescence was seen in nuclei in control cells. After stimulation of 10 mg/L VEGF,the proliferation value (A490) was elevated and the numbers of cell migration were increased in comparison with control group (t = -15. 191, P = 0. 000; t = -21. 274, P = 0. 000 ) , but the cellular apoptosis rate was lower than the control group (t - 10. 228, P = 0. 000 ) . After treated with various concentrations of IGFBP-rP +10% VEGF, the proliferation and migration of RF/6A cells were significantly decreased in comparison with only 10% VEGF group (F = 534. 158,P = 0. 000;F = 2742. 323,P = 0.000,respectively) ,and the inhibitory effects were gradually enhanced with the increase of IGFBP-rP1 levels (P<0. 05). The apoptosis rate of RF/6A cells in 50,100 and 200 mg/L + 10 mg/L VEGF groups increased by ( 1. 26±0. 04)% ,( 1. 50±0. 07)% and ( 1. 93±0. 27)% respectively,showing significant differences among different groups ( F = 274. 273, P = 0. 000). Conclusion IGFBP-rP1 inhibits the proliferation and activity of retina and choroid endothelial cells induced by VEGF at a concentration-independent manner. It appears to be as a novel endogenous inhibitory factor in retinal angiogenesis.

BackgroundThe immunotherapy for retinoblastoma(RB) is gradually concerned recent year.To seek relative immune-associated antigen is a basis of immunotherapy.NY-ESO-1 and NY-SAR-35 are two kinds of genes of cancer testis antigen( CTA ).To understand their expressions in RB tissue can offer index for relative study.ObjectiveThis study was to investigate the expressions of two CTA NY-ESO-1 and NY-SAR-35 in RB and explore the possibility of them as potentially promising targets for antigen-specific immunotherapy of RB.Methods The samples were obtained from 15 RB eyes,12 non-tumor retinopathy eyes and 22 normal eyes with other benign eye diseases.Reverse transcription polymerase chain reaction(RT-PCR) was used to detect the expressions of NY-ESO-1 mRNA and NY-SAR-35 mRNA in the samples.Genes of positive PCR results were sequenced randomly.The relevance of the expression of the two cancer-testis antigen genes with the clinical characteristics such as tumor stage,tumor size and clinical stage were analyzed.This study was approved by Ethic Committee of Guangxi Medical University.Written informed consent was obtained from each patient before operation. Results NY-ESO-1 mRNA was positively expressed in 6 RB samples and NY-SAR-35 mRNA was expressed in 9 RB samples.In the non-tumor retinopathy samples and normal eye tissues,NY-ESO-1 mRNA and NY-SAR-35 mRNA were absent.No significant relevances were found between the expressions of the NY-ESO-1 mRNA and NY-SAR-35 mRNA with clinical characteristics such as age ( P =0.426,0.822 ),gender ( P =0.180,0.464 ),pathological classification ( P =0.744,0.582 ),tumor size ( P =0.760,0.790),and clinical stage ( P =0.868,0.707 ).Conclusions NY-ESO-1 and NY-SAR-35 have high expressing frequencies in RB tissue and their expressions in RB have specificity.These results offer a clue for the identification of targets antigen of RB.

Endothelin(ET)is the most potent mammalian vasoconstrictor identified to data. As a pathogenic factor,ET is involved in the genesis of many diseases. In this study, a pair of primers was designed and synthesized according to the human ETB receptor gene (hETBR)sequence. A 394bp of DNA fragment was amplified by polymerase chain reaction(PCR) and labeled with a-32p-CTP using Random Primer-Labeling method. With this probe, rabbit lung cDNA library was screened by in situ hybridization and 11 positive clones were identified. Sequencing result showed that a complete reading frame of rabbit ETB receptor(rETBR)cDNA could be produced from three positive clones of eleven. By a series of subcloning,a recombinant plasmid including the 1326 bp of rETBR coding sequences, named pBlu Script-rETBR, was constructed. The deduced amino acid sequence indicated that the rETBR is 441 residues in length, with an expected molecular mass of approximately 49.44 kD. N-terminal 18 residues is the potential signal peptide (Score＝11.11)and therefore the molecular mass of mature rETBR is 47.65 kD with 423 amino acid residues. Analysis of the rETBR hydropathy profile indicates the presence of seven hydrophobic regions, putative transmembrane domains. Potential N-glycosylation sites are the 60th and the 118 th. The structure exhibits a significant sequence and topographical similarity with G protein-coupled receptors.

OBJECTIVETo analyze the expression and clinical values of HPV L1 capsid protein and p16INK4a protein in uterine cervical lesions.

METHODSFifty-four cervical intraepithelial neoplasias CIN1, 44 CIN2, 78 CIN3, and 48 squamous cell carcinoma were included in this study. All CIN and squamous carcinomas were stained with anti-HPV L1 capsid protein antibodies and anti-p16INK4a antibody. Forty-five CIN1 patients were followed up for 6 years.

RESULTSForty-five CIN1 patients were followed up for 6 years, among them 6 cases showed a progression (One case changed to CIN3, 5 cases to CIN2). L1 positivity was found in 50 cases which decreased with CIN increasing (χ(2) = 259.923, P < 0.001) while p16INK4a positivity was found in 177 cases which co-increased with CIN (χ(2) = 48.842, P < 0.001). L1(-)p16INK4a (-) or L1(+)p16INK4a(-) appeared mainly in CIN1 while L1(-)p16INK4a(+) appeared mainly in CIN2 lesions. No progression was found in the group of L1(-)p16INK4a(-) CIN1 patients. The risk of CIN1 progression in L1(-)p16INK4a(+) group was 66.7% while L1(+)p16INK4a(-) group was 9.5%, and L1(+)p16INK4a(+) group was 33.3%.

CONCLUSIONSThe expression of p16INK4a together with HPV L1 are different in various cervical lesions, and the combined detection of p16INK4a and HPV L1 can be helpful for estimating the biological potentiality of CIN lesions.

Adult ; Aged ; Capsid Proteins ; metabolism ; Carcinoma, Squamous Cell ; metabolism ; pathology ; virology ; Cervical Intraepithelial Neoplasia ; metabolism ; pathology ; virology ; Cyclin-Dependent Kinase Inhibitor p16 ; metabolism ; Disease Progression ; Female ; Follow-Up Studies ; Humans ; Middle Aged ; Oncogene Proteins, Viral ; metabolism ; Papillomavirus Infections ; Uterine Cervical Neoplasms ; metabolism ; pathology ; virology ; Young Adult

OBJECTIVETo introduce a method of three-column classifications for Pilon fractures and observe clinical utility on column fixation.

METHODSFrom June 2007 to March 2010,a total of 27 patients (29 ankles, 26 males and 1 female,ranging in age from 23 to 59 years, with an average of 33.1 years) with Pilon fractures were treated through column fixation by using semitubular plates or screws with anteromedial, anterior, posterolateral,posteromedial approach. And postoperative follow up were carried out.

RESULTSThe mean follow up was 17.5 months(ranged,5 to 33 months). According to the Mazur ankle grading system, the outcome was excellent in 20, good in 4 and fair in 5 ankles. Patients in this group did not have complications of wound dehiscence, deep infection, osteomyelitis, nonunion, ankylosis, and joint instability.

CONCLUSIONBased on the three-column classification, the clinical results for the treatment of Pilon fractures demonstrate the rationality and efficiency of this method.

Adult ; Female ; Humans ; Male ; Middle Aged ; Tibial Fractures ; classification ; diagnostic imaging ; surgery ; Tomography, X-Ray Computed

Chemical and Drug Induced Liver Injury ; etiology ; Drugs, Chinese Herbal ; toxicity ; Humans ; Liver ; drug effects

OBJECTIVETo investigate the induction of antitumor immune responses and therapeutic effects of 10-hydroxycamptothecinc-treated (HCPT) DC-Hepa fusion vaccines by DC fused with hepal-6 cell from hepatoma.

METHODSThe fused cells were isolated by magnetic cell sorting and adherent culture. Cell apoptosis was detected by Rhodamine123/PI double-labeled assay, CTL activity by 4 h (51)Cr releasing assay. Protective and therapeutic effects of the fusion vaccine to the tumor-bearing mice was also observed.

RESULTSThe apoptosis rate was 29.7%+/-4.1% when DC-Hepa fusion vaccine was treated with 50 microg/ml HCPT for 24 h. After treatment with the HCPT-DC-Hepa fusion vaccine, the tumor grew obviously slowly, survival period of the mice was prolonged, induced more potent CTL cytotoxicity, and resisted against the rechallenge of Hepal-6 cells.

CONCLUSIONVaccination with HCPT-DC-Hepa fusion vaccine could elicit potent antitumor responses, which will provide a new approach to the DC-mediated therapeutic antitumor immunity.

Animals ; Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; Camptothecin ; analogs & derivatives ; pharmacology ; Cancer Vaccines ; administration & dosage ; genetics ; immunology ; Cell Fusion ; Cytotoxicity, Immunologic ; Dendritic Cells ; immunology ; transplantation ; Female ; Liver Neoplasms ; immunology ; pathology ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Rats ; T-Lymphocytes, Cytotoxic ; immunology ; Tumor Cells, Cultured

Using of two-step integrating technology, transducted the H and L chain gene of humanized Fab fragment of anti-HB-sAg antibody into the genome of methylotropic yeast P. pastoris. Constructed a engineering yeast to produce humanized Fab fragment of the anti-HBsAg antibody. The Fab fragment was efficiently secreted into the medium at a concentration of 50-80 mg/L. The Fab fragment was purified from culturing supernatant of the recombinant yeas by affinity chromatography. The ELISA analysis showed the high affinity of the expressed humanized Fab fragment to the HBsAg.
Chromatography, Affinity ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; Hepatitis B Antibodies ; biosynthesis ; genetics ; isolation & purification ; Hepatitis B Surface Antigens ; immunology ; Humans ; Immunoglobulin Fab Fragments ; biosynthesis ; genetics ; isolation & purification ; Pichia ; genetics ; Recombinant Proteins ; biosynthesis ; isolation & purification