BACKGROUND: Some studies have demonstrated that the degradation of extracellular matrix (ECM), which matrix metalloproteinases (MMPs) participates in, plays a key step in the corneal neovascularization (CNV). Tissue factor pathway inhibitor 2 (TFPI-2), a new type serine proteinase inhibitor found recently, can effectively inhibit the activity of MMPs. Whether TFPI-2 gene transfection can influence CNV is unclear.OBJECTIVE: To investigate the effect of TFPI-2 gene transfection on CNV.DESIGN: Randomized controlled experiment.SETTING: Laboratory for Department of Surgery, Wuhan Union Hospital; Central Laboratory, the Affiliated Third Hospital of Sun Yat-sen University.MATERIALS: This study was carried out in the laboratory for Department of Surgery of Wuhan Union Hospital and State Central Laboratory of the Third Hospital Affiliated to Sun Yat-sen University between June 2004 and March 2006. Sixty healthy purebred adult New Zealand rabbits of either gender, weighing 2.5 to 3.0 kg, were involved. Preoperatively, no obvious anterior segment ocular lesion was found by slit-lamp examination. pBos-Cite-neo/TFPl-2 was kindly gifted by Dr. Zhong Ren (Department of Hematology, Union Hospital). Peroxydase blocking agent, nonimmune goat serum,mouse anti-human MMP-1, 2 and 3 monoclonal antibodies, biotin labeled goat-anti-mouse IgG second antibody (Santa cruz Company) were used in this study.METHODS: Experimental intervention: Experimental rabbit models of CNV were created in each group by silver nitrate cautery. Then, the rabbit models were randomized into 3 groups and 20 rabbits for each group. Different reagents were subconjunctivally injected via many points in each group: saline in the group Ⅰ, empty vector in the group Ⅱ, plasmid encoding TFPI-2 in the group Ⅲ. Experimental evaluation: CNV growth was observed under the slit-lamp biomicroscope.The expression of TFPI-2 in each rabbit model was detected by reverse transcription-polymerase chain reaction (RT-PCR) method 2 weeks after modeling; the expression of MMPs in corneal tissue was detected by immunohistochemical method at 3,5,7,9 and 14 days after modeling.TFPI-2 gene expression was significantly higher in the group Ⅲ than in the group Ⅱ and group Ⅰ (P ＜ 0.01); The MMP-1, 2, 3 expressions in the corneal tissue were significantly lower in the group Ⅲ than in the group Ⅱ and group Ⅰ,respectively, especially MMP-1, 3.

AIM: To study the antiproliferation of vitamin E succinate (VES) on pterygium fibroblasts in vitro and to find a potential agent for prevention and treatment of primary and recurrence pterygium.METHODS: Primary culture and subculture of pterygium fibroblasts were established in vitro ,and different concentrations of VES (0, 10 and 20mg/L) were added to subcultured fibroblasts, respectively. Influence of VES on the growth curve of fibroblast was observed at day 2, 4 and 7 after treatment of VES. 3- [4,5-Dimethylthiazolzyl]-2,5-Diphenyl Tetrazolium Bromide (MTT) assay at 490nm was used to evaluate the effect of the cells proliferation.RESULTS: The addition of VES to culture caused the marked descent of growth curve in comparison with the control group, and the inhibiting rate of 10 and 20mg/L of VES was 33.2% and 46.7%, 67.9%, and 76.8%, 81.7% at day 2,4 and 7, respectively. VES could obviously inhibit the fibroblast proliferation in dose-dependent manner by MTT assay.CONCLUSION: VES can significantly inhibit the proliferation of pterygium fibroblast in vitro.

BACKGROUND:Previous studies have suggested that achaete-scute homology 1 (ASCL1) plays a key role in the neuronal commitment. Therefore, somatic cels may directly differentiate into neurons by gene transfection ofASCL1, which wil provide new therapeutic strategies for optic nerve regeneration. OBJECTIVE:To construct a recombinant adenovirus vector expressing ratASCL1 gene for further research ofASCL1 gene function. METHODS:The ratASCL1 gene and advenovirus shuttle plasmid (pYr-adshuttle-4) which contained enhanced green fluorescent protein (EGFP) reporter gene were cleaved by restriction endonucleaseXhoI andEcoR I. The target gene fragments were connected together to generate a recombinant plasmid pYr-ads-4-rat-ASCL1 and then transfected into E.coliDH5α. The plasmid was confirmed to be constructed as expectation by enzyme digestion and sequence reaction. The plasmid pYr-ads-4-rat-ASCL1 and pAd/PL-DEST were reconstructed by homologous recombination processes to obtain rat ASCL1 recombinant adenovirus vector. The plasmid pYrAd-rASCL1 was linearized byPac I and subsequently transfected into HEK293 cels for packaging and amplification. RatASCL1 gene in the recombinant adenoviruses were identified by PCR. Virus titer was determined by tissue culture infectious dose 50. Infection efficiency was monitored by EGFP expression. RESULTS AND CONCLUSION:Restriction endonuclease digestion and DNA sequencing showed that the recombinant adenovirus vector pAd-rat-ASCL1 was constructed correctly. The positive amplification bands of 862 bp could be seen in PCR analysis. The virus titer reached 2×1010 pfu/mL. Infection efficiency of recombinant adenovirus in HEK293 cels was more than 80%. The results indicate that the recombinant the adenovirus vector containingASCL1 with high titer and infection efficiency has been successfuly constructed, which can be helpful for further research of the function and clinical application ofASCL1 gene for optic nerve regeneration.

ObjectiveTo investigate the effects of ginsenoside Rg1 on apoptosis in spinal dorsal horn in development of morphine tolerance in rats with arthritis.MethodsTwenty-four healthy male SD rats weighing 280-320 g in which intrathecal(IT) catheters were succcessfully implanted without complication were randomized into 4 groups ( n =6 each):group normal saline ( group C) ; group morphine ( group M ) ; group ginsenoside Rg1 (group G) and group morphine + ginsenoside Rg1 (group MG).Arthritis was induced with complete Freund adjuivant injected into the ankle joint of right hind limb according to the method described by Butler et al in all 24 animals.Chronic morphine tolerance was induced by IT morphine 10 μg twice a day for 7 consecutive days in groups M and MG.Ginsenoside Rg1 100 μg was given IT once a day for 7 consecutive days in groups G and MG.Paw withdrawal threshold to mechanical stimulation with yon Frey filaments (MWT) was measured before induction of arthritis (T1,baseline),and IT drug administration (T2) and at day 1,3,5,7 (T3-6) after IT administration.The rats were sacrificed after last MWT measurement and their lumbar segments of the spinal cord ( L3-5 ) were removed for detection of apoptosis in the spinal dorsal horn by TUNEL.ResultsMWT was significantly decreased after induction of arthritis at T2-6 compared with the baseline value before arthritis at T1 in all 4 groups.In group M IT morphine significantly increased MWT at T3.4 compared with the baseline at T2 but the MWT was decreasing at T5.6 indicative of development of morphine tolerance.In group MG,addition of ginsenoside Rgl to IT morphine significantly attenuate the decrease in MWT at T5.6.The number of apoptotic cells in spinal dorsal horn was significantly higher in groups M and MG than in group C,but was significantly lower in group MG than in group M.Concluson Ginsenoside Rg1 can prevent the development of morphine tolerance in rats with arthritis by inhibiting apoptosis in spinal dorsal horn.

BACKGROUND:Engineered hepatic tissue is considered a promising strategy for healing acute liver failure. But, there are series of hindrances in the construction of engineered hepatic tissues, including acquisition of vital hepatocytes, choice of scaffolds and culture system, and nutrition supply. OBJECTIVE:To construct three kinds of engineered hepatic tissues in hope to screen the optimal one for transplantation in acute liver failure. METHODS:After purification, amplification, bone marrow mesenchymal stem cells were induced to differentiate into hepatocyte-like cells which were co-cultured with acellular amniotic membrane, DACRON PATCH cardiovascular surgical patch, biological surgical patch, respectively to construct three kinds of engineered hepatic tissues. After 3 days of culture, morphological and functional detections were performed. RESULTS AND CONCLUSION:Rat bone marrow mesenchymal stem cells with higher purity were successful y harvested by using density gradient centrifugation and adherent methods, and then the cells were differentiated into hepatocyte-like cells. In the three kinds of engineered hepatic tissues, hepatocyte-like cells were found to be combined with the biological surgical patch to the maximum extent, and their combination exhibited stronger ability of urea synthesis and albumin secretion, which provides experimental basis for treatment of acute liver failure.

Debates over PBL have never stopped since its introduction to medical education in 1969 and are even becoming more heated.This paper expounds on the origin and definition of PBL,and then describes and analyzes the application of the clinical teaching in orthopedic surgery.

BACKGROUND:The degradation of extracellular matrix, which is mediated by matrix metal oproteinases, plays a crucial role in the corneal neovascularization. Tissue factor pathway inhibitor 2, a new type serine proteinase inhibitor, can effectively inhibit the activity of matrix metal oproteinases.
OBJECTIVE:To elucidate the effect of tissue factor pathway inhibitor 2 on the expressions of matrix metal oproteinases in keratocytes in vitro.
METHODS:Rabbit keratocytes were primarily cultured and subcultured in vitro. Plasmid vector pBos-Cite-neo/TFPI-2 was transfected into keratocytes with Lipofectamine 2000. The positive cells were selected using G418.
RESULTS AND CONCLUSION:The results of reverse transcription-polymerase chain reaction, western blot analysis and gelatinase zymography analysis showed that, expression of mRNA and protein of tissue factor pathway inhibitor 2 was upregulated in the transfected keratocytes (P<0.05), while activity of matrix metal oproteinase 1 and 2 was significantly decreased (P<0.05). Experimental findings indicate that that tissue factor pathway inhibitor 2 strongly inhibits the activity of matrix metal oproteinase 1 and 2 in keratocytes.

Objective To analyze the epidemic characteristics of mushroom poisoning incident in order to find the regularity of outbreak and provide the fundamental guidelines of prophylaxis,control,diagnosis and treatment.Methods According to the reported information from the Management Information System of Public Health Emergency in China mainland,the area-time distribution of mushroom poisoning incidents from 2004 to 2014 was analyzed,and the descriptive analysis of mushrooms poisoning incidents including causes,places,occupation of victims and incidents identification were made from 2010 to 2014.Results In China (excluding Hong Kong,Macao and Taiwan),the top five provinces of mushroom poisoning incidents were Yunnan,Guizhou,Sichuan,Guangxi and Hunan.The epidemic peak was reached in summer-autumn season.The major and significant incidents accounted for 76.56％ of overall mushroom poisoning incidents,and the fatality rate of 3 701 patients accounted for 21.24％ (786 deaths).The causes were mistaking poisonous mushrooms as edible mushrooms or purchasing poisonous mushrooms in the market by accident.About 87.50％ incidents happened at home.Farmers,workers,children and students were easily subjected to mushroom poisoning because of their large range of activities,strong curiosity and lacking related ability for distinguishing edible mushroom from poisonous mushrooms.No identification was done in 200 mushroom poisoning incidents from 2010 to 2014,which accounted for 92.59％ of mushroom poisoning incidents in the corresponding period.Standard species identification was carried out only in two poisonous mushroom incidents.Conclusions Mushroom poisoning incident was one of the most important causes of death in per-oral poisoning incidents.It should to cope with surveillance and meticulous management during high prevalence season and in high-risk provinces.At the same time,it should be strengthened to train doctors and health professionals with the knowledge of identification of mushroom poisoning in key areas as well as to develop the health promotion of mushrooms poisoning prevention.

Aged ; Castleman Disease ; complications ; Hepatitis B ; complications ; Humans ; Liver Neoplasms ; complications ; virology ; Lymphoma, Non-Hodgkin ; complications ; Male

In this study, the effect of heparin-derived oligosaccharide (HDO) on platelet-derived growth factor (PDGF) induced vascular smooth muscle cells (VSMCs) proliferation and the related signal transduction mechanisms were investigated. MTT assays were used to measure VSMCs proliferation. Cell cycle distribution was analyzed by flow cytometry. The level of key regulatory proteins in PKC, MAPK and Akt/PI3K pathways were determined by RT-PCR, Western blot and immunocytochemical methods. Meanwhile, mRNA expressions of some proto-oncogenes were assayed by RT-PCR method. Our data showed that HDO (0.01, 0.1 and 1 μmol · L(-1)) inhibited 30 ng · mL(-1) PDGF-induced VSMCs proliferation in a dose-dependent manner, blocked the G1/S transition and inhibited the level of key regulatory proteins and some proto-oncogenes (P < 0.05). The results showed that HDO may decrease the key regulatory proteins expression, hence suppress the transcription of proto-oncogene and G1/S transition, finally inhibiting VSMCs proliferation.