In order to improve the expression level of segment of GABAA receptor a1 subunit in E. coli, growth conditions of the recombatant, which influence the final yield of protein expression, including growth medium, inoculation ratio, temperature, pH, rotation speed, inducing time and concentration of IPTG and so on, were studied in shaking flasks. The results indicated that, with 3% inoculation ratio, cultured 3.5 hours at 37℃, and then induced 5 hours by IPTG at 32℃, the yield of GABAA receptor protein was 95mg/L and the biomass was 3.25 g/ L. In contrast, using a 16 L stirred fermentor instead of shaking flasks, the highest level of the protein expression, 136mg/L with 4.95g/L of biomass, was achieved after fermenting 5.5 hours.

?AlM:To observe the clinical effects of thelacrimal fistula excision combined with double silicone intubation in the treatment of chronic dacryocystitis with lacrimal fistula.? METHODS: Totally 25 cases ( 25 eyes ) of chronic dacryocystitis with lacrimal fistula were allocated into two groups:the double silicone intubation group of 13 cases (13 eyes) received lacrimal fistula excision combined with double silicone intubation, and the routine group of 12 cases (12 eyes) received routine dacryocystorhinotomy, we analyzed the curative effect.?RESULTS: The double silicone intubation group had a cure rate of 92. 3% (12 eyes), a effective rate of 100%, compared with the routine group had a cure rate of 91. 7%( 11 eyes ) , a effective rate of 100%, there were no statistically significant difference between the two groups (P>0. 05).?CONCLUSlON:Lacrimal fistula excision combined with double silicone intubation has a same effect with routine dacryocystorhinotomy in the treatment of chronic dacryocystitis with lacrimal fistula, but less invasive, no scar, less pain, and meet the patients' esthetic needs.

Objective To determine a possible role for Clara cell secretary protein(CCSP)during acute RSV infection.Method CCSP-deficient [CCSP (-/-)]and wild-type (WT) mice were intratracheally infected with RSV and the lung inflammatory response to RSV infection were assessed.Results RSV-F gene expression increased in the lungs of CCSP (-/-) mice compared to WT mice following RSV infection, consistent with increased viral persistence. Lung inflammation was significantly worsened in CCSP (-/-) mice compared to WT mice after RSV infection. Th2 cytokines and neutrophil chemokines increased in the lungs of CCSP (-/-) mice following RSV infection.Conclusion These findings suggest that lack of CCSP may promote the inflammatory and Th2 immune response to RSV infection.

Background Blepharitis is a common ocular surface disease.It is associated with the disorder of lipid secretion of meibomian gland.The change of tear film stability can cause dry eye symptoms,so blepharitis is thought to be one of the factors causing dry eye,but the relation between them is in study. Objective This study was to observe the morphology of meibomian gland in blepharitis patients and to investigate the correlation of morphology of meibomian gland with dry eye. Methods A series of case-observational study was designed in this study.A total of 83 eyes of consecutive 83 blepharitis patients were enrolled in Henan Eye Institute from October 2010 to April 2011.Blepharitis was diagnosed based on American Preferred Practice Pattern Guidelines.Some relevant ocular examinations were performed under the informed consent of the subjects,including the anterior segment manifestation by the slit lamp,such as meibography,lid margin abnormality,and the dry eye-relevant examinations,such as tear film break-up time (BUT),Schirmer test Ⅰ and corneal fluorescein staining also been carried out.Tear film shape was examined by film interference images and scored.Absent degree of meibomian gland was graded under a Noncontact Infrared Meibography.The correlations of absent degree of meibomian gland with ocular syndrome score,dry eye examination results were evaluated using Spearman rank correlation coefficients.Informed consent was obtained prior to this trail. Results No significant difference in the frequencies of blepharitis was found between male and female among different ages (x2 =2.69,P =0.75 ).Absent grading of the meibomian glands was positively correlated with age of blepharitis patients ( r =0.58,P =0.00 ),lid margin abnormality scores ( r =0.64,P =0.00 ),conjuntival hyperemia score ( r=0.50,P =0.00),tear film interference imaging grade ( r =0.23,P =0.04 ),corneal fluorescein staining score( r =0.50,P =0.00 ) but was negatively correlated with BUT ( r =-0.32,P =0.00 ).No significant correlation was found between meibography grading and gender( r =-0.09 ; P =0.99 ) or Schirmer test Ⅰ ( r =-0.05;P =0.69 ).No significant difference was found in meibography grading between male and female in different age groups(Z=-0.09,P=0.93). Conclusions Blepharitis can irriter dry eye symptom because of overevaporation of tear fluid and abnormality of secreting function of meibomian glands.The missing of the meibomain glands increases with age in the patients with blepharitis.Noncontact Meibography System is an assistant tool to the diagnosis of blepharitis.

Objective To determine the effects of~(99)Tc-MDP on joint inflammation and bone destruc- tion in collagen-induced arthritis(CIA)rats model and its effect on tumor necrosis factor alpha(TNF-?). Methods CIA was induced by immunization of male SD rats with an emulsion of collagen.~(99)Tc-MDP or placebo was intravenous infused to rats for 20 days.Joint inflammation was assessed by arthritis index.Lesions of bone were assessed based on the histological changes in ankle joints,radiographic analysis in hind paw with Larsen score.Systemic TNF-?level was measured by radioimmune assay.Results~(99)Tc-MDP suppressed joint swelling(P

Objective To evaluate the efficacy and safety of retroperitoneal laparoscopic pyelolithotomy ( RLP) combined with holmium laser lithotripsy under flexible cystoscopy in the treatment of complicated nephrolithiasis. Methods The retrospective analysis was made on the clinical data of 37 patients who underwent RLP and holmium laser lithotripsy under flexible cystoscopy for complicated nephrolithiasis from January 2013 to January 2014. The clinic parameters involved basic data of patients,operational time,blood loss,post-operative hospital stay,the status of stone-free,perioperative complications,and the follow-up data of patients were observed. Results No patient was converted to open surgery. The mean stone size was (2. 8 ± 0. 9) cm in diameter,operational time was (89 ± 24) min,blood loss was (21. 3 ± 7. 7) mL,post-operative hospital stay was (6. 8 ± 1. 7) d,the stone removal rate in one session was 94. 6%. One case occurred urinary leakage,1 case occurred fever after operation,who were all recovered through conservative treatment. All cases were followed up at the sixth months after operation. Conclusion RLP combined with holmium laser lithotripsy under flexible cystoscopy is effective and safe for the treatment of com-plicated nephrolithiasis.

Background Human limbal allograft transplantation or limbal autograft transplantation are the primary approaches to the severe corneal-blindness,but their application in clinic were limited because of the defects of donor material.With the development of tissue engineering technology,transplantation of in vitro cultured limbal epithelial stem cells is being an advanced management.Objective The aim of this work was to expand human limbal epithelial stem cells ex vivo under the guidance of confocal microscope and to lay the foundation for fabricating ex vivo cultured cell sheets.Methods Ten eyes of ten patients were examined with the Heidelberg Retina Tomography Ⅲ Rostock Cornea Module(HRT3-RCM)to elucidate the structure of the human corneoscleral limbus and to correlate limbal epithelial dimensions.According to the analysis of the images of limbal epithelia,the limbal tissues provided by Eye Bank of Henan Eye Institute were cut into suitable explants.Then,this study was conducted to expand limbal epithelial stem cells ex vivo on denuded amniotic membrane.The phenotypes of primary cultured cells were evaluated by morphology and immunofluorescent staining with antibodies for limbal epithelial stem cell markers (p63,cytokeratinl9)and differentiation markers(keratin 3,involucrin).This experimental procedure was approved by the Ethic Committee of Henan Provincial People's Hospital.The written informed consent was obtained from subjects before initiation of any examination.Results The palisade morphology of human limbus was imaged clearly on the laser scanning in vivo confocal microscopy and many hyperreflective cells were observed in palisade basal cells.The cell-island phenomenon was seen in the basement membrane under the laser scanning in vivo confocal microscopy.The oblique sections of limbus showed many papilla-like epithelial columns below the superficial limbal epithelia.Throughout the experiment duration,the epithelial cells grew well with the migration rates from limbal tissue (68.62± 16.94)％ and the migration time(5.83 ±2.04)days,which depended on the tissue freshness.Compared with the second and forth batch of tissue,the migration rates of the third and sixth batch of tissues were significantly higher(P＜0.05),and the migration time was evidently longer in the forth and sixth batch of tissue compared with the first,second,third and fifth batch(P＜0.05).The positively expressing rates in the cultured corneal stem cells were 4.05％ and 36.52％ for p63,26.07％ and 40.55％ for CK19,57.88％ and 40.81％ for K3,64.66％ and 59.19％ for involucrin.Conclusion Human limbal epithelial stem cells can be successfully and purposefully obtained from the limbal tissue based on the guidance confocal miscroscope.The cultured corneal stem cells can grow well on the denuded amniotic membrane

Condition optimization for immobilization of Fungal laccase with Nylon Net and glutaraldehyde and the nature of the immobilized enzyme were studied. The optimum conditions of the immobilization are: Nylon Net is crosslinked with 5% glutaraldehyde 15mL for 6 hours; the 30U laccase is added for immobilization for 8 hours. On this case, the recovery of enzyme activity was 50.3%. Compared with free enzyme, the thermal stability of immobilized enzyme was improved evidently but the optimal pH decreased slightly. 52% enzyme activity of immobilized laccase was hold after 8 cycles treatment with low concentration pulp wastewater.

Objective To demonstrate high mobility group box chromosomal protein 1(HMGBI) expression in synovium and joint,and to identify the role of HMGB1 in the pathogenesis of synovitis and joint destruction in adjuvant-induced arthritis(AA).Methods AA of 15 male rats were induced in SD rats by intradermal injection of 100?l Freud's complete adjuvant in the foot pad of the left hind paw.All rats were killed at the 18th day.Synovium and joints were collected for histopathology studies and determining the expression of HMGB1 by immunohistochemistry,and serum was collected for determining the expression of HMGB1 by western blotting analysis.Results Immunostaining of specimens from normal rats showed that HMGB1 was primarily confined to the nucleus of synoviocytes with occasional cytoplasmic staining.In contrast, inflammatory synovial tissues from AA rats showed a distinctly different HMGB1 staining pattern.Nuclear HMGBI expression was accompanied by a cytoplasmic staining in many mononuclear cells.The cytoplasmic HMGB1 expression in synovium of AA rats is significantly higher than that of normal rats.Additionally,HMGBI was highly expressed in the nuclei and cytoplasm of the subchondral chondrocytes and inflammatory cells in bone erosion in AA rats(P＜0.01),while fewer positive cytoplasmic staining of HMGB1 was found in chondrocytes and fewer positive nuclear staining was found in bone cells in normal rats.HMGB1 concentration was significantly higher in serum of AA rats than that in normal rats(P＜0.001).Conclusion The cytoplasmic HMGBI expression in synovium and joints is greatly upregulated;the level of HMGB1 in serum is increased in AA rats which suggests a patbogenetic role of HMGB1 in synovitis and bone destruction of adjuvant-induced arthritis.

Objective To construct and express the recombinant plasmid pET28α-Sj26GST-Sj32 of Schistosoma japonicum(Sj) in Escherichia coli BL21 (DE3). Methods Total RNA was extracted from Sj adult worms by ultrasound-breaking, Sj26GST and Sj32 antigen gene was respectively amplified by RT-PCR from the total RNA; Sj26GST-Sj32 fusion gene obtained with gene splicing by overlap extension(SOEing) was cloned into prokaryotic expression plasmid pET28α and transformed into Escherichia coli BL2 (DE3) to construct pET28α-Sj26GST-Sj32;BL21 (pET28α-Sj26GST-Sj32) was induced with isopropyl-β-D-thiogalactopyranosid (IPTG), and the expressed products were analyzed and identified by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE)and Western blotting. Results The 1991 bp Sj26GST-Sj32 fusion gene was successfully amplified by gene SOEing and cloned into pET28α by restriction analysis and PCR identification, the recombinant plasmid pET28α-Sj26GST-Sj32 was successfully constructed; the relative molecular mass of the expressed recombinant protein was approximately 69 × 103 by SDS-PAGE, and the amount of the expressed protein was 25% of the total bacterial proteins; the fusion protein could be recognized by sera from rabbits infected with Sj by Western blotting.Conclusions The recombinant plasmid pET28α-Sj26GST-Sj32 is successfully constructed and highly expressed in Escherichia coli in fused form with His-tag, and the expressed fusion protein shows specific antigenicity.