Background Efficient and lowcost way to isolate keratocytes is helpful for research on cornea.Either relatively expensive or inefficient is the shortage of those means now applied,while raising the keratocytes through passage will change the phenotype of them quickly.Our aim is to approach the way getting keratocytes effectively utilizing modified two step enzymatic digestion by type I collagenase. Objective To evaluate the effect of isolating the bovine keratocytes utilizing two step enzymatic digestion and observe the morphological changes of the keratocytes during cultivation in vitro. Methods Keratocytes were isolated from bovine corneas using 0.5 mg/mL and 1 mg/mL type I collagenase digestion.The harvesting rate and viability rate of the primary keratocytes were evaluated.During the primary cultivation in vitro,the morphological changes of the keratocytes and their F-action distribution were observed.Results(1)The extracellular matrix of the bovine corneas were almost dissolved by the two step enzymatic digestion,followed the keratocytes completely isolated from the solid matrix.The amount of the harvested keratocytes from each cornea was(2.11±0.15)X106 on average while the viability rate was(91.69±3.73)% and the inoculation rate Was(81.20±1.25)%.(2)The primary keratocytes attached and spreaded out with dendritic and stellate morphology.After 3 days cultured,the branches of the keratocytes were contacting and formed networks.The F-actin detected by phalloidin binding showed a limited cortical localization. Conclusion (1)The method of two step enzymatic digestion can make the extracellular matrix of bovine cornea stroma completely degraded with the advantages in high efficiency of harvesting keratocytes and high cell viability and relatively simple manipulation. (2) The primary bovine keratocytes have dendritic morphology and with limited F-action distribute in cellular cortex.

Objective To detect and analyze the mutation of fibroblast growth factor receptor 3(FGFR3) gene among a family with congenital achondroplasia(ACH).Methods Six blood samples of family member in this pedigree were cellected according to the informed consent process for genetic research,and 2 unralted healthy human blood sample were taken as controls.The mutation at nucleotide position 1 138 on FGFR3 gene was detected by using Polymerase chain reaction and single-strand conformation polymorphism(PCR-SSCP)and polyme-rase chain reaction and restriction endonuclease technology(PCR-RFLP) methods.Results Using PCR-SSCP method firstly,only the proband with ACH and his father in this family had the same abnormal band.The amplified products including 1 138 loci on FGFR3 gene further was analyzed by Sfe Ⅰ digestion,3 fragments including 164 bp,109 bp and 55 bp were detected in the proband and his father again,and the other members in the family and 2 controls just showed 164 bp band.It indicated that just 2 patients (proband and his father) showed heterozygous G→A transition mutation at nucleotide position 1 138 on the FGFR3 gene.The amplified products at 1 138 loci was also detected by MspⅠ digestion,just 1 band was observed in all members in this family and 2 controls.It showed that there was no G→C substitution at nucleotide position 1 138.Conclusions The G→A transition mutation at nucleotide position 1 138 in transmembrane domain of FGFR3 gene may be the main cause of achondroplasia in this family.In this pedigree,the proband showed's father a de novo mutation which was transferred to his child again.

Objective To study on the expression of the eukaryotic recombinant vector carrying HlyX gene of Leptospira serovar Lai in mammalian cell and explore the humoral immune response in BALB/c mice immunized with the recombinant plasmid. Methods The HlyX gene was amplified from Leptospira serovar Lai genomic DNA by PCR and inserted into pcDNA3.1 vector. After transformed into E. coli DH5α,the recombinant plasmid was assayed for identification by PCR analysis,restriction nuclease enzyme digestion and sequencing. The recombinant plasmid was transfected into COS-7 cells,then RT-PCR and Western blot were performed to test the expression of the target gene. The recombinant plasmid was injected intramuscularly into BALB/c mice for three times at intervals of two weeks,and the antibody titer was measured by ELISA. Results PCR showed the full length HlyX gene was about 1100 bp. PCR analysis,restriction nuclease enzyme digestion and sequencing indicated the recombinant vector was constructed successfully. After the plasmid Was transfected into COS-7 cells,a fragment about 1100 bp was found by RT-PCR and a specific band relative molecular mass(Mr)about 40×103,which was consistent with the expected size of the target proteins was showed by Western blot. ELISA showed the antibody titer in BALB/c mice immunized by the HlyX gene of Leptospira serovar Lai can elicit high-titer antibody in BALB/c mice,which has laid the foundation for the application of the DNA vaccine.

OBJECTIVETo observe the impacts of acupoint catgut embedding therapy and acupuncture-moxibustion therapy on the long-term efficacy and patient's life quality in the treatment of allergic rhinitis.

METHODSSixty-nine patients were randomized into the combined acupuncture-moxibustion and acupoint catgut embedding therapy group (combined therapy group, 36 cases) and an acupuncture-moxibustion group (33 cases). In the acupuncture-moxibustion group, acupuncture was applied at Yingxiang (LI 20), Shangyingxiang (EX-HN 8), Yintang (GV 29), Shangxing (GV 23), Tongtian (BL 7) and Zusanli (ST 36). Moxibustion was applied at Zusanli (ST 36), Feishu (BL 13), Dazhui (GV 14) and Fengmen (BL 12). In the combined therapy group, on the basis of the treatment as acupuncture-moxibustion group, the catgut embedding therapy was applied at Feishu (BL 13), Fengmen (BL 12), Pishu (BL 20), Shenshu (BL 23), Zhongwan (CV 12) and Qihai (CV 6). The treatment duration was 4 weeks in the two groups. The clinical efficacy of allergic rhinitis and rhinoconjunctivitis quality of life questionnaire (RQLQ) score were observed before and after treatment as well as in the 4-weeks follow-up after the end of treatment respectively.

RESULTSThe markedly effective rate was 72.7% (24/33) in the combined therapy group and 48.4% (15/31) in the acupuncture-moxibustion group after treatment. The efficacy was similar between the two groups (P > 0.05). It was 57.6% (19/33) in the combined therapy group and was 22. 6% (7/31) in the 4-week follow-up after treatment, indicating the long-term efficacy in the combined therapy group was superior to that in the acupuncture-moxibustion group (P<0. 05). Scores of RQLO after treatment and in 4-week follow-up after treatment in both groups were improved as compared with those before treatment (all P < 0.05). In 4-week follow-up, the improvements in sleep and affection in the combined therapy group were superior to the acupuncture-moxibustion group (3.27 +/- 3.23 vs 4.61 +/- 3.56, 3.48 +/- 3.67 vs 5.81 +/- 4.15, both P < 0.05).

CONCLUSIONThe acupoint catgut embedding therapy combined with acupuncture-moxibustion therapy are safe and effective in the treatment of allergic rhinitis and display the more roles in the long-term efficacy.

Acupuncture Points ; Acupuncture Therapy ; instrumentation ; Adolescent ; Adult ; Catgut ; utilization ; Child ; Female ; Humans ; Male ; Middle Aged ; Moxibustion ; Quality of Life ; Rhinitis, Allergic ; Rhinitis, Allergic, Perennial ; therapy ; Treatment Outcome ; Young Adult

By analyzing the epidemiological and clinical features of adenovirus in children with acute respiratory tract infection (ARTI), we provide a theoretical basis for early clinical diagnosis and treatment. Nasopharyngeal secretions were collected from 3480 children with ARTI, who were hospitalized at the No. 2 Hospital of Changzhou from January 2011 to December 2012. Adenovirus were detected using direct immunofluorescence assays. A total of 80 samples were positive for adenovirus (2.30%). The rate of adenovirus infection during 2011 was significantly higher than that in 2012, and the infection rate was higher in summer and autumn than in winter and spring. The infection rate was 1.14% among children aged < 1-year-old and the rates were higher among children in other age ranges. Adenovirus was found to be an important ARTI pathogen in children in Changzhou, mainly affecting children older than 1 year. ADV infections have various clinical presentations, but affected children tend to be severely ill with poor outcomes.
Acute Disease ; epidemiology ; therapy ; Adenovirus Infections, Human ; epidemiology ; therapy ; virology ; Adenoviruses, Human ; classification ; genetics ; isolation & purification ; Child ; Child, Preschool ; China ; Female ; Hospitalization ; Humans ; Infant ; Male ; Respiratory Tract Infections ; epidemiology ; therapy ; virology ; Seasons

Animals ; Humans ; Influenza A virus ; immunology ; Influenza Vaccines ; immunology ; Protein Engineering ; Viral Matrix Proteins ; chemistry ; genetics ; immunology ; metabolism

Objective To observe the effects of propofol on the hippocampal astrocytes and microglia in the nenotal mice . Methods 15 healthy mice from the same litters on postnatal 7 d were randomized into 3 groups:high dose propofol group ,low dose propofol group and 10% intralipid control group .All mice were treated with drugs on postnatal 7 d by intraperitoneal injection and were sacrificed at 24 h after drugs treatment .The high dose group was injected with propofol 60mg · kg -1 ;the low dose group was injected with propofol 30mg · kg -1 ;the control group was injected with the equal volume of 10% intralipid .The immunohistochem‐istry assay was used to detect the expression of glial fibrillary acidic protein (GFAP) and ionized calcium binding adapter molecular 1 (Iba1) for observing the effect of propofol on the astrocytes (AST ) and microglia in the hippocampus .Results Compared with the control group ,the number of GFAP‐labeled AST in the dentate gyms (DG) molecular layer of hippocampus in P7 mice of the high dose propofol group was significantly reduced (P<0 .01) ,while no obvious effect of the low‐dose propofol on the number of AST was observed ;high dose and low dose propofol all significantly decreased the number of Iba1‐labeled microglia .Conclusion Propofol can inhibit the growth of the hippocampal AST and microglia in a dose‐dependent manner .

Objective To investigate the role of oxidative stress-dependent Rasextracellular signal-regulated kinase (ERK1/2) signaling in aldosterone (ALDO)-induced mesangial cell proliferation. Methods The incorporation of 3H-thymidine (3H-TdR) and cell count were used as the measure of mesangial cell (MC) proliferation.Western blotting was used to detect the activation of Ki-RasA,c-Raf,MEK1/2,ERK1/2 and PI3K. Results Aldosterone significantly induced human mesangial cell proliferation,and anti-oxidant N-Acetylcysteine (NAC),catalase,and super oxide dismutase (SOD) significantly inhibited ALDO-induced mesangial cell proliferation (P＜0.01,respectively).Stimulation by ALDO for 3 h,Ki-RasA,c-Raf,MEK1/2,and ERK1/2 activity increased by 4.05-, 3.62-, 4.52-, and 3.40-fold compared with control group (P ＜0.01,respectively).NAC almost completely blocked ALDO-induced Ki-RasA,c-Raf,MEK1/2,and ERK1/2 activation (P＜0.01,respectively).Ki-RasA siRNA dose-dependently inhibited Ki-RasA expression, ALDO-induced Ki-RasA activation, and mesangial cell proliferation (P ＜0.01,respectively).c-Raf inhibitor GW5074 and MEK1/2 inhibitor PD98059 also reduced ALDO-induced mesangial cell proliferation by 65％ respectvely (P＜0.01).Ki-RasA siRNA had no effect on ALDO-induced PI3K phosphorylation.Combining LY294002 and PD98059 completely blocked ALDO-induced mesangial cell proliferation (P＜0.01). Conclusions ALDO-induced Ki-RasA-c-Raf-MEK-ERK signaling activation is dependent on reactive oxygen species (ROS) production,which mediates ALDO-induced mesangial cell proliferation.Inhibition of both ERK1/2 and PI3K signaling simultaneously completely blocks ALDO-induced mesangial cell proliferation.

Six years' English teaching in department of obstetrics and gynecology in Wenzhou Medical College were reviewed. Language and cultural differences are the main reasons hindering teaching quality. Rational use of a variety of teachers,preparing for lessons adequately,adoption of English image data,supplying and revising English teaching materials,using network auxiliary teaching and forming ef-fective education mode are conducive to improving English teaching quality in department of obstetrics and gynecology for foreign students.

Objective To explore the clinical feature of Kimura disease complicated with nephrotic syndrome in children. Methods The clinical data from 4 children with Kimura disease complicated with nephrotic syndrome were retrospectively analyzed. Results In all of the 4 male children, level of serum IgE was increased but level of eosinophils was not increased in peripheral blood. The renal pathological manifestations were different among them. Two cases had acute tubular injury, one case had pathological changes in repeated renal biopsy, and one case had a little of eosinophils infiltration in renal interstitium. All of them were sensitive to oral hormone treatment. Except that one case had no relapse yet in short follow-up period, the other 3 cases had relapsed and all manifested as positive urinary protein without lymphadenectasis. The 3 cases with recurrence were treated by combined immunosuppressive agents, the palindromia of two cases were reduced which were combined with tacrolimus. Conclusion The Kimura disease complicated with nephrotic syndrome in children has a long course and different renal pathological manifestations. It is sensitive to hormone treatment but easy to relapse, and the treatment should be combined with immunosuppressive therapy. Tacrolimus may have the effect on reducing relapse and delaying the progress of renal pathology.