OBJECTIVE:To develop a HPLC method for simultaneous determinatio n of ofloxacin and dexamethasone ac?etate in compound ofloxacin ear drops.METHODS:The analysis was carried on a XDB-C 8 ,the mobile phase was composed of methanol-potassium dihydrogen phosphate(0.02mol/L)by gradient elution,the flow rate was1.0ml/min,the column tem?perature was30℃and the detection wavelength was at240nm.RESULTS:The detectable concentrations of ofloxacin and dexamethasone acetate showed good linear correlation in the ranges of100～500?g/ml and10～30?g/ml respectively,the av?erage recoveries of ofloxacin and dexamethasone acetate were100.6%(RSD=0.46%,n=9)and101.3%(RSD=0.72%,n=9)respectively.CONCLUSION:The method is accurate and reproducible and can be used for determination of ofloxacin and dexamethasone acetate in compound ofloxacin ear drops.

Objective In order to study the pathogenesis of human papillomavirus(HPV) and seek for a therapeutic approach of the diseases caused by HPV, the construction of HPV18 E6E7 antisense RNA expressing recombinants was studied. Methods We amplified the HPV18 E6E7 816bp by PCR with HPV18 plasmid DNA as the template. pLNSX retroviruses were used as vectors,the HPV18 E6E7 retrovirus recombinants were constructed. And then the recombinants were cleaved with restriction endonuclease and hybridized with Southern blot for identifying the inserting direction and special check respectively. Results and conclusion The HPV18 E6E7 antisense RNA retrovirus expressing recombinants were screened and obtained,which had laid the foundation of studying the function of E6E7 genes further and explore whether the antisense technique can adjust and control the expression of E6E7 genes.

BACKGROUND:Human embryonic stem cels are able to self-renew indefinitely and have the capacity to differentiate into al three germ layers (ectoderm, endoderm and mesoderm). These properties imply great potential in the basic research and clinical application, including regenerative medicine, drug screening and toxins, early human embryo, cel transplantation, gene therapy,etc. However, it is a substantial chalenge to develop efficient techniques for their large-scale culture under defined conditions, and for controling and directing their differentiation. For therapeutic purposes, many scholars are trying to establish methods for maintaining pluripotency in defined xeno-free conditions and scalable culture systems. OBJECTIVE:To discuss the progress of serum-free culture systems in human embryonic stem cel research reported in recent years and to highlight the chalenges and advances being made towards the development of serum-free and xeno-free culture systems suitable for therapeutic applications. METHODS:A computer-based search of CNKI and PubMed academic database was performed for articles addressing serum-free culture systems of human embryonic stem cels published from 2008 to 2015. Repetitive and old articles were excluded. Finaly, 58 articles were summarized. RESULTS AND CONCLUSION:Several groups have attempted to exclude individual animal components by using feeder-free matrices, feeder cels of human origin, or defined xeno-free media, aiming to select a suitable matrix and medium that can minimize or not use heterologous components, in order to obtain cel lines at clinical level. However, the current cel products are far from clinical application. There are stil many problems to be solved, such as standardization, normalization and individualization of cel products. With the normative development of stem cel research and industry, human embryonic stem cel products are expected to be widely used in clinic.

In order to find an easy and rapid quantitative analytical method to detect rhamnolipid produced by Pseudomonas aeruginosa, three methods, H_ 2 SO_ 4 -anthrone analysis method, L-cysteine-H_ 2 SO_ 4 method and phenol-H_ 2 SO_ 4 method, were compared in the present paper, and the influence factors were also considered.The results showed that H_ 2 SO_ 4 -Anthrone analysis method was better than the others and its optimal reaction condition was obtained.The influence to the quantitative analysis of rhamnolipid from the residual glucose and the top clean liquid layer in the ferment solution could be ignored.But the influence from the bacterial body and the middle layer of the ferment solution reached a certain degree.Thus, the bacterial body should be removed before measuring.However, the influence from the middle layer of the ferment solution could be avoided by making a standard curve which was made by using a rhamnose mixed with the middle layer ferment solution.

Objective To prepare high-titer monoclonal antibodies against STX2A1 subunit of enterohemorrhagic E.coli(EHEC) O157∶H7.Methods BALB/c mice were immunized with GST-STX2A1 fusion protein and the spleen cells of BALB/c mice which were not immunized were used as feeder cells.Hybridoma technique,natural STX2A protein and ELISA test were used to prepare and screen the hybridoma cell lines of monoclonal antibodies against STX2A1.The ascites developed by injecting the hybridoma cells into abdominal cavity of the BALB/c mice and was purified with Protein A-Sepharose.The subclasses and isotypes were identified by mouse monoclonal antibody isotyping kit.The antigenic epitopes that can be recognized by STX2-1A3,STX2-1E10 and STX2-3A7 were analyzed by the ELISA additivity test.Results Three hybridoma cell strains were obtained and named as STX2-1A3,STX2-1E10 and STX2-3A7,respectively,all of which produced monoclonal antibodies specifically against STX2A1.The isotypes of the monoclonal antibodies were IgG1?,IgG1?,and IgG3? and the affinity constant was 5.76 ?109,1.21 ?109 and 3.97 ?108,respectively.Conclusion We have successfully prepared three hybridoma cell strains which secrete high-titer and highly specific monoclonal antibodies against STX2A1.Our study provides a basis for researching the early diagnosis,prevention and cure of the disease induced by EHEC O157∶H7.

AIM To observe the clinical efficacy of Maixuekang Capsules (hirudin) combined with female progestational hormone treating blood-stagnated cryptogenic hypomenorrhea and their mechanism of action.METHODS Ninty eligible patients were randomly and equally divided into treatment group (Maixuekang Capsules combine with female progestational hormone) and control group (female progestational hormone),treated for three menstrual cycles,the menstrual blood volume,blood D-dimer,maximum platelet aggregation rate,the thickness of endometrium,spiral artery blood flow peak systolic velocity (PSV),resistance index (RI) and pulsatility index (PI) were recorded during the three months period.RESULTS The menstrual blood volume of the two groups were all increased post-treatments (P ＜ 0.05),the treatment group was superior to the control group (P ＜ 0.05);blood D-dimer and maximum platelet aggregation rate of the treatment group had reduced post-treatments (P ＜ 0.05),there was no difference in the two values of the control group between post-treatment and pre-treatment (P ＞ 0.05);the thickness of endometrium of the two groups were both increased after the treatment (P ＜ 0.05),while there was no remarkable statistical difference between the two groups (P ＞ 0.05);RI in the treatment group had remarkable reduction post-treatments (P ＜ 0.05),but there was no remarkable difference of RI in the control group (P ＞ 0.05);there was no statistical difference of PSV and PI in both groups before and after the treatment (P ＞ 0.05).CONCLUSION The combination treatment of Maixuekang Capsules and female progestational hormone can obviously increase menstrual blood volume,improve uterine blood perfusion and endometrial microcirculation.It has more advantages than use hormones alone.

Objective To investigate the subtype distribution and sequence characteristics of HIV-1 strains prevalent among sexual infectors in Beijing. Methods We collected the blood samples from 100HIV sexual infectors in Beijing during 2008 and separated plasma specimens. RNA was extracted from the plasma and the gag gene was amplified by RT-PCR and nest-PCR. The PCR products were sequenced directly and phylogenetic analyses of gag gene was performed using the MEGA4 software. Results Among 100 HIV-1 plasma samples,84 gag gene fragments were amplified and analyzed. Eight HIV subtypes including B(22 strains), B'(8 strains),C( 1 strain) ,CRF01_AE (38 strains) ,CRF02_AG (2 strains) ,CRF07_BC(9 strains) ,CRF08_BC(3 strains) and C/CRF01_AE recombinant like strain( 1 strain) were identified circulating in Beijing. Conclusion CRF01 _AE and subtype B were predominant in Beijing account for 45.2% and 26.2% and the surveillance of HIV gene variation should be paid more attention.

Objective To investigate the role of annexin A1 (ANXA1) in human pulmonary arterial smooth muscle cells (PASMCs) proliferation induced by hypoxia.Methods Primary cultured human PASMCs were randomly divided into 5 groups ( n =60 each):normal oxygen group (group N) and hypoxia 2,8,12 and 24 h groups(groups H1-4).Group N was treated with 21％ O2 for 24 h,and groups H1-4 was treated with 3％ O2 for 2,8,12 and 24 h respectively.ANXA1 mRNA and protein expression was determined by RT-PCR and Western blot.Proliferation of PASMCs were determined by MTT and 3 H-TdR methods.Results ANXA1 mRNA and protein expression was down-regulated,proliferation of PASMCs was enhanced in groups H1-4 as compared with group N (P ＜ 0.05).ANXA1 mRNA and protein expression was down-regulated gradually,proliferation of PASMCs was enhanced gradually in groups H1-4 ( P ＜ 0.05 ).Conclusion The expression of ANXA1 is down-regulated in human PASMCs during hypoxia treatment,which might be the mechanism of human PASMCs proliferation induced by hypoxia.

Objective To evalute the effect of Ilizarov technique in the treatment of hypertrophic nonunion.Methods Form June 2008 to December 2010,12 patients with hypertrophic nonunion were treated with Ilizarov technique,including 10 males and 2 females with an average age of 46.5 years.The pathology sites of nonunion were kept as closed as possible without any bone graft during operation.As to patients who had ever been treated with plate or intramedullary nail,the hardware should be removed by minimal invasive approach.These procedures aimed to keep the vascularity of nonunion site intact.Ilizarov apparatus were preoperatively constructed.Distal segment and proximal segment of nonunion were mounted respectively with two external circle using the smooth wires and half pins.The two-circle stabilizing one segment was nominated with transosseous modules.Distal module and proximal one was connected with a pair of axial hinges.The pathology sites were gradually distracted from the seventh day postoperatively,0.25 mm/d.Accompanying with deformity correction,limb length discrepancy (LLD) also were restored simultaneously.Then,all the screws and nuts in the apparatus should be tightened,which was favourable to the callus consolidation.Results All 12 cases of nonunion healed without any bone graft.The fixator wearing time lasted 6-12 months,with an average of 8 months.Correction of deformity and LLD were achieved.The average lengthening was 3.0 cm (range,2.0-5.5 cm),the average correction angle was 23° (range,10°-30°).After 6-18 months follow-up,all the patients restored satisfactory function.Conclusion Hypertrophic nonunion can be treated successfully with Ilizarov technique.The key of successful callus distraction is strictly identifying the indications.

Objective To analyze the expression of Yes-associated protein and risk factors in differentiated thyroid carcinoma. Methods Clinical data of 152 patients with differentiated thyroid carcinoma and 27 cases of benign thyroid tumor from Changji Hui Autonomous Prefecture People’s Hospital of Xinjiang, were analyzed retrospectively. According to the expression levels of Yes-associated protein in differentiated thyroid cancer and benign thyroid tumor, univariate Chi-square test and multivariate Logistic regression methods were used to analyze the relationship between Yes-associated protein and gender, age, thyroid stimulating hormone(TSH) level, nodule size, capsule integrity, histological type and lymph node metastasis, in order to find out risk factors in differentiated thyroid cancer. Results The positive rate of expressed Yes-associated protein in benign thyroid tumor group was 66.7%(18/27), which was significantly higher than 31.58%(48/152) of differentiated thyroid cancer group, and the difference was statistically significant(χ2=12.127, P<0.01). Under an optical microscope, changes of Yes-associated protein were found to be mainly located in the nucleus and cytoplasm , and in benign thyriod tumor the degree of staining was deep, strong positive or moderately positive; differentiated thyroid carcinoma was lightly stained or no staining, weakly positive or negative. Chi-square test showed that the expression of Yes-associated protein was not affected by sex, age and pathological type(χ2= 0.419, 0.221, 0.315, all P >0.05); TSH level, nodule size, capsule integrity, lymph node metastasis had an impact on the expression of Yes-associated protein which was down regulated (χ2=4.020, 8.424, 4.386, 6.673, P<0.05 or<0.01). Logistic regression analysis showed that the nodule size was not a risk factor ( odds ratio , OR ) of Yes-associated protein expression (OR=1.929, P>0.05); TSH levels above 4.5 mU/L, lymph node metastasis and envelope incomplete were risk factors that down regulated the expression of Yes-associated protein (OR=2.167, 2.665, 3.048, all P<0.05). Conclusion Yes-associated protein is down regulated in differentiated thyroid cancer. Elevated TSH levels , incomplete capsule and lymph node metastasis are risk factors of Yes-associated protein down expression and differentiated thyroid cancer.