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.

Objective To explore the value of mast cell tryptase and brain natriuretic peptide(BNP)in the differential diagnostic of sudden death due to hypersensitivity and coronary atherosclerotic heart dis-ease.Methods Totally 30 myocardial samples were collected from the autopsy cases in the Department of Forensic Pathology, Shanxi Medical University during 2010—2015. All samples were divided into three groups:death of craniocerebral injury group, sudden death of hypersensitivity group and sudden death of coronary atherosclerotic heart disease group, 10 cases in each group. Mast cell tryptase and BNP in myocardium were detected by immunofluorescence staining and Western Blotting.Results Immunofluo-rescence staining showed that the positive staining mast cell tryptase appeared in myocardium of sudden death of hypersensitivity group and coronary atherosclerotic heart disease group. Among the three groups, the expression of mast cell tryptase showed significantly differences through pairwise comparison(P<0.05); The expression level of BNP in sudden death of coronary atherosclerotic heart disease group were significantly higher than the sudden death of hypersensitivity group and death of craniocerebral injury group(P<0.05). The difference of the expression level of BNP between the sudden death of hypersensitivity group and the death of craniocerebral injury group had no statistical significance(P>0.05).Conclusion The combined detection of the mast cell tryptase and BNP in myocardium is expected to provide help for the forensic differential diagnosis of sudden death due to hypersensitivity and coronary atherosclerotic heart disease.

Objective To establish a double-antibody sandwich ELISA for the rapid detection of shiga toxin typeⅡ ( StxⅡ) in shiga toxin-producing Escherichia coli ( STEC) infection. Methods A pool of murine hybridomas was used to screen out the optimal antibody pair for the establishment of double-anti-body sandwich ELISA. The established ELISA system was used to detect StxⅡin the culture supernatants of 16 clinical strains of STEC. Specificity and sensitivity of the established ELISA system were also evaluated. Results Two antibodies, S2D8 and S2C6, were successfully screened out, based on which the double-anti-body sandwich ELISA was set up. StxⅡand its variants rather than StxⅠwas detected in the culture super-natants of STEC with a lowest detection limit of 4 ng/ml. Its performance was consistent with that of commer-cial colloidal gold test kit, indicating the characteristics of good specificity and sensitivity. Conclusion The S2D8/S2C6-based ELISA laid a foundation for researches which designates the shiga toxin as a potential can-didate on the diagnosis and therapy of STEC infection.

Objective To evaluate the effect of moderate activities at moderate high altitude on acute mountain sickness (AMS) incidence .Methods Ninety-one healthy sea level residents traveled from sea level (345 m) to high altitude city (3200 m) ,by train within 48-hour .They walked 5 kilometers after 2-night stayed ,Lake Louis Score (LLS) Questionnaires ,blood pressure(BP) and oxygen saturation (SpO2 ) was administered before and after walking .Results Seven subjects were excluded because of incomplete data .The incidence of AMS before and after exercise was 20 .24% (n=17/84) and 11 .90% (n=10/84) respectively(P>0 .05) .Af-ter a 5 kilometer walking ,the heart rate increased from (73 .83 ± 9 .96)bpm to(84 .31 ± 12 .55)bpm (P<0 .05) ,Systolic BP and SpO2 level decreased from(128 .86 ± 13 .93)mm Hg to (124 .48 ± 13 .13)mm Hg ,(92 .80 ± 2 .25)% to (89 .94 ± 2 .45)% (P<0 .05) .Headache symptom improved after walking (P<0 .05) .Smoking was negative correlate with LLS score before and after ex-ercise(P<0 .05) .There is no relation between SpO2 and LLS scores .Conclusion Walking five kilometers at 3200 m improve head-ache symptom and tend to decrease AMS .

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 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.