Objective To establish an effective PCR assay for leptospirosis detection , and applicate the assay in tree shrew, mongolian gerbil and gray hamster .Methods Sequence of leptospira was obtained from the NCBI Genbank , and primers were designed based on the sequences .The positive amplified fragments were sequenced to verify the reliability of the method.The samples from tree shrew, mongolian gerbils and hamsters were tested using this PCR method .Results The PCR method for detection of leptospirosis was successfully established .The positive rate of Leptospira was 8.33% in 60 samples of conventional tree shrews , 100% in 104 samples of the conventional Mongolian gerbils , and 0% in 60 samples of clean gray hamsters.Conclusions The establishment of this PCR assay is useful in the detection of leptospirosis in tree shrew, mongolian gerbil and gray hamster .The results of our investigation of leptospira infection levels of the three new experimental animals may promote their application in biomedical research .

Objective To investigate the risk of permanent hair dye use for connective tissue disease ( CTD ) with positive antinuclear antibodies ( ANA ).Methods We conducted a population-based case-control study to analyze 440 CTD patients from the department of our hospital.Four hundreds and forty patients' relatives or friends who were free from any CTD were selected as controls.Patients and controls were matched in sex and age.A face-to-face interview was adopted to finish questionnaires about subjects' demographic data and the use of permanent hair dye.Data about the duration,frequency,times of usage and allergy related to hair dye were collected.Medical history was also reviewed,such as the diagnosis,disease course,primary symptoms,serum tests and organ involvements.Statistical analysis was performed by SPSS 17.0 software using x2 test and Logistic regression.Results The association between the use of permanent hair dye and CTD did not reach statistical significance (OR=1.282,95％CI 0.966-1.700,P=0.085 ),when the relationship of hair dye use with systemic lupus erythematosus (SLE) was tested,the same result (OR=1.092,95％CI 0.795-1.500,P=0.587 ) could be revealed.Conclusion This preliminary study has shown that the use of permanent hair dye couldn't induce the occurrence of CTD.

Objective To explore the application effect of action-oriented teaching method in senior midwifery practice teaching.Methods Selecting 30 students from class 1,grade 2011 of the midwifery professional for the experimental group,another 30 students from class 2 for the control group.We selected four core contents for the experimental teaching refbrm such as obstetric abdominal examination,external pelvimetry measurement,prenatal vulva washing disinfection and sterile towel covering before the delivery.The experimental group was taught by action-oriented teaching,in accordance with the procedures:putting forward the projects or work task,set goals and grouping,making work plans,implementing and evaluating.The control group was taught by traditional teaching.We used questionnaire smvey and skills examination to evaluate the teaching effects and conducted teaching satisfaction scores and scores for t test to two groups of students.Results (1)Questionnaires:The resuhs of the 18 issues on the teaching effect evaluation,professional technology level and nursing soft skills,etc.showed that the experimental group's evaluation of the action-oriented teaching is higher than the control group's evaluation of the traditional teaching(P＜0.05),action-oriented teaching is more helptul to improving the professional skill and nursing soft skills(P＜0.05).@The scores of skill examination:The skills scores of the experimental group(80.01 ± 9.36) were significantly higher than those of the control group(71.32 ± 8.85)(t=3.70,P=0.000).The difference was statistically significant (t=3.70,P=0.00).Conclusions Action-oriented teaching helps to raise practice teaching effecr,train students' clinical work ability and nursing soft skills.It is suitable for practice teaching of senior midwifery.

Objective To establish an effective PCR assay for detection of Bartonella, and application of this assay in tree shrew .Methods Sequence of Bartonella was obtained from NCBI Genbank .Three pairs of primers were designed based on this sequence .One pair of primers was determined through amplifying the major strains in China .Sixty tree shrew blood samples were tested with this PCR assay .The positive amplified fragments were sequenced to verify the reliability of this method .Results A PCR method for detection of Bartonella is successfully established , with a high specificity and the sensitivity was of 2.0 ×10 -5 μg/mL.Among the tested 60 blood samples , 15 positive cases were detected.Sequencing of the samples confirmed a 25%infection rate of Bartonella in the tree shrews, well consistent with the amplification results , and verified the applicability of this detection method .Conclusion The establishment of this method provides the basis for detection of Bartonella in tree shrew.

Objective To observe the nasal flush combined budesonide suspension liquid atomizing inhaled treatment of allergic rhinitis in infants,explore the allergic rhinitis treatments in infants. Methods 137 cases diagnosed as allergic rhinitis were collected and randomly divided into 70 cases as the treatment group and 67 patients as the control group. The patients in control group were washed the nasal cavity with 2.8% warm sodium chloride solution using 50ml and 0.5% metronidazole injection 30ml by turn. At the first week, 1 time/d, then one time every other day,while according to age,body mass the patients were given to loratadine, 1 time/d. Treatment group were used budesonide nasal inhalation of 1 ml at the base of treatment of the control group. Before and after the treatment , nasal congestion,sneezing, flow clear nose, sleep snoring and sleep quality score index were observed and compared.Results 70%of the treatment group the nasal congestion,sneezing,flow clear in 3 times after treatment with ease.The children sleep quality improved and the snoring fewer over night,only 56.7% of the control group of these symptoms improved. After the treatment the efficiency evaluation of treatment group and control group respectively was 95.1% and 77.7% ,there was statistically significant difference( x2 =9.83 ,P ＜0. 01 ). 137 cases of patients without a side effects. Conclusion curative effect of nasal flush combined budesonide suspension liquid nasal spray inhaled treatment of allergic rhinitis was distinct,infant effect-acting quickly,without side effects,easy to use.

Objective To evaluate the clinical value of contrast-enhanced ultrasound (CEUS) in preoperative diagnosis of anal fistula. Methods Forty-five patients with fistula in ano were evaluated by physical examination, then CEUS were peformed by injecting SonoVue through the external opening to enhance the detection of the fistulous track and the internal opening. The results of CEUS were matched with surgical features to establish their accuracy in preoperative assessment of anal fistula. Results Simple typing fistula was found in 19 of 45 patients and 26 patients had complex fistulas. The accurate diagnostic rates by conventional ultrasound of simple typing fistula and complex fistulas were 89. 5% and 61.5%,respectively,the accurate diagnostic rates by CEUS of simple typing fistula and complex fistulas were 94. 7% and 92.3%, respectively, there was no significant difference between conventional ultrasound and CEUS about simple typing fistula( P ＞0.05), and there was significant difference about complex fistulas ( P ＜0. 05). Conclusions CEUS has a good visibility and accurate rate for diagnosis of anal fistula,It plays an important role for operation.

Objectives To establish method for collecting haemocytes of Oncomelania hupensis and study its morphology and immunological importance. Methods Referring to the method of haemocytes collection from peripheral lymphoid organ, suspension technique was used for collection of haemocytes from snails, which were then Giemsa-stained and observed under microscope. Stained by gentian violet, number of haemocytes was counted and compared with that of conventional squashing method and needling method by ANOVA and Dunnett-t test. Supernatant from freeze thawing haemocytes was applied for the tests of immuno-precipitation, bacteriostasis, and phagocytosis. SDS-PAGE was used to analyze relative molecular mass of protein ingredients. Results Four kinds of haemocytes were found: round cells with filiform filopodia, acidophilic and basophilic round cells both without filiform filopodia, and spindle cells. The average diameter of the 4 type cells was 10.93, 6.13, 6.08, and 11:06?m, and occupied 50%, 30%, 5%, and 15% respectively. The mean of haemocytes received from suspension, squashing and needling methods was 15 000, 6 600 and 300/ml respectively. ANOVA analysis showed F=281.47, P

Objective To establish a rapid, simple, sensitive, and specific multiplex real-time PCR method for quantitative detection of Campylobacter jejuni, Salmonella and Shigella in tree shrews.Methods Specific primers and probes were designed, according to the HipO gene of Campylobacter jejuni, inV gene of Salmonella and ipaH gene of Shigella.The primers were confirmed by single pathogen quantitative PCR, and the sensitivity and specificity of the multiplex PCR were analyzed.Finall, the samples of experimental tree shrews were detected by this multiplex PCR method.Results The PCR element of TaqMan-MGB real-time PCR assay was able to quantitatively amplify the Campylobacter jejuni, Salmonella or Shigella.Appropriate standard amplification curves of Campylobacter jejuni, Salmonella and Shigella in the multiplex quantitative PCR were obtained.The sensitivity of this method was 1×103 ng/μL.There was no false positive detection from other bacterial strains.Conclusions This multiplex quantitative real-time PCR method has good application and development prospects in the detection of microorganisms in tree shrews.

Objective To investigate the effect of mesenchymal stem cells (MSCs) on subcutaneous xenograft tumors in mice with Lewis lung cancer. Methods MSCs isolated from bone marrow of C57BL/6 mice were made into single cell suspension and were cultured in vitro. The cells of the 4th to 5th passage were used for the subsequent experiments. Fifty six C57BL/6 mice were inoculated subcutaneously with Lewis lung cancer cells, and were grouped into Group D0 (MSCs were given simultaneously with inoculation)and Group D10(MSCs were given 10 d after inoculation). Group D0 included three subgroups (n=8): Group 1 with inoculation of tumor cells, Group 2 with inoculation of tumor cells and MSCs, and Group 3 with inoculation of tumor cells and tail intravenous injection of MSCs. Group D10 included four groups: Group 4 with inoculation of tumor cells and injection of MSCs in tumors, Group 5 with equivalent PBS (the control of Group 4), Group 6 with inoculation of tumor cells and tail intravenous injection of MSCs, and Group 7 with equivalent PBS (the control of Group 6). The time of tumor formation and the volume of tumor were observed and compared among the groups. ResultsIn Group D0, earlier onset of tumor development was observed in Group 2 as compared to Group 1 and Group 3 (P<0.05), while there was no significant difference on the volume of tumor in the three groups (P>0.05). In Group D10, the volume of tumors were larger in Group 4 compared to the control (P<0.05), while there was no significant difference on the volume of tumors between Group 6 and the control (P>0.05). Conclusion Inoculating mixture of MSCs and Lewis lung cancer cells accelerates tumor formation,and injection of MSCs in tumors stimulates the growth of tumors.

Objective To investigate the expression and aberrant methylation of Ptchl gene in hedgehog signal pathway in carcinogenesis of human gastric cancer.Methods The total RNA and genomic DNA were extracted from 10 human gastric carcinoma tissues,adjacent tissues(＞3 cm from cancerous tissue)and gastric cancer eell line AGS.Ptchl mRNA expression was detected by real-time quantitative reverse transcription PCR.The pattern of CpG island in Ptchl gene 5'regulation sequence was analyzed by software and its methylation extent was tested by bisulfite sequencing PCR.Results The analysis of CpG island(starting-3950 bases upstream of the Ptchl mRNAla transcription start site and ending 2050 bases downstream)revealed that there were two CpG islands in Ptchl gene 5' regulation sequence(first CpG:-1139 bp～+860 bp;second CpG:+875 bp～+1692 bp).Bisulfite sequencing PCR analysis of 19 CpG sites included in the first CpG island(-870 bp～+229 bp)showed that there was methylation present in all cell lines and the average extent of the methylation of these CpG sites was significantly higher in cancerous tissues(64%±32%,ranged 16%～100%)than that in adjacent tissues(13%±14%,ranged 0%～42%,P＜0.05).There was a negative correlation of the Ptchl methylation with its expression.Conclusion The high methylation of Ptchl gene that involves in the carcinogenesis of human gastric carcer will be a new biomarker for gastric carcer.