Objective To explore and establish the possible method of low-intensity pulsed ultrasound(LIPUS) irradiation directly combined with SonoVue microbubbles could enhance 5-azacytine (Aza) induced cardiomyogenic differentiation of human bone marrow mesenchymal stem cellin(MSCs)vitro,initially explore the appropriate irradiation condition,and to provide experimental basic data for ultrasound-mediated microbubble destruction application in MSCs transplantation for acute myocardial infarction.Methods (1) In order to screen out the appropriate ultrasound irradiation intensity and time without conspicuous harm to MSCs,stem cells were exposed to different ultrasound intensity irradiation (0.11,0.33,0.55,0.77,0.99 W/cm2) and time (0,30,60,90 s) and then cell proliferation determined by methyl thiazolyl tetrazolium(MTT).In order to screen out the appropriate microbubble-to-cell ratio without conspicuous harm to MSCs,stem cells of different microbubble-to-cell ratios (0,10,50,100,150) were exposed above the screened ultrasound irradiation intensity and time and then cell proliferation determined by MTT.(2) MSCs were divided into four groups and induced cardiomyogenic differentiation by different inducing systems in vitro as follows:no-treatment group,5-Aza inducing group(10 μmol/L),microbubble and ultrasound group,5-Aza inducing (10 μmol/L) with microbubble and ultrasound group,followed by four weeks of further culture.The morphological changes were studied daily under phase-contrast microscope,then the expression of cardiac troponin-T (cTnT) was detected by immunocytochemistry method to analyze and compare differentiation rate of cardiomyocyte-like cells in each group.Results (1)The direct ultrasound irradiation condition of no conspicuous harm to MSCs,but with destruction was intensity of 0.55 W/cm2,exposure time of 30 s and microbubble-to-cell ratio of 50.(2) This study confirmed the feasibility of direct ultrasound exposure of LIPUS on MSCs in vitro.(3) At 28 days after induction,the stem cells of 5-Aza inducing group and 5-Aza inducing with microbubble and ultrasound group expressed cTnT and the positive rate of the latter was higher than the former by immunocytochemistry (13.69％ vs 34.13％,P ＜0.05).Conclusions The appropriate ultrasound direct irradiation condition was intensity of 0.55 W/cm2,exposure time of 30 s and microbubble-to-cell ratio of 50.Appropriate conditions of LIPUS could enhance 5-Aza induced cardiomyogenic differentiation of MSCs in vitro.

BACKGROUND: As indicated by clinical research, if cerebral infarction could be effectively treated at early stage, especially normal specific therapy provided within 6 hours or even earlier after attack, the prognosis would be significantly better than delayed therapy. However, it is still unclear that whether the changes of cellular apoptosis-inducing or -inhibiting indicators could be used as criteria in the judgment of prognosis.OBJECTIVE: To test the content of cell apoptotic factor and to investigate the prognosis in hospitalized patients with cerebral infarction who received treatment at different time for further verification of the therapeutic timing for the disease.DESIGN: A same term randomized controlled study based on patients.SETTING: Department of neurology of a general hospital of a military area command of Chinese PLA.PARTICIPANTS: Totally 144 male patients admitted in the Second Department of Neurology, General Hospital of Jinan Military Area Command of Chinese PLA between 2000 and 2002 were divided into four groups including 6 hours, 24 hours, 72 hours and 96 hours group according to different time of therapy provided.METHODS: Oral administration of 400 mg Lumbrokinase, 2 tablets of heparin sodium, 60 mg of nimodipine, and 100 mg of vitamin E, three times a day. 150 mL of normal saline(NS) containing 52.5 mg of Ginkgo biloba L extractive(Jin Na Duo) and 150 mL of NS containing 10 mL of Cerebroprotein Hydeolysate were used through intravenous drop once a day. Ten days were set as one therapeutic course and 2 courses were given. 200 g/L of mannite was given to dehydrate for patients with large area infarction(＞ 7 cm2) . Platelet membrane Fas, Apo2.7 and Bcl-2 percentage and prognostic assessment were tested in patients of four groups before and after therapy.MAIN OUTCOME MEASURES: Peripheral platelet membrane Fas,Apo2.7 and Bcl-2 percentage in patients of different group and prognosis evaluation.RESULTS: Percentage of platelet membrane Fas, Apo2.7 and Bcl-2 of 6 hours group was significantly higher or lower after therapy than before therapy ( P ＜ 0.05 ), and moreover, the difference with other groups was significant( P ＜ 0.05), As revealed in the analysis of prognosis, the effectiveness of patients who received therapy within 6 hours was significantly better than that of 96 hours group and the mortality reduced significantly.CONCLUSION: Normal hospitalizing therapy provided within 6 hours after attack could surely improve the prognosis and reduce the disability rate, and the abnormity and extent in Fas, Apo2.7 and Bcl-2 are closely correlated with prognosis.

Objective: To explore influence of nursing health education on morning blood pressure surge (MBPS) fluctuations in aged patients with hypertension.Methods: A total of 100 outpatients and inpatients with non-dipper hypertension, who haven't been treated, were selected from Apr 2011 to Sep 2015.According to random number table method, patients were randomly and equally divided into routine treatment group and health education group (received nursing health education based on routine treatment group), both groups were treated for four weeks.MBPS, trough/peak(T/P) ratio and smoothness index (SI) were measured and compared between two groups after treatment.Results: Compared with routine treatment group after four-week treatment, there were significant reductions in systolic blood pressure (SBP) morning surge[(46.95±5.17) mmHg vs.(42.21±5.19) mmHg]and diastolic blood pressure (DBP) morning surge[(16.31±3.23) mmHg vs.(14.21±3.58) mmHg], and significant rise in SBP T/P[(59.22±6.65)% vs.(70.11±6.41)%], DBP T/P[(50.91±5.04)% vs.(56.97±5.12)%], SBP SI[(1.19±0.13) vs.(1.42±0.12)]and DBP SI[(1.11±0.09) vs.(1.29±0.08)]in health education group, P<0.05 all.Conclusion: Health education can effectively improve blood pressure fluctuations and may likely improve prognosis in patients with non-dipper hypertension, which is worth extending.

Objective To evaluate the reliability and validity of the Chinese version of the Newest Vital Sign(NVS). Methods The NVS was translated and back-translated. Cultural adaption of scale was performed by Delphi expert consultation and pilot study. The reliability and validity of the Chinese version of the NVS was tested in 451 Chinese residents. Results Chinese version of the NVS consisted of 6 items with Pearson correlation between item and total score of scale ranging from 0.50 to 0.71. Two factors were abstracted by exploratory factor analysis and explained 61.51% of total variance. Confirmatory factor analysis showed that the fitness of the model was acceptable:comparative fit index was 0.96, Tuker-Lewis index was 0.92, standardized root mean square residual was 0.04, root mean square error of approximation was 0.077. Cronbach α coefficient was 0.71, retest reliability was 0.92. Conclusions Chinese version of the NVS has good reliability and validity, which can be used to test the health literacy of residents in China.

Objective To research the homology and cross reaction characteristics of human papillomavirus(HPV)16 type L2 N-terminal(1-200)protein in clinical common HPV infection types.Methods The amino acid sequences of the common HPV infection types(6,11,16,18 ,etc.)were blasted and it was found that 1-200 N-terminal sequence of L2 protein was highly homologous.The gene of HPV16 L2(1-200)was amplificated from tissue sample of cervical cancer patient and inserted into the prokaryotic expression vector PGEX-4T-1 to construct the recombinant plasmid PGEX-4T-1-HPV16 L2(1-200).After sequencing identification,the recombinant plasmid was tranformed into E.coli BL21(DE3).Induced by IPTG,the fusion protein containing HPV16 L2(1-200)was expressed and analyzed by both SDS-PAGE and Western blot.Furthermore,the specific binding capacity of the fusion protein to the HPV 6,11,16 and 18 DNA positive patient serums were analyzed by Western blot.The fusion protein was purified with Ni-NTA Agarose Kit and coated with ELISA reaction plates.The specific serum IgG of 98 condyloma acuminatum patients,135 cervix cancer patients and 96 healthy control subjects were detected respectively by indirect ELISA.Results After comparing the amino acid sequences of the common HPV infection types(HPV6,11,16,18,etc.),We found that the homology of HPV L2(1-200)reached 52.7%-74.3%.The recombinant plasmid PGEX-4T-1-HPV 16 L2(1-200)was constructed successfully.Highly expressed HPV16 L2(1-200)fusion protein was obtained and the expression level was account for up to about 22.6% of total bacterial protein.The relative molecular mass(Mr)of the fusion protein is about 49×103,which matches up to the expected Mr Meanwhile,the serums of HPV 6,11,16,18 DNA positive patients were used as the first antibody and the specific band was detected respectively at about 49 × 103 by Western blot.Indirect ELISA showed that the A490 values of the specific IgG of condyloma acuminatum group,cervical cancer group and healthy control subjects were 0.753 ± 0.262,0.756 ± 0.274 and 0.178 ± 0.157 with the positive rate were 89.8%,88.9% and 9.4% respectively.There was no significance of the specific IgG between condyloma acuminatum group and cervical cancer group(P＞0.05),but it was significant among the three groups(P＜0.001).Conclusion The N-terminal 1-200 amino acids of HPV L2 has high homology and there exits cross reaction among the most common HPV infection types.

Objective To investigate the cross reaction characteristics of recombinant human papillomavirus 16 type L2 full-length fusion protein in HPV types of 6, 11, 18.Methods The serum samples of 108 condyloma acuminatum patients, 156 cervix cancer patients and 100 healthy control subjects were collected.The gene of full-length HPV16 L2 was amplificated from the tissue DNA of cervical cancer patient and inserted into the prokaryotic expression vector PGEX-4T-1 to construct the recombinant plasmid PGEX-4T-1-HPV16 L2.After sequencing identification, the recombinant plasmid was tranformed into E.coli BL21 (DE3).After induction by IPTG, the fusion protein containing HPV16 L2 was expressed and analyzed by both SDS-PAGE and WB.Furthermore, the specific binding capacity of the fusion protein to the HPV 6,11, 16 and 18 DNA positive patient's sera were analyzed by WB.The fusion protein was purified with NiNTA Agarose Kit and coated with ELISA reaction plates.The specific serum IgG were detected by indirect ELISA.Results The recombinant plasmid PGEX-4T-1-HPV16 L2 was constructed successfully. Highly expressed HPV16 L2 full-length fusion protein was obtained and the expression level was 27.2 %.The relative molecular mass(Mr) of the fusion protein is about 82 × 103, which matches up to the expected Mr.Meanwhile, the sera of HPV 6,11,16,18 DNA positive patients were used as the primary antibody and the Mr of the specific band was detected to be about 82 × 103 by WB.The results of indirect ELISA showed that the average levels of specific IgG in condyloma acuminatum group, cervical cancer group and healthy control subjects were 0.848 ±0.257, 0.822 ±0.247 and 0.173 ±0.143 with the positive rate of 92.6%, 94.2%and 8.0% respectively.There was no significance of the specific IgG levels between condyloma acuminatum group and cervical cancer group ( F = 0, P ＞ 0.05 ), but there was significant difference of specific IgG levels and positivity among the three groups ( F = 305.201 ,x2 = 253.178, P ＜ 0.01 ).Conclusions The HPV16 L2 full-length fusion protein has better antigenicity.However cross reactions with HPV6, 11 and 18 were found.It can be applied in serological screening reagents for HPV infection and associated cancer.

The amino acid contents of five floral sources Chinese honeys (jujube, rape, chaste, acacia, and lungan) were measured using reversed phase high-performance liquid chromatography (RP-HPLC). The results showed that proline was the main amino acid in most of the analyzed samples. Phenylalanine presents at the highest content in chaste honey samples, and the total amino acid contents of chaste honeys were also significantly higher than those of other honey samples. Based on the amino acid contents, honey samples were classified using chemometric methods (cluster analysis (CA), principal component analysis (PCA), and discriminant analysis (DA)). According to the CA results, chaste honeys could be separated from other honeys, while the remaining samples were correctly grouped together when the chaste honey data were excluded. By using DA, the overall correct classification rate reached 100%. The results revealed that amino acid contents could potentially be used as indicators to identify the botanical origin of unifloral honeys.

The amino acid contents of five floral sources Chinese honeys (jujube, rape, chaste, acacia, and lungan) were measured using reversed phase high-performance liquid chromatography (RP-HPLC). The results showed that proline was the main amino acid in most of the analyzed samples. Phenylalanine presents at the highest content in chaste honey samples, and the total amino acid contents of chaste honeys were also significantly higher than those of other honey samples. Based on the amino acid contents, honey samples were classified using chemometric methods (cluster analysis (CA), principal component analysis (PCA), and discriminant analysis (DA)). According to the CA results, chaste honeys could be separated from other honeys, while the remaining samples were correctly grouped together when the chaste honey data were excluded. By using DA, the overall correct classification rate reached 100%. The results revealed that amino acid contents could potentially be used as indicators to identify the botanical origin of unifloral honeys.

The aim of this study is to develop a rapid and accurately species typing method for Brucella isolates by using High Resolution Melting (HRM ) analysis .Six pairs of primers were used according to the reference for the sequence of pur‐pose gene .Nineteen biotypes of six species Brucella standard strains were identified by PCR‐HRM analysis and this analysis was used to detect the 35 clinical isolates .Results showed Brucella amplified specific melting curves were different from con‐trasted strains with primer Bspp .The six species Brucella standard strains have own characteristic curve shape from each oth‐ers by PCR‐HRM analysis with five pairs of primers .Thirty‐five clinical isolates of Brucella have entirely consistent with PCR‐HRM curve shape with Brucella melitensis standard strains .So ,PCR‐HRM analysis methods can accurately identify Brucella strains ,especially clinical isolated Brucella melitensis ,and may be used in clinical microbiology laboratories .

Objective To observe the influence of pioglitazone on the expression of adiponectin in the liver of high fat diet-induced obese rats.Methods Forty male SD rats were randomly divided into control group which received regular diet ( n =10),high fat diet group ( n =15 ) and high fat diet goup administened pioglitazone ( n =15 ) on high fat diet.After twelve weeks,fasting blood glucose,fasting serum insulin,triglyceride,total cholesterol,and the histomorphologieal changes in liver were observed;insulin sensitivity test was performed;the fatty experimental group are treated with Pioglitazone(30mg · kg-1 · d-1,oral gavage),treatment was administered daily for 2 weeks.The control group and fatty control group all received an equal volume of vehicle (saline).The protein expression of adiponectin in the liver of rats was determined by immunohistochemistry.Results The insulin sensitive index was significantly decreased in the high-fat-diet rats compared with the control group[(0.021 ±0.010),(0.015 ±0.007),P ＜0.05] ;The protein level of adiponectin in the liver of fatty,control group was significantly decreased compared with the control group[(2929.73 ± 157.45 ),(3814.21 ±211.42),P ＜0.05],adiponectin in the liver of fatty experimental group was significantly increased compared with the fatty control group[(2929.73 ± 157.45),(3657.68 ±217.31 ),P ＜0.05].Conclusion Pioglitazone could improve the expression of adiponectin,and improve the resistance of insulin.