Objective: To isolate Mycobacterium tuberculosis H37Rv and H37Ra genes especially,and to construct two cDNA libraries by using suppression subtractive hybridization (SSH).Methods: We used suppression subtractive hybridization (SSH) to clone the differential genomic genes between Mycobacterium tuberculosis virulence strain H37Rv and attenuated strain H37Ra.After two times of subtractive hybridization and two times of PCR,the products of last PCR amplification were inserted into pGEM-T Easy vector and be transformed into the E.coli DH5α and screened of blue and white clones of the transformants.The subtracted cDNA library of differentially expressed genes were identified by RT-PCR.Results:High or especially expressed genes as tester had been obtained by SSH in correctitude reaction (H37Rv as tester) and reverse reaction (H37Ra as tester),the cDNA libraries of A and B of Mycobacterium tuberculosis H37Rv and H37Ra were successfully constructed.90% of the colonies were white clones,the single band of the colonies was 75% and 80%.Conchision:The cDNA libraries of Mycobacterium tuberculosis H37Rv and H37Ra were successfully constructed using SSH technique,which lay a solid foundation for screening and cloning new specific differentially expressed genes in them.

Objective To investigate the relationship between the levels of total serum IgE and IL-4 in patients with seasonal contact dermatitis. Methods Total serum IgE was tested by immunoradiometric assay(IRMA) in 29 cases of seasonal contact dermatitis, 35 cases of allergic contact dermatitis and 24 cases of healthy controls. Production of IL-4 was detected by ELISA from peripheral blood mononuclear cells (PBMC) cultured with PHA for 48 hours. Results The levels of IL-4 secreted by PBMC and total serum IgE were significantly higher in patient with seasonal contact dermatitis than those in control group (P

Objective To investigate the status of social support and job satisfaction of community nurses and their relationship in Wuhan. Methods A total of 230 community nurses from 20 community health service centers in Wuhan were investigated by general information questionnaire, job satisfaction scale of community nurse and social support scale. Results The total score of job satisfaction was (96.14±14.76) points, the highest score of dimension was leadership and management (23.64±4.60) points, and the lowest was pay and welfare (10.83±3.00) points. The total score of social support was (37.17±7.05) points, the highest score of dimension was perceived social support (20.15 ± 4.48) points, the lowest was availability (8.05±1.57) points. There was a positive association between community nurses′social support and job satisfaction (r=0.248, P<0.05). Conclusions Social support is one of the predictors of job satisfaction. In order to make sure the sustainable development of community health services, it is necessary to know the status and take corresponding measures to improve the levels of social support and job satisfaction.

BACKGROUND: Focal ischemic model made by middle cerebral artery occlusion (MCAO) is much similar to the process of onset in human cere bral infarction, whereas in the process of modeling some problems such as selection of nylon strand with improper diameter and inserted depth would result in modeling failure. OBJECTIVE: To improve MCAO model of focal cerebral infarction. DESIGN: Single-factor design, animal experiment. SETTING: Department of Pediatrics, Affiliated Maternal and Children's Hospital of Guangzhou Medical College. MATERIALS: The experiment was conducted in the Experimental Center of Guangdong Province from January. 2002 to March 2004. A total of 24 healthy Wistar rats were randomly divided into sham-operation group and model group with 10 rats in each group.METHODS: In rats of the model group, the common carotid artery (CCA) and external carotid artery (ECA) were isolated and ligated. A strand was inserted via the incision on CCA near the furcation between CCA and ECA as deep as possible with the depth of (2.0±0.2) mm. The diameter of nylon strand was 0.2 mm, the top of nylon strand was treated by melting paraffin. The interrupted time of blood circulation was 3 hours. Rats in the sham-operation group were treated by slightly drawing back the nylon strand to the CCA immediately after inserting.MAIN OUTCOME MEASURES: ①Neurobehavioral rating: It was conducted at 3 and 12 hours after ischemia with the score ranged 0-4 points.The higher the score was, the severer the neural functional deficit was, 1-3 points signified successful modeling. ②Area of cerebral infarction: Rats wereexecuted at 12 hours after ischemia. Then, brains were quickly removed and stained with tetrazolium chloride (TTC). The percentage of cerebral infarction area was calculated. ③Observation on pathological changes under light microscope.RESULTS: All of 20 rats entered the final analysis.①Eight out of 10 rats in the model group represented contra lateral tumble or draw outward circles, positive Homer's syndrome can be seen in the ligated side (3 points);One rat was disable to completely extend the claws in the contra lateral side of ligation (1 point), one rat had no neural symptoms.②Some pathological changes can be seen in the model group such as swelled cerebral tissues in the ligated side, which were bigger than the contra lateral side and were in pale; The cortex, basal ganglia and thalamus in the ligated side after TTC staining were in pale, whereas the cerebral tissues in the sham-operation group were in red with clear border.③There was no infarct in the sham-operation group, and the percentage of cerebral infarction area in the model group was (22.40±4.52)%, the infarct area of rats in groups were fundamentally same.CONCLUSION: It is necessary to adapt the strand with appropriate diameter, inserted depth and interrupting time for successful modeling.

Objective To investigate the effects of a short hairpin RNA targeting epidermal growth factor recereceptor (EGFR-shRNA) on Colo-16 cell apoptosis and sensitivity to rapamycin. Methods The expression vector of EGFR-specific shRNA was constructed. Colo-16 cells were classified into 4 groups, normal control group remaining untreated, liposome group transfected with lipofectamine 2000, negative control group transfected with shRNA-NC/Iipofectamine 2000 and positive interference group transfected with the expression vector of shRNA-EGFR/Lipofectamine 2000. After additional culture, immunocytochemistry and Western blot were conducted to detect the protein expression of EGFR, and flow cytometry to measure the apoptosis in Colo-16cells. MTT assay was performed to measure the sensitivity of Colo-16 cells to rapamycin. Results Compared with the normal control group, the expression of EGFR was down-regulated by 43.3% in positive interference group (F= 44.6, P< 0.05), and the sensitivity to rapamycin was increased by 2.44 folds. The apoptosis rate in positive interference group was (12.65±0.091)%, significantly different from that in the normal control group (F = 2042.9, P < 0.05). Conclusion The plasmid expression vector containing shRNA targeting EGFR can effectively suppress the expression of EGFR by Colo-16 cells, enhance the sensitivity of Colo-16 cells to rapamycin and induce the apoptosis in Colo-16 cells.

Objective To estimate the effects ot rosiglitazone on cultured HaCaT human keratinocytes and their possible mechanism.Methods HaCaT cells were cultured and treated with different concentrations ( 10,20,40,80 μ mol/L) of rosiglitazone or solvent for 24,48,72 and 96 hours,respectively.Cell proliferation was detected with methyl thiazolyl tetrazolium (MTT) assay.Western blot was performed to measure the protein expression of β-catenin and cyclin D1.Results Compared with the solvent-treated cells,the proliferation of HaCaT cells was significantly inhibited by 18.9％,23.7％,35.1％ and 44.6％ (all P＜ 0.05) after treatment with rosiglitazone of 10,20,40 and 80 μmol/L,respectively,for 48 hours.The expressions of β-catenin and cyclin D 1 were significantly lower in rosiglitazone-treated HaCaT cells than in solvent-treated cells (all P ＜ 0.05).Conclusion Rosiglitazone could inhibit the proliferation of HaCaT cells,likely by downregulating the expressions of β-catenin and cyclin D 1.

Objective To investigate the health behavior level of elderly patients with coronary heart disease (CHD) in order to instruct nurses how to give health education to them. Methods By con-venient sampling, 71 elderly patients with CHD in four hospitals in Wuhan were surveyed with the ques-tionnaire of health promoting lifestyle profile Ⅱ of FANG Heng-ying. The investigation results were ana-lysed. Results The findings showed that mean total score of health behavior of elderly patients with CHD obtained from the questionnaire was only 2.40,the level of health behavior was low. There were statistically significant differences among scores of elderly patients with CHD at different levels of family income and at different occupations before retirement. Conclusions The general level of health behavior of elderly pa-tients with CHD was low, especially in the aspect of health responsibility. Moreover, Patients with lower family income, peasants, and as a worker before retirement have lower health behavior level. This survey suggests that nurses should enhance the health education of the elderly patients with CHD, especially the poor and elderly farmer patients with CHD to improve their health behavior level.

Objective To investigate the in vitro effect of sirolimus on the apoptosis of a cutaneous squamous cell carcinoma cell line, Colo-16 cells. Methods Cultured Colo-16 cells were treated with different concentrations (50, 100, 150, 200 nmol/L) of sirolimus for various durations ( 12, 24, 48, 72 hours). Subse-quently, cell proliferation was detected by MTT assay, and cell apoptosis by Annexin V-FITC and PI double staining. Morphological changes of the cells were observed with Hoechst 33258 fluorescent staining. Total RNA was extracted from Colo-16 cells treated with sirolimus for 48 hours, and subjected to reverse tran-scription (RT)-PCR for the detection of mRNA expression of B cell lymphoma/leukmia-2 (Bcl-2) and Bcl-2-associated X Protein (Bax). Results Sirolimus inhibited the proliferation of Colo-16 cells in a time-and dose-dependent fashion. The early apoptosis rate was 7.26%±0.26%, 8.34% ±0.19%, 9.86%±0.14%, 11.92% ±0.15% in Colo-16 cells treated with sirolimus of 50, 100, 150, and 200 nmol/L, respectively, signifi-candy higher than that in untreated cells (1.53%±0.09%, P < 0.05); a positive correlation was observed between the apoptosis rate and concentrations of sirolimus (r = 0.955, P = 0.000). Typical morphological changes of apoptosis, such as chromatin condensation and margination as well as nuclear fragmentation were observed by fluorescence staining. After treatment with sirolimus for 48 hours, a significant decrease was observed in the mRNA expression of Bcl-2, while an increase in that of Bax was noticed. Conclusion Sirolimus could induce Colo-16 cells apoptosis in vitro, which may be associated with the modulation of expression of apoptosis-regnlating genes, such as Bcl-2 and Bax.

Objective To explore the determination of dioxin in wastewater samples pretreated by solid phase microextraction(SPME). Methods The wasterwater samples were purified and enriched by SPME, then were determined with HRGC-HRMS for the concentration of dioxin in wastewater samples. Results The most suitable conditions of the pretreatment were 30 min microextraction at 45 ℃. The detection limit was 0.05 pg/?l. RSD was lower than 10%. The recovery rates were 99%-102%. The concentration of total dioxins in wastewater sample was 0.78 pg/?l. Conclusion The method of the determination of dioxins in wastewater by SPME was simple and quick, and presented a broad prospects for application.

Objective To evaluate effects of cucurbitacin Ⅰ on in vitro proliferation of HaCaT cells and the expression of keratin 17 (K17),signal transducer and activator of transcription 3 (STAT3) and vascular endothelial growth factor (VEGF) in cultured HaCaT cells.Methods In vitro cultured HaCaT cells were divided into 6 groups to be treated with cucurbitacin Ⅰ at different concentrations of 0.0125,0.025,0.05 and 0.1 μmol/L (0.0125,0.025,0.05 and 0.1 μmol/L cucurbitacin Ⅰ groups),DMEM containing the same volume of DMSO as 0.1 pmol/L cucurbitacin Ⅰ (DMSO group),DMEM (negative control group) and 10 nmol/L calcipotriol (positive control group),respectively.Cell counting kit-8 (CCK8) assay was performed to assess in vitro cellular proliferative activity after 12-,24-,36-hour treatment,reverse transcription (RT)-PCR to measure the mRNA expression of K17 and VEGF in HaCaT cells after 24-hour treatment,and Western blot analysis to determine the protein expression of K17,STAT3,phosphorylated-STAT3 (p-STAT3) and VEGF after 24-hour treatment.Statistical analysis was carried out by one-way analysis of variance (ANOVA),repeated measures ANOVA,Student-Newman-Keuls (SNK)-q test and Pearson correlation analysis.Results The proliferative activity of HaCaT cells started to decrease after 12-hour treatment with cucurbitacin Ⅰ at the concentration of 0.0125 μmol/L.When the concentration of cucurbitacin Ⅰ increased up to 0.1 μmol/L,the cell proliferation rates were inhibited by 43.00％ ± 2.11％ and 48.98％ ± 2.27％ after 24-and 36-hour treatment respectively.Compared with the negative control group,cucurbitacin Ⅰ at different concentrations all could inhibit in vitro proliferation of HaCaT cells (all P ＜ 0.05),and the inhibitory effects increased gradually with the increase of cucurbitacin Ⅰ concentration and treatment duration.After 24-hour treatment,the mRNA expression of K17 and VEGF and the protein expression of K17,VEGF and P-STAT3 were significantly decreased along with the increase of concentration of cucurbitacin Ⅰ (all P ＜ 0.05).Conclusion Cucurbitacin Ⅰ can inhibit in vitro proliferation of HaCaT cells,and down-regulate the mRNA expression of K17 and VEGF and protein expression of K17,VEGF and P-STAT3.