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 COX-2 antisense oligonucleotide (AsODN) on the proliferation and expression of COX-2 in human skin squamous cell carcinoma cell line Coio-16. Methods The COX-2 AsODN was synthesized artificially, and various concentrations (50, 100, 200, 400 nmol/L) of the AsODN were transfected into Colo-16 cells with lipofectin followed by additional culture for different durations. The transfection results were observed with fluorescence microscopy. Subsequently, MTT assay,Western blotting and reverse transcription PCR were used to detect the cell proliferation, protein and mRNA expression of COX-2 in Coio-16 cells, respectively. Restults Compared with untreated cells, the proliferation of Colo-16 cells was inhibited significantly at 24, 48, 72 and 96 hours after transfection with different concentrations of COX-2 AsODN (all P < 0.05), and the COX-2 AsODN of 400 nmol/L exerted the highest inhibition rate of 60.3% at 48 hour. The average gray scale was 0.763±0.070, 0.600±0.065, 0.430±0.074 and 0.251±0.045 for COX-2 protein, 0.778±0.025, 0.602±0.041, 0.417±0.031 and 0.297±0.051 for COX-2 mRNA in Colo-16 cells transfected with COX-2 AsODN of 50, 100, 200, and 400 nmol/L respectively,significantly lower than that in untreated Colo-16 cells (all P < 0.05); there was a significant difference in the expression of COX-2 protein and mRNA among the cells transfected with the four concentrations of COX-2 AsODN and untreated cells (F = 83.54, 132.48, respectively, both P < 0.05). Conehtsions COX-2 AsODN can inhibit the proliferation, as well as the expression of COX-2 protein and mRNA in Colo-16 cells, which suggests that COX-2 AsODN has a potential therapeutic effect on skin squamous cell carcinoma.

Objective To investigate the effects of recombinant human pigment epithelium derived factor (rhPEDF)on in vitro proliferation of and expressions of interleukin 6(IL-6), IL-8 and vascular endothelial growth factor (VEGF)in cultured human HaCaT keratinocytes. Methods Some cultured HaCaT cells were treated with rhPEDF at various concentrations(25, 50, 100 μg/L)for different durations, and some treated with RPMI 1640 medium only served as the control group. Cell counting kit-8(CCK8)assay was performed to evaluate cell proliferation after 24-, 48- and 72-hour treatment, reverse transcription (RT)-PCR to measure the mRNA expressions of IL-6, IL-8 and VEGF in HaCaT cells after 24-hour treatment, and Western blot to detect the protein expressions of IL-6, IL-8 and VEGF in HaCaT cells after 48-hour treatment. Statistical analysis was carried out by two- and one-way analysis of variance, Student-Newman-Keuls(SNK)-q test and Pearson correlation analysis. Results After treatment with rhPEDF of 25-100 μg/L for 24 - 72 hours, the proliferation of HaCaT cells was significantly inhibited to different extents compared with the control group(all P < 0.05), and the inhibition rate significantly increased with the increase in treatment duration and concentrations of rhPEDF(F = 1115, 329.9, respectively, both P < 0.001). Moreover, there was a significant decrease in the expressions of IL-6, IL-8 and VEGF mRNAs(at 24 hours)and proteins(at 48 hours)in HaCaT cells after treatment with rhPEDF of 25 - 100 μg/L compared with the control group(all P < 0.05). The expression levels of VEGF mRNA as well as IL-6 and IL-8 proteins all significantly decreased with the increase of rhPEDF concentrations (all P < 0.05). The mRNA expressions of IL-6 and IL-8 were significantly lower in the 100-μg/L rhPEDF group than in the 25-μg/L rhPEDF group (both P < 0.05), and the protein expression of VEGF was significantly weaker in the 100-μg/L rhPEDF group than in 25-and 50-μg/L rhPEDF groups (both P < 0.05), but similar between the 25- and 50-μg/L rhPEDF groups (P > 0.05). Conclusions rhPEDF can inhibit the proliferation of HaCaT cells, and down-regulate the mRNA and protein expressions of IL-6, IL-8 and VEGF.

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 study the effects of naked DN A encoding vascular endothelial growth factor 165 (VEGF 165 )on cerebra l infarction in rats. Methods Following establishme nt of a permanent middle cerebral artery occlusion (MACO) model by nylon suture embolization in Wistar rats, the puCCAGGS/hVEGF 165 was directly injected i nto the ischemic tissues through skull hole. Seven days later, the rats were sac rificed. The infarct volume was measured by 2% TTC staining technique, then the expression of VEGF 165 gene and vascular proliferation were measured by use of RT-PCR and immumohistochemistry methods. Results Expression of VEGF 165 mRNA and VEGF protein in the therapy group increas ed. Compared with the control group, the number of vessels of the therapy group was significantly higher (50.76/HPF vs 40.67/HPF)( P

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 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.