With the development of functional genomics, high throughput analysis of genes’ function has been the mainstream of research, and exogenous gene's over expression via Agrobacterium-mediated transformation is the most commonly used method in gene functional analysis.The versatile plant expression vector cassette named pBHT-5 was constructed by the method of site-specific mutagenesis based on pBI121. First of all, the restriction enzyme SfiI recognition site in trfA gene (X00713) which was relevant to plasmid replication and stability was replaced without changing its amino acid composition. And then the SfiIA,SfiIB sites were added between promoter CaMV35s and terminator NOS. The versatile plant expression vector cassette can be directly used to construct plant expression vector containing the full-length genes cloned by Clontech SMARTTM technology, which will raise the efficiency of vector construction. The result will provide basis of new genes’ high throughput screening and functional analysis, then get the new genes functioning in plant osmotic stress resistance.

Objective To evaluate the efficacy of 10-day compound allantoin containing quadruple regimen in the treatment of chronic gastritis with Helicobacter pylori (H.pylori) infection,and to compare with the bismuth-containing quadruple therapy.Methods Altogether 173 patients with H.pylori positive chronic gastritis confirmed by gastric endoscope were divided into 10-day compound allantoin containing quadruple regimen group (n =43),24-day compound allantoin containing quadruple regimen group (n =46),10-day bismuth-containing quadruple regimen group (n =42) and 24-day bismuth-containing quadruple regimen group (n =42).After the treatment,the eradication rate of H.pylori,the rate of gastrointestinal symptoms (epigastric pain,bloating and belching) relief and the adverse effects of each group were observed.Intention-to-treat (ITT),per-protocol (PP) statistical analysis and chi-square analysis were performed for statistical analysis.Results H.pylori eradication rates of 10-day compound allantoin containing quadruple regimen group,24-day compound allantoin containing quadruple regimen group,10-day bismuth-containing quadruple regimen group and 24-day bismuth-containing quadruple regimen group analyzed by ITT were 90.7％ (39/43),91.3％ (42/46),90.5％ (38/42) and 88.1％ (37/42),respectively; while analyzed by PP were 90.7％ (39/43),93.3％ (42/45),90.5％ (38/42) and 90.2％ (37/41),respectively.And there were no statistical differences between groups (all P＞0.05).Ten days after the treatment,the rates of epigastric pain relief of 24-day compound allantoin containing quadruple regimen group and 24-day bismuth-containing quadruple regimen group were 81.1％ (30/37) and 78.8％ (26/33),respectively,the rates of bloating relief were 82.4％ (28/34) and 71.0％ (22/31),respectively,and the rates of belching relief were 76.9％ (20/26) and 75.0％ (21/28),respectively.There were no statistical differences between the two groups (all P＞ 0.05).However after 24-day treatment,the rates of epigastric pain relief of 24-day compound allantoin containing quadruple regimen group and 24-day bismuth-containing quadruple regimen group were 91.9 ％ (34/37) and 87.9％ (29/33),respectively,the rates of bloating relief were 94.1％ (32/34) and 87.1％ (27/31),respectively,and the rates of belching relief were 96.2％ (25/26) and 85.7％ (24/28),respectively.There were no statistical differences between the two groups (all P＞0.05).And the rates of epigastric pain and bloating relief increased after 24-day treatment compared with those of 10-day treatment,however the differences were not statistically significant between the two groups (all P＞ 0.05).In 24-day compound allantoin containing quadruple regimen group,the rate of belching relief was higher after 24-day treatment compared with that of 10-day treatment,and the difference was statistically significant (x2=4.127,P=0.042).No severe adverse effects were observed in each group,and there were no adverse effects such as oral metal odor,tongue black and melena in compound allantoin containing quadruple therapy.Conclusions Ten-day compound allantoin containing quadruple therapy as first-line approach in the treatment of chronic gastritis with H.pylori infection can get better H.pylori eradication and the efficacy is similar to bismuth quadruple therapy.Meanwhile the symptom relief rate is high and no obvious adverse effects were found.

OBJECTIVETo investigate the factors associated with insulin resistance (IR) in patients with chronic hepatitis B virus (HBV) infection.

METHODSSixty-eight patients with mild chronic hepatitis B (MCHB) caused by HBV were recruited for study. Sixty-seven healthy individuals with no hepatitis virus infections and normal liver function were enrolled as controls. Demographic, anthropometric, clinical, and blood biochemical parameters were compared between the two groups. IR was determined by the homeostasis model assessment (HOMA-IR). The MCHB group was further divided into patients with IR (HOMA-IR: > 2.7) and patients without IR (HOMA-IR: less than 2.7). Demographic, anthropometric, clinical, and blood biochemical parameters were compared between the two sub-groups. Finally, the potential factors associated with IR were evaluated.

RESULTSCompared to the healthy controls, the MCHB patients had significantly higher serum insulin (Z = -5.451, P less than 0.01), alanine aminotransferase (ALT) (Z = -8.211, P less than 0.01) and HOMA-IR (Z = -5.631, P less than 0.01). IR was detected in 44.12% (30/68) of the MCHB patients. The levels of ALT and body mass index (BMI) were significantly different between the MCHB patients with IR and without IR (t = -2.358, and t = -3.566, P less than 0.05). There was a significant correlation between BMI, ALT, and HOMA-IR in the MCHB patients (r = 0.374, r = 0.282, P less than 0.05), but not with the HBV DNA loads (r = 0.015, P = 0.904). Binary logistic regression analysis indicated that BMI [Exp(B): 1.859, P less than 0.01] and ALT [Exp(B): 1.022, P less than 0.05] were independent risk factors of IR in MCHB.

CONCLUSIONThere is a high prevalence of insulin resistance in patients with mild hepatitis caused by chronic HBV infection. In these patients, IR is correlated with abnormal liver function and BMI, and not HBV load.

Adult ; Alanine Transaminase ; analysis ; Blood Glucose ; Body Mass Index ; Case-Control Studies ; Female ; Hepatitis B virus ; Hepatitis B, Chronic ; metabolism ; virology ; Humans ; Insulin ; blood ; Insulin Resistance ; Male ; Middle Aged ; Viral Load

Objective To investigate the expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) and its associated mechanism during the wound healing. Methods The animal model with the full-thickness skin injury was used in the study. Fifty male mice were involved in the study and divided randomly into control group (n = 25) and GM-CSF group (n = 25). Each group had five time points (5 mice per time point). All the mice received full-thickness skin defect (1 cm × 1 cm) through the panniculus camosus on the midline of the back near the neck after anesthesia. Recombinant human granulocyte-macrophage colony stimulating factor (RhGM-CSF) gel (10 μg/cm2) were applied in the GM-CSF group and gel matrix without RhGM-CSF applied in the control group. The wound healing time and rate were observed at days 3, 5, 7, 10 and 14 after injury. The wound specimens were collected to detect the histopathological change. The microvessel density of the wound was counted based on the results of CD31 immunohistochemistry. RT-PCR was employed to detect the expression changes of GMCSF, platelet-derived growth factor (PDGF) , vascular endothelial growth factor ( VEGF) and stromal cell derived factor-1 (SDF-1). Results RT-PCR results showed that the gene expression of GM-CSF reached the peak at day 3 after injury (P＜0. 01) and kept the high level at days 3-10 after injury (P＜ 0. 05) , followed by a sharp decrease to a normal level at day 14 after wound. The wound healing time was average (2.4 ±0. 3) days earlier than the control mice after application of rhGM-CSF, with significant increase of the wound healing rate during 7-14 days after injury ( P ＜ 0. 05 ). In the GM-CSF group, the early histology of trauma wound showed a small number of neutrophils, obvious epithelial cell proliferation in the wound margin, marked hyperplasia of the granulation tissue, high cell density with quantity of spindle-shaped and oval-shaped cells and increased number of new blood vessels. The microvessel density was also increased significantly (P ＜ 0. 05) at days 7-14 after injury. The gene expressions of VEGF and SDF-1 were significantly increased at day 7 and day 10 respectively after injury (P＜0.05) and the gene expression of pro-healing factor PDGF was significantly increased in every time point (at days 5, 7 and 10,P＜0.05;at day 14,P＜0.01). Conclusion GM-CSF expresses highly in the early stage after injury and can promote the wound healing, when the mechanism may relate to the up-regulated expressions of pro-angiogenic factors and pro-healing growth factors.

Objective To investigate the clinical cross infections of myeoplasma pneumoniae(MP)and other viruses in children,providing a reference for the diagnosis and treatment of respiratory disease.Methods Serum specimens of the children hospitalized with fever,respiratory symptom besides positive results of MP-Ab IgM detection were collected.And several common viruses popular in children were investigated within the specimens collected by ELISA kits or indirect immunofluorescence.Results (1)The PCT levels of 385 cases(81.7%)appear to be under 0.5 ng/ml.(2)In the 514 cases detected for Cox-IgG and Cox-IgM,the positive rates are respectively 40.3% and 35.6%.(3)2 cases(0.8%)appear to be irdluenza B virus positive.And the positive rates of parainfluenza virusl,2 and 3 are 0.8%,0,and 9%.4,84 cases(11.8%)are positive for EB-IgM and 451 cages(63.6%)positive for EB-IgG.Conclusion Cross infections rarely occur between MP and common respiratory viruses in Children.The cross-infection rate between Cox-virus and MP iS up to 35.6%.

Objective To study the B lymphocytes counts and the expression of TLR9 mRNA on B lymphocytes in peripheral blood from Chinese HIV-infected patients.Methods The cells from peripheral blood were stained with antibodies labeled with fluorescence and B lymphocytes were counted with flow cytometry.Using the method of magnetic activated cell sorting and real-time PCR,the expression of TLR9 mRNA was measured.Results The B lymphocytes counts in HIV/AIDS patients was significantly lower than that of healthy controls(P ＜0.01).The B lymphocytes counts in HIV/AIDS patients positively correlated with the CD4 +T cells counts(r =0.534,P = 0.006).The expression of TLR9 mRNA on B lymphocytes in HIV/AIDS patients was significantly lower than that of healthy controls(P =0.023),and positively correlated with the CD4 + T cells counts(r = 0.390,P = 0.040).Conclusion The B lymphocytes counts and the expression of TLR9 mRNA on B lymphocytes in Chinese HIV/AIDS patients were decreased due to HIV infection,which may correlate with disease progression.

To investigate the effect of HBV on the expression of Sterol regulatory element binding proteins( SREBP ) in the hepatocyte of patients with chronic hepatitis B (CHB) combined with hepatic fatty change. 55 cases diagnosed as CHB combined with hepatic fatty change in our department were selected and liver biopsies were carried out. The patients were dividied into 3 groups, group A: HBV DNA is less than or equal to 1000 copies/ml(15 cases), group B: 1000 copies/ml less than HBV DNA less than 100000 copies/ml (18 cases) and group C: HBV DNA is more than or equal to 100000 copies/ml (22 cases). 10 patients with HBV DNA in less than or equal to 1000 copies/ml after antiviral therapy with Nucleoside analogues were seen as group C1 (before treatment) and group C2 (after treatment) respectively; 12 patients with HBV DNA is more than or equal to 100000 copies/ml after antiviral therapy were classified as group C3 (before treatment) and group C4 (after treatment). Lipid droplets in the hepatic tissue were observed with oil red staining. Real time PCR were performed to detect the expressions of SREBP-1c and SREBP-2 mRNA in the liver. The protein expressions of SREBP-1c and SREBP-2 were detected with immunohistochemistry staining. Statistic data were analysed with SPSS11.5 software. (1) Red integrated optical densities (IOD) reflected by lipid drops in group A, B and C are 1004.27+/-218.63, 1937.01+/-401.47 and 4133.79+/-389.28 respectively, the degree of oil red O in each group was different (F = 385.69, P is less than to 0.01), which is increased as HBV DNA load increasing; Red IOD in group C1, C2 and C3, C4 are 4020.84+/-326.64, 1012.02+/-244.89, 4189.18+/-329.21 and 4121.76+/-304.09 respectively. Compared with group C1, the degree of oil red O in group C2 is decreased and the difference is statistically significant (t = 22.55, P is less than to 0.01); However, the difference of the degree of oil red O between group C4 and C3 is not statistically significant. (2) Compared with group A, the expressions of SREBP-1c mRNA in group B and C are raised by 1.218+/-0.130 and 1.798+/-0.118 times respectively, among group A, B, C, the expressions of SREBP-1c mRNA are statistically significant different ( F = 297.47, P is less than to 0.01). The expressions of SREBP-2 mRNA in group B and C are decreased by 0.956+/-0.118 and 0.972+/-0.153 times as compared to group A. However, the difference of SREBP-2 mRNA expression among the 3 groups is not statistically significant ( F = 0.568, P is more than to 0.05). Compared with group C1, SREBP-1c mRNA in group C2 is decreased by 0.714+/-0.081 folds (t=11.224, P is less than to 0.01), while SREBP-2 mRNA in group C2 is raised by1.034+/-0.155 times(t=0.692, P is more than to 0.05). SREBP-1c mRNA and SREBP-2 mRNA in group C4 are raised by 1.012+/-0.206 times and decreased by 0.998+/-0.183 times as compared to group C3 without difference found (t=0.196 or 0.031, P is more than to 0.05). (3) the expressions of SREBP-1c protein in group A, B and C are 36257.21+/-5709.79, 50413.47+/-4989.28 and 71025.83+/-6047.13 respectively, and the difference is statistically significant among the 3 groups (F = 178.26, P is less than to 0.01); the expressions of SREBP-2 protein in group A, B and C are 32913.52+/-3951.21, 32625.91+/-4025.06 and 34173.44+/-5316.25 respectively, but the difference is not statistically significant among the 3 groups ( F = 0.562, P is more than to 0.05), SREBP-1c protein levels in group C1, C2, C3, C4 are 69832.16+/-4941.36, 48735.47+/-5471.41, 70871.69+/-5083.14 and 68913.32+/-5343.22 respectively, the difference of SREBP-1c protein levels between group C1 and C2 is statistically significant (t=10.260, P is less than to 0.01); while the difference between group C3 and group C4 is not statistically significant(t=1.558, P is more than to 0.05). The expressions of SREBP-2 protein in group C1, C2, C3 and C4 are 33 980.21+/-4081.80, 34011.50+/-3859.27, 33610.12+/-4761.10 and 32915.66+/-5023.61 respectively, the difference of SREBP-2 protein levels in group C1 and group C2 is not statistically significant (t=0.038, P is more than to 0.05) and same result exists between group C3 and group C4 (t=0.459, P is more than to 0.05). HBV DNA may participate in the hepatic steatosis formation through interfering with the SREBP-1c expression.
Fatty Liver ; metabolism ; Hepatitis B, Chronic ; metabolism ; Hepatocytes ; metabolism ; Humans ; Sterol Regulatory Element Binding Protein 1 ; metabolism

OBJECTIVETo explore the mechanism of acupuncture for treatment of perimenopausal syndrome.

METHODSThe rats of perimenopausal syndrome were randomly divided into 3 groups, including an acupuncture group treated with acupuncture, a medication group with Gengnian'an, and a perimenopausal control group, with young rats used for a control group. Granulocyte apoptosis and expressions of Bcl-2 and Fas proteins in the ovary of the rat were detected.

RESULTSGranulocyte apoptosis increased significantly (P < 0.01), expression of Bl-2 proteins decreased significantly (P < 0.01) and expression of Fas proteins increased significantly (P < 0.01) in the ovary of perimenopausal rats as compared with the young rats; after acupuncture treatment, granulocyte apoptosis decreased significantly (P < 0.05), expression of Bel-2 proteins increased significantly (P < 0.05) and expression of Fas proteins decreased significantly (P < 0.01); after treatment of Gengnian'an, granulocyte apoptosis did not significantly change (P > 0.05), expression of Bcl-2 prteins increased significantly (P < 0.05) and expression of Fas proteins decreased significantly (P < 0.01).

CONCLUSIONAcupuncture can inhibit granulocyte apoptosis, up-regulate expression of Bcl-2 proteins and down-regulate expression of Fas proteins in the ovary of the perimenopausal rat.

Acupuncture Therapy ; Animals ; Apoptosis ; Female ; Granulocytes ; pathology ; Ovary ; metabolism ; pathology ; Perimenopause ; Proto-Oncogene Proteins c-bcl-2 ; analysis ; Rats ; Rats, Sprague-Dawley ; fas Receptor ; analysis

OBJECTIVETo observe the role of tumor necrosis factor α (TNF-α) in endothelial-mesenchymal transition (EnMT), and to explore the mechanism of fibrosis disease.

METHODSHuman umbilical vein endothelial cells (HUVEC) from umbilical cord of healthy fetus were isolated by enzymatic digestion and identified by immunofluorescence assay. The third to fifth generations of cultured HUVEC in logarithmic phase were harvested and seeded in 12-well plates and 6-well plates, and they were divided into control group (ordinary culture without any stimulation), 5, 10, 25, 50, and 100 ng/mL TNF-α groups (5, 10, 25, 50, 100 ng/mL of TNF-α was respectively added into the nutrient solution) according to the random number table, with three samples in each group. After being cultured for 72 hours, the cell morphology was observed under inverted phase-contrast microscope; the expression levels of coagulation factor VIII and α smooth muscle actin (α-SMA) were detected by immunofluorescence assay, and the ratios of numbers (absorbance values) of cells with expression of both factors were calculated. The mRNA expression levels of cadherin, α-SMA, and type I collagen were detected by RT-PCR (denoted as gray value ratio). Data were processed with one-way analysis of variance and LSD test.

RESULTS(1) The shape of primary HUVEC was round, short-spindle, or flat, and cells grew vigorously in cobblestone appearance after passages. After being subcultured for 1, 2, 3, 4, 5 passage (s), the positive rate of coagulation factor VIII of HUVEC was respectively (85.5 ± 1.8)%, (88.1 ± 5.0)%, (93.6 ± 3.7)%, (92.9 ± 4.8)%, (89.5 ± 1.1)%, and they were significantly higher than that of primary HUVEC [(81.4 ± 3.8)%, with F values all equal to 7.481, P values all below 0.05]. (2) As compared with that in control group, the appearance of cells in 5, 10, 25, 50, and 100 ng/mL TNF-α groups was gradually transformed from round, short-spindle, or flat shape to long-spindle shape with reduced intercellular junction and larger intercellular gap along with the increase in the concentration of TNF-α. (3) The ratios of numbers and the absorbance values of coagulation factor VIII and α-SMA double positive cells in control group (0.055 ± 0.015, 0.078 ± 0.017) were significantly lower than those in 5, 10, 25, 50, and 100 ng/mL TNF-α groups (0.257 ± 0.106, 0.280 ± 0.129, 0.505 ± 0.059, 0.817 ± 0.035, 0.929 ± 0.101 and 0.437 ± 0.040, 0.456 ± 0.097, 0.496 ± 0.082, 0.787 ± 0.131, 0.885 ± 0.087, with F value respectively 45.009, 50.099, P values all below 0.01). (4) The expression levels of cadherin mRNA in 5, 10, 25, 50, and 100 ng/mL TNF-α groups were 0.70 ± 0.05, 0.63 ± 0.06, 0.60 ± 0.10, 0.45 ± 0.16, and 0.26 ± 0.14, and it was significantly lower in the latter four groups than in control group (0.83 ± 0.03, with F values all equal to 11.593, P < 0.05 or P < 0.01). The mRNA expression levels of α-SMA and collagen I in 5, 10, 25, 50, and 100 ng/mL TNF-α groups were 0.45 ± 0.10, 0.51 ± 0.16, 0.49 ± 0.12, 0.60 ± 0.09, 0.76 ± 0.03 and 0.38 ± 0.18, 0.45 ± 0.15, 0.52 ± 0.12, 0.66 ± 0.17, 0.76 ± 0.20, and they were significantly higher in the latter three groups than in control group (0.37 ± 0.14, 0.31 ± 0.12, with F value respectively 7.839, 2.898, P < 0.05 or P < 0.01).

CONCLUSIONSTNF-α can obviously promote EnMT in a dose-dependent manner. EnMT may be another significant source of myofibroblasts that contributes to fibrotic tissue in scar formation.

Cell Differentiation ; Cells, Cultured ; Epithelial-Mesenchymal Transition ; drug effects ; Human Umbilical Vein Endothelial Cells ; cytology ; Humans ; Stromal Cells ; cytology ; Tumor Necrosis Factor-alpha ; pharmacology

OBJECTIVEThe aim of the study was to investigate the positivity of human papillomavirus (HPV) and the possible related risk factors for HPV infection in certain district government in Daqing city, Heilongjiang province.

METHODSA total of 2015 female staffs who participated cervical cancer screening were selected as subjects, in certain district government in Daqing city, Heilongjiang province, from June to October, 2010. First of all, a standardized questionnaire was used for collection of subject's demographic information and possible risk factors. Afterwards, cervical cytological and HPV DNA testing were applied to all participants. Subjects with suspected cervical intraepithelial neoplasia (CIN) or cervical cancer were recalled for taking biopsy under colposcopy for further histopathological diagnosis. Standardized HPV positivity by Segi's world population and compared the difference of HPV positivity of different types. The positivity of HPV between women with and without cervical abnormalities were compared by unconditional logistic regression. And the possible risk factors for HPV infection were also investigated.

RESULTSA total of 1759 out of 2015 subjects had gynecological examination, among which 152 subjects were HPV positive. The positivity of HPV was 8.64% (95%CI: 7.37% - 10.05%), and it was 8.47% (95%CI: 7.93% - 9.03%) after age standardization. Finally, 57 (3.24%) and 1702 (96.76%) subjects had abnormal (≥ CIN1 or low-grade squamous intraepithelial lesion (LSIL)) and normal cervix, respectively. The HPV positivity between women with and without cervical abnormalities were 73.68% (42/57) and 6.46% (110/1702), respectively. There was a significant difference (χ(2) = 307.23, P < 0.05) in HPV positivity between women with and without cervical abnormalities. The risk of cervical abnormalities for women with HPV positivity was 40.52 times (95%CI: 21.79 - 75.36) higher than that for women with HPV negativity. Among women with cervical abnormalities, the most three common HPV types, in descending order, were HPV16 (28.07% (16/57)), HPV52 (14.04% (8/57)) and HPV58 (12.28% (7/57)). While among women with normal cervix, the most three common HPV types were HPV52 (1.23% (21/1702)), HPV16 (1.00% (17/1702)) and HPV58 (0.71% (12/1702)). The positivity of HPV clade A9 among women with and without cervical abnormalities were 59.65% (34/57) and 3.23% (55/1702), which were higher than that of other clades. Analysis for risk factors of HPV infection showed that smoking (OR = 2.71, 95%CI: 1.00 - 7.33), late age (≥ 15 years old) of menarche (OR = 1.44, 95%CI: 1.00 - 2.05), early age (≤ 20 years old) of marriage (OR = 3.09, 95%CI: 1.30 - 7.35), multiple (≥ 2) sexual partners (OR = 2.69, 95%CI: 1.46 - 4.95), husband's extramarital sexual behaviors (OR = 2.77, 95%CI: 1.25 - 6.12) and multiple (≥ 2 times) parity (OR = 1.77, 95%CI: 1.03 - 3.03) would increase the risk of HPV positivity.

CONCLUSIONHPV positivity among women with cervical abnormalities was significantly higher than that among women with normal cervix. HPV16, 52, 58 were the major genotypes among the study population. Smoking, late age of menarche, early age of marriage, multiple sexual partners, husband extramarital sexual behaviors and multiple parity increase the risk of HPV infection.

Adult ; Aged ; Case-Control Studies ; Cervical Intraepithelial Neoplasia ; epidemiology ; virology ; Cervix Uteri ; pathology ; virology ; China ; epidemiology ; DNA, Viral ; isolation & purification ; Early Detection of Cancer ; Female ; Humans ; Middle Aged ; Papillomaviridae ; isolation & purification ; Papillomavirus Infections ; epidemiology ; virology ; Risk Factors ; Young Adult