Animals ; Asthma ; drug therapy ; immunology ; metabolism ; physiopathology ; Child, Preschool ; Humans ; Infant ; Leukotrienes ; immunology ; metabolism ; pharmacology ; Respiratory Sounds ; drug effects ; immunology ; physiopathology

Objective To obtain a lasting and purified cell line of vascular smooth muscle cells (VSMCs) by primary culture, and to provide these test materials related research. Methods The primary and transfer culture was done by tissue - piece inoculationand trypsin digestion,respectively. The cells were frozen in liquid nitrogen,and cultured cells were identified by phase contrast microscopy and immunohistochemical SP kit. Results 85% of inoculated tissue pieces survived. More than 90% of VSMCs regrew in transfered culture. The frozen cells could recover their proliferation by several culture transfers. The VSMCs showed the typical "peak and valley" characteristics under microscopy. Immunohistochemical staining with antibody against SM - a - actin expression in these cells was positive. Conclusion The tissue - piece inoculation is a simple method for obtaining satisfactory VSMCs in short term.

Apolipoprotein A5(ApoA5)level and other indices were determined in patients with type 2 diabetes mellitus and healthy individuals.Compared to control group,ApoA5 level in the diabetic group was lower (P

Objective To discuss the protective effect of bone marrow mesenchymal stem cell(BMSC)on lung injury induced by vibrio vulnificus sepsis and its mechanism. Methods BMSCs were isolated by whole bone marrow adherent culture from mouse. Male ICR mice were randomly divided into normal saline control group(NS group),normal saline+BMSC control group(NSB group),vibrio vulnificus sepsis group(VV group),vibrio vulnificus sepsis + BMSC group(VVB group)according to random number table,with 40 mice in each group. Sepsis mouse model was reproduced by injecting vibrio vulnificus(1×107 cfu/mL)5 mL/kg through the left side peritoneal cavity, and caudal intravenous injection of BMSC(4×105 cfu/mL)5 mL/kg for intervention after model reproduction. Ten mice in each group were sacrificed at 6,12,24 or 48 hours after injecting vibiro vulnificus,and their lung tissues were harvested. The lung wet/dry(W/D)ratio was calculated. The expression of nuclear factor-κBp65(NF-κBp65)in nucleus was measured by Western Blot. The levels of tumor necrosis factor-α(TNF-α)and interleukins(IL-1β, IL-6)in lung tissue were detected by enzyme-linked immunosorbent assay(ELISA). The pathological changes in lung tissue were observed after hematoxylin-eosin(HE)staining and uranyl acetate-lead citrate staining. Results After vibrio vulnificus injection,lung W/D ratio,the expression of NF-κBp65 in nucleus,and the levels of TNF-α, IL-1β,IL-6 in the lung tissues were significantly increased in VV group compared with those in NS group at all the time points,and peaked at 12 hours. Compared with the VV group,the VVB group had significantly decreased levels of lung W/D ratio,NF-κBp65 expression,and the levels of TNF-α,IL-1β,IL-6,with significant differences at all the time points〔VV group vs. NS group at 12 hours:lung W/D ratio 7.22±0.03 vs. 5.21±0.02,NF-κBp65 expression (glay scale)1.86±0.74 vs. 0.75±0.07,TNF-α(ng/L)433.24±3.23 vs. 106.57±1.21,IL-1β(ng/L)35.64±0.15 vs. 10.64±0.48,IL-6(ng/L)58.84±0.55 vs. 17.69±1.35,all P<0.05;VVB group vs. VV group at 12 hours:lung W/D ratio 6.49±0.06 vs. 7.22±0.03,NF-κBp65 expression(A value)1.16±0.08 vs. 1.86±0.74,TNF-α(ng/L)357.22±3.25 vs. 433.24±3.23,IL-1β(ng/L)27.77±0.59 vs. 35.64±0.15,IL-6(ng/L)38.68±1.29 vs. 58.84±0.55,all P<0.05〕. There were no significant differences in above indexes between NS group and NSB group. In the NS and NSB groups pathological changes were not obvious under light microscopy,in the VV group lung tissue hyperemia and edema was significant,the edema fluid,red blood cells and inflammatory cells also could be seen, and in the VVB group lung damage that mentioned above could be alleviated. In the NS and NSB groups epithelial cell structure of type Ⅰ and type Ⅱ was completed,and the changes were not obvious under the transmission electron microscopy. In the VV group the alveolar walls were damaged significantly,with type Ⅰ epithelial cell cytoplasm swelling,bubbling and rupture,with type Ⅱ epithelial cells visible cytoplasm decrease,cavitation,addiction to osmium lamellar corpuscle emptying,lysosome hyperplasia,microvilli reduction,and in the VVB group the above damage was alleviated. Conclusion Vibrio vulnificus sepsis can cause acute lung damage and edema,and BMSC can down regulate inflammatory cytokines,reduce lung injury caused by vibrio vulnificus sepsis.

OBJECTIVETo evaluate the relationship between leukotriene expression in blood polymorphonuclear leukocytes (PMNL) and the efficacy of inhaled corticosteroids (ICS) in children with asthma.

METHODSThirty-two children with asthma (5-12 years) and ten healthy children (control group) were enrolled. The asthmatic children were subdivided into ICS well-controlled and ICS poorly-controlled groups based on their clinical symptoms and lung function. The level of leukotriene C4 synthase (LTC4S) mRNA in PMNL was detected by fluorescence quantitative polymerase chain reaction. The level of LTC4S mRNA was expressed by the value of qCt, and the value of qCt was diversely correlated with the level of LTC4S mRNA expression. The concentration of urinary leukotriene E4 (LTE4) was measured using ELISA.

RESULTSThe expression of LTC4S mRNA in PMNL was significantly higher in children with asthma (qCt: 1.12+/-0.27) than that in the control group (qCt: 1.42+/-0.12; P< 0.05). The expression of LTC4S mRNA in PMNL in the ICS poorly-controlled group (qCt: 1.03+/-0.17) was significantly higher than that in the ICS well-controlled group (qCt: 1.24+/-0.33; P< 0.05) and the control group(1.42+/-0.12; P< 0.01). There was no significant difference in the level of urinary LTE4 among the the ICS poorly-controlled, the ICS well-controlled and the control groups.

CONCLUSIONSLTC4S mRNA expression in PMNL in asthmatic children increases, and the LTC4S mRNA expression in the ICS poorly-controlled group is higher than that in the ICS well-controlled group. This suggests that an increased leukotriene expression might be associated with poorly-controlled asthma.

Administration, Inhalation ; Adrenal Cortex Hormones ; administration & dosage ; Asthma ; drug therapy ; Child ; Child, Preschool ; Female ; Glutathione Transferase ; genetics ; Humans ; Leukotriene E4 ; urine ; Male ; RNA, Messenger ; blood

To investigate the effect of Yunkang Oral Liquid on preventing lipopolysaccharide(LPS)-induced abortion and regulating immune tolerance in mice,sixty normal ICR mice were randomly divided into normal group,model group,Yunkang Oral Liquid high,middle and low dose groups and progesterone group.Abortion model was established by tail vain injection of LPS(0.1μg/mouse)on the 7th day of pregnancy.Since the first day of pregnancy,the same volume of distilled water,Yunkang Oral Liquid at the dose of 36,18 and 9 m L·kg~(-1)·d~(-1),and progesterone at the dose of 0.038 g·kg~(-1)·d~(-1)were given in corresponding groups.The mice were sacrificed at the 9th day of pregnancy,and the embryo loss of each group was calculated.The levels of Th1 type cytokines(TNF-α,IFN-γ)and Th2 type cytokines(IL-4,IL-10)in uterus homogenate were detected by ELISA.HE staining was performed to examine the histopathological changes in the decidua.The expression levels of CD14,F4/80 in macrophages of uterus were detected by immunohistochemistry.Western blot was used to investigate the protein expression of TLR4,MyD88 and NF-κB in uterine decidua.In our study,all Yunkang Oral Liquid groups could significantly reduce the embryo absorption rate of mice,while high dose group can significantly increase the levels of IL-10 and IL-4;both medium and high dose groups can significantly decrease TNF-α,and IFN-γlevelsin the uterus of model mice,reduce the protein expression of NF-κB,MyD88 and TLR4 in uterine decidua tissue.Various treatment groups could reduce the counts of F4/80,CD14 macrophages and decrease expression area in uterine tissue.Our results showed that Yunkang Oral Liquid could prevent LPS-induced abortion,and the mechanism may be associated with inhibiting the TLR4/MyD88/NF-κB signaling pathway and regulating the balance of Th1/Th2 immune factors,which could improve the endometrial receptivity of mice,and promote the development of decidua and implantation of embryo.
Abortion, Induced ; Animals ; Female ; Immune Tolerance ; Lipopolysaccharides ; Mice ; Mice, Inbred ICR ; NF-kappa B ; Pregnancy

OBJECTIVETo study the solubility peak and dielectric requirement of the Buyang Huanwu docoction materials, and provide theoretical and experimental foundation for selecting extraction solvent for extracting traditional Chinese drugs (TCD).

METHOD11 types of solvents were employed as single or complex solvent systems, whose solubility parameter and dielectric constant were from 14.11 to 47.86, dielectric requirement from 1 to 80 respectively, to lixiviate Buyang Huanwu decoction (5 g per samples) in nearly saturate volume as V0 for materials at 25 degrees C. The apparent solubilities of extracts were determined and calculated out according to the section of determination of extract in the appendix of 'Chinese Pharmacopoeia'.

RESULTThe saturate solvent V0 for materials powder were 0.21, 0.31, 0.49, 0.36, 0.77, 0.93, 0.86, 0.92, 1.08, 1.00, 1.14 mL x g(-1), respectively. The apparent solubility of Buyang Huanwu docoction for each solvent system were 114.0, 101.3, 73.40, 109.4, 210.7, 295.0, 501.4, 437.0, 355.6, 423.1, 210.6 g x mL(-1), respectively, among which the max apparent solubility, illustrated as solubility peak, was carried out by methanol-water (68: 32) with 47.5 corresponding to the Buyang Huanwu docoction dielectric requirement.

CONCLUSIONThe apparent solubilities of (TCD) and their formula are controlled by dielectric constant of extraction solvent, and are in accordance with stable dielectric requirement.

Drugs, Chinese Herbal ; chemistry ; Solubility ; Solvents ; chemistry

OBJECTIVETo study the association of human epidermal growth factor receptor family molecules expression in gastric cancer tissues with the prognosis of patients with gastric cancer.

METHODSClinical data of 161 patients with gastric cancer undergoing gastrectomy in Jiangsu Cancer Hospital between January 2006 and January 2007 were analyzed retrospectively. The expression of HER1, HER2, HER3 and HER4 was detected by immunohistochemistry. Association of the expression of HER family with the prognosis of patients was examined. Kaplan-Meier method was used to analyze the survival.

RESULTSHigh expression rates of HER1, HER2, HER3 and HER4 were 46.0% (74/161), 10.6% (17/161),55.9% (90/161) and 68.3% (110/161) respectively. Univariate analysis revealed that high expression of HER3 was associated with tumor invasion depth, lymph node metastasis, stage, neurovascular invasion, and overall 4-year survival. High expression of HER4 was associated with tumor distant metastasis and stage. High co-expression of HER2 and HER3 was associated with overall 4-year survival (P=0.023). Multivariate analysis revealed that high expression of HER3 and stage were prognostic independent factors.

CONCLUSIONUp-regulated expression of HER3 is associated with the poor prognosis in gastric cancer patients.

Adult ; Aged ; Aged, 80 and over ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Male ; Middle Aged ; Prognosis ; Receptor, Epidermal Growth Factor ; metabolism ; Receptor, ErbB-2 ; metabolism ; Receptor, ErbB-3 ; metabolism ; Receptor, ErbB-4 ; Retrospective Studies ; Stomach Neoplasms ; metabolism ; pathology

OBJECTIVETo investigate the effect of hyperthermic intraoperative intraperitoneal chemotherapy(HIIC) and postoperative nutritional support on the intestinal permeability and the cellular immunity function in patients with advanced gastric cancer.

METHODSAll the patients diagnosed as advanced gastric cancer in the Gastric Tumor Diagnosis and Treatment Center of Jiangsu Cancer Hospital were randomly divided into three groups using random digit table:(1)EN group treated with enteral nutrition during postoperative period; (2)HIIC+EN group treated with HIIC combined with postoperative enteral nutrition;(3)HIIC+PN group treated with HIIC combined with postoperative parenteral nutrition. Index of lactulose/mannitol(L/M) ratio was used to evaluate the permeability of intestinal mucosa. The percentage of CD4(+), CD8(+) and NK cell, the ratio of CD4/CD8 T cell in peripheral blood were tested by flow cytometry. The time points of these measurements were the day before surgery, postoperative days (POD) 3, 7, and 12.

RESULTSCompared with the day before surgery(POD-1), the ratio of L/M on POD+3 increased significantly in all the three groups(0.1235±0.0611 vs. 0.0280±0.0183, 0.1648±0.0571 vs. 0.0305±0.0208, 0.1702±0.0628 vs. 0.0298±0.0229)(P<0.05) and then decreased gradually. The L/M ratio of EN(0.0278±0.0217) and HIIC+EN(0.0336±0.0235) groups recovered to the baseline on POD+12, however HIIC+PN group still had elevated L/M ratio(0.0616±0.0430). The percentage of CD4(+)T cell and the ratio of CD4/CD8 in HIIC+EN group and HIIC+PN group were significantly lower than those in EN group(P=0.033, P=0.002, respectively). Compared with the POD-1,the percentage of CD4(+)T cell and the ratio of CD4/CD8 in HIIC+EN group and EN group on POD+12 were increased significantly(P<0.05).

CONCLUSIONSHIIC may cause significant increase in intestinal permeability and inhibit cellular immunity in patients undergoing radical resection for advanced gastric cancer. Mucosal permeability can be reversed by enteral nutrition.

Abdominal Cavity ; Adult ; Aged ; Chemotherapy, Cancer, Regional Perfusion ; Female ; Humans ; Immunity, Cellular ; Intestinal Mucosa ; immunology ; physiopathology ; Intraoperative Care ; Male ; Middle Aged ; Nutritional Support ; methods ; Permeability ; Postoperative Period ; Stomach Neoplasms ; immunology ; physiopathology ; therapy

The argE gene from Escherichia coli coding for N-acety-L-ornithine deacetylase(NAOase), the key enzyme involved in the L-arginine biosynthesis, had been cloned in pET22b and transformed into BL21 (DE3). With 32.5% expression level in the optimal fermentation medium at 37 degrees C, most NAOase was expressed as inclusion bodies. The soluble and active proportion could be slightly increased when expressed at low temperature. The specific activity of soluble NAOase purified by Ni-NTA resin chromatography was 1193.2u/mg. The species and proportions of whole cell proteins varied with induction conditions. The inclusion bodies expressed at 37 degrees C was more pure than 22 degrees C after gradient wash with urea. Inclusion bodies could be partly refolding and reactivated by dilution and dialysis. Low protein concentration and suitable rate of oxidant/reducing agents were important to renaturation. In the optimal conditions 17.78% of Urea-denatured NAOase could be refolding and reactivated by dilution. The purified fusion protein was obtained after wash, solubilization and Ni-NTA resin affinity chromatography purification of inclusion bodies.
Amidohydrolases ; chemistry ; genetics ; metabolism ; Biocatalysis ; drug effects ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; enzymology ; genetics ; Escherichia coli Proteins ; genetics ; metabolism ; Gene Expression Regulation, Bacterial ; Gene Expression Regulation, Enzymologic ; Inclusion Bodies ; enzymology ; Protein Folding ; drug effects ; Recombinant Proteins ; chemistry ; isolation & purification ; metabolism ; Urea ; pharmacology