Objective To explore the cell frequency , phenotypes and in vitro cytotoxic effects of circulating CD56+T cells in the patients with chronic HCV infection .Methods Peripheral blood mononu-clear cells (PBMCs) were isolated from 33 patients with HCV chronic infection and 21 healthy subjects. Multi-color flow cytometry was used to analyze cell frequency , expressions of activating receptors ( NKG2C, CD16 and NKp46) and inhibitory receptors (NKG2A and CD158a) on CD56+T cells.The functional mark-er for cytotoxic effects (CD107a) on circulating CD56+T cells and their cytokines expression (IFN-γand TNF-α) with or without stimulation of K 562 human Leukemia cell line were also analyzed .Then the correla-tions among the expressing levels of CD 107 a, IFN-γand TNF-αwere investigated .Results The frequency of CD56+T cells in periphery lymphocytes were significantly decreased in the patients with chronic HCV in -fection as compared with that in healthy controls ( P=0.018 ).The expressions of activating receptors (NKG2C, CD16 and NKp46) on CD56+T cells from HCV infected patients were decreased (P=0.015 for NKG2C, P=0.036 for CD16 and P=0.001 for NKp46), while there was no significant change in the ex-pressions of inhibitory receptors (P>0.05 for both CD158a and NKG2A).The concentrations of IFN-γand TNF-αsecreted by CD56+T cells in the patients with chronic HCV infection were significantly decreased with or without K562 stimulation (P<0.0001).However, in the presence of K562 cells CD107a expression on CD56+T cells were sharply decreased in the patients (P<0.0001).In absence of K562 cells, there was no significant change in CD107a expression on CD56+T cells from patients and healthy controls (P>0.05). The expressions of CD107a, IFN-γand TNF-αwere closely related under the stimulation of K562(r>0.80, P<0.0001).Conclusion The frequency of CD56+T cells was reduced in patients with chronic HCV infec-tion.Moreover, cytotoxic effects and cytokines production mediated by CD 56+T cells were also significantly impaired, indicating that the dysfunction of circulating CD 56+T cells might be associated with the persist-ence of chronic HCV infection .

OBJECTIVETo study the effect of osthole (Ost) on adrenocortical function in Y1 mouse adrenocortical tumor cells.

METHODSY1 mouse adrenocortical tumor cells were taken as subjects in this experiment. In 10.0%, 1.0%, and 0.1% serum DMEM-F12 medium, Y1 cells were treated with 1, 10, 25, 50, 100, and 200 micromol/L Ost for 24 and 48 h. 0.1% Dimethyl Sulfoxide (DMSO) was taken as negative control group and 1 mmol/L (Bu) 2cAMP as positive control group. Cell growth morphology was observed under inverted microscope. Contents of corticosterone were tested by ELISA. Expression levels of steroids synthase such as Star, Cyp11a1, Cyp21a1, Hsd3b2, Cyp11b1, Cyp11b2, Cyp17a1, and Hsd17b3 mRNA were detected by Real time quantitative PCR (RT-qPCR).

RESULTSY1 cell proliferation was obviously inhibited by 100 and 200 micromol/L Ost, and its inhibitory effect was more significant in 0.1% serum medium. Compared with the negative control group, gene expressions of Star, Cyp11a1 , Cyp21a1, Hsd3b2, Cyp11b1, Cyp17a1, and Hsd17b3 were significantly enhanced in the posi- tive control group (P < 0.05). Y1 cell corticosterone levels significantly increased in 50 micromol/L Ost treatment group after 24-and 48-h intervention (P < 0.05). Contents of corticosterone increased more obviously in 25 and 50 +/- mol/L Ost treatment groups after 48-h intervention, as compared with 24-h intervention (P < 0.01). After 24-h intervention, expression levels of Star, Cyp21a1, and Hsd3b2 genes were significantly up-regulated in 25 and 50 lLmol/L Ost groups (P < 0.05). Star gene expression was further enhanced after 48-h intervention (P < 0.05). However, Ost showed no effect on Cyp11a1 (P > 0.05). Additionally, gene expressions of Cyp11b1 and Cyp17a1 were significantly enhanced by 10, 25, and 50 pLmolIL Ost after treatment for 24 and 48 h (P < 0.05). Ost showed no obvious effect on Cyp11b2 and Hsd17b3 expressions.

CONCLUSIONOst could regulate adrenal cortex function and promote corticosterone synthesis and secretion through strengthening gene expressions of steroidogenic enzymes.

Adrenal Cortex ; drug effects ; Adrenal Cortex Neoplasms ; pathology ; Animals ; Corticosterone ; biosynthesis ; Coumarins ; pharmacology ; Gene Expression ; Mice ; RNA, Messenger ; metabolism ; Tumor Cells, Cultured

Arachidonic acid is an essential fatty acid in human nutrition and a biogenetic precursor of the biologically active prostaglandins and leukotrienes. ?~5 desaturase catalyzes the ?~5 dehydrogenation of di-homo-?-linolenic acid to form arachidonic acid in biosynthetic pathway of arachidonic acid. Using real time PCR technology,the transcriptional expression levels of gene encoding ?5 desaturase in three Mortierella alpina strains M10,M6 and M23,and in different growth phase of high arachidonic acid yielding strain M6,were determined. Results showed that there was a distinct corelationship between mRNA transcript level of ?~5 desaturase gene and biosynthesis of arachidonic acid. Results indicated that ?~5 desaturase plays an important role in arachidonic acid biosynthesis.

Adult ; Anti-HIV Agents ; therapeutic use ; Familial Primary Pulmonary Hypertension ; HIV ; pathogenicity ; Hepacivirus ; pathogenicity ; Hepatitis B virus ; pathogenicity ; Humans ; Hypertension, Pulmonary ; drug therapy ; virology ; Male ; Sulfonamides ; therapeutic use

Objective To detect the mutations of ATP2C1 gene in patients with Hailey-Hailey dis- ease (HHD).Methods PCR and direct sequencing were performed in 17 patients and 120 healthy controls to screen the mutations in the exons of ATP2C1 gene.Results Eight mutations were identified in nine probands, including three deletion mutations (nt1464-1487 del/nt1462-1485del,1523delAT,2375delTTGT),three splice site mutations (360—2A→G,1415—2A→T,2243+2T→C) and two missence mutations (C920T and G1942T).None of the above mutations was found in the controls.Conclusion Eight specific novel mutations were identified in nine probands of HHD,which could be causative factors of the disease.

Objective To study the sources and the relationship between the management and the outcome of hemorrhage after cephalic pancreatoduodenectomy.Methods The clinical data of 370 patients who underwent pancreatic resection at the Lihuili Hospital and the Second Affiliated Hospital of Zhejiang University were retrospectively analyzed.Results Postoperative bleeding occurred in 35 patients with 11 deaths.Among those intraabominal bleeding occurred in 14 cases and gastrointestinal hemorrhage occurred in 22,with one case suffering from both.Bleediug developing within 72 hours after operation in 12 cases (early-stage group),which was caused by improper intraoperative homeostasis.In other 23 cases,bleeding 72 hours after operation(later stage group)was caused by the erosion following pancreatic and/or bile leakage.Relaparotomy was performed in 13 cases and endoscopic homeostasis was performed in 3. Relaparotomy or endoscopic homeostasis was superior to that of conservative therapy in the early-stage group (P0.05).Pancreatic or bile leakage was identified as the significant risk factors for the postoperative bleeding.Conclusions In order to prevent the postoperative hemorrhage and to reduce the mortality of pancreatic resection,skillful techniques,expeditious homeostasis,proper management of stump pancreas and the prevention of pancreatic and bile leakage are essential.

Based on the interaction between supramolecule of traditional Chinese medicine and enterobacteria, the material basis of Rhei Radix et Rhizoma and Coptidis Rhizoma was explored. Scanning electron microscopy (SEM) and dynamic light scattering (DLS) were used to characterize the morphological differences of Rhubarb single decoction, Coptis single decoction and Rhubarb and Coptis co-decoction. An in vitro antibacterial model (E. coli, E. faecium and B. subtilis) was established to evaluate the damage effect of the combination of Rhei Radix et Rhizoma and Coptidis Rhizoma on enterobacteria. Ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to analyze the changes of chemical components of single decoctions and co-decoctions. The co-decoction of Rhei Radix et Rhizoma and Coptidis Rhizoma was turbid after decocting. The spherical particles of 300-400 nm were observed under SEM, and the co-decoction was more uniform and stable than that of single decoction. The interaction between supramolecules formed after the combination of Rhei Radix et Rhizoma and Coptidis Rhizoma and enterobacteria was significantly different from that of single decoction. In the process of interaction between supramolecules and enterobacteria, the spherical state was maintained, and the medicinal ingredients in Coptidis Rhizoma or Rhei Radix et Rhizoma were blocked, which could effectively alleviate the damage to enterobacteria. This study provided a reference for subsequent studies on the regulation of intestinal flora homeostasis by the combination of Rhei Radix et Rhizoma and Coptidis Rhizoma.

Objective To construct RhoA siRNA plasmid expression vector.Methods According to the computer aided design,RhoA-specific siRNA gene was synthesized and cloned into the RNAi-Ready Pgenesil-1 Vector.The constructed RhoA-RNAi plasmid were transfected into human HEPG2 cell.Western blot was used to detect the effect of RhoA-RNAi plasmid.Results The recombinant was cloned and the se- quence was obtained.RhoA-RNAi plasmid can down-regulate the expression of RhoA in human hepatocel- lular carcinoma cell line HEPG2.Conclusion Successfully cloning the recombinant makes it possible for searching new mechanism of RhoA in hepatocellular carcinoma.

Animals ; Cadherins ; metabolism ; Catenins ; metabolism ; Estradiol ; pharmacology ; Estrogen Receptor Modulators ; pharmacology ; Female ; Ovariectomy ; Rats ; Uterus ; drug effects ; metabolism

OBJECTIVETo extract corosolic acid from loquat leaves for medical use.

METHODSLoquat leaves were boiled in water to remove the water-soluble substances followed by 3 cycles of extraction with 25% aqueous methanol for 30 min and then by 95% aqueous methanol for 1 h at 80 degrees celsius;. After cooling at room temperature and filtration, the extract was treated with activated carbon to remove chlorophyll, and the liquid was filtered and concentrated to allow precipitation. The sediment was washed to obtain the total crude triterpene acid, which was further dissolved with methanol and purified by high-performance liquid chromatography (HPLC). The fractions including corosolic acid were collected, concentrated with vacuum distillation, and dried to obtain corosolic acid product, which was analyzed with HPLC.

RESULTSHPLC analysis of the extracts showed that the percentages of corosolic acid were 4.66%, 2.42%, and 24.18% in crude corosolic acid extracted with methanol, boiling water, and 95% aqueous methanol, respectively. After purification with HPLC, the purity of corosolic acid in the product exceeded over 80%.

CONCLUSIONThe optimal extraction method, which is convenient and cost-effective, is established for extracting corosolic acid from loquat leaves for medical use.

Chromatography, High Pressure Liquid ; methods ; Eriobotrya ; chemistry ; Plant Leaves ; chemistry ; Technology, Pharmaceutical ; Triterpenes ; isolation & purification