Objective To construct eukaryotic expression plasmid pIRESneo3-pigment epithelium-derived factor (PEDF) and detect its transient expression in SP2/0 cells. Methods Specific primers were designed based on the mature peptide sequence of human PEDF cDNA in the GenBank. Human PEDF gene was cloned into the eukaryotic expression vector pIRESneo3. The PEDF DNA was transfected into SP2/0 with LipofectamineTM 2000. The recombinant human PEDF protein expressed in SP2/0 cell culture supernatant was identified by Western blot and enzyme-linked immunosorbent assay. The biological activity of the recombinant human PEDF was measured by 3-(4,5-dimethylthiazol-z-y1)-2,5-diphenytetrazolium bromide(MTT) method. Results PCR amplification, restriction enzyme digestion and DNA sequencing confirmed that the mature peptide sequence of human PEDF cDNA was successfully cloned into the eukaryotic expression vector pIRESneo3. And the plasmid was transfected into SP2/0 cells, which could secret PEDF. Western blot analysis showed that there was only one obvious band at the position of relative molecular weight of 50 000, and it is equivalent to the expected value. Enzyme-linked immunosorbent assay suggested that the content of PEDF began to rise after transfection, and peaked at 36 h [(0.92±0.04) μg/ml]. The proliferation of human umbilical vein endothelial cell line was significantly inhibited by supernatant after transfection of 36 h (P<0.05). Conclusions The eukaryotic expression plasmid pIRESneo3-PEDF had been successfully constructed and active human PEDF was transiently secreted, which made a foundation for further study of stable expression and purification of PEDF. This protein could be a potential medication for preventing and managing retinopathy of prematurity.

Objective To investigate the effects of mild pefioperative hypothermia on cellular immune function in patients undergoing surgery for rectal cancer.Methods Fifty ASA Ⅰ or Ⅱ patients aged 30-64 yr undergoing surgery for rectal cancer were randomly divided into 2 groups ( n =25 each):mild hypothermia group and normal body temperature group.Anesthesia was induced with midazolam,fentanyl,etomidate and vecuronium and maintained with propofol,remifentanil and vecuronium.The patients were mechanically ventilated after tracheal intubation.PET CO2 was maintained at 35-45 mm Hg.The venous blood samples were taken from peripheral vein at 1 h before anesthesia (T1 ),at the end of operation (T2),at 24 h after operation (T3),and on 7th day after operation (T4) to measure the serum Th1 and Th2 cytokines levels and Th1/Th2 cytokines balance was observed.Results Compared with the baseline value at T1,the serum Th2 cytokines level was significantly decreased and Th1/Th2 ratio was significantly increased at T4 in normal body temperature group,and the serum Th1 cytokines level and Th1/Th2 ratio were significantly decreased and the serum Th2 cytokines level was significantly increased at T2.3 in mild hypothermia group ( P ＜ 0.05).Compared with normal body temperature group,the serum Th1 cytokines level and Th1/Th2 ratio were significantly decreased and the serum Th2 cytokines level was significantly increased at T2-4 in mild hypothermia group ( P ＜ 0.05).Conclusion Mild perioperative hypothermia can depress cellular immune function in patients undergoing surgery for rectal cancer.

Background and purpose: Because of the aggressive nature of hilar cholangiocarcinoma and the absence of effective adjuvant therapy, surgical radical resection offers hilar cholangiocarcinoma patients the only choice. Research focus include preoperative assessment, the use of preoperative biliary drainage, the range of hepatic resection, and the range of lymphadenectomy. To investigate the clinical experience and efifcacy of combined hepatectomy in the treatment of hilar cholangiocarcinoma. Methods: Two hundred and seven patients with hilar cholangiocarcinoma treated surgically in the First Afifliated Hospital of Kunming Medical University form Jan. 2007 to Oct. 2013 were retrospectively analyzed. Results:Of the 207 patients, 125 patients who received radical resection (R0 resection) and the curative resection rate was 60.4%. One hundred and iffty-six cases were treated in combined hepatectomy group, 51 cases in non-hepatectomy group, the rate of R0 resection was 70.5%in hepatectomy group and 29.4%in non-hepatectomy group, and the difference was signiifcant (P<0.01). Two patients died perioperatively, the main postoperative complications included hepatic function insufifciency and bile leakage. One hundred and seventy-two patients were followed up, the median survival time of the 102 patients who received R0 resection was 45 months, and the 1, 3, 5 year survival rates were 96.1%, 59.1%and 17.2%. The median survival time of the 70 patients who received R1-2 resection was 26 months, and the 1, 3 year survival rates were 81.3%and 19.2%, and none of the patient survived for over 5 years. The survival rate of patients who received R0 resection was signiifcantly higher than those who received R1-2 resection (χ2=39.121, P<0.01). In the hepatectomy group was awarded the R0 resection in patients with postoperative 1, 3, 5 year survival rate was 97.8%, 63.9% and 18.0%, in non-hepatectomy group received R0 resection in patients with postoperative 1, 3, 5 year survival rate was 83.3%, 20.8%and 8.3%. There were signiifcant differences in the postoperative survival rate between both group (χ2=5.988, P=0.014). Conclusion:Radical excision is the key to improve the long term survival. Combined hemihepatectomy and standardized lymph node resection has signiifcantly improved the radical resection rate and the efifcacy of treatment for hilar cholangiocarcinoma.

Objective To investigate the clinical effect of Ethibond suture and anchor fixation by arthroscopic technique for the treatment of avulsion fracture of the anterior cruciate ligament (ACL) tibial insertion.Methods Twenty patients with avulsion fracture of the ACL tibial insertion hospitalized between July 2013 and June 2014 were collected retrospectively.There were 12 males and 8 females,aged 18-41 years (mean,25.3 years).All patients were identified with type Ⅱ and type Ⅲ fractures according to the Meyers-McKeever classification.Results of Lachman test and anterior drawer test were both positive.All patients accepted the operation that avulsion fracture of the ACL tibial insertion was treated with the Ethibond suture and anchor fixation by arthroscopic technique within 3 weeks after injury.Follow-up X-ray examinations were carried out to evaluate the bone union.Lysholm and international knee documentation committee (IKDC) scores were used to evaluate the function of knee joint postoperatively.Results Operation time was 45-70 min (mean,50 min).Blood loss was 5-15 ml (mean,10 ml).Follow-up was conducted for 12-24 months (mean,16.3 months).Postoperative X-ray showed the reduction was satisfactory.Lachman test and anterior drawer test were both negative after operation.Knee functions were recovered to normal.Lysholm and IKDC scores were 89-96 points [(93.5 ± 2.3) points]and 84-96 points [(91.0 ± 3.9)points] respectively at the final follow-up,improved compared to the preoperative 42-61 points [(51.1 ± 6.2) points] and 46-68 points [(55.2 ± 7.0) points] (both P ＜0.05).Conclusion Arthroscopic Ethibond suture and anchor fixation for avulsion fracture of the ACL tibial insertion is an effective procedure with the advantages of small trauma,reliable fixation,good functional recovery and satisfactory clinical effect.

White spot syndrome virus (WSSV) is a major pathogen in aquaculture penaeid shrimp, which caused catastrophic economic losses in the worldwide. No adequate treatments against WSSV are available. In order to study infection mechanism of WSSV, a phage display scFv cDNA library against WSSV was constructed and a neutralizing antibody of scFv P1D3 was selected in our lab previously. In this study, scFv P1D3 was expressed successfully in yeast. Firstly, the original expression vector of P1D3, M13 phagmid, was used as a template to design primers with restriction sites of SnaB I and EcoR I . Then the gene of P1D3 was amplified by PCR. After digested by SnaB I and EcoR I , the fragment of scFv P1D3 with E-tag was inserted into yeast and E. coli shuttle plasmid pPIC9k. The recombinant plasmid pPIC9k-scFv P1D3-Etag was linearized with Bgl II and then transformed into Pichia pastoris GS115 by electroporation. Positive clones were selected and verified by PCR and DNA sequencing. The scFv PID3 was induced to express in yeast by methanol. The results of ELISA demonstrate that scFv P1D3 expressed in yeast still has high specificity to bind on WSSV and the binding activity is higher than that expressed in E. coli TG1. After several optimizing experiments, the results show that the expression amount of scFv P1D3 can reach to 302 mg/L in yeast culture supernatant. This experiment has offered a new source of antibody for the researches on passive immunology for shrimp.
Animals ; Antibody Specificity ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; drug effects ; Methanol ; pharmacology ; Penaeidae ; virology ; Pichia ; drug effects ; genetics ; Single-Chain Antibodies ; analysis ; biosynthesis ; genetics ; immunology ; Temperature ; White spot syndrome virus 1 ; immunology

Objective To study the mechanism of marcosomia by investigating insulin-like growth factor 2(IGF_2)imprinting status,expression level and the promoter usage in the placenta of macrosomia. Methods We selected heterozygous cases for Apa Ⅰ polymorphism in exon 9 of IGF_2 gene and then analyzed its imprinting status in 168 placentas of macrosomia and normal pregnancies.IGF_2 transcription levels and promoter usages in macrosomic and normal placenta were evaluated by using semi-quantitative RT- PCR assay.Results Thirty specimens of macrosomic placenta and 30 of normal placenta were identified as heterozygous for IGF_2.All of the heterozygous specimens showed maintenance of imprinting.The expression of placental IGF_2 mRNA(2.2?1.2)was significantly higher in macrosomia than that of normal weight group (1.6?0.6,P 0.05).Conclusion It is possible that over expression of IGF_2 in placenta contributes to macrosomia while the promoter usage and imprinting status are not associated with macrosomia.

BACKGROUNDInterleukin-13 (IL-13) has been implicated to be responsible for recruitment of inflammatory cells from the blood to the lung, regulation of matrix metalloproteinase and induction of mucin production and secretion in chronic obstructive pulmonary disease (COPD). We determined plasma IL-13 levels in patients with COPD and investigated its association with common polymorphisms of IL-13 gene in a case-control study.

METHODSWe genotyped 160 cases and 175 control subjects in a local hospital using Mass-Array(TM) Technology Platform then tested the association of four SNPs in IL-13 (rs1295685, rs1800925, rs1881457, rs20541) with COPD, and then determined plasma IL-13 levels in patients with COPD and controls.

RESULTSAssociation was found between IL-13 gene SNPs (rs20541 and rs1800925) and an increased risk of COPD. By linkage disequilibrium (LD) analysis, two blocks (rs1881457 and rs1800925; rs20541 and rs1295685) were found. The risk of COPD was found associated with the IL-13 gene polymorphism among southern Chinese Han population. Plasma IL-13 level was increased in COPD patients compared with controls.

CONCLUSIONSThe polymorphism of the IL-13 gene is associated with an increased risk of COPD in southern Chinese Han population. Plasma IL-13 levels were found elevated in patients with COPD.

Adult ; Aged ; Asian Continental Ancestry Group ; genetics ; Case-Control Studies ; Gene Frequency ; genetics ; Genetic Predisposition to Disease ; genetics ; Genotype ; Haplotypes ; genetics ; Humans ; Interleukin-13 ; genetics ; Linkage Disequilibrium ; genetics ; Male ; Middle Aged ; Polymorphism, Single Nucleotide ; genetics ; Pulmonary Disease, Chronic Obstructive ; genetics

OBJECTIVETo determine plasma imatinib concentration, intracellular imatinib concentration, and hOCT1 and ABCB1 mRNA expression in bone marrow cells of CML patients to further evaluate the potential usefulness of these measures as markers of imatinib efficacy and their clinical relationships.

METHODSEighty CML patients in chronic phase receiving imatinib were enrolled in this study, including 56 males and 24 females with a median age of 39.5 (6 - 76) years. Imatinib was administered at a median dose of 400 (200 - 800) mg/d orally per day with a median course of 24 (3 - 90) months. The intracellular imatinib concentrations in bone marrow cells of 28 patients were simultaneously determined. Real-time quantitative PCR with a taqman probe was used to assess hOCT1 and ABCB1 mRNA expression on bone marrow cells of 36 patients. Imatinib trough concentration was determined by high-performance liquid chromatography-tandem mass spectrometry with a detectability of 2 - 10 000 µg/L. Serum α1-acid glycoprotein (AGP) was measured by immune turbidimetry on a BNProspec protein analyzer (Dade Behring, USA). All patients were divided into MMR, CCyR, PCyR or drug-resistant groups according to response.

RESULTSPlasma imatinib trough concentration of 80 patients was (1274.1 ± 559.1) (109.0 - 3400.0) µg/L. The plasma imatinib trough concentration of 59 (73.8%) patients with a dose of 400 mg/d was (1252.0 ± 569.5) (109 - 3400) µg/L, including 37 (62.7%) patients with concentrations of more than 1000 µg/L and 9 (15.2%) patients more than 800 µg/L. Plasma imatinib trough concentration in the MMR group \[(1531.9 ± 634.1) µg/L\] was significant higher than in the PCyR \[(812.8 ± 480.3) µg/L\] or drug-resistant group \[(875.2 ± 243.1) µg/L\] (P < 0.05). Plasma imatinib trough concentration in the CCyR group \[(1288.4 ± 498.2) µg/L\] was significant higher than in the PCyR group (P = 0.027). There was no significant difference between CCyR and MMR groups with regard to plasma imatinib trough concentration (P = 0.136). The intracellular imatinib concentration in bone marrow cells in the CCyR group \[12.6 (2.4 - 90.4) µg/L\] was significantly higher than drug-resistant \[6.6 (2.6 - 111.0) µg/L\] or PCyR \[2.7 (2.4 - 4.7) µg/L\] groups (P = 0.013). The hOCT1 mRNA expression on bone marrow cells in the CCyR group \[25.9(0.7 - 123.9) × 10(-5)\] was significantly higher than in drug-resistant \[7.8 (2.5 - 33.5) × 10(-5)\] or PCyR \[4.2 (1.4 - 11.9) × 10(-5)\] groups (P = 0.036). The ABCB1 mRNA expression on bone marrow cells in drug-resistant group \[136.7 (15.0 - 1604.9) × 10(-5)\] was significantly higher than in CCyR \[129.1 (12.9 - 783.3) × 10(-5)\] or PCyR \[34.4 (2.2 -108.2) × 10(-5)\] groups (P = 0.013). Plasma imatinib trough concentration was positively correlated with AGP (r = 0.446, P = 0.000) or dose (r = 0.346, P = 0.002). There were no significant correlations between plasma imatinib trough concentration and height, weight or body surface area (P > 0.05). There were no significant differences among different courses with regard to plasma imatinib trough concentration (P > 0.05).

CONCLUSIONClinical responses in CML patients were correlated with plasma and intracellular imatinib trough concentrations. Imatinib concentration was regulated by AGP and the activities of hOCT1 and ABCB1.

ATP Binding Cassette Transporter, Sub-Family B ; ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Adolescent ; Adult ; Aged ; Benzamides ; blood ; pharmacokinetics ; therapeutic use ; Bone Marrow Cells ; metabolism ; Child ; Female ; Humans ; Imatinib Mesylate ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; blood ; drug therapy ; metabolism ; Male ; Middle Aged ; Organic Cation Transporter 1 ; metabolism ; Piperazines ; blood ; pharmacokinetics ; therapeutic use ; Plasma ; metabolism ; Pyrimidines ; blood ; pharmacokinetics ; therapeutic use ; Treatment Outcome ; Young Adult

OBJECTIVETo study the relationship between human herpesvirus 6 (HHV-6) and oral squamous cell carcinoma.

METHODSThe serum anti-HHV-6 antibody titers from oral squamous cell carcinoma patients and control subjects were detected by indirect immunofluorescence assay. HHV-6 DNA in peripheral blood mononuclear cells from oral squamous cell carcinoma patients and control subjects was amplified by PCR with primers from sequence of HHV-6 and the specificity was confirmed by Southern-blot hybridization with an internal probe oligonucleotide. An immunohistochemical staining using rabbit anti-HHV-6 antibody was used to detect HHV-6 antigen in oral tumor tissues from oral squamous cell carcinoma patients.

RESULTSSignificantly higher proportion of patients with oral carcinoma (16/16) had IgG antibody to HHV-6 in sera compared with those (12/16) in control subjects, and geometric mean titer of these two groups was 1:118 and 1:64 respectively (P less than 0.05). The detectable rate of HHV-6 DNA in peripheral blood mononuclear cells for the above groups was 10/16 and 6/16 respectively (P less than 0.05). HHV-6 antigens were positive in 9 out of 12 oral tumor cases and in only 2 out of 8 pericancerous tissues the difference between these two groups was also significant (P less than 0.05).

CONCLUSIONThese results demonstrated the frequent presence of HHV-6 in oral squamous cell carcinoma, therefore, HHV-6 possibly play a role in the pathogenesis of oral squamous cell carcinoma.

Antibodies, Viral ; blood ; Carcinoma, Squamous Cell ; virology ; DNA, Viral ; blood ; Herpesviridae Infections ; complications ; virology ; Herpesvirus 6, Human ; genetics ; immunology ; isolation & purification ; Humans ; Immunoglobulin G ; blood ; Infant ; Mouth Neoplasms ; virology

An HPLC-UV method has been developed for the determination of valibose, miglitol, voglibose and acarbose, the four anti-diabetic drugs. The separation was accomplished successfully by using reversed phase chromatography (Prevail carbohydrate column, 250 mm x 4.6 mm, 5 microm) with a gradient acetonitrile-phosphate buffer solution (pH 8.0) at a wavelength of 210 nm. Furthermore, the method of a high-performance liquid chromatography coupled with ESI-MS in positive ionization mode has been established. These two methods were successfully applied to the assay and qualitative detection of four alpha-glucosidase inhibitors in the potential counterfeit anti-diabetic drugs.
1-Deoxynojirimycin ; analogs & derivatives ; analysis ; Acarbose ; analysis ; Chromatography, High Pressure Liquid ; methods ; Chromatography, Reverse-Phase ; Glycoside Hydrolase Inhibitors ; Hypoglycemic Agents ; chemistry ; Inositol ; analogs & derivatives ; analysis ; Spectrometry, Mass, Electrospray Ionization ; methods ; Spectrophotometry, Ultraviolet ; alpha-Glucosidases ; analysis