Objective To investigate the relationship between activity of plasma platelet-activating factor acetylhydrolase (PAF-AH)and insulin resistance (IR) in patients with polycystic ovary syndrome (PCOS). Methods From Oct. 2006 to Jan. 2008, 105 PCOS patients undergoing treatment in Department of Obstetrics and Gynecology of Second Hospital affiliated to West China University were enrolled in the study, among 53 cases with homeostatic model assessment IR (HOMA-IR) exceed or equal 2.77 as IR-PCOS group and 52 cases with HOMA-IR less than 2.77 as non IR-PCOS group. In the mean time, 53 infertile women due to fallopian tube or husband factors were chosen as control group. Plasma PAF-AH activity, high-density lipoprotein-associated PAF-AH (H-PAF-AH) activity, low-density lipoprotein-associated PAF-AH (L-PAF-AH) activity, the ratio of L-PAF-AH to H-PAF-AH activity were measured and compared among three groups. The method of Pearson correlation analysis and stepwise multiple regression analysis were used to study the relationship between the ratio of L-PAF-AH to H-PAF-AH activities and hormonal and metabolic parameters. Results (1) Plasma PAF-AH activity [ (0.055±0.012) mmol· L~(-1)·min~(-1)], L-PAF-AH activity [(0.052±0.012) mmol·L~(-1)·min~(-1)], and the ratio of L-PAF-AH to H-PAF-AH activity (23±6) in the IR-PCOS group were significantly higher than those at control group [(0.050±0.009) mmol·L~(-1) ·min~(-1), (0.047±0.009) mmol·L~(-1)·min~(-1) and (18±4)] and non-IR-PCOS group [(0.050±0.0012) mmol·L~(-1) ·min~(-1), (0.048±0.012) mmol·L~(-1)·min~(-1) and (18± 5 ), P <0.05 ]. The ratio of L-PAF-AH to H-PAF-AH activities at IR-PCOS group was also significantly higher than those at Non IR-PCOS and control group after correction for body mass index (P<0.01). (2) Pearson correlation analysis showed that the ratio of L-PAF-AH to H-PAF-AH in PCOS patients was positively correlated with atherogenic index, age, body mass index, waist-to-hip ratio, HOMA-IR, triglyceride levels, fasting insulin levels, fasting glucose levels, low density lipoprotein cholesterol and total cholesterol (r=0.644, 0.247, 0.296, 0.212, 0.356, 0.587, 0.377, 0.375, 0.292 and 0.199, respectively, P<0.05), and negatively correlated with high density lipoprotein cholesterol (r=-0.510, P<0.05). The multiple stepwise regression analysis showed that atherogenic index and triglyceride were significant variables for the ratio of L-PAF-AH to H-PAF-AH activity (standardized coefficient, β=0.515 and 0.201, respectively, P<0.05). Conclusion The elvated L-PAF-AH activity may be associated with IR in PCOS patients, and the increased ratio of L-PAF-AH to H-PAF-AH activities may be a potential marker of inflammation in the patients.

Objective To investigate the effect of small direct‐current electrical stimulation on migration and invasion related MMPs/TIMPs expression of trophoblast cells .Methods The trophoblast cells were exposed to the direct current electrical field at 150 mV/mm for 5 and 10 hours .Cell images were recorded with continuous photographing and analyzed by image analyzer .The ex‐pression levels of MMP2 ,MMP9 ,TIMP1 and TIMP2 were measured using quantitative RT‐PCR and Western blot .Results In non‐electrical field culture trophoblast cells migrated slowly with random directions .Trophoblast cells cultured in media containing 10% calf serum with the application of 150 mV/mm direct current electrical stimulation ,showed marked cathodal migration (P<0 .01) ,the cell body stretched ,perpendicular to the direction of the electric field .Compared with the non‐electrical field stimulation controls ,trophoblasts under the electrical field stimulation had the increased MMP2 mRNA and protein expression (P< 0 .05) , while MMP9 ,TIMP1 and TIMP2 had no obvious changes of mRNA or protein expressions .Conclusion Physiological direct‐cur‐rent electrical fields might induce directed migration and perpendicular orientation of trophoblast cells .The enhanced MMP2 expres‐sion may play an important role in the migration and invasive activity of trophoblast cells in small electrical field .

AIM: To investigate apolipoprotein A-Ⅰ gene (Apo A-Ⅰ) polymorphism and its relationship with serum HDL subclasses in patients with hyperlipidemia (HL). METHODS: Apo A-Ⅰ genotype was assayed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The subclasses of serum HDL in 118 patients with hyperlipidemia and 109 healthy subjects were determined by two-dimensional gel electrophoresis conjunction with immunodetection method. RESULTS: Both in HL group and the control group, G/G and C/C genotypes were the most frequent at -78 bp and +83 bp of Apo A-Ⅰ gene, respectively. The frequency of rare A allele at -78 bp in HL group was significantly higher than that in control group. In HL group, subjects with G/A mutation had higher serum levels of TG, Apo C-Ⅲ, pre ?_1-HDL and HDL_ 3a , and lower levels of HDL_ 2a and HDL_ 2b compared to the subjects with G/G genotype. CONCLUSION: The G/A transition in the -78 bp position of the Apo A-Ⅰ gene promoter in patients with hyperlipidemia is associated with HDL subclasses. There is a general shift toward smaller sized HDL, which, in turn, indicates that HDL maturation might be abnormal.

AIM: To study the effects of oxidative modification lipoproteins on blood coagulation and fibrino (lysis) in vitro. METHODS: Normal human plasma VLDL, LDL and HDL, which were isolated by density gradient ultracentrifugation method, were oxidatively modified by Cu~(2+) and HOCl method. N-VLDL, Ox-VLDL, N-LDL, Ox-LDL, N-HDL, Ox-HDL were added to the reaction system which consisted of mixed fresh normal plasma respectively, prothrombin time (PT), activated partial thrombplastin time (APTT), plasminogen activator inhibitor 1 (PAI-1), tissue plasminogen activator (t-PA) and platelet aggregation were measured according to the direction of the kits. RESULTS: The relative electrophoretic mobility (REM), absorbance at 234nm and TBARS of oxidized VLDL, LDL and HDL mediated by HOCl or Cu~(2+) were significantly higher than that of the control group (P

?AIM: To study the association of the single nucleotide polymorphism ( SNP) rs1157699 in the calcitonin receptor-like receptor ( CRLR ) gene with primary angle closure ( PAC) in a Han Chinese population.?METHODS: All samples, involved 232 PAC cases and 306 controls, were obtained from an epidemiologic survey conducted in Funing, Jiangsu Province, China. Genotyping were carried out by TaqMan-MGB probe using the real time quantitative polymerase chain reaction system to study the relationship between SNP of rs1157699 in CRLR gene and PAC.?RESULTS: The prevalence of CRLRrs1157699 genotype was 67.4%, 30.0%, 2.6% for CC, CT, TT in cases, and 71.3%, 27.0%, 1.7% in controls respectively.There was no difference between the two groups in the distribution of genotype and allele frequencies of rs1157699 (P>0.05).?CONCLUSION:Our results do not support a significant role for rs1157699 in CRLR with PAC.

Objective　 To investigate apolipoprotein(apo) E polymorphism and its relationship with serum lipids and apolipoproteins in Chinese patients with endogenous hypertriglyceridemia(HTG). Methods　 apoE genotype was assayed by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP), serum lipids were determined by enzyme method, and apolipoproteins were measured by radial immunodiffusion assay in 225 endogenous HTG patients whose fasting serum TG levels were ≥2.26 mmol/L and in 230 healthy subjects whose fasting serum lipids levels were TG<1.82 mmol/L, TC<6.21 mmol/L from a population of Chinese Han nationality in Chengdu area. Results　 Compared with the controls, the values of BMI,TG, TC, nHDLC, apoB100,CⅡ, CⅢ, E and TG/HDLC in HTG patients were significantly increased and the values of HDLC, apoAⅠ and apoE/apoCⅢ were significantly decreased(P<0.001). apoE3/3 genotype and allele ε3 frequency in HTG group and the control group were both the highest, and allele ε2 frequency in HTG group tended to increase than that in the control group(P>0.05). Both in HTG group and control group, the genotype of apoE2 had higher serum TG and apoE levels, lower LDLC level and decreased apoE/apoCⅢ ratio as compared with the genotype of apoE3 or apoE4(P<0.001). Conclusion　 Allele ε2 of apoE gene was associated with higher serum TG and apoE levels and lower serum LDLC level, and the lower ratio of apoE/apoCⅢ was associated with the higher serum level of TG in endogenous HTG.

OBJECTIVETo establish the method of detecting the concentrations of chlorpyrifos in air of workplace with high performance liquid chromatographic (HPLC).

METHODSAccording to standards of methods for determining the chemical substances in workplace air, chlorpyrifos in the air was collected by silicone tube, then dissolved by acetonitrile and determined by high performance liquid chromatography with UV-detector.

RESULTSThere was a linear relationship within the range of 0 ∼ 10.0 µg/ml, and regression equation was y = 5206.1x - 104.7, correlation coefficient was 0.9999, the detection limit was 0.006 µg/ml. The lowest detected concentration was 0.001 mg/m(3) (sampling volume 4.5 L). The average recoveries was 98.3% ∼ 102.5%. The within-run precision was 1.96% ∼ 4.39%, the between-run precision was 2.76% ∼ 5.87%. The desorption efficiencies were 99.0% ∼ 103.3% and the sampling efficiencies were 94%. The samples in silicone tube could be stored for 15 days at room temperature.

CONCLUSIONThe present method could meet with the requirements of Guide for establishing occupational health standards-Part 4 Determination methods of air chemicals in workplace and be feasible for determination of chlorpyrifos in workplace air.

Air Pollutants, Occupational ; analysis ; Chlorpyrifos ; analysis ; toxicity ; Chromatography, High Pressure Liquid ; methods ; Limit of Detection ; Reproducibility of Results ; Workplace

OBJECTIVECord blood (CB) provides a rich source of stem cells for transplantation. CB transplantation has been used widely after myeloablative therapy. One major disadvantage of CB transplantation is delayed platelet engraftment. The aim of this study was to hasten platelet engraftment by investigating the ability of different hematopoietic growth factor combinations to generate large numbers of megakaryocyte (Mk) from CB and by evaluating the biologic characteristics and function of the expanded Mk.

METHODSCB samples were obtained at the end of normal full-term deliveries with informed consent. Mononuclear cells (MNCs) were isolated from CB using Ficoll density centrifugation. MNC population was positively selected for CD(34) expression by magnetic cell sorting (MACS). CD(34)(+) cells were cultured in serum-free and stroma-free medium containing the following two different cytokine combinations: thrombopoietin (TPO) + stem cell factor (SCF) + interleukin (IL) -3 + IL-6 and TPO + SCF. Cultures were characterized after 3, 7, 10 and 14 days by flow cytometry, colony forming unit-megakaryocyte (CFU-Mk) and maturation evaluation (Mk ploidy). The expanded Mk function was examined by the platelet activation in vitro and severe combined immunodiffiency (SCID) mice transplantation in vivo.

RESULTSDifferent results were observed with different culture conditions. With the first cytokine combination optimal expansion of CD(41)(+) cells was observed on day 10, but the optimal expansion of Mk progenitors (CD(34)(+)CD(41)(+)) was observed on day 7, with a median 121 and 44-fold increase at the starting cell dose. This result was also proven by CFU-Mk. The largest numbers of CFU-Mk were also observed on day 7. The degree of maturation of Mk cells also increased as suggested by DNA content of CD(41)(+) cells, which means that CD(34)(+) cells cultured for 3 - 7 days were richer in primitive Mks, while those cultured for 10 - 14 days had greater numbers of more differentiated Mks. For the second cytokine combination, CD(41)(+) and CD(34)(+)CD(41)(+) cells were fewer than the first one, but it produced 36 and 85-fold CD(34)(+)CD(41)(+) and CD(41)(+) respectively on day 7. Platelet activation test confirmed that the expanded Mks had normal function. Therefore, the expanded Mks could be transplanted into the SCID mice bone marrow and produce human platelet in the peripheral blood of the mice.

CONCLUSIONEx vivo expanded Mk might facilitate CB transplantation and help shorten the period of post-transplant thrombocytopenia.

Animals ; Antigens, CD34 ; Cell Culture Techniques ; methods ; Cells, Cultured ; Culture Media ; Fetal Blood ; cytology ; Humans ; Leukocytes, Mononuclear ; cytology ; Megakaryocytes ; cytology ; Mice ; Mice, SCID

The interaction among collagen, von Willebrand factor (vWF) and glycoprotein Ib axis is the first step in hemostasis and thrombosis, especially under high shear condition. To develop a new remedy of anti-thrombosis, mRNA from endothelial cells was extracted, and reverse transcription PCR was adopted to amplify DNA of interest. After sequencing, recombinant expression vector was constructed. The amplified DNA fragment of vWF domain A3 was inserted into expression vector with 6 x his taq, pET20b(+), the recombinant was transformed into E coli (strain DE3) and induced by IPTG. Recombinant vWF-A3 was designated as a recombinant fragment comprising residues 918 - 1114 of mature vWF subunit. It was purified through Ni-NTA resin column and refolded in Tris buffer containing GSH and GSSG. The results showed that rvWF-A3 was expressed successfully in E coli (strain DE3), accounting for 46% of total bacterial protein with its purity of over 95%. It was identified that rvWF-A3 is capable to bind collagen and inhibit the wild vWF binding to collagen by competition. It is concluded that rvWF-A3 fragment might be an effective antithrombotic agent for preventing arterial thrombosis.
Cloning, Molecular ; Collagen ; metabolism ; Escherichia coli ; genetics ; Humans ; Protein Structure, Tertiary ; Recombinant Proteins ; biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA ; von Willebrand Factor ; chemistry ; genetics ; metabolism

OBJECTIVETo investigate the polymorphism of acyl-coenzyme A: cholesterol acyltransferase 1(ACAT1) gene and its relationship with endogenous hypertriglyceridemia (HTG) in Chinese population.

METHODSA total of three hundred and seventy-two subjects (105 endogenous hypertriglyceridemics and 267 healthy controls) from a population of Chinese Han nationality in Chengdu area were studied using PCR-restriction fragment length polymorphism (RFLP).

RESULTSThe frequency of C allele in normal Chinese at rs1044925 locus was 0.137, which was lower than that reported in the population of central and Southern Europe (0.354) (P< 0.05). The frequency C allele was 0.153 in HTG group. No significant difference between normal control and HTG group. In control group, subjects with genotype AA had a higher serum mean concentrations of low density lipoprotein-cholesterol (LDL-C) and non-high density lipoprotein-cholesterol(nHDL-C) when compared with those of C allele carriers (AC and CC genotype carriers), respectively [(3.25+/- 0.68) mmol/L vs (3.03+/- 0.87) mmol/L, P< 0.05; (3.80+/- 0.71) mmol/L vs (3.23+/- 0.82) mmol/L, P< 0.05]. In HTG group, subjects with genotype AA had a higher high density lipoprotein-cholesterol (HDL-C) level compared with those of C allele carriers [(1.00+/- 0.28) mmol/L vs (0.87+/- 0.17) mmol/L, P< 0.05].

CONCLUSIONThese results suggest that rs1044925 polymorphism in ACAT1 gene is not only associated with serum LDL-C and nHDLC levels in healthy Chinese subjects in Chengdu area, but also with HDL-C level in subjects with endogenous hypertriglyceridemia in this population.

Adult ; Aged ; Asian Continental Ancestry Group ; Female ; Gene Frequency ; Genotype ; Humans ; Hypertriglyceridemia ; genetics ; Male ; Middle Aged ; Polymerase Chain Reaction ; Polymorphism, Genetic ; genetics ; Polymorphism, Restriction Fragment Length ; Sterol O-Acyltransferase ; genetics ; Young Adult