Objective To investigate the effect of lipopolysaccharide(LPS) on the lactose metabolism in mammary epithelial cells of dairy cows(MAC-T) and its mechanism,so as to provide a new idea for the selection of a more reasonable immune metabolism regulation scheme to ensure the health of mammary glands of dairy cows.Methods MAC-T cells were treated with 10 μg/mL LPS for 0,3,6,12 and 24 h,which was detected for the activities of glycolytic enzymes(hexokinase,phosphofructokinase,pyruvate kinase) and the contents of lactic acid,glucose and lactose by ultraviolet light spectrophotometry,for the expression and phosphorylation of hypoxia-inducible factor-1α(FIF-1α) and protein kinase B(also known as AKT) by Western blot and for the transcription levels of HIF-1α,AKT and glucose transporter 1(GLUT1) by fluorescence quantitative RT-PCR. Results Compared with 0 h,the activities of hexokinase and phosphofructokinase reached the peak at3 h(F=4.197 and 6.941,respectively,each P <0.01),and then decreased gradually;The hexoki-nase activity decreased significantly at 24 h(F=4.987,P <0.01) while the phosphofructokinase activity maintained at a relatively high level within24 h;The activity of pyruvate kinase increased significantly in the first 6 h,then increased very significantly,and increased to 27 times of that at 0 h after treatment for 24 h(F=26.95,P <0.001).During 24 h treatment with LPS,the lactose content decreased significantly;Glucose content increased significantly at 3 h(t=4.294,P <0.001),then showed a significant downward trend,and decreased significantly at 24 h(t=5.218,P <0.001);The lactic acid content increased significantly at 12 and 24 h.The expression levels of HIF-1α mRNA and protein increased significantly 6 h after LPS treatment(F=5.355 and 5.466,respectively,P<0.001),and then decreased gradually;The expression level of total AKT mRNA and protein and p-AKT(Ser473) protein all increased significantly 24 h after LPS treatment(F=6.207,6.368 and 6.253,respectively,each P <0.01);The mRNA transcription level of GLUT1 increased significantly at 3,6 and 12 h after LPS stimulation(F=6.549,5.821 and 5.553,respectively,each P <0.01).Conclusion LPS treatment results in increased glucose uptake by MAC-T cells,and the glucose intake has shifted from mostly for lactose synthesis to mostly for glycolytic metabolism and inflammatory response,leading to significant decrease of lactose synthesis in cells.

Objective To understand the feeling of mothers of children with osteogenesis imperfecta during the accompany of children's diagnosis and treatment.Methods A total of 20 mothers of children with osteogenesis imperfecta participated the semi-structured interview.The data were analyzed by Colaizzi's method of phenomenology.Results Three themes were acquired including heavy psychological burden,facing multiple pressure and mother's support through analysis,reorganization and extraction of the subjects.Conclusions Mothers of children with osteogenesis imperfecta needed humanistic concern and emotional support.Nursers should adopted personalized care measures to improve their quality of life.

Objective To evaluate the efficacy of prevention of recurrent femur fracture in children with osteogenesis imperfecta.Methods Data of 35 patients (19 boys,16 girls) who were treated for femur deformity due to osteogenesis imperfecta at our hospital from Jun 2009 to Jun 2013 were retrospectively analyzed.The average age of the patients was 9 years 3 months,ranging from 3 years 6 months to 13 years 1 month.All the patients had suffered from recurrent femur fractures before which led to femoral deformity.The mean angulation angle was 55° (range,10°-90°).10 patients were operated at one side;the other 25 patients were treated bilaterally.None of them received operation before for fractures.The average incidence of femur fracture before operation was 2.4/year (range,2-4).According to the modified Sillence classification system,9 were type Ⅲ OI,14 type Ⅳ,and 12 type I.The curved deformity of the femurs were corrected by osteotomy and stabilized by telescopic intramedullary rods in 35 patients.Results All the 35 patients were followed up for a mean period of 62 months (range,36-72).The average healing time of the osteotomy site of the femur was 8.5 weeks (range,7-12).The patient was encouraged to begin weightbearing and walking when the Xray film showed healing of the osteotomy site.By the latest follow up,80％ of the patients could stand and walk independently.The incidence of femur fracture decreased significantly to the level of 0.3±0.1/year,compared with the level of 2.4± 1.3/year before operation.All the parents of the children were satisfied with the result of deformity correction.At the latest follow up,the Barthel index score increased from 71.82 (range,51-92) before operation to 92.32 (range,82-100) postoperatively,while the WeeFIM score increased from 53.32 (range,42-72) before operation to 78 (range,70-86) postoperatively.The difference of Barthel index score and WeeFIM score between that before and post operation was significant.The children's selfcare and motion ability improved obviously after operation.During follow up,22 patients suffered from recurrent fracture of the femur by various degree,7 of them were treated by open reduction and telescopic rodding surgery,while the other 15 patients were treated conservatively because the fracture displaced or angulated minimally and the fracture healed uneventfully.Conclusion The telescopic intramedullary rod can maintain the correction of the femur deformity and improve the quality of the bone,thus prevent the recurrent fracture of the femur in children with osteogenesis imperfecta effectively.

Objective To Investigate the concentration of VILIP-1 and NR2 peptide in the serum of patients with ischemic stroke , and to explore their clinic value in early diagnostic of ischemic stroke patients.Methods The levels of VILIP-1 and NR2 peptide were examined by ELISA ( enzyme linked immunosorbent assay ,ELISA) with suspicious TIA ( defined as a neurological deficit that resolved within 24 hours) or acute ischemic stroke patients ( within 72 hours of onset of symptoms ) 340 cases,102 healthy controls,98 patients with vascular risk factors and 35 patients with hemorrhagic stroke.Among all the groups , VILIP-1 and NR2 peptide level were analyzed using the nonparametric Wilcoxon test.Diagnostic performance were analyzed among the groups with the two biomarkers independently and combinedly .Results Serum levels of VILIP-1 and NR2 peptide in patients with ischemic stroke (IS) were 9.80 (1.90-14.22) μg/L, 14.40 (5.60-27.91) μg/L respectively,which was higher than that of the healthy control group [VILIP-1:0.02 (0.01-0.09),NR2:0.33 (0.02-1.15),χ2 were 5.61 and 9.54,P<0.001],the group with vascular risk factors [VILIP-1:0.03 (0.02-0.16),NR2:0.27 (0.01-1.54),χ2 were 6.74 and 10.62,P<0.001], the group of patients non-stoke [VILIP-1:0.04 (0.03-0.19),NR2:0.53 (0.45-1.21),χ2 were 3.78 and 7.63, P <0.001 ].The levels of VILIP-1 and NR2 peptide was significantly increased in IS patients presenting within 3 h of symptom onset.When differentiating IS from patients with hemorrhagic stroke ,NR2 had a AUC of 0.934,showing a strong distinguishing effectiveness.Differentiating IS from healthy controls , patients with vascular risk factors and non-stroke patients,the AUC of combination of VILIP-1 and NR2 was 0.974,which was higher than the AUC of either VILIP-1(0.849) or NR2(0.862) alone(P <0.05). Conclusions VILIP-1 and NR2 peptide are very sensitive and specific biomarkers to the early diagnosis of IS.The combination of VILIP-1 and NR2 peptide has higher value of clinical applications than one of them independently.

Objective To investigate the applicability of Cys C-based formulas for prediction of GFR in Chinese patients with CKD.Methods A total of 176 adult patients with CKD including 90 males and 86 females collected from 4 hospitals located in different geographic regions of China (Beijing,Shanghai,Dalian and Changsha) were enrolled in this study from September 2007 to July 2009.The rGFR was measured using 99mTc-DTPA clearance rate two-sample method.Cystantic C was measured by PETIA and PENIA respectively.The results of eGFR in the Larsson formula,Grubb formula,Hoek formula,Filler formula,Stevens formula and Hojs formula were compared with the rGFR to evaluate the calculation coherence,bias,precision,accuracy and the performance of correct phasing of the formulas.Results The mean 99mTc-DTPA clearance was [40.70 ( 19.09 - 86.49)] ml · min-1 · ( 1.73m2 ) -1.Significant difference was witnessed in the evaluation of GFR estimation formulas calculated by PETIA and PENIA (P ＜0.01).ICC and Spearman correlation analysis revealed a significant correlation between eGFR and rGFR.The ICCs of eGFR and rGFR ranged from 0.874 to 0.938.Compared with rGFR,the 30％ accuracy of all the eight evaluation formulas using PETIA and PENIA method were lower than 60％.The percentages of correct phasing in all the 5 stages of CKD were not ideal.With these formulas,percentages of correct phasing from CKD stage 2 to CKD stage 4 were lower than 65％.The eGFRs were underestimated by formulas evaluated by PENIA in CKD stage 1.All the eGFRs were overestimated remarkably by all equations in CKD stage 5.Conclusions None of the eight Cys C based formulas are ideal for estimation of GFR in Chinese CKD patients,and they can not be applied to Chinese patients directly.For this patient population,further studies will be needed to develop a more accurate Cys C-based GFR estimation formula that includes ethnicity,age and other factors.

Objective To evaluate specific clinical and imaging features of osteogenesis imperfecta V and to improve diagnostic accuracy of this disease. Methods Data of 15 patients with osteogenesis imperfecta type V were retrospectively analyzed for their clinical and imaging features. There were 10 males and 5 females, aged from 1 year and 30 years old (median age,12.5 years ). All 15 patients had plain X-ray, and 4 of 15 had CT. All data were analyzed by 3 experienced deputy chief doctors in OI according to OI V standard. Results X-ray:calcification of the interosseous membrane between radius-ulna was detected in all patients and calcification of the interosseous membrane between tibia-fibula was detected in 2 of 15 patients. Dislocation of the radial head was seen in 13 of 15 patients,bilateral in 9 and utilateral in 4.All patients showed restriction in the pronation and supination of the forearm and restricton in the flexion and extention of the elbow joint. Patients with dislocation of raidal head were associated with large coronoid process and olecranon of the ulna. Hyperplastic callus of the extremities were detected in 7 of 15 patients (7 at femur , 3 at humerus, 1 at tibia.In early stage, hyperplastic callus showed thin cortice, and clear boundares with the diaphysis showing and low density, irregular, mesh-like lamellation inside. In the later stage, there were dense calcification inside hyperplastic callus, and no difference in density with the diaphysis. Diaphysis surrounded by hyperplastic callus had clear boundaries with the hyperplastic callus. No cortical destruction was detected. CT:there were sparse needle-dot calcification inside hyperplastic callus, with the patterns of granular, ring-and-arch,irregular streaky mineralization. The cross section of proximal femoral shaft showed irregular shape , flat square shape and tiny medullary cavity, with no calcification on the edge of hyperplastic callus. CT value:-91 HU inside hyperplastic callus; 283 HU in femoral shaft. Conclusions Interosseous membrane between radius-ulna or tibia-fibula, hyperplastic callus ,dislocation of the radial head are specific features in osteogenesis imperfecta V. X-ray can make a definitive diagnosis of osteogenesis imperfecta V. CT scan is helpful in the differential diagnosis of osteogenesis imperfecta V from osteosarcoma.

AIM: To illustrate the expression of hypermethylation p15 gene in 53 non-Hodgkin’s lymphoma (NHL). METHODS: The methylation of p15 gene in 53 cases of non-Hodgkin’s lymphoma was detected by using methylation-specific PCR (MSP) technique. RESULTS: 18.9% (10/53) in NHL were methylation in p15 gene. p15 gene was frequently in high malignant NHL patients (27.3%) compared with in low malignant patients (0%). CONCLUSION: The result suggested that the hypermethylation of p15 may play an important role in non-Hodgkin’s lymphoma.

Objective To validate the analytical performance of three Cys C reagents with particle-enhanced turbidimetric immunoassay(PETIA) method used on the automatic biochemistry analyzer for preliminary clinical application.Methods The performance of three Cys C reagents (labeled as A, B, C) with PETIA method from Shanghai Jing Yuan Co., Beijing Leadman Co. and Beijing Jiuqiang Co. on OlympusAU2700 automatic biochemistry analyzer were assessed.According to the standard of CLSI EP6-A, EP15-A and EP7-P, the precision, linearity range, disturbance (bilirubin, hemoglobin, chyle) were assessed, and compared with those of Cys C reagent based on particle-enhanced nephelometric immunoassay(PENIA) from Dade Behring Co.. The reference ranges for Cys C in serum of 120 healthy individual were evaluated.Results The within-run CVs of the three reagents (A, B and C) were 3.08%-3.2%, 2.3%-4.15% and 1.38%-1.53% respectively.The total CV in A, B and C were 3.29%-3.44%, 2.65%-5.18% and 1.67%-1.69% respectively, lower than the stated.Limits of quantitative determination (LOQ) of the three reagents were 0.41, 0.23 and 0.07 mg/L, basically meeting the testing requirement.The linearity range was 0.22-7.26 mg/L(r=0.996), 0.20-7.72 mg/L(r=0.999)and 0.20-7.62 mg/L(r=0.997)in the three reagents, which demonstrated a sound linear correlation. For interference tests, no remarkable interference (<±10%) of reagent C was detected when bilirubin≤684 μmol/L, hemoglobin≤9.7 g/L and Chyle turbidity≤6 200 FTU; and no significant interference of reagent B was found when bilirubin≤684 μmol/L, hemoglobin≤6.79 g/L and Chyle turbidity≤6 200 FTU; when bilirubin≤684 μmol/L, hemoglobin≤4.85 g/L and Chyle turbidity≤1 240 FTU reagent A was not interfered significantly. The comparison afte and before the high-speed centrifugation reveals that the average percentage of bias for reagents A, B, C measured Cys C in chylous serum samples of patients was -8.31%, 1.52%, 1.32%, respectively.In method comparison tests, the regression equations of the three reagents compared with Dade Behring PENIA Cys C reagent were as follows:Y=0.787X+0.492 (R2=0.976), Y=1.098X+0.137 (R2=0.982) and Y=1.037X+0.249 (R2=0.996), respectively. Agreement rates of the high Cys C in reagent A, B, C and Dade Behring Cys C reagent were 80% (Kappa=0.615,P=0.000), 100% (Kappa=1.000,P=0.000), 91.2% (Kappa=0.824,P=0.000); While for reference range of preliminary clinical assessment, diagnosis coincidence rate of reagent A increased to 98.8% (Kappa=0.974,P=0.000). Conclusions When used in automatic biochemical analyzer, the three Cys C reagent with PETIA showed high precision,sensitivity, and sound correlation with Dade Behring PENIA reagents.The three reagents are all able to meet clinical test requirements, nevertheless, anti-interference capability were diffierent and the reference range should be further validated.

Objective:To investigate the relationship between Toll-like receptor and the immune regulation about inflammation by Sertoli cell in vitro.Methods:Here we examined the expression and potential functions of TLR family in rat Sertoli cells.Using our well-characterized urealyticum(UU) induced model we tested the expression changes of TLR2 and TLR6 at 12~(th),24~(th),36~(th) hours after UU infection in vitro.Results:We demonstrated that TLR2-8 are highly expressed;TLR9 and TLR10 are expressed at relatively low level;the expression of TLR1 and TLR5 are not detected in normal rat Sertoli cells.Comparing with control group,Sertoli cells express more TLR2 and TLR6 after infected by UU.Conclusion:There is some relationship between the activation of TLRs and the immune regulation about inflammation by Sertoli cell.

[ ABSTRACT] AIM:To investigate the role of hydrogen sulfide ( H2 S) on impaired wound healing in ob/ob mice and the underlying mechanism.METHODS:The ob/ob mice were randomly divided into 3 groups, including vehicle, in-sulin and NaHS for treatment.C57BL/6 mice were treated with vehicle as control.Full-thickness punch biopsy wounds were created on the mice.Firstly, H2 S concentrations in the skins and granulation tissues were measured.The mRNA ex-pression of cystathionineγ-lyase ( CSE) was detected by RT-qPCR.The protein expression of CSE and MMP-9 were deter-mined by Western blot.The neutrophil and monocyte/macrophage infiltration was analyzed by immunohistochemistry me-thod.The levels of tumor necrosis factor (TNF)-αand interleukin (IL)-6 were measured by ELISA.Collagen formation was measured by Masson staining.RESULTS:The H2 S levels in the skin and granulation were significantly decreased in ob/ob mice and increased in the NaHS-treated mice ( P<0.05 ) .CSE expression at mRNA and protein levels was significantly decreased in ob/ob mice compared with the control mice (P<0.05).The wound healing period was significantly shorter in NaHS group than that in vehicle-treated ob/ob mice group (P<0.05), in which the insulin group had no difference with vehicle ob/ob mice group.The neutrophil and monocyte/macrophage infiltration, and TNF-αand IL-6 levels were signifi-cantly increased in ob/ob groups, but were decreased in NaHS group ( P<0.01 or P<0.05 ) .Meanwhile, NaHS in-creased collagen formation in the granulation tissues of ob/ob mice.CONCLUSION:H2 S/CSE down-regulation contributes
to impaired wound healing in diabetes, which is alleviated by exogenous H2 S possibly through anti-inflammation.