Objective To obtain abundant human betacellulin(BTC) with biological activity. Methods The whole mature protein coding sequence of BTC gene was amplified by polymerase chain reaction(PCR) method applied to human pancreatic ?-cell tumors cDNA.The fragment was cloned into prokaryotic expression vector pET32a(+) plasmid.The recombinant plasmid was transformed into E.coli BL21 and the fusion protein was expressed under isopropyl-beta-D-thiogalactopyranoside(IPTG).The fusion protein was purified by Ni2+ affinity chromatography.SDS-PAGE and Western blot were employed to determine the expression and purification of the expected protein.BTC was added to culture NIH3T3 cells for 5 days,and cell proliferation was detected by MTT. Results Lots of fusion protein were produced,and the purified protein can stimulate the proliferation of NIH3T3 cells. Conclusion The human BTC can be successfully obtained from the pET32a(+) system with the biological activity of stimulating the proliferation of NIH3T3 cells.

Objective To construct eukaryotic expression vector of human pancreatic duodenal homeobox 1(PDX-1) gene,and to detect its expression in NIH3T3 cell lines. Methods The whole coding sequence of PDX-1 gene was amplified by polymerase chain reaction(PCR) from human pancreatic-cell tumors cDNA.The fragment was inserted into eukaryotic expression vector pcDNA3.1 plasmid.The recombinant plasmid was verified by double digestion and DNA sequencing.The expression of PDX-1 gene in NIH3T3 cells was assayed by Western blot. Results The length of specific fragment amplified by PCR was 852 bp,and the recombinant plasmid pcDNA3.1-PDX-1 showed two bands of 5.5 kb and 852 bp by digestion using respective restriction enzymes BamHⅠand EcoRⅠ.The sequence of PDX-1 gene was approved or confirmed by blasting to GenBank.It was suggested that PDX-1 gene had been cloned into pcDNA3.1 vector correctly.Western blot showed that PDX-1 gene was expressed,which was detected 24 h after pcDNA3.1-PDX-1 plasmid was transfected into NIH3T3 cells. Conclusion The recombinant eukaryotic expression vector pcDNA3.1-PDX-1 was successfully constructed and expressed in NIH3T3 cell lines.

AIM:Xilei Powder,a traditional Chinese prescription,has been used to treat wounds for hundreds of years,but the mechanism has not been fully understood.METHODS:The effects of Xilei Powder on fibroblast proliferation,collagen accumulation,matrix metalloproteinases-2,9(MMP-2,9)activities and tissue inhibitor of metalloproteinase 1(TIMP-1)production were investigated by MTT,chloramine T method,gelatin zymography and enzyme-linked immunosorbent assays(ELISA),respectively.RESULTS:The aqueous extract of Xilei Powder significantly promoted fibroblasts proliferation in a time and concentration manner,the population doubling time(125 ?g/mL)was 33.8 h,it also significantly(P

AIM:Xilei Powder,a traditional Chinese prescription,has been used to treat wounds for hundreds of years,but the mechanism has not been fully understood.METHODS:The effects of Xilei Powder on fibroblast proliferation,collagen accumulation,matrix metalloproteinases-2,9 ( MMP-2,9 ) activities and tissue inhibitor of metalloproteinase 1 (TIMP-1) production were investigated by MTT,chloramine T method,gelatin zymography and enzyme-linked immunosorbent assays ( ELISA),respectively.RESULTS: The aqueous extract of Xilei Powder significantly promoted fibroblasts proliferation in a time and concentration manner,the population doubling time (125 μg/mL) was 33.8 h,it also significantly (P ＜0.05 ) promoted collagen production.Both of the aqueous and alcoholic extracts could significantly ( P ＜ 0.05 ) increase MMPo2 activity,and also very significantly ( P ＜ 0.01 )promote TIMP-1 production.CONCLUSION: Xilei Powder could promote fibroblasts proliferation,collagen and TIMP-1 production,this might be parts of mechanism to promote wound healing.

Objective To investigate feasibility and effect of external fixator plus steel plate in treatment of open tibiofibular fractures combined with tibial defect.Methods The study involved 21 patients with open fractures of tibia and fibula (15 patients with type Gastilo ⅢA,five with type Gastilo ⅢB and one with type Gastilo ⅢC) with concurrent tibial defect of 2-6 cm.External fixator plus fibular steel plate was performed at the first stage,followed by iliac bone grafting for bone defect at the second stage.Results All patients showed successful reconstruction of the tibial defects with length difference between affected and healthy extremities less than 2 cm in follow-up for (14.0 ± 10.5) months (range,8-24 months).Meanwhile,no talipes equinovarus existed.Conclusions External fixator plus steel plate is an effective method for treating open tibiofibular fractures combined with tibial defect.The length and function of the extremities of patients with open tibiofibular fractures combined with tibial defect of less than 6 cm can be successfully restored.

OBJECTIVE: To investigate the epidemiological features in patients with allergic rhinitis (AR) in Yichang city, and put forward effective prevention and control measures. METHOD: Collecting the data of allergic rhinitis in city proper from 2010 to 2013, input the data into the database and used statistical analysis. RESULT: In recent years, the AR patients in this area increased year by year. The spring and the winter were the peak season of onset. The patients was constituted by young men. There was statistically significant difference between the age, the area,and the gender (P < 0.01). The history of allergy and the diseases related to the gender composition had statistical significance difference (P < 0.05). The allergens and the positive degree in gender, age structure had statistically significant difference (P < 0.01). CONCLUSION Need to conduct the healthy propaganda and education, optimizing the environment, change the bad habits, timely medical treatment, standard treatment.
Allergens ; China ; epidemiology ; Female ; Humans ; Male ; Rhinitis, Allergic ; epidemiology ; Seasons

Objective: To analyze the clinical characteristics of recurrent thrombosis in patients with polycythemia vera (PV) and essential thrombocythemia (ET) to probe the risk factors for recurrent thrombosis in patients with ET and PV. Methods: The clinical data of 104 ET and PV patients with thrombosis in Beijing Anzhen Hospital from February 2001 to November 2016 were retrospectively analyzed. Thrombosis reoccurred in 38 patients. Statistical analyses were performed by multivariate logistic regression for risk factors of recurrent thrombosis in ET and PV patients. Results: Recurrent thrombosis occurred in 36.5% of patients with ET/PV, the total incidence rate in ET and PV patients was 9.8% patient-years, 12.3% patient-years and 5.7% patient-years in ET and PV respectively. There were a total of 56 re-thrombotic events, and 42.1% of events occurred within 1 year after the first thrombosis. The arterial re-thrombosis was 97.4% (most of acute coronary syndrome, ACS), and venous events was 2.6%. The most common cases of re-thrombosis were ACS in ET patients (18 cases, 64.3%), and cerebral infarction in PV patients (7 cases, 70.0%). The number of PV patients with 2 times or more re-thrombotic events was significantly higher than that of ET patients (9 cases, 90.0% vs 7 cases, 25.0%). The proportion of the patients with WBC>12.5×10(9)/L or Hct>45%, and thrombosis history or splenomegaly and high risk thrombotic events were higher than those with a single thrombus (52.6% vs 31.8%; 50.0% vs 30.0%; 86.8% vs 13.6%; 84.2% vs 33.3%; 52.6% vs 15.2%; 94.7% vs 53.0%; P values were 0.036,0.046, <0.001, <0.001, <0.001 and <0.001, respectively). Logistic regression analysis showed that thrombosis history (OR=13.697, P=0.025), splenomegaly (OR=13.301, P=0.034) and high risk stratification of thrombotic events (OR=44.618, P=0.025) were independent risk factors for recurrent thrombotic events. Conclusions: ET and PV patients had a higher risk of re-thrombosis. The incidence of re-thrombosis in ET was higher than in PV, ACS was more common cases of re-thrombotic events; but PV patients were more susceptible to multiple re-thromboses than ET ones, also with more cerebral infarction. Previous thrombus history, splenomegaly and high risk stratification of thrombotic events were independent risk predictors for re-thrombosis of ET and PV patients.
Humans ; Polycythemia Vera ; Retrospective Studies ; Risk Factors ; Thrombocythemia, Essential ; Thrombosis

To explore the influence of calculus bovis on the function of primary cultured mice oral fibroblasts, we determined the effects of calculus bovis on the fibroblast proliferation, collagen production, matrix metalloproteinases-2, -9 activities and tissue inhibitor of metalloproteinase-1 production by MTT assay, chloramine T method, gelatin zymography and enzyme-linked immunosorbent assays respectively. The results showed that calculus bovis could significantly inhibit the proliferation of fibroblasts and collagen synthesis in a concentration dependent manner, could significantly (P<0.05) suppress matrix metalloproteinases-2 activity and very significantly (P<0.01) inhibit the production of tissue inhibitor of metalloproteinase-1. In conclusion, the major function of calculus bovis in the process of ulcer healing is not to promote tissue regeneration, the mechanism that calculus bovis inhibits collagen synthesis may be partly due to its ability to very significantly (P<0.01) suppress the production of tissue inhibitor of metalloproteinase-1.
Animals ; Cattle ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cholelithiasis ; chemistry ; veterinary ; Collagen ; drug effects ; metabolism ; Fibroblasts ; cytology ; physiology ; Materia Medica ; pharmacology ; Mice ; Mice, Inbred BALB C ; Mouth Mucosa ; cytology ; Tissue Inhibitor of Metalloproteinase-1 ; drug effects ; metabolism

Objective To explore whether hypertonic saline would partake in regulating Notch signaling in microglia in experimentally induced cerebral ischemic rats.Methods Male SD rats were randomly divided into sham group, cerebral ischemia group, normal saline group ( NS group ) , 10%hypertonic saline group (10%HS group) , the model of cerebral ischemia were established in all rats except the sham group by using middle cerebral artery occlusion ( MCAO) .After 2 hours of MCAO, the rats were through reperfusion for 24 h.In addition, rats in the normal saline group and 10% HS group were respectively treated with a continuous intravenous injection of normal saline (0.3 mL/h) and 10%HS (0.3 mL/h) by tail vein for 24 h.Immunofluorescence methods, RT-PCR and Western blot were used to detect the expression of Notch1 and intracellular Notch receptor domain ( NICD) .All data was analyzed by one-way analysis of variance ( ANOVA) , The intergroup comparisons were analyzed by the least-significant-difference (LSD) tests.Differences were considered statistically significant if P<0.05.Results Immunofluorescence showed that the expression of Notch1 and NICD were significantly increased in the microglia around peri-ischemia area in cerebral ischemia group and normal saline group compared to sham group;the expression of Notch1 and NICD in the microglia around peri-ischemia area were significantly reduced in 10% HS group compared to ischemia group and NS group.RT-PCR showed that the mRNA expression of Notch1 was significantly increased in ischemia group and NS group compared to sham group ( sham group: 1.000 ± 0.076; ischemia group: 2.203 ±0.283; NS group: 1.616 ±0.185; P <0.01 ); however, it was significantly reduced in 10% HS group compared to ischemia group and NS group ( ischemia group:2.203 ±0.283; NS group: 1.616 ±0.185; 10%HS group: 1.202 ±0.177; P <0.05 ) .Western blot showed that the protein expression of Notch1 was significantly increased in ischemia group and NS group compared to sham group ( sham group: 0.290 ±0.079; ischemia group: 0.750 ±0.029; NS group:0.765 ±0.182;P<0.01);but was significantly reduced in 10%HS group compared to ischemia group and NS group ( ischemia group:0.750 ±0.029; NS group:0.765 ±0.182;10%HS group:0.390 ±0.195;P<0.05 ) .The protein expression of NICD was significantly increased in ischemia group and NS group compared to sham group ( sham group: 0.401 ±0.196; ischemia group: 0.906 ±0.359; NS group:0.847 ±0.153;P<0.01);but was significantly reduced in 10%HS group compared to ischemia group and NS group ( ischemia group:0.906 ±0.359; NS group:0.847 ±0.153;10%HS group:0.561 ±0.165;P<0.05 ) .Conclusion Our results suggest that HS markedly suppresses Notch signaling in microglia around the ischemia tissue area in experimental induced cerebral ischemic rats.

BACKGROUNDMultidrug resistance (MDR) is a main reason for paclitaxel (TAX) treatment failure. Indirubin-3'-monoxime (IRO) and Matrine are traditional Chinese medicines, which may reverse the resistance of tumor cells to some chemotherapy drugs, but the relationship between paclitaxel resistance and Matrine is still unclear. The aim of this study was to explore the potential molecular mechanism of IRO and Matrine in reversal of TAX resistance.

METHODSIn this study, MTT assay was used to measure the non-cytotoxic dosage of IRO and Matrine on NCI-H520/TAX25 cells and determine the reversal extent of TAX resistance under non-toxic doses. In addition, RT-PCR and Western blotting were used to evaluate the mRNA expression and the protein level of survivin, Oct-4, and Sox-2 in NCI-H520/TAX25 cells using semi-quantitative methods.

RESULTSThere was no obvious inhibition on sensitive cell strains and drug-resistant strains, when the final concentration was at lest 4 µmol/L for IRO and 100 µmol/L for Matrine. So 4 µmol/L of IRO and 100 µmol/L of Matrine were considered as the reversal dosage. When 4 µmol/L of IRO or 100 µmol/L of Matrine were used together with TAX, the sensitivity to TAX increased evidently in NCI-H520/TAX2 cells; the reversal rate of IRO and Matrine was about 1.92 (43.56/22.6 nmol/L) and 1.74 (43.56/25.0 nmol/L), respectively. The mRNA expression and the protein level of survivin, Oct-4, and Sox-2 in NCI-H520/TAX25 decreased significantly (P < 0.05) after addition of IRO or Matrine in TAX treatment, compared to that of TAX treatment alone.

CONCLUSIONThe decrease in both mRNA expression and protein level of survivin, Oct-4, and Sox-2 might be the molecular mechanism, by which IRO and Matrine mediate the reversal of TAX resistance.

Alkaloids ; pharmacology ; Blotting, Western ; Cell Line, Tumor ; Drug Resistance, Neoplasm ; Humans ; Indoles ; pharmacology ; Inhibitor of Apoptosis Proteins ; genetics ; metabolism ; Octamer Transcription Factor-3 ; genetics ; metabolism ; Oximes ; pharmacology ; Paclitaxel ; pharmacology ; Quinolizines ; pharmacology ; SOXB1 Transcription Factors ; genetics ; metabolism