Purpose:To construct replication-defective adenovirus which was recombinated with Egr-1 promoter and to Smad7, and to study whether it can express exogenous Smad7 protein in cytoplasm.Methods:Based on Adeno-X~(TM) expression system, the CMV promoter of the pShuttle vector was replaced by Egr-1 promoter, and the Smad7 cDNA was subcloned into the MCS(multiple cloning site) of pShuttle. The recombinant pShuttle was then subcloned into the Adeno-X~(TM) genome, which was transformed into E.coli to get recombinant Adeno-X~(TM) plasmid DNA. The recombinant adenovirus was made and amplified in the transfected HEK 293 cells before it was purified and tested for viral titer. Then the Smad7’s location in cells was determined by immunocytochemistry.Results:Identified by restriction endonuclease analysis and PCR, recombinant adenovirus with Egr-1 promoter and Smad7 cDNA was constructed successfully, with a viral titer of 1.0?10~(11) TCID50/ml. Both endogenous and exogenous Smad7 protein was found to be located in cytoplasm of fibroblast cells.Conclusions:With Adeno-X~(TM) expression system, utilizing the techniques of molecular cloning, recombinant adenoviral vector could be quickly and efficiently constructed which could be packaged into replication-defective Adenovirus and amplified in HEK293 cells. The recombinant adenovirus could express exogenous Smad7 protein in fibroblast cells.[

Objective To explore the role of serotonin(5-HT) in obsessive compulsive disorder(OCD) and the difference in platelet 5-HT content between OCD and healthy controls, the obsession and the compulsion subgroup. Methods The concentration of serotonin (5-HT) in twenty-seven patients with OCD and twenty-seven patients without OCD were detected in the study. Results Platelet serotonin level in patients with OCD ( ( 139 ±172 ) μg/L) was lower than that in patients without OCD ( ( 248 ± 215 ) μg/L), and the differences were significant (P＜0.05). Conclusion The present results support the hypothesis that 5-HT hypofunctionality contribute to OCD. And the differences between the obsession and the compulsion subgroup in the role of 5-HT are significant.

Objective To study whether the expression Smad 7 protein by the recombinant adenovirus with Egr-1 promoter and Smad 7 cDNA in fibroblast cell can block the signal transduction pathway of transforming growth factor-beta1 (TGF-?1) under irradiation thereby inhibiting collagen synthesis in vitro. Methods The location of endogenous Smad 7 and exogenous Smad 7 protein in recombinant adenovirus infected fibroblast cells(3T6) were determined by immunocytochemical method. The infected 3T6 cells were irradiated and then cultured with TGF-?1 4 hours after irradiation. The activity of preventing radiation-induced fibrosis by expression Smad 7 protein was evaluated by the amount of collagen synthesis and proliferation of 3T6 cells. The amount of collagen synthesis was shown by the coruscant per minute (cmp) through the 3?H-Proline incorporation technique. Results The endogenous Smad 7 and exogenous Smad 7 protein both were located in the cytoplasm. When cultured with TGF-?1 4 hours after irradiation, the amount of collagen synthesis in the 3T6 cells infected with the recombinant adenovirus was significantly less than that in the cells without infecting adenovirus after irradiation(P=0.001), But, there was no difference in the proliferation of 3T6 cells between those with and without adenovirus infection (P= 0.312 ). Conclusions The Egr-1 promoter in the recombinant adenovirus can regulate the expression of downstream Smad 7 cDNA in 3T6 cells. The expression Smad 7 protein could block the TGF-?1 signal transduction pathway thereby inhibiting the collagen synthesis. The mechanism of inhibiting the collagen synthesis may be accomplished at the transcription level.

BACKGROUND:As current studies on isolation, culture andcryopreservation of human amniotic epithelial cells (hAECs) are relatively scattered, it is difficult to form a comprehensive and effective solution to meet the clinical needs of stem cells for transplantation in future.OBJECTIVE:To establish the technology of isolation, culture and cryopreservation of hAECs, and to study the biological characteristics of hAECs.METHODS:Orthogonal method was used to study the effects of different factors on the separation, culture and cryopreservation, and range method was adopted to analyze the data to optimize the separation, culture and cryopreservation. We performed cell primary and passage cultures, morphology observed by microscope, drawn cell growth curve and flow cytometry assay, immunofluorescence staining, hepatocyte like cell differentiation to study the biological characteristics of hAECs.RESULTS AND CONCLUSION:(1) The optimal hAECs separation conditions were as follows:trypsin digestions were conducted at a concentration of 0.25%, four times, once for 20 minutes digestion; optimal conditions of culture were 4×108/L cell seeding density, 10 μg/L epidermal growth factor, 5% serum; optimal conditions of cryopreservation were 1×1010/L cell cryopreservation density, 10% dimethyl sulfoxide, 80% serum. (2) The primary cells were adhered to the wall in 2-3 days, exhibiting irregular polygon, paving stone-like growth. Cell adherence and growth rate were accelerated after subculture, and the growth and proliferation ability of passage 2 cells were not significantly decreased after cryopreservation and resuscitation. (3) Immunofluorescence staining showed that the primary cells strongly expressed SSEA-4 and CK19, but did not express Vimentin, CD45 and HLA-DR. The immunophenotype statistics of the primary and passage 4 cells showed the epithelial mesenchymal transition of hAECs in culture process. (4) Immunofluorescence staining showed that the liver cell marker expression of ALB, CK18 was significantly increased after hAECs were induced to differentiate into hepatocyte-like cells. Glycogen staining revealed glycogen synthesis in hAECs after 3 weeks of induction. To conclude, hAECs are easy to obtain and have strong proliferation ability in vitro, and express surface markers for undifferentiated embryonic stem cells.

Objective To study the effect of different dose fractionation on overall survival in patients with limited-stage small cell lung cancer (LS-SCLC). Methods LS-SCLC patients treated with radical combined chemotherapy and radiotherapy (RT) between January 2001 and Dec 2007 were analyzed retrospectively. According to the dose fractionation schemes, patients were divided into three groups:conventional fractionated RT (1. 8 -2.0 Gy,once daily), hyperfractionated RT (1.4 Gy, twice daily) and hypofractionated RT (2. 5 Gy,once daily). Overall survival, disease free survival and pattern of failures of the three groups were compared. A total of 177 patients were enrolled, including 63 patients in conventional fractionated RT group, 79 in hyperfractionated RT group and 35 in hypofractionated RT group. Results The overall follow-up rate was 96. 6%. The patient numbers with follow-up of more than 2 and 5 years were 153 and 92, respectively. The median survival time of the entire group was 22. 4 months, and the 2-and 5-year survival rates were 43.4% and 23. 5%, respectively. The 2-year survival rates for three groups were 31%, 46% and 59% (x2 =7.94,P=0.019), respectively. The 2-year disease free survival for three groups were 20%, 31% and 40% ( x2 = 4. 86, P = 0. 088 ), respectively. In the pairwise comparisons,patients in hypofractionated RT group have better survival than those in conventional fractionated RT group ( x2 = 7. 81, P = 0. 005 ), the effect of hyperfractionated RT group lies between the hypo-and the conventional fractionated RT groups, but no significant differences were detected ( x2 = 2. 31, P = 0. 128; x2 = 2. 95, P =0. 086). The mildest side effect was found in the hypofractionated RT group. No statistically significant differences were found in the patterns of first failure. Conclusion The hypofractionated RT scheme showed potential survival benefits for patients with LS-SCLC and should be considered in the setting of randomized clinical trials.

OBJECTIVE:To investigate clinical efficacy and safety of meglumine adenosine cyclophosphate in the treatment of acute myocardium infarction. METHODS:A total of 80 patients with acute myocardium infarction in our hospital during May 2015-Jan. 2016 were selected and divided into control group and observation group according to random number table,40 cases in each group. Control group was given conventional treatment. Observation group was additionally given Meglumine adenosine cyclo-phosphate injection 120 mg added into 5% Glucose injection 250 mL,ivgtt,qd,on the basis of control group. Both groups re-ceived treatment for 7 d. Clinical efficacies as well as the levels of serum hs-CRP and NT-proBNP before and after treatment were observed in 2 groups,and the occurrence of ADR was compared between 2 groups. RESULTS:Total response rate of observation group(92.50%)was significantly higher than that of control group(75.00%),with statistical significance(P<0.05). Before treat-ment,there was no statistical significance in the serum levels of hs-CRP or NT-proBNP between 2 groups(P>0.05). After treat-ment,the serum levels of hs-CRP and NT-proBNP in 2 groups were decreased significantly,and the observation group was signifi-cantly lower than the control group,with statistical significance(P<0.05). There was no statistical significance in the incidence of ADR between 2 groups(P>0.05). CONCLUSIONS:Meglumine adenosine cyclophosphate shows significant therapeutic efficacy for acute myocardium infarction,reduces serum levels of hs-CRP and NT-proBNP significantly with good safety.

Objective To observe the changes of microRNA‐214 (miR‐214) expression in rat pulmonary artery smooth mus‐cle cells (PASMCs) induced by different hypoxia time ,and lay the foundation to explore the effect and mechanism of regulation of miR‐214 on PASMCs proliferation .Methods The primary cultured PASMCs were cultured under hypoxic 0 h ,6 h ,12 h ,24 h ,48 h ,respectively .The real time quantitative PCR was used to detect miR‐214 expression in each group PASMCs .Results The ex‐pression of miR‐214 in hypoxia group PASMCs was sustained as time increased ,apart from hypoxic hypoxia 6h group and 0h group ,the expression of miR‐214 was no significant difference (P>0 .05);the expression of miR‐214 among other groups PASMCs was significantly different (P<0 .05) .Conclusion The expression of miR‐214 in PASMCs increased after induction of hypoxia .We speculated that miR‐214 may be involved in the regulation of hypoxia induced PASMCs proliferation .

BACKGROUND:The research of dental stem cells in the fields of regenerative medicine and tissue engineering has been deepening,bringing hope for the repair of tooth-related tissues and the treatment of systemic diseases.However,there is a lack of systematic research and analysis on the biological characteristics of dental stem cells in different age groups. OBJECTIVE:To explore the biological characteristics of the human deciduous tooth and permanent tooth pulp stem cells cultured in umbilical cord blood platelet lysate to provide a reliable basis for human platelet lysates to replace fetal bovine serum. METHODS:The pulp tissues of deciduous teeth,juvenile permanent teeth and adult permanent teeth were taken out and cultured in DMEM/F-12 medium supplemented with 10%fetal bovine serum or different concentrations(5%,10%and 15%)of human platelet lysates.Cell proliferation in the four groups was detected by cytometry.The optimal concentration of human platelet lysates was selected for subsequent experiments.Under the optimal concentration of human platelet lysates,human deciduous tooth and juvenile and adult permanent tooth pulp stem cells were cultured in vitro.The cell growth status was observed under the microscope.The specific antigen on the cell surface was detected by flow cytometry.The cell proliferation ability was tested by the cell counting method and CCK-8 assay.The cell differentiation ability in vitro was observed by a three-line differentiation assay. RESULTS AND CONCLUSION:(1)The cell proliferation rate of the 10%human platelet lysate group was the highest.(2)In all three groups,fusiform fibrous cells grew and expanded from around the tissue block.There was no significant difference between deciduous teeth and juvenile permanent tooth cells,but the adult permanent tooth cells were larger than the deciduous and juvenile permanent tooth cells of the same generation.(3)The results of flow cytometry showed that deciduous teeth,juvenile permanent teeth and adult permanent teeth conformed to the phenotypic characteristics of mesenchymal stem cells.(4)The proliferative capacity of adult permanent dental pulp stem cells was significantly lower than those of deciduous teeth and juvenile permanent dental pulp stem cells(P<0.01).(5)mRNA expressions of osteoblast-related genes alkaline phosphatase and bone morphogenetic protein 2,lipoprotein lipase and peroxisome proliferator-activated receptor γ2,mRNA expressions of chondroblast related gene type II collagen α1 and cartilage oligomeric matrix protein in adult pulp stem cells of permanent teeth were significantly lower than those of deciduous teeth and juvenile permanent teeth pulp stem cells(P<0.01).(6)Compared with adult dental pulp stem cells,human deciduous teeth and juvenile permanent teeth dental pulp stem cells have the stronger proliferative capacity and multidirectional differentiation potential,and are more suitable for clinical research and disease treatment.

Resource utilization is an effective way to cope with the rapid increase of kitchen waste and excess sludge, and volatile fatty acids produced by anaerobic fermentation is an important way of recycling organic waste. However, the single substrate limits the efficient production of volatile fatty acids. In recent years, volatile fatty acids produced by anaerobic co-fermentation using different substrates has been widely studied and applied. In this paper, we analyze the characteristics of fermentation to produce acid using kitchen waste and excess sludge alone or mixture. Influences of environmental factors and microbial community structure on the type and yield of volatile fatty acids in the anaerobic fermentation system are discussed in detail. Moreover, we propose future research directions, to provide a reference for recycling kitchen waste and excess sludge.
Anaerobiosis ; Bioreactors ; Fatty Acids, Volatile ; Fermentation ; Hydrogen-Ion Concentration ; Microbiota ; Organic Chemicals ; Sewage