Objective:To clone the encoding sequence of human EBF3 gene,construct recombinant eukaryotic expression plasmid vector pEGFP/EBF3,and study the effect of EBF3 on HepG2 cell cycling.Methods:Total RNA was isolated from placental tissue.Full-length human EBF3 cDNA was amplified by RT-PCR,cloned into eukaryotic expression plasmid vector pEGFP-N1 and sequenced.The expression and sub-cellular localization of the fusion protein EBF3-EGFP in HepG2 cells were analyzed by Western blot.Cell cycles were analyzed with flow cytometry analysis.Results:Obtained full encoding sequence of early B cell factor 3 was identical with that included in GeneBank,and the eukaryotic expression plasmid vector pEGFP/EBF3 was constructed correctly.24 h after transfected by pEGFP/EBF3,the fusion protein EBF3-EGFP was observed mainly in the cellular nucleus under the inverted fluorescence microscope.Western blot analysis confirmed that the EBF3-EGFP fusion proteins of Mr 87 000 were detected in both cytoplasmic and nuclear protein of the HepG2 transfected by pEGFP/EBF3 for 24 h or 48 h.Flow cytometry analysis revealed that the percentage of cells in the S phase was markedly increased in HepG2 cells transfected by pEGFP/EBF3 as compared with that in pEGFP-N1 transfected cells.These findings suggested that transfection of EBF3 gene into HepG2 induced cell proliferation by increasing the number of cells from G1 phase to G2 phase.Conclusion:The recombinant eukaryotic expression plasmid vector pEGFP/EBF3 is successfully established.The percentage of cells in the S phase is markedly increased in pEGFP/EBF3 transfected cells as opposed to pEGFP-N1 transfected cells.It is likely that EBF3 promotes HepG2 cells proliferation through DNA replication.

Child ; Child, Preschool ; Computer Systems ; Humans ; Phylogeny ; Polymerase Chain Reaction ; methods ; Respiratory Syncytial Viruses ; genetics ; isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; methods

Objectives To establish real-time fluorenscence quantitative polymerase chain reaction ( RFQ-PCR ) for measurement of the expression level of guanylyl cyclase-C(GC-C) in the Peripheral blood mononuclear cell(PBMC)in 30 blood donors, 10 cases colorectal cancer tissue and 1 case T84 human colon cancer cell line. Methods Specific primers and TaqMan probe have been designed,and fluorenscence of the PCR product was detected continuously during amplification. According to the standard curve created by plasmid DNA, the expression level of GC-C in clinical samples has been determined using software, and the results were presented as the ratios of GC-C mRNA to?2-microgluobulin(?2M)mRNA. Results The detection range of the assay was from 101 pg/ml to109pg/ml,the coefficient of variation values of both intra-experimental and inter- experimental reproducibility were 6. 87% to 11. 12% and 8. 86% to 15. 19% . None of 30 blood donors and 11 benign intestinal patients expressed GC-C mRNA,it was expressed in 31/37 colorectal cancer patients. The expression level of GC-C mRNA in colorectal cancer patients was 0. 88?0.06,and the expression level of its in colorectal carcinoma tissue and T84 cells were 0. 86?0.07/ug tissue and 0.0082/per cell. Conclusions This assay had high sensitivity,specificity and reproducibility.

Objective To investigate the expression and clinical value of miR-127-3p in plasma of patients with breast cancer .Methods 80 cases of breast patients , 70 cases of benign breast tumor patients and 70 cases of normal control group were recruited .A real-time fluorescent quantitative reverse transcription polymerase chain reaction ( RT-PCR ) method for detecting miR-127-3p was established; Liner, reproducibility, and specificity were evaluated.In addition, correlations between the relative expression of plasma miR-127-3p and the concentrations of CEA and CA 153 were assessed.the relationship of miR-127-3p expression and clinicopathological features was further determined by Mann-Whitney test.Results The method for detection of plasma miR-127-3p was established.The relative plasma expression of miR-127-3p in breast patients [ 10.561 ( 5.424 -16.465 ) ] was significantly higher than that in benign breast tumor patients [3.015 (1.987-5.035)] (P=0.000 6) and healthy controls [2.375 (1.173-4.370)] (P=0.000 2).However, there was no significant difference between benign tumor patients and healthy control group (P=0.143).Positive relationship was found between the relative expression of miR-127-3p and the concentration of CA153 (R2 =0.457, P=0.003).The area under the ROC curve (AUC) of miR-127-3pwas 0.763, which was higher than that of CEA and CA 153.No significant difference was found between plasma miR-127-3p expression and clinicalpathological features including tumor size , differentiation and tumor node metastasis stage (P>0.05).Conclusions miR-127-3p was increased in breast cancer patients and may be an important diagnostic index for breast cancer .

Objective To explore the effectiveness and adverse effect of high frequency oscillation ventilation (HFOV) combined with Sildenafil (SIL) treatment on newborns with persistent pulmonary hypertension (PPHN).Methods A total of 89 cases of PPHN infants collected from Chengdu Women and Children's Central Hospital from Sep.2010 to Sep.2012 were randomly divided into HFOV group,constant mechanical ventilation (CMV) group,HFOV combined SIL group (HFOV + SIL group) and CMV combined with SIL group (CMV + SIL group).The arterial blood gas,pulmonary artery pressure (PAP) and adverse reactions were monitored before and 3 days after treatment.SNK multiple comparison method andx2 test were performed for data before and after treatment among groups for continuous variables and categorical variables,respectively.Results The levels of pa (O2) [(79.1 ± 13.7) mmHg (1 mmHg =0.133 kPa),(77.9 ±14.6) mmHg,(85.4 ±15.2) mmHg],Sa(O2) [(87.8 ±13.4)％,(88.4±15.6)％,(96.1±15.9)％],pa(CO2)[(42.5±11.3) mmHg,(40.2 ±10.5) mmHg,(35.6 ±8.7) mmHg] and PAP [(31.1 ± 8.1) mmHg,(30.4 ± 9.5) mmHg,(25.8 ± 7.3) mmHg] were all improved significantly in CMV + SIL group,HFOV group and HFOV + SIL group compared with those in CMV group[(69.9 ± 12.3) mmHg,(81.1 ± 14.9)％,(48.1 ±9.5) mmHg,(35.6 ±8.9) mmHg] (F =4.629 3,3.673 2,5.865 3,4.849 5,P ＜0.05),especially for HFOV + SIL group(P ＜ 0.05).No significant difference in such indicators was observed between CMV + SIL group and HFOV group (P ＞ 0.05).The effective rate in HFOV + SIL group (90％) was the highest among the 4 groups (x2 =7.938,P ＜ 0.05).During the treatment,all neonates have no adverse reaction.Conclusion The combined use of SIL and HFOV might be a more effective and safer method in the treatment of PPHN of neonate.

Objective To investigate the clinical characteristics of postinfectious irritable bowel syndrome (PI-IBS) in Qingdao. Methods Two hundred and four PI-IBS and 2068 non-PI-IBS patients were investigated with questionnaire including general information, symptoms and quality of life scores with microecological study before and after therapy. Results (1) The morbidity rate of PI-IBS in female was 2. times of that in male, which was similar to that in non-PI-IBS. (2) Brainwork labors dominated in both PI-IBS and non-Pl-lBS patients. (3) As to the simultaneous presence of extra-gastrointestinal symptoms,there was no statistical difference between the rate of physical symptoms in PI-IBS and non-PI-IBS patients (X<'2>= 10. 5, P>0.05) ,but the rate of mental symptoms was higher in PI-IBS than in non-PI-IBS patients, and the difference was significant(X<'2>= 28.7, P<0.05). (4)The alteration of intestinal microflora rate in PI-IBS was obviously higher than that in non-PI-IBS patients. (5) The quality of life scores in PI-IBS was improved after treatment with Birid Triple Viable , and there was significant difference(t =3. 8, P<0.01),but there was no statistical difference in non-Pl-IBS (t = 1.5, P>0.05). Conclusion There was some difference in certain clinical characteristics between PI-IBS and non-PI-IBS patients in Qingdao.

AIM:To study the type, degree and axial distribution of low vision astigmatism in preschool children.METHODS:A group of 3-6 years old children were selected for astigmatism screening, and statistical analysis was performed on the detected 445 eyes of 308 people.RESULTS:With more than 0.50D astigmatism criteria, astigmatism examination of 308 people, accounting for 36.2%, of which 137 eyes astigmatism, astigmatism 171 monocular.The five types of astigmatism were compound hyperopia 40.7%, mixed 35.5%, compound myopia 8.5%, myopia 8.3%, simple hyperopia astigmatism degree 7.0%;69.0% were mild, 16.6% moderate, 14.4% severe.Astigmatism axial distribution was with the rule for 54.9%, against the rule 28.8%, oblique 16.6%.In binocular astigmatism eyes, axial symmetry was in 35.8%, asymmetry in 64.2%.CONCLUSION:The main type of astigmatism in preschool children are compound hyperopia and mixed astigmatism.Astigmatism degree is mainly mild.With the increase of age, the detection rate of moderate and high astigmatism increased.

Objective To investigate the effects of electroacupuncture on cell apoptosis(CA)and the ex- pression of insulin like growth factor 1(IGF_1)in the hippocampal CA1 region of rats'brains after cerebral ischemic- reperfusion(CIR).Methods Middle cerebral artery obturation(MCAO)was established by the suture embolic method.CA and the expression of IGF_1 in the hippocampal CA1 region were detected by immunohistochemical meth- ods and TUNEL staining,respectively.Results Compared with those in the normal and sham operation groups, apoptotic cells were significantly increased in the hippocampal CA1 region of the model group(P＜0.01),while the expression of IGF_1 was slightly enhanced and plasma staining was also slightly positive(P＜0.05).Apoptotic cells in the CA1 region in the electroacupuncture group were obviously fewer in comparison with the normal group(P＜0.01),while the expression of IGF_1 was distinctly increased and the plasma staining was also obviously positive(P＜0.01).Conclusion Electroacupuncture treatment has preventive and therapeutic effects on ischemia-reperfusion injury,and its mechanism might be related with up-regulating the expression of IGF_1 and inhibiting CA.

Objective To investigate the protective effects of electroacupuncture on the endothelial tissues of microvessels in the basal ganglia in the rats with cerebral ischemia-reperfusion.Methods The MCAO model was established by using Longa's method.The immunohistochemistry SABC(strepto-avidin-biotin-peroxidase complex) method was employed to detect the expression of ICAM-1,P-selectin in the microvessel of rats'ipsilateral basal gan- glia.Results The number of positive ICAM-1 and P-selectin endothelial cells of model group were significantly in- creased,as compared to normal group and sham operated group(P

Objective To explore the mechanism of lupeol in the treatment of rheumatoid arthritis(RA)based on network pharmacology and molecular docking.Methods The target of lupeol was obtained by Swiss and TCMSP analysis platform,and the RA related target was obtained by searching"rheumatoid arthritis"in GeneCards database,which was transformed into the corresponding standardized gene name by UniProt database.The common targets of lupeol and RA were mapped by R-package,and the cross genes were obtained.The cross genes were introduced into the string database to construct protein protein interactions(PPI)network.The clusterProfiler database was used to analyze the enrichment of GO and KEGG pathways.The binding of lupeol with AR,CASP3 and CCNB1 was evaluated by molecular docking.The RA mouse model was established and the foot volume of each group was measured.HE staining for pathological changes,ELISA kit for detecting mouse serum TNF-α,IL-1β and IL-6 expression levels.The protein expression levels of Bcl-2,Bax,CASP3 and CASP9 were detected by Western blot.Results Forty targets of lupeol,4734 related targets of RA disease and 27 common targets of lupeol RA were obtained.The top three genes of PPI network were AR,CASP3 and CCNB1.291 GO and 20 KEGG pathways were enriched.Molecular docking showed that lupeol had good affinity with AR,CASP3 and CCNB1.The foot volume of the model group was significantly higher than that of the normal control group on the 8th,12th,16th and 20th day(P<0.05).Compared with the model group,the foot volume ofthe lupeol group was significantly lower on the 8th,12th,16th and 20th day(P<0.05),andthat ofthe diclofenac group was significantly lower onthe 12th,16th and 20th day(P<0.05).The HE staining results showed that compared with the model group,the lupine alcohol drug group significantly improved the pathological state.Compared with the model group,the level of TNF-α,IL-1β and IL-6 in serum of mice in the Lupeol drug group decreased(P<0.01).WB results showed that compared with the normal group,the protein expressions of Bax,CASP3 and CASP9 in the model group were significantly decreased(P<0.05),and the protein expression of Bcl-2 was significantly increased(P<0.05).Compared with the model group,the protein expression of Bax,CASP3 and CASP9 in the positive control group and drug group were significantly up-regulated(P<0.05),and the protein expression of Bcl-2 was significantly down regulated(P<0.05).Conclusion Lupeol plays a therapeutic role in RA by regulating Bax,Bcl-2,Casp3 and Casp9 in the p53 signaling pathway.