Objective To study the effects of ?-catenin-dependent lymphoid enhancer factor(LEF-1) isoforms on biological behavior of HeLa cells.Methods ?-catenin-dependent LEF-1 genes were obtained by PCR from human lymphoid node cDNA library and inserted into pcDNA3.1/V5-His vector to construct the eukaryotic expression plasmid pcDNA3.1-F-LEF-1.Using lipofectamineTM 2000,the plasmid pcDNA3.1-F-LEF-1 was transfected into Hela cells.Then we screened the stable cell lines that expressed the truncated LEF-1 isoforms by G418 and identified the expression of target gene with Western blot.Then we analyzed the proliferation,apoptosis,cell clone formation and capability of tumor formation in vivo of transfected cell lines.Results We successfully constructed the ?-catenin-dependent LEF-1 eukaryotic expression plasmid and obtained the stable HeLa cell lines that expressed the full-length LEF-1 isoforms.The proliferation and capability of tumor formation in vivo of transfected cells were increased while apoptosis was decreased.Conclusion The overexpression of ?-catenin-dependent isoforms can stimulate the malignant biological behavior of HeLa cells.

Objective To investigate the effects of four interior-warming drugs( galangal,cinnamon,evodia rutaecarpa,and dried ginger)on the tension of ileum smooth muscle and Ca2+-ATPase on the cell membrane in rabbits. Methods The effects of galangal,cinnamon,evodia rutaecarpa,and dried ginger were examined on normal ileum smooth muscle,in vitro intestinal muscle contraction caused by acetylcholine(ACh),barium chloride(BaCl2 )and histamine(His), and ACh-induced calcium release by using BL-420E+ biological signal collection and processing system.The average tension was measured within 1 min before delivery and within 3 minutes after the treatment,and the inhibition rate was calculated according to the average tension value.The effects of sera containing galangal,cinnamon,evodia rutaecarpa,and dried ginger on Ca2+-ATPase activity on the cell membrane of the intestinal smooth muscle were examined by phosphorus method. Results Galangal,cinnamon,evodia rutaecarpa,and dried ginger at high concentrations could restrain in vitro intestinal contraction in normal circumstances(P<0.05 or P<0.01).Significant inhibitory effects on intestinal contraction caused by ACh,His and BaCl2 were found in low,medium and high concentration groups(P<0.01).There was a dose-effectiveness relationship between the inhibition rate and final drug concentrations.The ACh-induced intracellular and extracellular calcium dependent contraction were significantly inhibited by the four interior-warming drugs( P < 0. 05 or P < 0. 01). The Ca2+-ATPase activities were( 0. 384 ± 0.070),(0.302±0.016),(0.307±0.016),(0.296±0.016),(0.313±0.003)U·mg-1 ,respectively,in intestinal smooth muscle in normal control group and high concentration groups of galangal,cinnamon,evodia rutaecarpa,and dried ginger(P<0.01). Conclusion Interior-warming drugs may relax intestinal smooth muscle by reducing the intracellular calcium release and the extracellular calcium inflow via receptor-controlled calcium channels,and inhibiting the Ca2+-ATPase activity in smooth muscle.

Objective:To investigate the effect of pidotimod in reducing pulmonary infection in patients with lung cancer undergoing chemotherapy.Methods:One hundred and twenty patients with lung cancer in the Fifth People′s Hospital of Dalian City from July 2017 to July 2018 were selected. The patients were divided into control group and pidotimod group by random digits table method with 60 cases each. The patients were treated with standard two drugs chemotherapy containing platinum drug according to the pathological type, and the patients in pidotimod group were combined with pidotimod. The number of pulmonary infections during chemotherapy, number of completed scheduled chemotherapy and adverse reaction were observed. The correlation between pulmonary infection and pidotimod was analyzed by multivariate orderly Logistic regression.Results:The incidence of pulmonary infection in pidotimod group was significantly lower than that in control group: 18.33% (11/60) vs. 40.00% (24/60), and there was statistical difference ( χ2 = 6.845, P<0.01). The rate of completed scheduled chemotherapy in pidotimod group was significantly higher than that in control group: 55.00% (33/60) vs. 36.67% (22/60), and there was statistical difference ( χ2 = 4.062, P<0.05). Multivariate orderly Logistic regression analysis result showed that pidotimod could reduce the risk of pulmonary infection ( OR = 0.210, 95% CI 0.072 to 0.606, P = 0.004), and help to complete the scheduled chemotherapy ( OR = 2.323, 95% CI 1.080 to 5.003, P = 0.031). In pidotimod group, no obvious adverse reaction related to pidotimod application was detected, and chemotherapy was not affected. Conclusions:Application of pidotimod can reduce the chance of pulmonary infection in patients with lung cancer undergoing chemotherapy and help patients complete scheduled chemotherapy.

With the rapid development of genome sequencing technology and bioinformatics in recent years,it has become possible to measure thousands of omics data which might be associated with the progress of diseases,i.e."high-dimensional data".This type of omics data have a common feature that the number of variable p is usually greater than the observation cases n,and often has high correlation between independent variables.Therefore,it is a great statistical challenge to identify really meaningful variables from omics data.This paper summarizes the methods of Bayesian variable selection in the analysis of high-dimensional data.

Objective: To analyze the expression of c-MET and its prognostic correlation in patients with lung adenocarcinoma. Methods: The clinical data and pathological specimen of patients with lung adenocarcinoma in Dalian 5th People′s Hospital from January 2006 to December 2011 were retrospectively analyzed. The expression difference of c-MET between lung adenocarcinoma tissue and normal adjacent tissues was compared. The correlation of c-MET with the pathology and clinical factors was also analyzed. Results: A total of 82 patients were retrospective analyzed, including 82 pathological specimens of lung adenocarcinoma and 45 specimens of normal adjacent tissues. Among 53 patients with stage Ⅰ-Ⅱ lung adenocarcinoma, 31 cases had low expression of c-MET and 22 cases had high expression of c-MET. Among 29 patients with stage Ⅲ lung adenocarcinoma, 10 cases had low expression of c-MET and 19 cases had high expression of c-MET. There was a correlation between TNM stage and c-MET positive expression in lung adenocarcinoma (P=0.037). The positive expression rate of c-MET was not significantly correlated with age, sex and differentiation degree (P > 0.05). Among 82 cases of lung adenocarcinoma, 39 cases had low expression of c-MET and 43 cases had high expression of c-MET; 45 cases of para-carcinoma tissuehad low expression of c-MET. The positive expression rate of c-MET in lung adenocarcinoma tissues was significantly higher than that in para-carcinoma tissue (P < 0.01). Kaplan-Meier survival curve analysis showed that the median disease-free survival was 41.5 months in c-MET high-expression group and 55.3 months in low-expression group. The expression level of c-MET was closely related to disease-free survival in lung adenocarcinoma patients (P < 0.05). Conclusions The expression of c-MET is significantly increased in patients with lung adenocarcinoma. The expression level has relevance with TNM staging and prognosis.

OBJECTIVETo detect the expressions of CXCR4 and Nrf2 in non-small cell lung cancer (NSCLC) tissues and analyze their association with the clinicopathological features of NSCLC.

METHODSWe investigated the expressions of CXCR4 and Nrf2 in 66 NSCLC and corresponding distant normal tissue specimens using immunohistochemistry and real-time PCR.

RESULTSThe expressions of CXCR4 protein and mRNA were significantly higher in NSCLC tissue specimens than in the distant normal tissues, while the expression of Nrf2 protein and mRNA increased significantly in NSCLC tissues compared to those in the distant normal tissues (P<0.01). A high expression level of CXCR4 was positively correlated with a large tumor size (P=0.048), poor differentiation (P=0.024), advanced TNM stage (P=0.018), lymph node metastasis (P=0.004), and distant metastasis (P=0.016). The expression of Nrf2 protein was positively correlated with a large tumor size (P=0.008), advanced TNM stage (P=0.028), lymph node metastasis (P=0.038), and distant metastasis (P=0.023). A strong correlation was found between CXCR4 and Nrf2 expressions in NSCLC tissues (r=0.324, P<0.01), and the co-expression of CXCR4 and Nrf2 was strongly correlated with lymph node metastasis and distant metastasis.

CONCLUSIONAbnormal expressions of CXCR4 and Nrf2 may contribute to the progression and malignant biological behavior of NSCLC.

Carcinoma, Non-Small-Cell Lung ; metabolism ; pathology ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Lung Neoplasms ; metabolism ; pathology ; Lymphatic Metastasis ; Male ; Middle Aged ; NF-E2-Related Factor 2 ; metabolism ; Neoplasm Staging ; Receptors, CXCR4 ; metabolism ; Signal Transduction

ObjectiveTo observe the effect of Jianpi Yangzheng Xiaozheng decoction （JYXD） on the proliferation and stemness of the human gastric cancer （GC） cell line HGC-27 by inhibiting aerobic glycolysis， and explore the underlying mechanism. MethodMethyl thiazolyl tetrazolium （MTT） assay was employed to determine the survival rate and chemotherapy sensitivity of HGC-27 cells treated with JYXD （0.25， 0.5， 1， 2， 4， 8， 16， 32 g·L^-1）. Colony formation assay was employed to detect the effect of JYXD （2， 4， 8 g·L^-1） on the colony formation of the cells. The aerobic glycolysis level of HGC-27 cells after treatment with JYXD was measured by glucose assay kit and lactic acid assay kit. The proportion of stem cell subsets in HGC-27 cells was detected by flow cytometry. Western blot was employed to determine the expression of glycolysis-associated proteins such as lactate dehydrogenase （LDH）， hexokinase 2 （HK2）， glucose transporter 1 （GLUT1）， and pyruvate kinase isozyme M2 （PKM2）， and the expression of stemness-associated proteins such as octamer-binding transcription factor 4 （OCT4）， SRY-box transcription factor 2 （SOX2）， and Nanog. ResultJYXD （0.5， 1， 2， 4， 8， 16， 32 g·L^-1） inhibited the activity of HGC-27 cells （P<0.05， P<0.01）， with the inhibitory concentration 50（IC₅₀） of 4.83 g·L^-1， and it improved the sensitivity of HGC-27 cells to cisplatin chemotherapy. Compared with the control group， JYXD （2， 4， 8 g·L^-1） reduced the colony formation number of HGC-27 cells （P<0.01） in a concentration-dependent manner. Flow cytometry showed that compared with that in the control group， the proportion of CD44⁺CD24⁺ALDH⁺ population in the cells treated with JYXD （2， 4， 8 g·L^-1） decreased （P<0.05）. In addition， JYXD （2， 4， 8 g·L^-1） inhibited the glucose uptake and lactic acid production of HGC-27 cells. Western blot showed that compared with the control group， JYXD （2， 4， 8 g·L^-1） down-regulated the expression levels of SOX2， Nanog， OCT4， PKM2， LDH， GLUT1， and HK2 （P<0.05， P<0.01） in a concentration-dependent manner. ConclusionJYXD may inhibit the proliferation and reduce the stemness of HGC-27 cells by regulating the aerobic glycolysis.

Objective: To investigate the effects and mechanism of digoxin on atrium electrical remodeling and susceptibility of atrial fibrillation (AF) in aged rabbits. Methods: Twenty aged male New Zealand rabbits were divided into aged group and aged plus digoxin group (n=10 each). Electrical parameters including heart rate (HR), RR and QT interval, ST segment and P wave dispersion from normal Ⅱ electrocardiogram, and the maximum upstroke velocity (Max_dv/dt), plateau potential (plateau P), action potential duration of 10%, 20% and 90% (APD₁₀, APD₂₀, APD₉₀) from recording of monophasic action potential (MAP), as well as atrial effective refractory period (AERP₂₀₀) and dispersion (dERP₂₀₀) with 200 ms of basic cycle length (BCL), and frequency self adaptation of AERP with 300 ms and 150 ms of BCLs (fERP) were recorded and compared between the 2 groups. BCLs and inducibility of AF post programmed electrical stimulation and Burst-pacing in left atrium tissue of rabbits in vivo were also analyzed. The L-type calcium current (I_Ca-L) in 2 groups were recorded via whole-cell patch clamp technique, and the fluorescence intensity of intracellular free Ca²⁺ was detected with Flup-3/AM loading by the laser scanning confocal microscope in enzymatically dissociated single rabbit atrial myocytes. Results: Compared with aged group, the heart rate was faster, RR and QT interval were obvious shorter, ST segment was raised and P wave dispersion was significantly increased in aged plus digoxin group (all P<0.05). Moreover, compared with aged group, the Max_dv/dt and plateau P were obviously increased, APD₁₀ and APD₂₀ were significantly prolongated, and APD₉0 was significantly shorter in aged plus digoxin group (all P<0.01). Otherwise, the fERP was markedly increased (0.81±0.15 vs. 0.67±0.05), and the induced rate of AF was obviously higher in aged plus digoxin group than in aged group (6/8 vs. 4/9) (all P<0.01). With voltage clamp model, digoxin significantly increased I_Ca-L of atrial myocytes of aged rabbits, When command potential was 10 mV, the current densities of I_Ca-L were significantly higher in digoxin group than that in aged group ((15.45±2.38) pA/pF vs. (7.03±1.69) pA/pF, P<0.01). Otherwise, the I-V curve of I_Ca-L was downward shifted of all I-V curves in digoxin perfused aged atrial cells of rabbits. Moreover, the fluorescence intensities of intracellular free Ca²⁺ was significantly higher in aged plus digoxin group than in aged group ((1 748±173) μmol/L vs. (478.13±87.63) μmol/L, P<0.01). Conclusion Digoxin could aggravate the atrial electrical remodeling in atrium of aged rabbits, facilitate susceptibility of atrial fibrillation in aged rabbit, increased current density of I_Ca-L and concentration of intracellular free Ca²⁺, followed Ca²⁺ overload and oscillations might be part of the underlying mechanisms.

OBJECTIVE: To investigate whether the Runx2 gene can induce the differentiation of human amniotic mesenchymal stem cells (hAMSCs) to ligament fibroblasts in vitro and promote the tendon-bone healing in rabbits. METHODS: hAMSCs were isolated from the placentas voluntarily donated from healthy parturients and passaged, and then identified by flow cytometric identification. Adenoviral vectors carrying Runx2 gene (Ad-Runx2) and empty vector adenovirus (Ad-NC) were constructed and viral titer assay; then, the 3rd generation hAMSCs were transfected with Ad-Runx2 (Ad-Runx2 group) or Ad-NC (Ad-NC group). The real-time fluorescence quantitative PCR and Western blot were used to detect Runx2 gene and protein expression to verify the effectiveness of Ad-Runx2 transfection of hAMSCs; and at 3 and 7 days after transfection, real-time fluorescence quantitative PCR was further used to detect the expressions of ligament fibroblast-related genes [vascular endothelial growth factor (VEGF), collagen type Ⅰ, Fibronectin, and Tenascin-C]. The hAMSCs were used as a blank control group. The hAMSCs, hAMSCs transfected with Ad-NC, and hAMSCs were mixed with Matrigel according to the ratio of 1 : 1 and 1 : 2 to construct the cell-scaffold compound. Cell proliferation was detected by cell counting kit 8 (CCK-8) assay, and the corresponding cell-scaffold compound with better proliferation were taken for subsequent animal experiments. Twelve New Zealand white rabbits were randomly divided into 4 groups of sham operation group (Sham group), anterior cruciate ligament reconstruction group (ACLR group), anterior cruciate ligament reconstruction+hAMSCs transfected with Ad-NC-scaffold compound group (Ad-NC group), and anterior cruciate ligament reconstruction+hAMSCs transfected with Ad-Runx2-scaffold compound group (Ad-Runx2 group), with 3 rabbits in each group. After preparing the ACL reconstruction model, the Ad-NC group and the Ad-Runx2 group injected the optimal hAMSCs-Matrigel compunds into the bone channel correspondingly. The samples were taken for gross, histological (HE staining and sirius red staining), and immunofluorescence staining observation at 1 month after operation to evaluate the inflammatory cell infiltration as well as collagen and Tenascin-C content in the ligament tissues. RESULTS: Flow cytometric identification of the isolated cells conformed to the phenotypic characteristics of MSCs. The Runx2 gene was successfully transfected into hAMSCs. Compared with the Ad-NC group, the relative expressions of VEGF and collagen type Ⅰ genes in the Ad-Runx2 group significantly increased at 3 and 7 days after transfection ( P<0.05), Fibronectin significantly increased at 3 days ( P<0.05), and Tenascin-C significantly increased at 3 days and decreased at 7 days ( P<0.05). CCK-8 detection showed that there was no significant difference ( P>0.05) in the cell proliferation between groups and between different time points after mixed culture of two ratios. So the cell-scaffold compound constructed in the ratio of 1∶1 was selected for subsequent experiments. Animal experiments showed that at 1 month after operation, the continuity of the grafted tendon was complete in all groups; HE staining showed that the tissue repair in the Ad-Runx2 group was better and there were fewer inflammatory cells when compared with the ACLR group and the Ad-NC group; sirius red staining and immunofluorescence staining showed that the Ad-Runx2 group had more collagen typeⅠ and Ⅲ fibers, tending to form a normal ACL structure. However, the fluorescence intensity of Tenascin-C protein was weakening when compared to the ACLR and Ad-NC groups. CONCLUSION Runx2 gene transfection of hAMSCs induces directed differentiation to ligament fibroblasts and promotes tendon-bone healing in reconstructed anterior cruciate ligament in rabbits.
Pregnancy ; Female ; Humans ; Rabbits ; Animals ; Vascular Endothelial Growth Factor A/metabolism* ; Fibronectins/metabolism* ; Collagen Type I/genetics* ; Tenascin/metabolism* ; Collagen/metabolism* ; Anterior Cruciate Ligament/surgery* ; Mesenchymal Stem Cells ; Tendons/metabolism* ; Fibroblasts/metabolism*