AIM:To investigate the effect of the N-terminal 24-amino acid (N24) overexpression in p55γre-gulatory subunit of phosphatidylinositiol 3-kinase ( PI3K) on the cell adhesion of human gastric carcinoma cell MGC 803. METHODS:MGC803 cells, which stably expressed GFP-N24 fusion protein and GFP alone , were generated by transfec-tion with pEGFPN-24 plasmid and control plasmid pEGFP-C1, respectively.The morphological change of the cells was ob-served under inverted microscope .The expression of GFP-N24 fusion protein was detected by Western blot .The adhesion of the cells was determined by cell adhesion assay .The effects of GFP-N24 fusion protein on the expression of E-cadherin andβ-catenin were analyzed by Western blot .The expression and secretion of matrix metalloproteinase 9 (MMP9) and u-rokinase-type plasminogen activator ( uPA ) in the MGC803 cells were detected by gelatin zymography .RESULTS:MGC803/GFP-N24 cell line steadily expressed GFP-N24 fusion protein and MGC803/GFP cell line steadily expressing GFP were successfully established , but the expression of fusion protein GFP-N24 was lower than that of the control protein GFP.The morphological changes of the transfected cells from paving stone to fibroblast cell form after gene transfection , and the cytoplasm secretory granules were increased significantly .The cell adhesion to fibronectin and collagen decreased after GFP-N24 transfection .GFP-N24 fusion protein increased the expression of cell adhesion molecule E-cadherin and de-creased the wnt signal pathway molecule β-catenin in the MGC803 cells.However, it did not affect the expression and se-cretion of tumor metastasis-related proteins MMP9 and uPA.CONCLUSION:Overexpression of N24p55γinhibits cell ad-hesion by influencing the expression of E-cadherin and β-catenin in the MGC803 cells.

Objective To evaluate the effect of electro-acupuncture pretreatment on endoplasmic reticulum stress during global cerebral ischemia-reperfusion (I/R ) in rats .Methods One hundred and forty-four adult male Sprague-Dawley rats ,weighing 400-500 g ,were randomly divided into 3 groups ( n=48 each) using a random number table:sham operation group (group S ) , I/R group and electro-acupuncture pretreatment group (group EA) .Global cerebral I/R was induced by 4-vessel occlusion method .Bilateral vertebral arteries were permanently occluded by cauterization and bilateral carotid arteries were occluded for 5 min , followed by reperfusion .Electro-acupuncture of Dazhui and Baihui acupoints lasting for 30 min was performed once a day for 5 consecutive days starting from 5 days before ischemia in group EA .At 6 ,12 ,24 and 48 h of reperfusion ,12 rats were sacrificed in each group and hippocampi were removed for microscopic examination and for determination of apoptosis rate (by TUNEL ) and expression of growth arrest-and DNA damage-inducible gene 153 (GADD153 ) protein (by Western blot ) .Results Compared with group S ,the apoptosis rate was significantly increased and the expression of GADD153 protein was up-regulated at 6 ,12 ,24 and 48 h of reperfusion in I/R and EA groups ( P<0.05) .Compared with group I/R ,the apoptosis rate was significantly decreased and the expression of GADD 153 protein was down-regulated at 6 ,12 ,24 and 48 h of reperfusion in group EA ( P< 0.05) .The pathological changes were significantly attenuated in group EA as compared with group I/R .Conclusion Electro-acupuncture pretreatment can down-regulate the expression of GADD 153 protein , reduce endoplasmic reticulum stress , and decrease cell apoptosis ,thus attenuating global cerebral I/R injury in rats .

Objective:To study the expressions of serum levels of intedeukin-12 (IL-12),interferon-γ (IFN-γ),erythropoietin (EPO) and ferritin in patients with acute leukemia and its clinical significance.Methods:76 patients with acute leukemia who were treated in our hospital from July 2015 to July 2016 were selected as the observation group,including 31 cases of newly diagnosed group,25 cases of remission group and 20 cases of relapse group.And another 76 cases who had taken the physical examination in our hospital were selected as the control group.Then the levels of serum IL-12,IFN-γ,EPO and ferritin in patients were observed and compared between the two groups.Results:The levels of IL-12 and IFN-γ in the observation group were significantly lower than those of the control group,and the levels of EPO and ferritin were significantly higher than those of the control group (P ＜0.05).The levels of serum IL-12 and IFN-γ in the untreated group and the relapse group were significantly lower than those of the remission group [(84.21± 5.43)pg/mL,(98.7± 7.98)pg/mL VS(112.43± 10.21) pg/mL,(38.54± 3.56)pg/mL,(49.87± 4.02)pg/mL VS(108.32± 8.43)pg/mL](P ＜0.05),and the levels of EPO and ferritin were significantly higher than those of the remission group [(402.32± 42.31) mIU/mL (321.58± 30.21)mIU/mL VS (98.21 ± 9.45) mIU/mLM (653.21 ± 54.24) ng/mLM (512.87 ± 43.45)ng/mL VS (342.15 ± 25.12)ng/mL] (P＜0.05).Conclusion:The serum levels of IL-12 and IFN-γ in patients with acute leukemia were lower,and the expression of EPO and ferritin was higher,and the disease and prognosis could be evaluated by monitoring the changes of these indexes.

OBJECTIVETo explore the effect of zoledronate (ZOL) on the osteoclast adhesion and expression of integrin α(v) and β3 in vitro.

METHODSMice RAW264.7 cells were used for osteoclast differentiation in vitro, and osteoclastogenesis was examined by tartrate-resistant acid phosphatase (TRAP) staining and dentin resorption lacunae examination. The cells were then divided into 2 groups, the control group and ZOL treatment group (treated with 1 x 10(-6) mol · L(-1) ZOL for 2 d). The adhesion ability of osteoclasts and mRNA and the protein expressions of integrin α(v) and β3 were examined by crystal violet staining, real-time fluorescence quantitative polymerase chain reaction, Western blot analysis, and immunofluorescent chemistry.

RESULTSTRAP staining and dentin resorption lacunae examination revealed the formation of multi-nuclear osteoclasts. ZOL treatment significantly decreased the adhesion ability of osteoclasts (P < 0.01). In the ZOL-treated group, the mRNA levels of integrin α(v) and β3 were 0.66 ± 0.05 and 0.59 ± 0.08, respectively. In the control group, the mRNA levels of integrin α(v) and β3, were 1.01 ± 0.01 and 1.01 ± 0.02, respectively; these values were higher than those in the ZOL-treated group (P < 0.01). The protein level of integrin α(v) and β3 in the ZOL-treated group (31,934.84 ± 112.91 and 18,812.79 ± 194.13) was downregulated by approximately 39.19% and 40.17%, respectively, compared with those in the control group (52,517.81 ± 211.72 and 31,441.93 ± 456.87) (P < 0.01). Immunofluorescent examination showed that the fluorescent intensities of integrin α(v) and β3 in the ZOL-treated group (9.491 ± 0.748 and 4.744 ± 0.759) were also significantly decreased compared with those in the control group (15.159 ± 1.143 and 11.418 ± 1.095) (P < 0.01).

CONCLUSIONZOL significantly inhibits osteoclast adhesion and downregulates integrin α(v) and β3, expression, thus contributing to the ZOL-induced inhibition of osteoclast- mediated bone resorption.

Animals ; Bone Resorption ; Diphosphonates ; Gene Expression ; Imidazoles ; Integrin alphaV ; Mice ; Osteoclasts ; RNA, Messenger

ObjectiveTo investigate the correlation of hippocampal synaptic plasticity with spatial learning and memory under normal and pathological condition,and provide experimental evidence for the coincidence of hippocampal late-phase long-term potentiation (L-LTP) and behavioral experiments.Methods 38 SD rats were randomly divided into two groups,control and AD model.First,Morris water maze was used to test the ability of spatial learning and memory of rats.The escape latencies for rats to search for an underwater platform in 5 days of navigation tests and the swimming time percentage in target qtuadrant on the 6th day after withdrawing the platform in probe trails were recorded.Then,in vivo hippocampus L-LTP of field excitatory postsynaptic potential (fEPSP)in CA1 region was recorded after delivering high frequency stimulation (HFS).ResultsBilateral intrahippocampal injection of 4 nmol amyloid β peptide ( Aβ 25-35 ) significantly impaired spatial learning and memory of rats in water maze tests,as well as in vivo hippocampal L-LTP.In control group,there was a significant negative correlation between the amplitude of fEPSP and the escape latency ( r =-0.8306,P ＜ 0.01 ) and a significant positive correlation between the amplitude of fEPSP and the swimming time percentage in target quadrant ( r=0.7709,P＜0.01 ).In AD model group,similar correlations were found,with a correlation coefficient of r =-0.7675 (P ＜0.01 ) and r =0.8049 (P ＜ 0.01 ),respectively.When putting all data from the two groups together,the hippocampal L-LTP was more correlated with escape latency ( r =-0.9124,P ＜ 0.01 ) and swimming time percentage ( r=0.9745,P＜0.01).ConclusionThere is very close correlation between the hippocampal L-LTP and the spatial learning and memory behavior in rats,suggesting that the hippocampal L-LTP may be involved in the electrophysiological mechanism of spatial learning and memory in rats,and the impairment of L-LTP could partly represent the deficits in cognitive function of animals.

BACKGROUND:Studies have shown that bisphosphonates inhibit osteoclast resorption, but whether cathepsin K, a key cytokine of bone resorption, plays an effect has rarely been reported.
OBJECTIVE:To study the effect of bisphosphonate on capthesin K and bone resorption function during osteoclast differentiation.
METHODS:Osteoclasts were cultured by mouse monocyte-macrophage cellline-RAW264.7. The cells were divided into two groups:control group, treated with 100μg/L receptor activator of nuclear factorκB ligand factor;alendronate group, treated with 100μg/L receptor activator of nuclear factorκB ligand factor+10-7 mol/L alendronate. Osteoclastogenesis and resorption function of osteoclasts were examined at 7 days of culture and gene expression of capthesin K was detected by immunofluorescence method at 72 hours of culture. Western blot assay was used to detect capthesin K protein expression at 72 hours of culture.
RESULTS AND CONCLUSION:Tartrate-resistant acid phosphatase positive multinuclear cells were observed and resorption lacunae formed in two groups. Control group showed the higher number of tartrate-resistant acid phosphatase positive multinuclear cells and larger size of resorption lacunae than the alendronate group (P<0.01). Immunofluorescence showed expression of capthesin K was higher in the control group than the alendronate group (P<0.01);furthermore, the protein expression of capthesin K was also lower in the alendronate group than the control group (P<0.01). These findings indicate that bisphosphonates could strongly inhibit osteoclastogenesis and its resorption function by inhibiting gene expression of capthesin K.

BACKGROUND:Tartrate-resistant acid phosphatase is a specific marker for osteoclast differentiation and bone resorption, which is a sign of osteoclast maturity.
OBJECTIVE:To study the effect of alendronate on tartrate-resistant acid phosphatase related to osteoclast differentiation and bone resorption.
METHODOsteoclasts were cultured by mouse monocyte-macrophage cellline-RAW264.7. The cells were divided into two groupcontrol group, treated with 100μg/L receptor activator of nuclear factorκB ligand factor;alendronate group, treated with 100μg/L receptor activator of nuclear factorκB ligand factor+10-7 mol/L alendronate. Osteoclastogenesis and resorption function of osteoclasts were examined at 7 days of culture. Gene expression of tartrate-resistant acid phosphatase was detected by immunofluorescence method. Western blot assay was used to detect protein expression of tartrate-resistant acid phosphatase.
RESULTS AND CONCLUSION:Tartrate-resistant acid phosphatase positive multinuclear cells were observed and resorption lacunae formed in two groups. Control group showed the higher number of tartrate-resistant acid phosphatase positive multinuclear cells and larger size of resorption lacunae than the alendronate group (P<0.01). Immunofluorescence showed expression of tartrate-resistant acid phosphatase was higher in the control group than the alendronate group (P<0.01);furthermore, the protein expression of tartrate-resistant acid phosphatase was also lower in the alendronate group than the control group (P<0.01). These findings indicate that bisphosphonates could strongly inhibit osteoclastogenesis and its resorption function by inhibiting protein expression of tartrate-resistant acid phosphatase.

BACKGROUND: During the process of isolating and culturing neural stem cells, trypsin is used to digest tissues and cells, but it is difficult to control the digestion time.OBJECTIVE: To isolate and cultivate neural stem cells from fetal brain of Kunming mice by trypsinization combined with mechanical isolation and to identify by the immunohistochemical method.DESIGN, TIME AND SETTING: In vitro observation of cytology. The experiment was performed at Basic Medical Laboratory of Guangxi Medical University from October 2006 to September 2007.MATERIALS: Kunming mice of pregnant 14 to 16 days were provided by the Laboratory Animal Center of Guangxi Medical University.METHODS: Brain tissues were isolated from the fetal brain of Kunming mice, pipettad mechanically after trypsinization, and incubated in serum-free DMEM/F12 medium containing B27, basic fibroblast growth factor and epidermal growth factor.MAIN OUTCOME MEASURES: Immunohistochemistry was performed to identify the cultured neural stem cells.RESULTS: After incubation in serum-free DMEM/F12 medium for 24 hours, the cells were suspensions with the manner of little neurospheres. After incubation for 48 hours, the cultured cells grew much larger, formed typical neurospheres, with no marked process, and survived well after passaged. In addition, immunocytochemical method showed nestin-positive.

Objective:To explore the influence of new labor standards on the indications in the birth process and the prognosis of mothers and infants.Methods:186 cases treated in our hospital from January,2015 to January,2016 were divided into the observation group (85 cases) and the control group (101 cases),the observation group received new labor standards,the control group adopt Friedman labor standards.The clinical indications,pregnant complications,pregnant outcome,neonatal-perinatal outcome were compared between two groups.Results:The cesarean delivery rate,number of using oxytocin,forceps delivery rate of observation group were significantly lower than those of the control group(P＜0.05);the duration time of both first and second stage of labor were obviously longer than those of the control group (P＜0.05);the duration time of active phase,bleeding volume in birth process in both groups showed no statistical difference (P＞0.05);there was no adverse maternal and infant events in both groups;the incidence rate of pregnancy complications,fetal distress in uterus,asphyxia neonatorum and neonatal body weight were of no statistical difference (P＞0.05).Conclusion:The new labor standards prolong the duration time of birth and give women fully trial opportunities,could effectively reduce the rate of cesarean section,reduce the over intervention production.

OBJECTIVETo investigate the effect of alendronate on the expressions of osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) in mouse osteoblasts.

METHODSMouse calvarial osteoblasts cultured in vitro were identified by alkaline phosphatase (ALP) staining and immunofluorescence assay of OPG and RANKL expressions. The second passage of the osteoblasts were treated with different concentrations of alendronate (10(-4) to 10(-7) mol/L) for 48 h, and the changes in OPG and RANKL mRNA and protein expressions were examined using real-time PCR and Western blotting, respectively.

RESULTSThe isolated osteoblasts were positive for ALP and expressed OPG and RANKL. Real-time PCR and Western blotting showed that at the concentration of 1×10(-4) mol/L, alendronate caused an obvious down-regulation of OPG and RANKL expressions in the cells, whereas at lower concentrations, alendronate increased the expressions of both genes with the highest expressions occurring after treatment with 1×10(-5) mol/L.

CONCLUSIONHigh concentrations of alendronate (>1×10(-4) mol/L) decrease the expressions of OPG and RANKL, whereas low concentrations (1×10(-5) to 1×10(-7) mol/L) increase their expressions in mouse osteoblasts cultured in vitro.

Alendronate ; pharmacology ; Animals ; Cells, Cultured ; Mice ; Mice, Inbred BALB C ; Osteoblasts ; drug effects ; metabolism ; Osteoprotegerin ; metabolism ; RANK Ligand ; metabolism