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Objective:To prepare specific anti- Fusarium yolk immunoglobulin (IgY) and investigate its tolerance to temperature and pH and verify its antifungal effect against Fusarium. Methods:Eighteen 22-week-old Leghorn laying hens were selected and randomized into negative control group and experimental group according to the random number table method, with 9 hens in each group.The 2×10 7 colony forming units (CFU)/ml suspension of inactivated hyphae of Fusarium and Freund complete adjuvant was mixed in a 1∶1 ratio and emulsified.The hens in the experimental group were injected with 1 ml of the mixture to immunize and received 1 ml of Freund incomplete adjuvant as booster injection at two weeks after the initial injection.The egg yolk was collected from the 5th to 16th week after immunization.Specific anti- Fusarium IgY protein was prepared by salting out method using ammonium sulfate.The obtained protein solution was put into a freeze dryer and made into freeze-dried powder stored at 4 ℃.The hens in the negative control group were injected with 0.9% sodium chloride to prepare the non-specific antibody as the negative control.Bradford method was used to determine the concentration of specific egg IgY protein and the indirect enzyme-linked immunosorbent assay (ELISA) was employed to measure its titer.The 1×10 5 CFU/ml and 1×10 3 CFU/ml Fusarium suspension were cultured with different concentrations of IgY and phosphate buffered saline (PBS) for 4 days, and the absorbance value at 600 nm was measured.The co-incubated PBS/negative IgY with Fusarium solution was set as blank control/negative control accordingly.The concentration-killing curve of anti- Fusarium IgY against Fusarium was obtained.The specific IgY solution was diluted to 0.02 mg/ml with PBS pH 7.4, and the diluted specific IgY solution was placed into the water bath for 30 minutes at 30, 40, 50, 60, 70, 80, 90 ℃, respectively, and was cooled down to room temperature.The specific IgY solution was diluted to 0.02 mg/ml with PBS pH 1, pH 2, pH 3, pH 4, pH 5, pH 6, pH 7, pH 8, pH 9, pH 10, pH 11, pH 12, respectively, and the diluted specific IgY solution was placed at 4 ℃ for one hour.The activity of diluted specific IgY solution by different methods was measured by indirect ELISA, and the tolerance of IgY to various temperatures and pH was evaluated.Twelve 8-week-old SPF female C57BL/6 mice were selected and randomized into the PBS control group and specific IgY treatment group according to the random table method, with 6 mice in each group.The right eyes of the 12 mice were infected with Fusarium to establish mice model of fungal keratitis.One day after modeling, 200 mg/ml of anti- Fusarium IgY was dropped to the right eyes of mice in the specific IgY treatment group, and PBS was dropped to the right eyes of mice in the PBS control group.The corneas of mice in the two groups were observed under the slit lamp microscope at 1, 3 and 5 days following modeling, and the corneal ulcer was scored according to the grading scale for inflammation score.The use and care of experimental animals followed the Association for Research in Vision and Ophthalmology statement.This study protocol was approved by an Ethics Committee of The Affiliated Hospital of Qingdao University (No.QYFYWZLL26168). Results:The IgY protein concentration from the 5th to 16th week after immunization was 1.57, 2.89, 24.98, 25.09, 23.89, 25.78, 21.57, 21.37, 18.98, 15.78, 14.67, 12.67 mg/ml, respectively.The titer of IgY was increased from the 5th week, and it reached the highest titer 1∶10 000 at the 7th week, which could be maintained until the 12th week after immunization before it dropped gradually.The concentration-killing curve showed that compared with the blank control group and negative control group, Fusarium grew slowly in the specific IgY treatment group.The specific IgY with a titer greater than 1∶10 000 had thermal stability below 60 ℃.The activity of specific IgY was highest at pH 4 to 6, which could be maintained above 70% at pH 3 to 9 and was further reduced with the decrease or increase of pH.At 1, 3 and 5 days after Fusarium infection, the inflammation scores were 3.50±0.55, 7.33±0.82, 4.00±0.63 in the PBS control group, and 3.33±0.82, 4.17±0.75, 2.50±0.55 in the specific IgY treatment group.There was a statistically significant overall difference in inflammation scores at various time points between the two groups ( Fgroup=247.35, P<0.05; Ftime=23.19, P<0.05). At 3 and 5 days after Fusarium infection, there was a smaller ulcer area and decreased inflammation scores in the specific IgY treatment group compared with the PBS control group, and the differences were statistically significant (all at P<0.05). Conclusions:The high titer specific IgY can be successfully prepared by salting out method using ammonium sulfate, which is with high stability, tolerance to temperature and pH.Moreover, it can alleviate the severity of corneal ulcers and reduce inflammation scores in the mouse model of fungal keratitis.
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Objective To examine the practical value of early detection of heart-type fatty acid binding protein (H-FABP)for risk stratification and prognosis assessment in cardiac troponin T (cTnT)-negative acute coronary syndrome(ACS)patients.Methods From March 2010 to March 2012,55 patients with chest pain and negative cTnT were selected from 232 ACS patients at the General Hospital of PLA.Expression levels of cTnT and H-FABP were detected within 6 h of the onset of clinical symptoms.H-FABP and cTnT values at 12,24,and 48 h from the onset of clinical symptoms were continuously measured to monitor the dynamic changes.Based on prognosis,patients were divided into two groups,levels of H-FABP were compared,and its predictive value for prognosis was assessed with the ROC curve.Results Within 6 h of the onset of clinical symptoms,cTnT levels in cTnT-negative ACS patients increased gradually as disease progressed and reached the peak value at 12 h before decreasing slowly and arriving at 50％ of the peak value at 48 h.Meanwhile,HFABP levels reached the peak within 6 h,decreased slightly(12.8％) at 12 h,and then decreased rapidly at 48 h (about 79％).Of 55 patients,24 had acute myocardial infarction during hospitalization.The H-FABP level within 6 h was a good predictor for cTnT-negative ACS patients.The area under ROC curve was 0.946 and the cutoff value was 15.47 μg/L.The prediction sensitivity was 87.5 ％,with a specificity of 90.3％.Eleven patients had cardiovascular events after a 12-month follow-up.Levels of H-FABP were different in patients with or without cardiovascular events,[(38.08±8.43) μg/L vs.(18.96 ± 2.85) μg/L (t =2.438,P＜0.05)].ROC curve analysis showed that the area under the curve was 0.772 and the prediction cutoff value was 44.71 μg/L.The rates of cardiovascular events were markedly different between patients with high(≥44.71 μg/L)and those with 1ow(＜44.71 μg/L)H-FABP levels(54.5％ vs.11.4％).Conclusions For ACS patients with negative cTnT,H-FABP is a good index for early risk stratification and prognosis assessment.
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Objective To investigate the effect of netropsin on migration and invasion ability of gastric cancer cells and its mechanisms.Methods To determine if netropsin inhibits migration and invasion of gastric cancer cells, Transwell migration and invasion assay was performed.Then Western blot was performed to detect expression of E-cadherin and vimentin in gastric cancer cells with or without presence in medium netropsin.Finally, immunofluorescence was performed to detect changes in the cellular localization of β-catenin to validate whether Wnt/β-catenin pathway was suppressed or not.Results Netropsin with the concentration of 25 μmol/L had minimal inhibition effect on cell proliferation and was able to suppress ability of migration and invasion by inhibiting EMT in gastric cancer cells(P<0.05).Meanwhile netropsin was able to down-regulate the expression of epithelial markers E-cadherin and up-regulate the expression of mesenchymal marker vimentin.Finally,immunofluorescence showed that netropsin was able to block translocation of β-catenin from cytoplasm to nuclear.Conclusions Netropsin can inhibit EMT thereby suppressing migration and invasion of gastric cancer cells.The mechanism is that netropsin can compete with HMGA2 for transcription factor binding site thereby suppressing the Wnt/β-catenin pathway.
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AIM:To explore the effect of FOXQ1 gene silencing on angiogenesis and proliferation ability of co-lon cancer cells induced by Sonic hedgehog (Shh).METHODS:Lentivirus expressing different FOXQ1-shRNA or nega-tive cantrol (NC)-shRNA was used to infect the SW480 cells.The best silencing condition was screened and used in the following experiments .The SW480 cells were divided into interfered group ( FOXQ1-shRNA) and control group ( NC-shR-NA) .The MTT assay was used to observe the doubling time and cell activity .Tube formation assay was performed to detect the ability of angiogenesis.Meanwhile, the expression of vascular endothelial growth factor (VEGF)-A, matrix metallopro-teinase ( MMP) 2 and cyclin D1 at mRNA and protein levels was determined by real-time PCR and Western blot .After in-duction of the cells by recombinant Shh proteins , the changes of angiogenesis and proliferation ability in each group were detected.At the same time, the transformation of related gene was examined .RESULTS:Compared with control group , the angiogenic ability in interfered group was decreased , and no obvious difference of proliferation ability was observed .The expression of VEGF-A and MMP2 was declined significantly , and the expression of cyclin D 1 was not obviously changed . Recombinant Shh proteins improved the expression of FOXQ1 gene.Compared with NC-shRNA group, after induction, the angiogenic ability of FOXQ1-shRNA group was decreased , and the proliferation ability was not obviously changed .CON-CLUSION:FOXQ1 gene mediates the angiogenic ability but does not affect the proliferation ability of SW 480 cells.Mean-while, it may be regulated by shh pathway .
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Objective:To explore the effect of calcium release-activated calcium channel modulator 1 (ORAI1) on the migration and invasion of colon cancer cell line SW480 and its mechanism. Methods:The SW480 cells were infected with ORAI interference lentivirus. The expression of ORAI1 mRNA and protein was confirmed by quantitative real-time polymerase chain reaction and Western blot. Transwell chamber, adhesion, angiogenesis, and vasculogenic mimicry experiments were conducted to detect the ability of cell invasion, migration, and angiogenesis and the intercellular adhesion of homogeneous and heterogeneous cells among each group. Confocal microscopy was employed to detect the difference of store-operated Ca2+entry (SOCE) in each group. Western blott was used to detect the expression of ERK1/2, p-ERK1/2, MMP-2, VEGF, and E-cadherin protein. Results:After the infection of SW480 with the ORAI1 interference lentivirus for 72 h, significant fluorescence expression was observed. Compared with the empty vector group and control group, the expression of ORAI1 was lower in the interference group (P<0.01). Invasion and migration ability decreased (P<0.01); the intercellular adhesion ability of homogeneous cells increased (P<0.05); the intercellular adhesion ability of heterogeneous cells decreased (P<0.05);the angiogenesi and vasculogenic mimicry were enhanced (P<0.01);the internal flow peak of SOCE was low (P<0.05); the expression of p-ERK1/2, MMP-2, and VEGF proteins decreased (P<0.01); and the expression of E-cadherin protein increased (P<0.01). Conclusion:ORAI1 may promote the migration and invasion of SW480. This mechanism may be associated with the increase of SOCE.