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Background@#and Purpose Intravenous tenecteplase (TNK) efficacy has not been well demonstrated in acute ischemic stroke (AIS) beyond 4.5 hours after onset. This study aimed to determine the effect of intravenous TNK for AIS within 4.5 to 24 hours of onset. @*Methods@#In this pilot trial, eligible AIS patients with diffusion-weighted imaging (DWI)-fluid attenuated inversion recovery (FLAIR) mismatch were randomly allocated to intravenous TNK (0.25 mg/kg) or standard care within 4.5–24 hours of onset. The primary endpoint was excellent functional outcome at 90 days (modified Rankin Scale [mRS] score of 0–1). The primary safety endpoint was symptomatic intracranial hemorrhage (sICH). @*Results@#Of the randomly assigned 80 patients, the primary endpoint occurred in 52.5% (21/40) of TNK group and 50.0% (20/40) of control group, with no significant difference (unadjusted odds ratio, 1.11; 95% confidence interval 0.46–2.66; P=0.82). More early neurological improvement occurred in TNK group than in control group (11 vs. 3, P=0.03), but no significant differences were found in other secondary endpoints, such as mRS 0–2 at 90 days, shift analysis of mRS at 90 days, and change in National Institutes of Health Stroke Scale score at 24 hours and 7 days. There were no cases of sICH in this trial; however, asymptomatic intracranial hemorrhage occurred in 3 of the 40 patients (7.5%) in the TNK group. @*Conclusion@#This phase 2, randomized, multicenter study suggests that intravenous TNK within 4.5–24 hours of onset may be safe and feasible in AIS patients with a DWI-FLAIR mismatch.
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Since the pathogenesis of depression is complicated, the therapeutic effects of western medicine are poor accompanied by severe side effects. Chinese medicine has unique advantages in the treatment based on syndrome differentiation and contains many effective components against depression, including flavonoids, terpenes, phenylpropanoids, quinones, and alkaloids. These chemical components can delay the course of the disease, improve the curative effect, and reduce side effects of western medicine by regulating the biochemical abnormalities of monoamine neurotransmitters, brain tissue protein content, and internal environment as well as energy metabolism to make the treatment of Chinese medicine highlighted and recognized. This study systematically reviewed the effective components and mechanisms of anti-depressive Chinese medicine to inspire the rational development and utilization of new Chinese medicines against depression.
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Humans , Drug-Related Side Effects and Adverse Reactions , Drugs, Chinese Herbal/therapeutic use , Medicine, Chinese Traditional , SyndromeABSTRACT
Objective To evaluate the effect of Oncomelania hupensis snail control of cutting the beach group in the south of Shaobo Lake. Methods The general situation of the project of cutting the beach was surveyed, and the snail distribution was surveyed and the results were compared before and after cutting the beach in the beach group. Results The area of cutting beach was 928.33 hm2, the cubic meter of earthwork was 1 717.00 m3, the area of dumping ground was 425.76 hm2, the beach surface elevation was 3.2 m after cutting the beach, and the beach surface was fallen to 1.0 m under the ordinary water level. The area with snails was 44.69 hm2 before cutting the beach in 2011 but the area with snails was 1.78 hm2 after cutting the beach in 2018. The area with remaining snails was declined by 96.02% in 2018 as compared with that in 2011, and surviving snails were distributed on the uncut beach face. Conclusion In Shaobo Lake, the O. hupensis snail breeding environment is eliminated by raising or lowering the beach, so it is an effective measure of snail control in lake regions.
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Objective To evaluate the effect of Oncomelania hupensis snail control of cutting the beach group in the south of Shaobo Lake. Methods The general situation of the project of cutting the beach was surveyed, and the snail distribution was surveyed and the results were compared before and after cutting the beach in the beach group. Results The area of cutting beach was 928.33 hm2, the cubic meter of earthwork was 1 717.00 m3, the area of dumping ground was 425.76 hm2, the beach surface elevation was 3.2 m after cutting the beach, and the beach surface was fallen to 1.0 m under the ordinary water level. The area with snails was 44.69 hm2 before cutting the beach in 2011 but the area with snails was 1.78 hm2 after cutting the beach in 2018. The area with remaining snails was declined by 96.02% in 2018 as compared with that in 2011, and surviving snails were distributed on the uncut beach face. Conclusion In Shaobo Lake, the O. hupensis snail breeding environment is eliminated by raising or lowering the beach, so it is an effective measure of snail control in lake regions.
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OBJECTIVE@#To explore the significance of lymphocyte to monocyte ratio (LMR) in the disease progress of primary gastrointestinal diffuse large B-cell lymphoma (PGI-DLBCL).@*METHODS@#The clinical data of 43 patients diagnosed as PGI-DLBCL in our hospital from January 2011 to December 2015 were collected, and the disease progress was followed up.@*RESULTS@#According to the ROC curve, the threshold value of LMR for 2 years PFS (%) of PGI-DLBCL patients was 2.6. Unvariate analysis showed that LMR (P＜0.05), large enclosed mass lesion (P＜0.01) and IPI (P＜0.05) were prognostic factors affecting PFS, the COX regression model multivariate analysis showed that LMR＜2.6 [ (risk ratio (RR)=3.083, 95%CI 1.828-8.313, P＜0.01], and large enclosed mass lesions (RR=2.718, 95%CI 1.339-6.424, P＜0.05) were the independent adverse prognostic factor for two years PFS.@*CONCLUSION@#Both LMR＜2.6 and large enclosed mass lesions relate with the progress of PGI-DLBCL.
Subject(s)
Humans , Leukocyte Count , Lymphocytes , Lymphoma, Large B-Cell, Diffuse , Monocytes , Prognosis , Retrospective StudiesABSTRACT
<p><b>OBJECTIVE</b>To evaluate biological effects of OCT4A gene on K562 cells and explore the molecular mechanism of K562 cell apoptosis.</p><p><b>METHODS</b>Two recombinant lentiviral vectors were constructed, which could stablely up- regulate and down- regulate OCT4A protein. Recombinant lentivirus was generated by co-transfection of three-plasmids and transfec-ted into K562 cells. The experiments were divided into 5 groups: normal, pLVX-OCT4A-ZsGreen1, pLVX vector control, PLB-OCT4A shRNA and non-specific shRNA groups. Western blot was applied to detect the expression of OCT4A protein, the cell counting kit-8 was applied to evaluate the effect of OCT4A on proliferation of K562 cells. The apoptosis and differentiation of K562 cells were detected by flow cytometry with AnnexinV/7-AAD double staining. The mRNA expressions of caspase-3,BIM,BCL-xL,BAX in K562 cells were determined by real time PCR.</p><p><b>RESULTS</b>The OCT4A fragment was amplified by reverse transcription polymerase chain reaction(RT-PCR), the 2 lentiviral vectors were successfully constructed. In comparson with those in the control group, the expression of OCT4A protein of pLVX-OCT4A-ZsGreen1 group was significantly increased, but decreased in PLB-OCT4A shRNA group. CCK-8 assay showed that the higher the content of OCT4A protein, the faster the cell proliferation. The apoptosis rate was (3.48±0.52)% of pLVX-OCT4A-ZsGreen1 group, which was lower than that of control group, while the apoptosis rate PLB-OCT4A shRNA group was (7.25±0.57)%, which was higher than that of control group (P<0.05), however, the K562 cells differentiation was not influenced(P>0.05). Compared with control group, the gene expression of Caspase-3,BIM and BAX was down-regulated(P>0.05), but a significant up-regulation of BCL-xL gene expression was observed(P<0.05).</p><p><b>CONCLUSION</b>Two lentiviral vectors have been successfully constructed, which can stably up- and down- regulate the expression of OCT4A in K562 cells respectively. OCT4A can promote the K562 cell proliferation and inhibit the apoptosis, the mechanism may be related with up-regulation of BCL-xl expression.</p>
Subject(s)
Humans , Apoptosis , Cell Proliferation , Genetic Vectors , K562 Cells , Lentivirus , Octamer Transcription Factor-3 , Genetics , TransfectionABSTRACT
To observe metabolic abnormalities, histology changes and eNOS expression of aorta in type 2 diabetes rat.And to observe intervention effect of rosiglitazone.Methods 80 male Wistar rats were randomized to control group, high diet group, diabetes group, and rosiglitazone treatment group (diabetes plus rosiglitazone treatment).Type 2 diabetes models were developed and rosiglization group was treated with rosiglitazone .Six weeks and twelve weeks after treatment with rosiglitazone, blood glucose, endothlin and nitric oxide were tested.Histology changes of aorta in different groups were observed under microscopy .Meanwhile the protein and mRNA expression of eNOS in aorta were examined.Results 1)Compared with control group, ET in high fat diet group, diabetes group and rosiglitazone group increased significantly , and the level of NO decreased significantly at 6 week and 12 week.At 12 week, ET in diabetes group increased, and NO decreased significantly than that of high fat diet group and rosiglitazone group.2)Histology changes were observed in high fat diet group , diabetes group and rosiglitazone group at 12 week.3)Compared with control group, protein and mRNA expression in high fat diet group , diabetes group and rosiglitazone group were down regulated at 6 week and 12 week.And protein expression in diabetes group was down regulated than that in rosiglitazone group .Conclusions Rosiglization can ease the endothelial dysfunction in type 2 diabetes rat.
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Objective To assess left atrial reservoir function based on left atrial filling volume,the left atrial expansion index and left atrial ejection fraction (LAEF) were measured in patients with left ventricular non-compaction cardiomyopathy (LVNC),using real-time three-dimensional echocardiography (RT3DE),to determine the value for LVNC diagnosis.Methods The study included 20 patients diagnosed with LVNC,including 3 patients,aged 4-16 years;8 patients,aged 17-35 years;9 patients,aged 36-55 years;and 20 healthy age-matched control subjects.All patients underwent two-dimensional echocardiography and RT3DE.Results Two-dimensional echocardiography showed no differences between groups (P ＞ 0.05).RT3DE analysis showed that the average left atrial filling volume/BSA,left atrial expansion index,and LAEF were significantly higher in the LVNC group.Conclusion LVNC is associated with increased left atrial reservoir function.RT3DE shows promise for the diagnosis of LVNC.
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Objective To investigate the changing characteristics of myocardial movement in patients with dilated cardiomyopathy by three?di?mensional speckle tracking echocardiography(3D?STE). Methods The peak systolic global longitudinal train(GLS),global radial strain(GRS), global circumferential stain(GCS)and global area strain(GAS)of left ventricle were measured by 3D?STE technology in 69 patients with dilated cardiomyopathy. According to the left ventricular ejection fraction(LVEF),all patients were divided into group A(35%≤LVEF<50%)and group B (LVEF<35%). The differences of measurements were compared between two groups. The correlation between global myocardial strain in all direc?tions and left ventricular ejection fraction was analyzed. Results The GLS,GRS,GCS and GAS were significantly higher in group A than those in group B(P<0.01). The GLS,GRS,GCS and GAS were correlated with LVEF in group A(r=-0.871,0.610,-0.423,-0.797;P<0.05).The GCS,GRS and GAS were correlated with LVEF in group B(r=-0.517,0.368,-0.438;P<0.05). There was no significant correlation between GLS and LVEF in group B. Conclusion 3D?STE technology can be applied to evaluate the change of the myocardial movement. GLS is a promis?ing marker of the prognosis in patients with DCM.
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<p><b>OBJECTIVE</b>To establish a mouse model of Phacute lymphoblastic leukemia (ALL) for providing a valuable tool to facilitate the researches on PhALL.</p><p><b>METHODS</b>CML-like mice were generated by transfection to bone marrow cells of BABL/c with Mig190 retrovirus. The PhALL mouse model was established by infusion of sorted CML like mouse-derived BCR-ABLB cells into the mice of same linege. Immonophenotypes, BCR-ABL transcription and expression of these leukemic cells were detected by flow cytometry, RT-PCR and Western blot respectively.</p><p><b>RESULTS</b>CML-like presentation appeared in the mice after Mig190 virus transfection. After infusion with sorted BCR-ABLB cells, all the receipted mice eventually presented the clinical signs of acute leukemia. Flow cytometry analysis showed that these leukemic cells were positive for CD19; both RT-PCR and Western blot indicated that this mouse model is consistent with PhALL phenotypecally and molecalarlly.</p><p><b>CONCLUSION</b>A novel method to establish PhALL mouse model has been developed successfully, and this model can provide useful tool for the study of PhALL.</p>
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<p><b>OBJECTIVE</b>To investigate the effect of metformin on proliferation, differentiation and apoptosis of THP-1 cells and explore its possible mechanism.</p><p><b>MEHODS</b>THP-1 cells were cultured with different concentrations of metformin for 24 h and 48 h. The cell proliferation was evaluated by CCK-8, the cell apoptosis was analyzed by Annexin V/7-AAD double labeling, the expression of CD14 and CD11b (surface differentiation antigens on THP-1 cells) was evaluated by flow cytometry, the BCL-XL, BAX, BIM and caspase-3 mRNA expressions of THP-1 cells were detected by real time quantitative PCR.</p><p><b>RESULTS</b>Metformin could significantly inhibit the growth of THP-1 cells in a time- and dose- dependent manner. After treated with 20 mmol/L metformin for 24 h, the expressions of CD14 and CD11b in THP-1 cells didn't change much (P>0.05), the early apoptosis rates in exprimental and control groups were (2.02±0.85)% and (4.46±1.33)% respectively, the late apoptosis rates in experimental and control groups were (1.43±0.83)% and (3.31±0.59)% respectively. In process of inducing effect of 20 mmol/L metformin on THP-1 cells, the expressions of BCL-XL and BIM did not significantly changed, while the expressions of BAX and caspase-3 significantly increased (P<0.01).</p><p><b>CONCLUSION</b>Metformin can effectively inhibit proliferation and induce apoptosis of THP-1 cells. However, it has no significant effect on differentiation of THP-1 cells, its mechanism inducing apoptosis maybe related with up-regulating BAX and caspase-3.</p>
Subject(s)
Humans , Apoptosis , Caspase 3 , Cell Differentiation , Cell Line, Tumor , Cell Proliferation , MetforminABSTRACT
Purpose To analyze the echocardiographic features of peripartum cardiomyopathy (PPCM) using Logistic regression, and to screen the indexes which can be used for the prognosis of PPCM. Materials and Methods Fifty patients who were diagnosed as PPCM by echocardiography were divided into recovered group (30 cases) and non-recovered group (20 cases), all the patients underwent echocardiography, left ventricular end-diastolic diameter (LVEDd) was measured in the parasternal long axis view, left ventricular ejection fraction (LVEF) was measured using Simpson biplane method in the apical four-chamber and two-chamber view to look for the evidence of left ventricular thrombosis, if complicated with pulmonary hypertension, continuous wave Doppler was used for measuring the peak velocity of tricuspid valve regurgitation, and estimating of pulmonary artery systolic pressure, Logistic regression model was established and receiver operating characteristic (ROC) curve was generated to evaluate the prediction value of Logistic regression model. Results Compared with non-recovered group, there was statistically significant difference of LVEDd, LVEF, left ventricular thrombosis, pulmonary hypertension and re-checked LVEF (t= -4.33, 7.64 and 11.54, P0.05). Three sonographic features LVEDd, LVEF and left ventricular mural thrombus) could be used for the establishment of Logistic model (χ2=5.14, 11.59 and 14.58, P<0.05). The prediction accuracy of the model was 90.0% (45/50, P<0.001) and the area under ROC curve was 0.945±0.030 (P<0.001). Conclusion Logistic regression analysis can be applied for the screening of ultrasound index for PPCM, LVEDD, LVEF and left ventricular wall thrombus can predict the prognosis of PPCM accurately.
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OBJECTIVE@#To study the correlation between miR-21 and Treg/Th17 ratio in the microenvironment of rats with hepatocellular carcinoma.@*METHODS@#Diethylnitrosamine was used to build the hepatocellular carcinoma model of rats; the content of Treg cells and Th17 cells and the expression of miR-21 in the peripheral blood of rats with hepatocellular carcinoma were detected. The statistical analysis was performed on the correlation between miR-21 expression and Treg/Th17 ratio.@*RESULTS@#Hepatocellular carcinoma model of rats was successfully constructed. The proportion of Th17 cells among all CD4(+)T cells in the peripheral blood of rats with hepatocellular carcinoma was 5.319%, which was higher than the control group; while the proportion of Treg cells was 9.472%, which was higher than the control group. Treg/Th17 ratio in the model group was 1.781, compared with 1.478 in the control group. The expression of miR-21 was increased in the peripheral blood of rats with hepatocellular carcinoma and it showed a positive correlation with the ratio of Treg/Th17.@*CONCLUSIONS@#There is a positive correlation between the expression level of miR-21 and the ratio of Treg/Th17.
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OBJECTIVE@#To discuss the abnormal expression of Wnt inhibitory factor (WIF1) in hepatocellular carcinoma cells and its regulating effect on the hepatocellular carcinoma invasion and metastasis factors of tissue inhibitor of matrix metalloproteinases-3 (TIMP-3) and caveolin-1.@*METHODS@#RT-PCR and Western blot were employed to detect the expression of WIF1 in six hepatocellular carcinoma cell lines of HepG2, Hep3B, Huh7, PLC/PRF/5, SMMC-7721 and MHCC97 and the immortalized human liver cell line THLE-3. Besides, Lipofectamine 2000 was employed to transfect the eukaryotic expression vector pcDNA3.1-WIF1 and blank plasmid pcDNA3.1 into hepatocellular carcinoma cell lines. Transwell assay was used to detect the effect of WIF1 on the invasion ability of hepatocellular carcinoma cells; Western blot was used to detect the effect of WIF1 on the expression of TIMP-3 and caveolin-1 in hepatocellular carcinoma cells, it also discussed the effect on the expression of β-catenin.@*RESULTS@#The expression of WIF1 in hepatocellular carcinoma cell lines was lower than that in the normal liver cell lines (P < 0.01); while there was basically no expression of WIF1 in the human highly metastatic cell line MHCC-97 and moderate expression in HepG2 and SMMC-7721. Therefore, HepG2 and SMMC-7721 were chosen as the further experimental cell lines. After transfecting the eukaryotic expression vector pcDNA3.1-WIF1 and blank plasmid pcDNA3.1 into hepatocellular carcinoma cell lines, compared with the blank plasmid group, the cell viability and invasion ability in the WIF1 group were all reduced (P < 0.01), the expression of TIMP-3, caveolin-1 and mRNA were all down-regulated (P < 0.01), and the expression of β-catenin was decreased (P < 0.01).@*CONCLUSIONS@#Because of down-regulation or missing of expression of WIF1 in hepatocellular carcinoma cell lines, the up-regulation of WIF1 expression can significantly inhibit the invasion and metastasis of HepG2 and SMMC-7721 of hepatocellular carcinoma cell lines, which are related to the up-regulated expression of TIMP-3 and down-regulated expression of caveolin-1 and may be realized through the Wnt/β-catenin signaling pathway.
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Objective To discuss the diagnostic value of two-dimensional speckle tracking imaging in the evaluation of left ventricular rotation and torsion in pre-apical hypertrophic cardiomyopathy(PAHCM)patients. Methods A total of 26 patients with PAHCM,26 patients with hyperten-sive left ventricular hypertrophy(HLVH)and 26 healthy volunteers were recruited for the study. Two-dimensional echocardiographs were performed carefully. Two-dimensional images were obtained from the left ventricular apical four-chamber section,two-chamber section and two short-axis sec-tions,including mitral valve and apical levels. The peak subendocardial rotation(endo-rot),epicardial rotation(epi-rot),transmural torsion(mural-tor)and bulk rotation(bulk-rot)of each short-axis section were measured and analyzed using QLAB 9.1 software. Results In the mitral level, there was no statistically significant difference in endo-rot,epi-rot,mural-tor and bulk-rot between the PAHCM group and normal control group (P>0.05). There was statistically significant difference in endo-rot,epi-rot,mural-tor and bulk-rot between the PAHCM group and HLVH group (all P0.05). There was statisti-cally significant difference in endo-rot,mural-tor and bulk-rot between each two groups(P<0.05). There was statistically significant difference in G-tor between each two groups(P<0.05). The receiver operating characteristic curve analysis showed that the accuracy of differential diagnosis of G-tor PAHCM was high(the area under the receiver operating characteristic curve is 0.89),the sensitivity was 73.08%,and the specificity was 92.31%. Conclusion Two-dimensional speckle tracking imaging could accurately and quantitatively measure the left ventricular rotation and tor-sion in PAHCM patients. The G-tor could accurately screen and identify between PAHCM and HLVH.
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Objective: To discuss the abnormal expression of Wnt inhibitory factor (WIF1) in hepatocellular carcinoma cells and its regulating effect on the hepatocellular carcinoma invasion and metastasis factors of tissue inhibitor of matrix metalloproteinases-3 (TIMP-3) and caveolin-1. Methods: RT-PCR and Western blot were employed to detect the expression of WIF1 in six hepatocellular carcinoma cell lines of HepG2, Hep3B, Huh7, PLC/PRF/5, SMMC-7721 and MHCC97 and the immortalized human liver cell line THLE-3. Besides, Lipofectamine 2000 was employed to transfect the eukaryotic expression vector pcDNA3.1-WIF1 and blank plasmid pcDNA3.1 into hepatocellular carcinoma cell lines. Transwell assay was used to detect the effect of WIF1 on the invasion ability of hepatocellular carcinoma cells; Western blot was used to detect the effect of WIF1 on the expression of TIMP-3 and caveolin-1 in hepatocellular carcinoma cells, it also discussed the effect on the expression of β-catenin. Results: The expression of WIF1 in hepatocellular carcinoma cell lines was lower than that in the normal liver cell lines (P < 0.01); while there was basically no expression of WIF1 in the human highly metastatic cell line MHCC-97 and moderate expression in HepG2 and SMMC-7721. Therefore, HepG2 and SMMC-7721 were chosen as the further experimental cell lines. After transfecting the eukaryotic expression vector pcDNA3.1-WIF1 and blank plasmid pcDNA3.1 into hepatocellular carcinoma cell lines, compared with the blank plasmid group, the cell viability and invasion ability in the WIF1 group were all reduced (P < 0.01), the expression of TIMP-3, caveolin-1 and mRNA were all down-regulated (P < 0.01), and the expression of β-catenin was decreased (P < 0.01). Conclusions: Because of down-regulation or missing of expression of WIF1 in hepatocellular carcinoma cell lines, the up-regulation of WIF1 expression can significantly inhibit the invasion and metastasis of HepG2 and SMMC-7721 of hepatocellular carcinoma cell lines, which are related to the up-regulated expression of TIMP-3 and down-regulated expression of caveolin-1 and may be realized through the Wnt/β-catenin signaling pathway.
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Objective: To study the correlation between miR-21 and Treg/Th17 ratio in the microenvironment of rats with hepatocellular carcinoma. Methods: Diethylnitrosamine was used to build the hepatocellular carcinoma model of rats; the content of Treg cells and Th17 cells and the expression of miR-21 in the peripheral blood of rats with hepatocellular carcinoma were detected. The statistical analysis was performed on the correlation between miR-21 expression and Treg/Th17 ratio. Results: Hepatocellular carcinoma model of rats was successfully constructed. The proportion of Th17 cells among all CD4
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This study was purposed to investigate the effect of bromodomain-containing protein 4 (BRD4) inhibitor GSK525762A on the proliferation and apoptosis of chronic myeloid leukemia blast crisis KU812 cells and its mechanism. KU812 cells were treated with different concentrations of GSK525762A (100, 250, 500, 1 000, 2 500 and 5000 nmol/L) and the inhibitory effects of drug on KU812 cell proliferation after 48 and 72 hours were detected by using CCK-8 assay. KU812 cells were treated with 3 different concentrations of GSK525762A (1.0, 2.5 and 5 µmol/L) and the cell apoptosis after 72 hours were assayed by using flow cytometry. KU812 cells were treated with DMSO and 2.5 µmol/L GSK525762A, and the mRNA levels of C-MYC, BCL-2, CDK6, BCL-xL, BAK and BAX were determined by using quantitative reverse transcription polymerase chain reaction (qRT-PCR). The results showed that GSK525762A could significantly inhibit the proliferation of KU812 cells and the inhibitory effect on KU812 cell proliferation was dependent on the dose-course and time-course of GSK525762A treatment. GSK525762A treatment could induce apoptosis of KU812 cells in a dose-dependent manner. After GSK525762A treatment, the mRNA levels of proliferation-promoting genes ( C-MYC and CDK6) and pro-survival genes ( BCL-2 and BCL-xL) decreased, while the transcription level of pro-apoptosis genes BAK and BAX increased, as compared to that of the control group. It is concluded that GSK525762A can inhibit the proliferation of KU812 cells and induce cell apoptosis possibly through depressing the transcription of C-MYC, BCL-2, CDK6 and BCL-xL gene, and down-regulating BAK and BAX transcription.
Subject(s)
Humans , Apoptosis , Benzodiazepines , Pharmacology , Cell Line, Tumor , Cell Proliferation , Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Nuclear Proteins , Proto-Oncogene Proteins c-bcl-2 , Transcription FactorsABSTRACT
<p><b>OBJECTIVE</b>To establish a high efficient human coagulation factor VIII (FVIII) eukaryotic stable expression system using lentiviral vector, and determine its biosafety.</p><p><b>METHODS</b>Lentiviral transfer plasmid carrying human B-domain-deleted FVIII(BDDhFVIII)-IRES-GFP(BDDhFVIII/pXZ9)or IRES-GFP(pXZ9) was constructed. Lentivirus particles were produced by transiently co-transfected 3-plasmids into 293FT cells and further concentrated via ultracentrifugation. CHO cells were infected, 72h later, the FVIII antigen (FVIII:Ag) concentration in the medium was examined by ELISA, the activity was detected via one stage coagulation,and the transcription of FVIII in the infected CHO cells was determined by RT-PCR.Virus infection ability in the medium and the gag gene in CHO cells were determined to evaluate the model's biosafety.</p><p><b>RESULTS</b>Lentiviral transfer plasmid BDDhFVIII-IRES-GFP(BDDhFVIII/pXZ9)carrying human B-domain-deleted FVIII or IRES-GFP (pXZ9) was successfully constructed, and high titer lentiviruses has been prepared. The lentivirus could infect CHO cells efficiently, after an additional 72 h, the FVIII:Ag concentration had up to (1724.9±283.7) mU/ml, the FVIII:C level increased to (10.58±1.55)%, and transcripts of BDDhFVIII mRNA could be measured by RT-PCR. Neither the gag gene nor the virus in the supernatant was detected.</p><p><b>CONCLUSION</b>Lentivirus-mediated human coagulation factor VIII could be expressed efficiently in CHO cells. The system couldn't produce offspring virus, proving a good biosafety.</p>
Subject(s)
Animals , Cricetinae , Humans , CHO Cells , Cricetulus , Factor VIII , Genetics , Gene Expression , Genetic Vectors , Lentivirus , Genetics , Plasmids , TransfectionABSTRACT
This study was aimed to construct the targeting AATF shRNA eukaryotic expression vector and establish the stably transfected U937 cell lines. The sequence of AATF mRNA was obtained from GenBank. After excluding homology, three plasmid expression vectors coding shRNA targeting 228 ∼ 249, 303 ∼ 324 and 443 ∼ 464 of AATF gene sequence were synthesized. Two terminals of shRNA carried BamHI and HindIII restriction sites. The selected nucleotides were cloned into the plasmid pSilencer 3.1-H1 neo respectively, and the resultant recombinant plasmids were named as pSA-1, pSA-2, pSA-3. The sequences of the recombinant plasmids were identified by DNA sequencing. The recombinant plasmids were transfected into the cell line U937 by electroporation with Neon(TM) Transfection System. The transfected cells were persistently screened under G418 (500 mg/L), and isolated with a limited dilution for 8 weeks. The inhibition of AATF mRNA and protein expression was respectively detected by RT-PCR and Western blot. The results indicated that RNAi eukaryotic expression vectors targeting AATF had correct reading frame and nucleotide sequence. Real-time PCR revealed that AATF shRNA effectively silenced mRNA expression of AATF. Western blot analysis found that AATF shRNA obviously suppressed protein expression of AATF (P < 0.05). It is concluded that the shRNA eukaryotic expression vector has been successfully constructed which can inhibit the expression of AATF, and the establishment of stably transfected U937 cell lines provide a original route for exploring the mechanism of AATF in human Leukemia further.