ABSTRACT
Objective To explore the expression of Octamers binding transcription factor 4 (Oct4) and its relationship with clinicopathological parameters in the non-small cell lung cancer (NSCLC) tissues .Methods From September 2013 to December 2016 ,100 cases of NSCLC patients with lung cancer (group NSCLC ,n=100) and their corresponding normal paracancerous tissues (the control group ,n=100) were selected as the samples .The expression of Oct4 mRNA and protein in two groups of tissue samples were detected by fluores-cence quantitative PCR ,immunohistochemistry and western blot .The relationship between the level of Oct4 expression and the clinicopathological parameters of the patients was analyzed ,and the prognosis of the Oct4 positive and negative patients in the NSCLC group was compared .Results The expression of Oct4 mRNA in NSCLC group was (3 .21 ± 0 .18) ,protein expression was (0 .74 ± 0 .11) and the positive rate was 74 .00％ , which were significantly higher than those of control group [(1 .47 ± 0 .11) ,(0 .10 ± 0 .03) ,10 .00％ ,P<0 .05] .The positive rate of Oct4 expression in NSCLC patients was not related to sex ,age ,smoking index and tumor size (P>0 .05) ,but related to pathological type ,degree of differentiation ,and TNM staging ,in which the positive rates of Oct4 in patients with adenocarcinoma ,low differentiation and TNM stage Ⅲ stage were significantly higher than those of non adenocarcinoma ,middle-high differentiation degree ,Ⅰ - Ⅱ TNM stage patients (P< 0 .05) .In NSCLC patients ,compared with Oct4 negative patients ,the distant metastasis rate (64 .86％ ) ,pulse tube infiltration rate (47 .30％ ) in Oct4 positive patients were significantly higher ,and the 1 year survival rate (67 .57％ ) decreased (P<0 .05) .Conclusion The expression of Oct4 in NSCLC tissue is closely related to its clinicopathological parameters .Oct4 is highly expressed in adenocarcinoma ,and with thedecrease of differentiation and progression of tumor stages ,the expression increases and affects the prognosis . Oct4 can be used as a specific diagnostic marker of NSCLC .
ABSTRACT
Objective In order to study the biological characteristics of macrophages and provide the materials to study the survival mechanism of intracellular parasites, we conducted this study to establish a high-purity alveolar macrophage isolation and culture method.Methods Goat lungs were lavaged with normal saline in sterile environment several times, and cells were collected and then goat alveolar macrophages were purified by density gradient centrifugation using peripheral blood mononuclear cells (PBMC) solution.The isolated goat alveolar macrophages were cultured in cell culture medium containing 10% fetal bovine serum and cell morphology was observed under an inverted microscope every day,and the phagocytic activity of the cells was detected by chicken red blood cell phagocytosis test.Flow cytometry was used to detect CD14, a characteristic monocyte-macrophage surface marker.Results The adherent cells were characterized by typical macrophage morphology, pseudopodia and protrusions, showing round and irregular shape, rich cytoplasm, and large cell body.Of the cultured macrophages, 54.5% could phagocytize chicken erythrocytes and showed good phagocytic activity.After one month of in vitro culture, 93.7% of the cells were able to express CD14 antigen, which had a macrophage-specific immunophenotype.Conclusions The alveolar macrophages obtained in this study have high purity and good bioactivity, thus provide a cell model for studying the immune mechanism of intracellular parasites.
ABSTRACT
Objective To observe the effect of Simiaoyongan decoction with Shengmai decoction on apoptosis of hypoxia/reoxygenation model of primary cultured neonatal rat cardiomyocytes. Methods The combination of trypsin and collagenase was used to isolate neonatal rat myocardial cells. Hypoxia/reoxygenation model of cardiomyocytes was made by high purity nitrogen gas. Primary cultured neonatal rat cardiomyocytes were randomly divided into control group, model group and treatment group (treated with Simiaoyongan decoction and Shengmai decoction). The myocardial apoptotic rates were detected by Annexin V-FITC/PI double-staining method. Results Compared with the control group, the apoptotic rate of model group and treatment group was high (P<0.05). Drug intervention lowered the apoptotic rate compared with the model group, the difference was significant (P <0.05). Conclusion The alcohol sedimentation solution of Simiaoyongan decoction with Shengmai decoction played a protective role on hypoxia/reoxygenation cardiomyocytes.
ABSTRACT
Interleukin-21 is a type I cytokine mainly produced by activated CD4+ T cells that acts as a regulator of immune system. In this work, hIL-21cDNA was amplified from human peripheral blood lymphocytes by RT-PCR, and then inserted into pPIC9K. The recombinant vector pPIC9K-hIL21cDNA was linearized by Sac I, and transformed into Pichia pastoris strain GS115 by electroporation. Transformants were selected by G418 and confirmed by PCR. The recombinant protein was expressed and secreted into the supernatant after inducing by methanol. SDS-PAGE analysis indicated the molecular weight of rhIL-21 was about 16 kD. ELISA results show that the yield of rhIL-21 reach 229.28 mg/L, rhIL-21 was purified from culture supernatants, and it was purified to about 95% purity with ion-exchange chromatography. When co-stimulate with Con A, rhIL-21 can promote the proliferation of human lymphocytes. This is the first expression of bio-active rhIL-21 in Pichia pastoris. It lays a foundation for further research in immunotherapy and cancer therapy.
Subject(s)
Humans , Electroporation , Genetic Vectors , Genetics , Interleukins , Genetics , Pichia , Genetics , Metabolism , Recombinant Proteins , GeneticsABSTRACT
Promoter-trap strategy for enriching targeted colonies has been usually used to elevate the gene targeting efficiency in somatic cells. Knocking out Prnp in animals by gene targeting can render them resistant to Prion diseases. We constructed a bovine Prnp promoter-less targeting vector BoPrPneo, then transfected the linearized vector into the bovine fetal fibroblasts BFF through electroporation. After selecting in cell culture medium with 250 microg/mL G418, we obtained 99 drug-resistant cell colonies, 4 of them were positive for targeted events after PCR screening, and the targeted colonies were further confirmed by sequencing and Southern blotting. This suggests that one allele of Prnp has been successfully knocked out in bovine fetal fibroblasts. This research supplies a simple, safe and effective method to targeting bovine Prnp.
Subject(s)
Animals , Cattle , Electroporation , Fetus , Fibroblasts , Metabolism , Gene Knockout Techniques , Methods , Prion Diseases , Genetics , Prions , Genetics , Promoter Regions, Genetic , Genetics , TransfectionABSTRACT
Objective To study the effect of Simiaoyongantang on human umbilical vein endothelial cells(HUVECs) injury induced by hypoxia.Methods The HUVECs were divided into normal group,model group and Simiaoyongantang group,and the hypoxia model was established at the latter two groups.The livability of HUVECs was measured by MTT assay.The transudation of LDH in the medium was measured by biochemistry and the positive expression of VEGF was detected by immunofuorescence chemistry.Results The level of LDH was reduced significantly and the cell activity indicated by MTT was increased.The number of the cells stained by VEGF increased in the treatment group of Simiaoyongantang.Conclution Simiaoyongantang has protective effect on hypoxia injured HUVECs,which may be related to its effect of increasing the cells livability,reducing the injury induced by hypoxia,up-regulating expressions of VEGF and promoting angiogenesis.
ABSTRACT
Objective To investigate the inhibitory effect of RNA interference(RNAi)on WT1 gene expression in HL-60 cell line.Methods Several pairs of small interfering RNA(siRNA)for WT1 gene was designed and transfected into HL-60 cells.WT1 gene expression was assessed by RT-PCR,and WT1 protein expression was tested by Western blot.Flow cytometry was used to measure the cell apoptosis.Results The expression rates of WT1 mRNA and the expression rates of WT1 protein were(23.62%?1.28%),(23.20%?1.04%),respectively in HL-60 WT1 siRNA-transfected cells,significantly lower than those of control cells(P