ObjectiveTo explore a simple and rapid pathological slices method to observe the porous structure and the composition distribution of composite materials. MethodsTaking polyurethane/small intestinal submucosa (PU/SIS) composite as an example, PU/SIS was OCT-embedded and sliced into sections by frozen section technology, after which general observation of the section integrity was carried out. After dyed with water-soluble eosin in alcoholic solution, the staining effect and the porous structure of the composite were observed under light field microscope. Sections were sealed with five different sealing methods. Group A: sealing piece using glycerogelatin method; group B: anhydrous alcohol dehydration→transparency using TO transparent reagent→sealing piece using neutral quick drying glue; group C: color separation using deionized water→air-drying→sealing piece using neutral quick drying glue; group D: air-drying→transparency using TO transparent reagent→sealing piece using neutral quick drying glue; group E: air-drying→sealing piece using neutral quick drying glue. Then, the morphology and the components distribution of the composite were observed under light field microscope, and the simple and feasible method was selected as optimum method. ResultsFrom general observation, the frozen section of the PU/SIS composite, which was 6 μm in thickness, was complete and continuous. Although the outline of the material and the porous structure in the sections could be observed clearly under light field microscope, the two components still could not be identified by using eosin staining method. After sealing piece, the material components in groups A, B, and C still could not be identified or be dissolved and deformed; the morphology of the material in groups D and E were preserved and the two components in the composite were clearly visible. ConclusionThe morphology and the components distribution of PU/SIS frozen sections can be characterized after soluble eosin staining and neutral quick drying glue sealing.
ObjectiveTo explore the effects of exogenous estrogen receptor β1 (ERβ1) gene on the expression of human telomerase reverse transcriptase (hTERT) as well as the changes of proliferation ability in MDA-MB-231 cell line by transfecting recombinant eukaryotic expressing vector containing ERβ1 cDNA into human breast cancer MDA-MB-231 cell. MethodsRecombinant eukaryotic expressing vector containing ERβ1 cDNA was transfected into human breast cancer MDA-MB-231 cell by using cationic liposome as transfecting agent (acted as pcDNA3.1ERβ1 transfection group), empty vector group and non-transfection group acted as controls. The expression levels in both the mRNA and protein of both the ERβ1 and hTERT were tested by real-time PCR and Western blot, respectively. The change of proliferation ability in MDA-MB-231 cell was displayed by cell growth curve, and the change of cell apoptosis was detected by flow cytometry. ResultsThe expression level of ERβ1 mRNA in the pcDNA3.1-ERβ1 transfection group (0.449±0.077) significantly increased as compared with the nontransfection group (0.153±0.035) or the empty vector group (0.160±0.020), P=0.001 or P=0.000. The expression level of ERβ1 protein in the pcDNA3.1-ERβ1 transfection group (0.847±0.065) significantly increased as compared with the non-transfection group (0.356±0.050) or the empty vector group (0.390±0.030), P=0.001 or P=0.000. The expression level of hTERT mRNA in the pcDNA3.1-ERβ1 transfection group (0.127±0.020) significantly decreased as compared with the non-transfection group (0.283±0.025) or the empty vector group (0.283±0.049), P=0.001 or P=0.002. The expression level of hTERT protein in the pcDNA3.1-ERβ1 transfection group (0.147±0.023) significantly decreased as compared with the non-transfection group (0.783±0.025) or the empty vector group (0.802±0.019), P=0.001 or P=0.002. The rate of cell apoptosis in the pcDNA3.1-ERβ1 transfection group 〔(6.15±0.94)%〕 was higher than that in the non-transfection group 〔(1.41±0.42)%〕， P=0.001. Cell proliferation curve showed that proliferation ability significantly decreased in the pcDNA3.1-ERβ1 transfected groups as compared with the non-transfection group (Plt;0.05). ConclusionERβ1 could inhibit cell growth of human breast cancer MDA-MB-231 cell by down-regulating the expression of hTERT.
To screen new tuberculosis diagnostic antigens and vaccine candidates, we predicted the epitopes of Mycobacterium tuberculosis latent infection-associated protein Rv2004c by means of bioinformatics. The homology between Rv2004c protein and human protein sequences was analyzed with BLAST method. The second structures, hydrophilicity, antigenicity, flexibility and surface probability of the protein were analyzed to predict B cell epitopes and T cell epitopes by Protean software of DNAStar software package. The Th epitopes were predicted by RANKPEP and SYFPEITHI supermotif method, the CTL epitopes were predicted by means of combination analyses of SYFPEITHI supermotif method, BIMAS quantitative motif method and NetCTL prediction method. The peptide sequences with higher scores were chosen as the candidate epitopes. Blast analysis showed that Rv2004c protein had low homology with human protein. This protein had abundant secondary structures through analysis of DNAStar software, the peptide segments with high index of hydrophilicity, antigenicity, surface probability and flexibility were widely distributed and were consistent with segments having beta turn or irregular coil. Ten candidates of B cell epitopes were predicted. The Th epitopes of Rv2004c protein were located after the 200th amino acid. Of 37 Th cell epitopes predicted, there were more epitopes of HLA-DRB1*0401 and HLA-DRB1*0701 phenotypes, and the MHC restrictive types of some Th cell epitopes exist cross overlap. Of 10 CTL epitopes predicted, there were more number and higher score of HLA-A2 restricted epitopes. Therefore Mycobacterium tuberculosis Rv2004c protein is a protein antigen with T cell and B cell epitopes, and is expected to be a new target protein candidate for tuberculosis diagnosis and vaccine.
ObjectiveTo optimize the culture method of human primary pancreatic ductal adenocarcinoma (PDAC) cells and cancer associated fibroblasts (CAFs) and investigate the effect of CAFs on the growth of primary PDAC cells in vitro and tumor formation in patient-derived xenograft (PDX) model.MethodsThe PDAC specimens were collected and primarily cultured. In order to observe the effect of CAFs on the growth of primary PDAC cells in vitro, the CAFs were co-cultured with primary PDAC cells consistently and the alone cultured primary PDAC cells served as the control. Then, these cells were injected into the shoulder blades of NOG mice in order to develop the PDX model.ResultsWhen the primary PDAC cells separated from the CAFs, the proliferation capacity of the primary PDAC decreased rapidly in the passage culture in vitro, and the most cells were terminated within 5 generations. By contrast, when the CAFs co-cultured with the primary PDAC cells, the proliferation capacity of primary PDAC cells were preserved, which could be stably transferred to at least 10 generations. The tumors of NOG mice were detected during 2–3 weeks after injecting the mixed cells (primary PDAC plus CAFs), while had no tumor formation after injecting CAFs alone. The rate of tumor was 92.9% (13 cases) in the primary PDAC plus CAFs group, which was higher than that of the CAFs alone group (64.3%, 9 cases), but there was no statistical difference because of the small sample size. The volume of tumor in the primary PDAC plus CAFs group at 2, 4, 6, and 8 weeks after the tumor cells injection was significantly larger than that in the CAFs alone group at the corresponding time point, the differences were statistically significant (P<0.01).ConclusionsThe CAFs could promote the growth of primary PDAC cells in vitro. This new method of co-culture CAFs with primary PDAC could improve the success rate of primary PDAC cells culture and improve the success rate of PDX model in NOG mice.
A great number of studies have demonstrated functional abnormalities in children with attention-deficit/hyperactivity disorder (ADHD), although conflicting results have also been reported. And few studies analyzed homotopic functional connectivity between hemispheres. In this study, resting-state functional magnetic resonance imaging (MRI) data were recorded from 45 medication-naïve ADHD children and 26 healthy controls. The regional homogeneity (ReHo), degree centrality (DC) and voxel-mirrored homotopic connectivity (VMHC) values were compared between the two groups to depict the intrinsic brain activities. We found that ADHD children exhibited significantly lower ReHo and DC values in the right middle frontal gyrus and the two values correlated with each other; moreover, lower VMHC values were found in the bilateral occipital lobes of ADHD children, which was negatively related with anxiety scores of Conners' Parent Rating Scale (CPRS-R) and positively related with completed categories of Wisconsin Card Sorting Test (WCST). Our results might suggest that less spontaneous neuronal activities of the right middle frontal gyrus and the bilateral occipital lobes in ADHD children.