ABSTRACT
【Objective】 To establish a magnetic bead enrichment strategy for the detection of human immunodeficiency virus deoxyribonucleic acid (HIV DNA) in peripheral blood, and to verify the improvement of the sensitivity of this method for the detection of HIV DNA in HIV infected patients after early antiretrovital treatment (ART). 【Methods】 Peripheral whole blood was collected at 4 timepoints in one ART HIV window period (WP) patient. Peripheral blood mononuclear cells (PBMCs) were isolated on a Ficoll gradient. CD4+ T lymphocytes were enriched from total PBMCs by negative sorting. HIV DNA concentration in magnetic beads enriched group and whole blood group was detected by HIV DNA detection kit. 【Results】 CD4+ T cells were isolated by magnetic beads and identified by FCM for purity at (96.4 ± 2.6)%. The viability was (95.9 ± 2.9)%, as demonstrated by trypan blue staining. The person on continued ART treatment in this study had significantly greater reduction in HIV viral load and undetectable HIV plasma RNA at follow up timepoint 4. No HIV DNA was detected in the whole blood group at all 4 timepoints. The quantitative results of HIV DNA in the CD4+ T lymphocyte group of the magnetic bead enrichment group were 73.4, 429.3, 137.1, 449.9 copies/106 CD4+ T cell′s respectively. 【Conclusion】 The magnetic bead enrichment method can be more sensitive in detecting the limit low copy HIV DNA in blood samples, and provide early confirmatory data for HIV WP infection and breakthrough infection after ART treatment.
ABSTRACT
BACKGROUND: Bladder cancer stem cells could promote the recurrence and drug resistance of bladder cancer. Numerous studies have shown that keratin 6B (KRT6B) is involved in the production and progression of tumors, and is closely related to the prognosis of tumors. OBJECTIVE: To observe the expression of keratin 6B in CD44+ bladder cancer stem cells and to show the influence of keratin 6B on proliferation, migration, and self-renewal of bladder cancer stem cells, and to further explore the effect of keratin 6B expression on the prognosis of bladder cancer patients. METHODS: (1) CD44+ 5637 bladder cancer stem cells were isolated by magnetic active cell sorting. Cancer stem cell-related gene expression of SOX2, OCT4, and NANOG was detected via real-time polymerase chain reaction. The spheroid formation assay was used to detect the ability of self-renewal of cancer stem cells in CD44+ cells. Keratin 6B expression was detected in CD44+ bladder cancer stem cells by real-time polymerase chain reaction. (2) The CD44+5637 bladder cancer stem cells were divided into two groups. In the keratin 6B siRNA group, keratin 6B small interfering RNA was transfected into CD44+ bladder cancer stem cells. Untransfected CD44+ bladder cancer stem cells were used as the black control group. Cells were collected at 2 days post-transfection. The proliferation, migration, and self-renewal capacity of keratin 6B siRNA CD44+ bladder cancer stem cells were detected by the colony and wound healing assay and spheroid formation respectively. (3) Totally 24 bladder cancer tissues were used by immunohistochemistry to analyze the expression of CD44v6 and keratin 6B. (4) ONCOMINE database was used to analyze the effect of keratin 6B expression on the overall survival of bladder cancer. RESULTS AND CONCLUSION: (1) Cancer stem cell-related genes (SOX2, OCT4, NANOG) and keratin 6B expression was higher in CD44+ cells isolated by magnetic active cell sorting compared with CD44- cells (P < 0.05). Cell proliferation, migration, and in vitro spheroid formation were significantly increased (P < 0.05). Keratin 6B small interfering RNA down-regulated the expression of keratin 6B in CD44+ bladder cancer stem cells (P < 0.05). (2) Compared with the blank control group, the proliferation and migration of CD44+ bladder cancer stem cells after transfection of keratin 6B small interfering RNA (P < 0.05), and the number of tumorsphere significantly diminished (P < 0.05); the expression of Notch1 and Hes1 mRNA increased (P < 0.05). (3) Keratin 6B and CD44v6 were significantly different in bladder cancer tissue (P=0.006). The overall survival rate of bladder cancer patients with high expression of keratin 6B was lower than that of patients with low expression of keratin 6B. (4) The results showed that keratin 6B was highly expressed in CD44+ bladder cancer stem cells, and could promote the proliferation, migration, and self-renewal capacity of bladder cancer stem cells. The high expression of keratin 6B contributes to improving the survival of bladder cancer patients.
ABSTRACT
OBJECTIVE: To develop and verify a magnetic beads-based extraction combined with quantitative PCR(q-PCR)method for determination of residual host cell DNA in recombinant human albumin products expressed in Pichia pastoris. METHODS: The residual Pichia pastoris host cell DNA in samples were extracted by magnetic beads-based extraction method and then determined by Taqman probe-based q-PCR. The residual DNA content was calculated according to the standard curve. The developed method was verified for accuracy and precision with different derivation albumin matrixes and concentrations, and the residual DNA of 3 batches of recombinant human albumin products expressed in Pichia pastoris were detected. RESULTS: The minimum detection limit of Pichia pastoris residual DNA by the developed method was 3 fgμL-1, the linear range was 3 fgμL-1-300 pgμL-1, and the correlation coefficient(r2) was 0.998 3. The recovery rates of spiked samples in rHA matrix were 93.58%(RSD 19.6%, n=4)at 100 fgμL-1 and 215.56%(RSD 42.9%, n=4) at 10 fgμL-1, respectively. The recovery rates of spiked samples in HSA matrix were 67.09%(RSD 6.9%,n=3)at 100 fgμL-1 and 113.40%(RSD 11.1%, n=3) at 10 fgμL-1, respectively. The residual Pichia pastoris DNA contents in 3 batches of recombinant human albumin products expressed in Pichia pastoris determined by the developed method were 5.98, 4.16, 4.49 fgμL-1(n=7) respectively and not more than 1 ng per 10 g protein. CONCLUSION: Magnetic beads extraction method combined with fluorescence quantitative PCR method solves the technical problem of quantitative determination of trace DNA in recombinant human albumin products with ultra-high concentration protein. The method is accurate and reproducible, and can be used for quantitative determination of DNA residue in recombinant human albumin expressed by Pichia pastoris.
ABSTRACT
Objective@#To search for the methods of isolating, purifying and culturing corpus cavernosal endothelial cells (CCECs) from SD rats, observe their growth characteristics, and providing seed cells for the study of erectile dysfunction (ED).@*METHODS@#The corpus cavernosal tissue from the SD rat was digested with 0.1% elastase, followed by purification of CCECs with immunomagnetic beads. After further amplification, monoclonal CCECs were sorted out with the cloning cylinder and their morphological and proliferative characteristics were observed. The von Willebrand factor (VWF) in the CCECs was identified by immunofluorescence staining, the CD31 molecule detected by immumohistochemistry, the purity of the CCECs determined by flow cytometry, and the proliferation of the cells measured with CCK-8 and growth curves.@*RESULTS@#After 7 days of purification and culture, the CCECs were fused into a monolayer under the inverted phase-contrast microscope, arranged like flagstones. The growth curves showed that the CCECs were in latency with a low growth rate at 1－2 days, in the logarithmic growth phase with a rapid rate at 3－4 days, and into the platform phase around the 6th day. VWF was positively expressed in the CCECs with much green fluorescence, and so was CD31 with a large number of brownish particles. The positive rate of the CCECs which were labelled with the VWF purified with magnetic beads combined with cloning cylinders was up to (91.9±3.75)%.@*CONCLUSIONS@#High-purity rat CCECs can be cultured successfully using immunomagnetic beads combined with cloning cylinders, with stable proliferation and passage in the endothelial cell medium.
Subject(s)
Animals , Humans , Male , Rats , Cell Culture Techniques , Cell Movement , Cell Proliferation , Cells, Cultured , Endothelial Cells , Chemistry , Cell Biology , Physiology , Erectile Dysfunction , Pathology , Flow Cytometry , Immunomagnetic Separation , Penis , Cell Biology , Platelet Endothelial Cell Adhesion Molecule-1 , Rats, Sprague-Dawley , Sincalide , von Willebrand FactorABSTRACT
Objective To evaluate the clinical application of a novel hepatitis B virus YMDD mutation DNA diagnostic kit (magnetic beads method kit).Methods A total of 324 HBV clinical serum samples was tested with the magnetic beads method kit and another kind of fluorescence diagnostic kit (boiling method).Accuracy, specificity, and sensitivity were compared.Results The consistency of positive detection rate of two kits was 100％ (95％ CI : 98.0％ ～ 100％), negative consistency was 97.12％ (95％ CI : 92.8％ ～99.2％) and the total consistency was 98.76％ (95％ CI : 96.9％ ～99.7％).Four cases of discrepant samples were confirmed by sequencing, and statistical analysis performed by Kappa test (Kappa =0.975) shows good consistency between the two methods.Conclusions The magnetic beads method kit has good consistency compared to the regular boiling method kit, and the polymerase chain reaction (PCR) detection system contains an internal positive control (internal control) to avoid a false negative resuit, which is more suitable for clinical diagnosis.
ABSTRACT
Objective: The present study aimed the comparison between the automated magnetic bead and silica column techniques for DNA extraction of human bones. Material and methods: Both techniques were performed on 25 human femur bones evaluating: I) the amount of extracted genetic material; II) the amount of amplified profiles; and III) the necessary time range to perform the techniques. Results: The automated magnetic bead technique recovered larger amount of DNA in 88% of the studied bone sample in comparison with the silica column technique. The automated magnetic bead technique also achieved a high level of amplifications (9/16loci) in 68% of the sample, while the silica column technique reached equal level of amplifications only in 36% of the sample. The time range elapsed for performing the automated magnetic bead technique was approximately 3 hours for processing 12 samples, while the silica column technique performed the same samples in 81 hours. Conclusion: Based on that, the automated magnetic bead technique presented optimal outcomes and faster performance compared to the silica column technique, revealing a valuable tool for forensic DNA extraction.
Objetivo: O presente objetiva comparar as técnicas de partículasmagnéticas e de coluna de sílica para a extração de DNA a partirde ossos humanos. Materiais e métodos: Ambas as técnicas foramaplicadas em 25 fêmures humanos, visando avaliar: I - a quantidadede material genético extraído; II - a quantidade de material amplificado;e III - o tempo decorrido para a aplicação de cada técnica.Resultados: A técnica de partículas magnéticas viabilizou maiorquantidade de material extraído em 88% da amostra. A mesmatécnica alcançou também maior quantia de material amplificado(9/16 loci) em 68% da amostra. O tempo decorrido para a aplicaçãoda técnica de partículas magnéticas consistiu num período de 3horas para o processamento de 12 amostras, enquanto a técnica decoluna de sílica realizou o mesmo procedimento em 81 horas. Conclusão:Desta forma, a técnica de partículas magnéticas apresentouresultados mais satisfatórios dentro de um desempenho mais rápidoquando comparada com a técnica de coluna de sílica, revelandoser uma eficaz ferramenta forense.
ABSTRACT
Objective To study DNA quantification and STR typing of samples pre-treated with pyrami-don. Methods The blood samples of ten unrelated individuals were anticoagulated in EDTA. The blood stains were made on the filter paper. The experimental groups were divided into six groups in accor-dance with the storage time, 30 min, 1 h, 3 h, 6 h, 12 h and 24 h after pre-treated with pyramidon. DNA was extracted by three methods: magnetic bead-based extraction, QIAcube DNA purification method and Chelex-100 method. The quantification of DNA was made by fluorescent quantitative PCR. STR typing was detected by PCR-STR fluorescent technology. Results In the same DNA extraction method, the sample DNA decreased gradually with times after pre-treatment with pyramidon. In the same storage time, the DNA quantification in different extraction methods had significant differences. Sixteen loci DNA typing were detected in 90.56% of samples. Conclusion Pyramidon pre-treatment could cause DNA degradation, but effective STR typing can be achieved within 24 h. The magnetic bead-based extrac-tion is the best method for STR profiling and DNA extraction.
ABSTRACT
Background & objectives: At present, the diagnosis of nephrotic syndrome (NS) requires a renal biopsy which is an invasive procedure. We undertook this pilot study to develop an alternative method and potential new biomarkers for diagnosis, and validated a set of well-integrated tools called ClinProt to investigate serum petidome in NS patients. Methods: The fasting blood samples from 49 patients diagnosed with NS by renal biopsy, including 17 mesangial proliferative glomerulonephritis (MsPGN), 12 minimal change nephrotic syndrome (MCNS), 10 focal segmental glomerulosclerosis (FSGS) and 10 membranous nephropathy (MN), were collected and screened to describe their variability of the serum peptidome. The results in NS group were compared with those in 10 control healthy individuals. Specimens were purified with magnetic beads-based weak cation exchange chromatography and analyzed in a MALDI-TOF MS. Results: The results showed 43, 61, 45 and 19 differential peptide peaks in MsPGN, MCNS, MN and FSGS groups, respectively. A Genetic Algorithm was used to set up the classification models. Cross validation of healthy controls from MsPGN, MCNS, MN and FSGS was 96.18, 100, 98.53 and 94.12 per cent, respectively. The recognition capabilities were 100 per cent. Interpretation & conclusions: Our results showed that proteomic analysis of serum with MALDI-TOF MS is a fast and reproducible approach, which may give an early idea of the pathology of nephrotic syndrome.
Subject(s)
Adolescent , Adult , Chromatography, Ion Exchange/methods , Humans , Immunomagnetic Separation/methods , Middle Aged , Nephrotic Syndrome/blood , Nephrotic Syndrome/pathology , Peptides/blood , Pilot Projects , Proteomics/methods , /methods , Tandem Mass Spectrometry/methodsABSTRACT
Objective To determine the urinary polypeptide patterns of glomerulonephritis by magnetic bead separation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) technology. Methods Urinary samples of 29 healthy volunteers and 34 patients with glomerulonephritis, including 10 cases of IgA nephropathy (IgAN), 10 cases of membranous nephropathy (MN), 9 cases of minimal change disease (MCD) and 5 cases of lupus nephritis (LN), were collected and separated by magnetic bead,and were screened for polypeptide patterns with a novel high throughput method, MALDI-TOF MS. Results Under the relative molecular weights 10 000 Da, 85 protein peaks were detected in healthy controls group and 109 protein peaks were detected in glomerulonephritis group. Six peaks of 3371.5 Da, 4026.35 Da, 4085.32 Da, 4116.96 Da, 4126.32 Da and 9527.31 Da were up-regulated,while 8 peaks of 861.28 Da, 1205.41 Da, 1642.52 Da, 1913.15 Da, 1976.52 Da, 2087.74 Da, 2193.47 Da and 3015.57 Da were down-regulated by more than 2 folds (P<0.01) in glomerulonephritis group as compared to healthy controls. Urinary polypeptide patterns in different diseases differed significantly from each other, indicating specific disease pattern of polypeptide excretion. Conclusions MALDI-TOF MS is a fast, convenient and high throughput analyzing method capable of screening some relative specific, potential biomarkers from the urine of glomerulonephritis patients thus it possesses better clinical value.
ABSTRACT
Objective To analyze the serum proteomic pattern of the cervical cancer patients,to develope diagnostic model and to evaluate its clinical significance.Methods WCX magnetic bead and MALDI-TOF were used to detect the serum proteomic pattern of 77 patients with cervical squanmous cell carcinomas,13 patients with CINⅢ and 52 healthy women.Biomarker Wizard software was used to detect protein peaks and potential difference between cervical cancer and controls.The model was developed by Biomarker Patterns software.Results A diagnostic pattern consisting of three differential protein peaks was established with 100%(32/32)sensitivity and 93.8%(30/32)specificity.A sensitivity of 77.8%(35/45)and a specificity of 75%(15/20)in blind test were obtained.The diagnostic model also could discriminate CINⅢ and SCC-Ag negative patients from controls.Conclusion The diagnostic pattern combining 3974,3398,13732m/z protein peaks can discriminate not only cervical squamous cell cancer but also CINⅢ and SCC-Ag negative patients from controls.
ABSTRACT
Objective To explore the in vitro isolation of ?2m-/Thy-1+ bone marrow-derived liver stem cells(BDLSCs) which bear double features of stem and liver cells from bone marrow stem cells(BMSCs)as so to provide suitable donor cells for the treatment of liver diseases by cellular transplant. Methods ?2m-/Thy-1+ BDLSCs were isolated by magnetic bead cell sorting(MACS) method from cholestatic rats in vitro,and cell purity was detected using flow cytometry.Liver associated phenotype markers were characterized by RT-PCR and immunofluorescence staining. Results BDLSCs isolated by MACS were purified and viable,and possessed hepatocyte-like features at gene and protein levels. Conclusion ?2m-/Thy-1+ BDLSCs are special subsets of BMSCs which may have promising potentials in the stem cell-based treatment of liver diseases.