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Objective To investigates the diagnostic value of combined detection serum CCL18, CXCL1 antigen, C1D, TM4SF1, FXR1, TIZ IgG autoantibody by suspension array for ovarian cancer. Methods Suspension array was used to detect CCL18, CXCL1 antigen, C1D, TM4SF1, FXR1, TIZ IgG autoantibody in 120 cases of healthy women, 204 cases of patients with benign pelvic tumors, 119 cases of pelvic malignant tumor patients, and 40 cases with breast cancer, lung cancer oroliver cancer, respectively. Constructed diagnosis model of combined detection six biomarkers for diagnosis of ovarian malignant tumor. Constructed diagnosis model of combined detection autoantibodies to diagnose epithelial ovarian cancer. Analysed the value of detecting six biomarkers for diagnosis of ovarian malignant tumor and detecting autoantibodies for diagnosis of epithelial ovarian cancer. Analysed diagnostic value of detecting six biomarkers to diagnose stageⅠandⅡepithelial ovarian cancer. Compared diagnostic value of detecting six biomarkers in diagnosis of tissue types and pathologic grading with that of CA125. Results Model of combined detecting six biomarkers to diagnose ovarian malignant tumor was logit(P)=-11.151+0.008×C1D+0.011 × TM4SF1+0.011 × TIZ-0.008 × FXR1+0.021 × CCL18+0.200 × CXCL1. Model of combined detection autoantibodies to diagnose epithelial ovarian cancer was logit(P)=-5.137+0.013 × C1D+0.014 × TM4SF1+0.060 × TIZ-0.060 × FXR1. Sensitivity and specificity of detecting six biomarker to diagnose ovarian malignant tumor was 90.6% and 98.7%. Sensitivity and specificity of detecting autoantibodies to diagnose epithelial ovarian cancer was 75.8%and 96.7%. Combined detection for six biomarkers to diagnose serous and mucinous ovarian cancer was statistically no better than those of CA125 (P=0.196 and P=0.602, respectively);there was significantly difference in diagnosis of ovarian cancer (P=0.023), and there was no significantly difference in diagnosis of different pathological grading (P=0.089 and P=0.169, respectively). Conclusions Constructing diagnosis model of combined detection six biomarker to diagnose ovarian malignant tumor and constructed diagnosis model of combined detectionautoantibodies to diagnose epithelial ovarian cancer. Combined detection six biomarkers to diagnose serous and mucinous ovarian tumors is better than that of CA125.
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Recent studies find that cancer cells which express CD133,CD90,epithelial cell adhesion molecule,CD13,chemokine receptor 4,CD44 and ATP-binding cassette transporters play important roles on growth,recurrence and metastasis of liver cancer.Liver cancer stem cells constitute by cells which have different phenotype.Mentioned above cells may be important parts of liver cancer stem cells,which bring difficulties for the further development and practical application of theory about liver cancer stem cells.
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BACKGROUND:Infrapatelar fat pad is often partialy resected in the knee surgery, which can be used as an important source of adipose-derived mesenchymal stem cels. OBJECTIVE: To explore the strategies of isolation, culture, and identification of adipose-derived mesenchymal stem cels from the infrapatelar fat pad and to detect the expression of cel surface markers of human adipose-derived stem cels. METHODS: Infrapatelar fat pad was obtained from patients undergoing knee arthroscopy surgery, and attached cels were obtained from adipose tissue by using colagenase I. Cels were cultured in 10% low-sugar DMEM. Stem cels proliferation was detected by means of MTT and then, cel growth curve was made. The obtained cels were induced and differentiated into adipocytes and osteocytes. Expressions of cel surface markers CD29 and CD44 were detected. RESULTS AND CONCLUSION:A few of attached cels were observed after cultured 24 hours. Cels proliferated faster and exhibited spindle shape after 1 week. Cel adherence and proliferation were speeded up after subculture. Growth curve of cels exhibited that the passages 5 and 2 cels had higher reproductive activity than passage 8 cels. The obtained cels can be induced and differentiated into adipocytes and osteocytes. Results from flow cytometry showed that 96.8% passage 5 cels expressed CD29 and 97.6% expressed CD44. These findings indicate that high-purity adipose-derived mesenchymal stem cels with high reproductive ability are easy to be isolated from the infrapatelar fat pad, which may be a kind of ideal seed cels for cartilage tissue engineering.
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Objective To knockout and identify the Antigen 43 (Ag43) in the Escherichia Coli JM109 .Methods Mutation group Ⅱ introns RNA protein complexes (RNP) gene sequence was obtained by Sigma Company′s TargeTron Gene Knockout Sys‐tem and Ag43 gene specific designed PCR primers amplification ,then ,to acquired Ag43 specific recombinant RNP plasmid pACD4K‐Ag4 ,this gene sequence was inserted into the plasmid pACD4K‐C of RNA′s expression .Finally ,pEGFP‐Ag43 was trans‐formed into JM109 and inserted the group Ⅱ intron into the Ag43′s locus by IPTG inducing expression .Results The best insertion locus was between 1 812 and 1 913 .Through the agarose electrophoresis gel ,the RNP gene sequence was consistent with the expec‐ted value (350 bp) .The pEGFP‐Ag43 vector was correctly constructed which was proofed by endonuclease Nhe Ⅰ and Hind ⅡI di‐gestion as predicted products (3 646 and 4 029 bp;7 000 and 550 bp ,respectively ) .The PCR and gene sequence results indicated that the group Ⅱ intron was inserted into the locus between 1 812 and 1 913 in the Ag43 gene .Conclusion Successful knockout of the Ag43 in Escherichia Coli JM109 found basis to further study the Ag43′s function and regard the coli as host bacteria of Ag43 chimeric protein recombinant .
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BACKGROUND:Bone marrow mesenchymal stem cells are considered as commonly used seed cells to construct tissue-engineered for repair of bone and cartilage defects. It is of great significance for cytology and tissue engineering experiments to study the common problems existing in the basic operation and how to avoid these problems in a timely manner. OBJECTIVE:To summarize the common problems existing in the process of operation in order to provide reliable methods about separation, culture and identification of bone marrow mesenchymal stem cells for beginners and researchers. These can reduce or avoid some errors and problems during operation. METHODS:Sixteen New Zealand white rabbits were selected as experiment objects, and bone marrow mesenchymal stem cells were separated from rabbits by iliac puncture, purified and augmented by using density gradient centrifugation combined with adherent culture method. Then cellmorphology was observed by inverted phase contrast microscope, growth curve detected by MTT method and cellphenotype identified by flow cytometry. RESULTS AND CONCLUSION:We encountered some problems in the process of separation and culture, when we operated the first five rabbits. After careful y summarizing and analysis of the reasons, the operation was successful y completed on the rest 11 rabbits. Bacteria pol ution and cellaging were not found in the process of cellculture. What is more, the cells at passage 3 appeared with high-expression of CD29, and CD44, but low expression of CD14 and CD34. The cellgrowth curve showed that the proliferation activity of cells at passages 3 and 5 was higher than that at passage 10. Although the technology of separation, culture and identification of bone marrow mesenchymal stem cells is mature, the failure wil be happen if we do not pay attention to the details of operation. By strictly carrying out normal operations, we can get high purity of bone marrow mesenchymal stem cells, which lays a good foundation for celland animal experiments in the future.
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BACKGROUND:The surface antigen CD80/CD86 on mature dendritic cells can activate helper T (Th) cells, reduce the differentiation of Th cells toward Th1 cells, and promote the differentiation of Th cells toward Th2 cells. OBJECTIVE:To investigate the effect of smal interfering RNA (siRNA) inhibiting the expression of surface antigens CD80/CD86 from asthmatic murine mature dendritic cells on Th1/Th2 type cytokines, interferon-γand interleukin-4. METHODS:Asthmatic model of mice was established;then bone marrow-derived mature dendritic cells were separated and cultured. The expression of CD11c, CD80 and CD86 on mature dendritic cells were examined by flow cytometry. The siRNA was transferred into mature dendritic cells of asthmatic mice, and the CD80/CD86 mRNA and protein expression before and after interference were determined by fluorescent quantitative PCR and flow cytometry. The mature dendritic cells in non-siRNA group, siRNA group and negative siRNA group were co-cultured with T cells. The interferon-γand interleukin-4 productions were measured by enzyme-linked immunosorbent assay. RESULTS AND CONCLUSION:(1) The expression of CD80/CD86 on the mature dendritic cells of asthmatic group was significantly higher than that in normal control group (al P<0.05). (2) After siRNA was transferred into mature dendritic cells, the expression level of CD80/CD86 mRNA and protein in siRNA group was significantly lower than other groups (al P<0.05). (3) After siRNA transfection, the level of interferon-γfrom the supernatant of mature dendritic cells and T cells co-culture system was significantly increased in the siRNA group compared with other groups (al P<0.05), while interleukin-4 production in the siRNA group was significantly decreased (al P<0.05). These findings suggest that high expression of CD80/CD86 on mature dendritic cells of asthmatic mice is observed, specific siRNA can effectively inhibit the expression of CD80/CD86, thus increasing interferon-γproduction and decreasing interleukin-4 production, which contributes to regulate the Th1/Th2 imbalance.
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BACKGROUND:Human amniotic epithelial cells are an important source of cells in regenerative medicine as its multipotentation, but new studies mainly focused on differentiation features and there were little research oneffect of culture in vitro on biological property of amniotic epithelial cells. OBJECTIVE:To analyze the effects of in vitro culture on growth, cellphenotype and differentiation capacity of human amniotic epithelial cells into cardiomyocyte-like cells, and explore the correlation of primarily cultured human amniotic epithelial cells marker SSEA-4 expression level and the change of biological characteristics of human amniotic epithelial cells. METHODS:Primarily cultured human amniotic epithelial cells were obtained from amniotic tissues by using the same separation protocol. Human amniotic epithelial cells were cultured in vitro. The proliferation, cellphenotype and the differentiation capacity of human amniotic epithelial cells into cardiomyocyte-like cells were evaluated by means of cellcounting kit-8, flow cytometry and real-time PCR. RESULTS AND CONCLUSION:The SSEA-4 positive cells in primarily cultured human amniotic epithelial cells from different fetal tissues were between 26.7%-97%, which indicated that there was great individual difference among amniotic tissue samples. Moreover, with passage, the SSEA-4 expression in human amniotic epithelial cells decreased significantly, which did not correlate with the SSEA-4 expression in primarily cultured human amniotic epithelial cells. Results indicated that there was great individual difference in SSEA-4 expression level in primarily cultured human amniotic epithelial cells from different amniotic tissue samples. Thus, it is necessary to set up clinical screening indexes to get samples with higher SSEA-4 expression stably and to control the quality of human amniotic epithelial cells. In addition, during culture period, SSEA-4 expression level was affected by culture conditions. The culture conditions of human amniotic epithelial cells should be optimized to maintain SSEA-4 expression at a high level. In addition, the differentiation capacity of human amniotic epithelial cells into cardiomyocyte-like cells was also affected by individual difference among different samples and culture conditions, which wil be further studied in the future.
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The authors report a case of superficial acral fibromyxioma (SAF) in a 74-year-old male who presented with a painless mass in a periungual dorsoradial region of the right thumb. It is a rare benign neoplasm, which was recently described, that arouse on the skin and subcutaneous tissue of the hands and feet, especially in the proximity to the ungual region of male adults. Surgical treatment was performed with the excision in blocks of the margins of the lesion and fragmentation of the nail and nail matrix, according to the literature recommendation. Although there may be local recurrence in 22% of the cases, the patient presents no symptoms, deformities or functional limitations. In addition, there was no sign of tumor recurrence 18 months after the surgery. We are not aware of a similar case report in the Brazilian literature.
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Humans , Female , Aged , Antigens, Surface , Fibroma/diagnosis , Hand/surgery , Thumb/surgeryABSTRACT
Objective To investigate the methods of isolation and culture in vitro and detect the surface markers of human umbilical cord mesenchymal stem cells.Methods Human umbilical cord Wharton' s jelly was separated and cut up as small as possible,and then cultured with α-MEM.Human umbilical cord mesenchymal stem cells could be obtained by culturing the tissue block adhered the bottle wall.And the cells were passaged at a certain density.The surface markers of human umbilical cord mesenchymal stem cells were detected by FACS when the cells were in Generation Three.Results Human umbilical cord mesenchymal stem cells were obtained from Wharton' s jelly conveniently,with fibroblast shape and stable proliferation and passage.CD29,CD44,CD105 were strongly expressed on human umbilical cord mesenchymal stem cells.But CD45,CD34,HLA-DR,HLA-G,CD80,CDs6 were not expressed.Conclusion Human umbilical cord mesenchymal stem cells can be obtained effectively from the culture of the tissue block,which provides a rich source of cells for tissue engineering.
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Objective To establish the methodology of flow cytometry for detecting human cells in human/goat chimerisra.Methods Human hemopoietic stem/progenitor cells (CD+34 cells) or MIG-tranadueed-GFP CD+34 cells were transplanted into the peritoneal cavity of fetal goats in utero to obtain human/goat chimera modeL The peripheral blood cells from the chimeras were labeled with multiple mouse anti-human antibodies and the monoelonal antibodies that were specific for human but had not or only minimal cross-reaction with goat were screened as the primary antibodies for routine analysis in flow cytometry.Human cord blood was proportionally (25% ,50% ,75%,100%) added into the blood of the untransplanted goats and the cells were labeled with CD+34 monoclonal antibody.The region and size of the "gate" were chosen based on to the distribution of CD+34 cells or human cord blood.One human/goat chimera marked with GFP (MIG goat) was sacrificed and the substantial liver cells from its perfused liver were analyzed for the GFP+cells percentage and DNA contents by flow cytometry.Results CD7,CD15,CD38,CD45CD20CD34CD14and GPA monoclonal antibodies were chosen as the primary antibodies in rou tine detection by flow cytometry.The size and area of the "gate" were also defined.29.1% (29100/100 000 ) of the substantial liver cells from the MIG goat expressed GFP.DNA content analysis showed that the GFP+ cells obtained from the liver of MIG goat mainly manifested two peaks that were correspond to those of human.Conclusions Flow cytometry is rapid,simple and effective for the investigation of differentiation,homing and biological characteristics of stem cells in vivo.The selections of suitable surface antibodies and the "gate" are very important for detecting human cells accurately in the human/goat chimerism.
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Objective: To determine whether there exist the cells expressing both Clara cell antigen (CCA) and surfactant protein C (SP-C) in different lung tissues from mouse, rat, and human by dual immunofluorescent labeling technique. The CCA+ and SP-C+ cells were named as the double positive cells (DPCs) which may provide the basis for further investigation of lung stem cells. Methods: The normal lung tissues were isolated from adult and newborn mice and rats. The human lung squamous carcinoma and adenocarcinoma tissues and para-cancerous lung tissues were isolated and subjected to cryosection and dual immunofluorescent staining. The DPCs in different lung tissues were identified by using laser confocal microscopy. For each specimen, four to six visual fields were selected and totally 100-200 cells were counted to calculate the percentage of DPCs. The data were analyzed by the SPSS 11.0 software. Results: The DPCs were found in normal lung tissues from adult and newborn mice and rats. DPCs were first discovered in human lung adenocarcinoma tissues but not found in lung squamous carcinoma tissues. Furthermore, the number of DPCs in the paracancerous tissues was significantly increased than the corresponding lung cancer tissues (P < 0.05). Conclusion: We not only validate the wide distribution of DPCs in the normal lung tissues, but also discover the existence of DPCs in the lung adenocarcinoma tissues and the number of DPCs in para-cancerous tissues is significantly higher than that in lung cancer tissues. Our study provide the experimental basis for further isolation and identification of DPCs and investigation of the biological features of normal lung stem cells and lung cancer stem cells.
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Se realizó un estudio descriptivo en pacientes con antígeno de superficie (HBsAg) positivo diagnosticados en el hospital de Mampong, Ghana, durante un año hasta agosto de 1999, los datos fueron procesados utilizando los programas Microstat. El 80 % del universo tenía de 15 a 46 años de edad, la malaria y el SIDA fueron los antecedentes en el 58 % y el 23, 7 % respectivamente, dentro de los riesgos el más importante fue el de los tratamientos parenterales (70 %). La fiebre, el cansancio, la pérdida de apetito, el dolor abdominal, la coluria y la ictericia fueron las principales manifestaciones clínicas. El 35 % tuvo un examen físico normal.
A descriptive study was performed in patients with psoitive surface antigen (HbsAg) diagnosed in Mampong, Ghana hospital, during one years until August 1999; data were processed using MICROSTAT 46 years old, malaria and AIDS were the antecedents with 58 % and 23, 7 % respectively, among risk the most important was that of parenteral treatments (70 %) fever, tiredness lack of apetite, abdominal pain, choluria and jaundice were the main clinical manifestations, 35 % had a normal physical exam.
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Objective To investigate the effect of FK506 on cell-surface antigen expression in porcine thymocytes.Methods After FK506 administration at a dose of 1 mg./kg body weight intramuscularly for 21 days the pigs were sacrificed and the thymus were removed.Cells isolated from the thymus were stained for two-color flow cytometric analysis in two-steps:(1)incubation with a combination of monoclonal antibodies,and(2)staining with isotype-specific conjugates(FITC/PE).Results There was a significant decrease of T-lymphocytes(SWC1+SWC3-)from 90.25% to 44.50% and an increase of the SWC￣/SWC3￣population from 3.56% to 46.13%.The mean percentages of CD1￣CD5￣,CD2￣CD5￣and CD4￣CD8￣thymocytes were 43.63%,60.20% and 67.38% after FK506 treatment,respectively,while 6.01%,2.02% and 28.78% in the controls.Besides the decrease of CD1+CD5+ and CD2+CD5+subsets,a significant decrease of CD4+CD8+phenotypes could be detected in the FK506 group.Conclusion FK506 treatment might lead to inhibition of maturation and differentiation of porcine thymic pregenitots from CD4￣CD8￣(double-negative)to CD4+CD8+(double positive)in thymus.
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0.05). There was significant difference between G_1, G_2and G_3(P0.05). Low expression levels in G_4 (Ⅲ,Ⅳ) were observed.Significant differences were noted between expression levels in G_4 (Ⅲ,Ⅳ) and in G_2, G_3 or G_4 (Ⅰ,Ⅱ; P
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Objetivo. Describir la prevalencia de maracadores serológicos de los virus de la hepatitis en una población de embarazadas. Material y métodos. Se estudiaron 1 500 sueros de embarazadas en los que se determinaron: anticuerpos IgG contra el virus de la hepatitis A (anti-VHA); anticuerpos IgG contra el antígeno central del virus de la hepatitis B (ant-HB-c), y su antígeno de superficie (AgsHB); así como anticuerpos contra el virus de la hepatitis C (anti-VHC). En los casos positivos al AgsHB se buscaron anticuerpos contra el virus de la hepatitis D (anti-VHD) y el antígeno e del virus de la hepatitis B (AgeHB). Todas las determinaciones se realizaron por la técnica de ELISA. Resultados. El 93.3 por ciento de los sueros estudiados tuvo anti-VHA IgG positivos. La prevalencia del AgsHB fue del 0.26 por ciento y de anti-VHC del 0.53 por ciento. No hubo pacientes con positividad para anti-VHD ni para el AgeHB. Conclusiones. Se encontró una prevalencia del AgsHB superior a la de otros estudios en embarazadas mexicanas. Consideramos que el escrutinio del AgsHB debe formar parte de los exámenes de control prenatal
Objective. To determine the seroprevalence of hepatitis A, B, C and D virus infection among pregnant women attending a perinatal care hospital. Material and methods. A prospective study was carried out to determine the seroprevalence of hepatitis A virus IgG antibodies (anti-HAV), hepatitis B virus markers (anti-HBcAg and HBsAg) and hepatitis C virus antibodies (anti-HCV) in pregnant women. In HBsAg positive cases, HBeAg and hepatitis D virus antibodies (anti-HDV) were investigated. All analyses were performed with the ELISA technique. Results. Of the 1500 pregnant women studied, 93.3% were positive for anti-HAV IgG. The HBsAg seroprevalence was 0.26% and anti-HCV seroprevalence was 0.53%. There were no patients with HBeAg or antiHDV. Conclusions. A higher seroprevalence of HBsAg was found in this study than in other studies of pregnant Mexican women. We propose that HBsAg screening become a routine prenatal test.
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Humans , Female , Pregnancy , Pregnancy Complications, Infectious/virology , Prevalence , Hepatitis/epidemiology , Hepatitis Viruses/immunology , Antigens, Surface , Prenatal Care , BiomarkersABSTRACT
Objective To investigate melanocyte membrane antigen associated with vitiligo,for further purification and cloning.Methods Sera from patients with vitiligo were screened by using a live cell enzyme-linked immunoabsorbent assay,98strongly positive sera were obtrained.Melanocyte antigens were immunoblotted after splitting of cultured normal human melanocytes.Thirty strong positive vitiligo sera were detected.Results There were positive bands in all screened sera as shown by immunoblotting.Whereas,positive band was seen in only one normal human sera.The antibody bound many antigens with different molecular weights(150kd,90kd,75kd,50kd,40~45kd).Positive rates for individual antigens were70%,60%,83%,16%and23%,respectively.Conclusions Antibody directed to melanocyte membrane antigens of melanocytes are present in the sera of patients with vitiligo.The molecular weights of the antigens are mainly150kd,90kd and75kd,the antigens with small molecules discovered previously maybe the degradation products of big molecules.
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Objective To screen the HBV SPⅡ promoter DNA-binding protein, and to investigate its potential role in the regulation of replication and expression of HBV DNA. Methods By using HBV SPⅡ biotinylated promoter DNA as a selective molecule, the T7 select human liver cDNA library was biopanned and the positive clones were selected. After screening, amplification of positive plaques was performed for inserted DNA fragment and then they were cloned into the pGEM-Teasy vector. Results Four positive plaques were chosen for DNA sequencing. The binding protein of HBV SPⅡ promoter was demonstrated as nicotinamide adenine dinucleotide (NADH) dehydrogenase 4 by BLAST. Conclusion The result suggests that this approach may provide a new tool for the study of replication and expression mechanism of HBV DNA.