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Objective To compare sensitive difference of docetaxel between the triple negative breast cancer(TNBC)cell line CAL-51 and non TNBC line T47D and analyze mechanisms underlying docetaxel resistance in former cells. Methods Cell activi?ty was determined by MTT method and IC50 value was calculated;Wright-Giemsa stain was used to analyze the effect of docetaxel in the morphology of CAL-51 and T47D cell lines. Flow cytometry(FCM)was performed to determine cell cycle distribution and apoptosis. Realtime fluorescence quantitative PCR was used to compare the relative gene expression levels.The anti-apoptosis protein Bcl-2 and caspase family protein expression levels were determined by Western blot. Results Wright-Giemsa stain showed significant morpholo?gy change in T47D cells by docetaxel treatment. Further flow cytometry results confirmed that docetaxel could significantly induce apoptosis in T47D cells compared to CAL-51 cells(P<0.01). The result of realtime fluorescence quantitative PCR revealed that anti-apoptosis protein Bcl-2 was significantly higher expressed in CAL-51 cells(P<0.05). Immunoblot analysis revealed docetaxel treat?ment induced the instrinsic pathways in both CAL-51and T47D cells,but the activated pathway of executioner caspase was different. Conclusion Our present study shows that docetaxel induces different intrinsic apoptosis pathway in CAL-51 and T47D cell lines. An?ti-apoprosis protein Bcl-2 is highly expressed,which might be the underlying mechanism of docetaxel resistance in TNBC cell line-CAL-51.
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Objective To clone and express Mycobacterium tuberculosis fusion antigen ESAT-6 and CFP-10 ( EC) and to evaluate the biological activity of the fusion antigen EC in inducing specific cytokines secretion from THP -1 cells. Methods The fusion antigen EC gene was cloned into pET-30 a prokaryotic expression vector and expressed highly in E.coli BL21.Then, the THP-1 cells were stimulated with purified fusion antigen EC of different concentrations (10 and 20 μg/ml).Culture supernatants were collected after 12 h and 24 h, respectively.The secretion levels of IL-2, IL-4, IL-6, IL-8, IL-10, GM-CSF, TNF-αand IFN-γin THP-1 cell culture supernatants were detected using Bio-PlexProTM Assays kit.Results The M.tuberculosis fusion antigen EC was cloned and expressed successfully .The secretion levels of IL-6, IL-8 and TNF-αin EC infected THP-1 cells were significantly higher than those in THP-1 cells (P<0.05).The secretion levels of other cytokines did not change significantly .Conclusion The obtained M.tuberculosis fusion antigen EC has biological activity in inducing the THP-1 cells to secrete a higher level of IL-6,IL-8 and TNF-α.
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Objective To establish a key technological system for spider fibroin gene code tandem connection , vector construction , prokaryotic expression and purification using genetic engineering in order to achieve MaSp 1 heterologous ex-pression in Escherichia coli and its separation and purification .Methods Isocaudarner ligation method was used to connect synthetic spider fibroin gene monomer code in tandem , and a recombinant clone concatemer was obtained .The identified recombinant clones were connected with prokaryotic expression vector pET 28a(+), and then transformed into E.coli BL21 (DE3).After being induced by IPTG for 6 hours, the expression product was identified by SDS-PAGE and Western blot-ting.Engineering bacteria were fermented in high density , and the obtained protein was purified through ammonium sulfate fractionation.Results and Conclusion The expression plasmids of MaSp1concatemers were successfully constructed , and the induced expression genetic engineering MaSp 1 protein was of the expected relative molecular mass .In addition, the pu-rity of the purified protein was above 80%.This study has developed crucial technologies for mass production of genetic en-gineering spider silk proteins .
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Objective To provide the candidate antigens for immunological diagnosis by analyzing the expression of nu -cleoprotein ( NP) of Ebola virus. Methods BioSun software was used to predict the NP epitopes. The bridging-PCR was used to synthesize the NP gene. The pBVIL1 vector was used to clone and express the NP gene. Results The 360-739 aa of NP was confirmed to be the dominant antigen by BioSun software. The recombinant NP dominant antigen was expressed in E.coli with molecular weight of 58 ×103.The specificity of ELISA based on recombinant NP was 99.24% (130/131) in negative samples. Conclusions The dominant NP antigen can be potentially used for developing Ebola virus diagnostic reagent.
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Objective To develop an antigen retrieval method for detection of human mammaglobin ( hMAM) immuno-histochemcal staining in old paraffin-embedded specimens .Methods The tissue sections in test group were put into dis-tilled water after deparaffinization and then moved into citric acid buffer ( pH 3.5) for 10-15 min.The other two meth-ods,microwave method and high pressure cooker method ,were compared as control groups at the same time .Finally, immu-nohistochemistry SP method was used to check the antibody in the sections .Results The color appearance in the test group (pH 3.5 citric solution) was better than that of microwave oven and high pressure cooker groups .In the test group, tissue sections were not easily cast off from the slices .Conclusion In this study,we have established a new and simple antigen retrieval method which will contribute to immunohistochemistry technology .
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@#ObjectiveTo test and verify whether Ba-alginate-Poly-L-Ornithine-Alginate microcapsules(B-PLO-A) can improve the physical properties and biocompatibility of the traditional BPA microcapsules.MethodsThe B-PLO-A and Ba-alginate-Poly-L-lysine-alginate（B-PLL-A） microcapsules were made by the static generator. The physical property of the microcapsules was evaluated by observing the morphological changes of the microcapsules in the hypotonic environment, changes in diameter of microcapsules in vitro culture and calculating broken microcapsules ratio by shaking method. The biocompatibility was observed by transplanting into peritoneal cavity of rat.ResultsB-PLO-A microcapsules are stronger and more stable in a hypotonic environment than B-PLL-A microcapsules. After 96 h mechanism shaking, the unbroken microcapsules ratio of B-PLO-A and B-PLL-A microcapsules were (99.3±1.0)% and (96.2±1.5)% respectively. The microcapsules were retrieved from peritoneal cavity of rat at 2, 4 and 8 weeks after transplantation, most of the microcapsules were of integrity, rotundity, and surface smooth without obviously bundled by connective tissue. 8 weeks after transplantation the intact microcapsules ratio of B-PLO-A and B-PLL-A microcapsules were (97.3±2.1)% and (95.4±2.4)% respectively.ConclusionB-PLO-A microcapsules as a whole have bettermechanical strength compared with B-PLL-A microcapsules, while maintaining a good biocompatibility.
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@#Objective To reconstruct tissue-engineered 3D bronchial model using human bronchial epithelial cells and human embryo lung fibroblast as seeding cells, and liquid collagen mixed Matrigel as scaffold. Methods Human bronchial epithelial cells and human embryo lung fibroblast were mixed with liquid collagen supplementing with matrigel and casted in 12-wells plate to reconstruct cells-collagen sheet. Macroscopic observation, phase-contrast microscopy observation, routine HE staining and immunohistochemistry staining(CK ets) were employed to assess the engineered 3D model. Results We reconstructed engineered 3D bronchial model successfully in vitro by tissue engineering techniques and exerted static stretch onto the collagen sheet. From Macroscopic observation, we gained contracted well sheet. We also observed network structure in phase-contrast microscopy meanwhile the viability of cells was fine. HE staining showed the formation of 3D network structure. The immunohistochemistry staining of CK and Vimentin were positive.Conclusion We reconstructed engineered 3D bronchial model successfully in vitro and seeding cells could implement polarity growing in the scaffold materials then gained the network structure.
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In order to study the chondrogenic phenotype differentiation of adult sheep bone marrow-derived mesenchymal stem cells (MSCs) in a defined medium as potential seed cells for cartilage tissue engineering. MSCs were isolated by density centrifugation with Percoll solution from bone marrow aspirated from sheep iliac crest. The third passage of MSCs were induced with H-DMEM containing TGF-beta3, IGF-I, Dexamethasone and VitC. The shape and ultrastructure of cells were observed, toluidine blue stain for GAG and immunohistochemistry for type II collagen were applied for chondrogenic phenotype identification. After 14 days of induction, MSCs changed from a spindle-like appearance to a polynal shape, a large amount of endoplasmic reticulum, Golgi complex and mitochondria were observed, and the differentiation of MSCs chondrogenic phenotype was verified by positive staining of toluidine blue and immunohistochemistry. MSCs derived from bone marrow can differentiate to chondrogenic phenotype when induced in vitro and can be used as optimal seed cells for cartilage tissue engineering.
Subject(s)
Bone Marrow Cells/cytology , Cell Differentiation , Cell Separation , Cells, Cultured , Chondrocytes/cytology , Chondrogenesis , Mesenchymal Stem Cells/cytology , Phenotype , Sheep , Tissue EngineeringABSTRACT
In order to study the chondrogenic phenotype differentiation of adult sheep bone marrow-derived mesenchymal stem cells (MSCs) in a defined medium as potential seed cells for cartilage tissue engineering. MSCs were isolated by density centrifugation with Percoll solution from bone marrow aspirated from sheep iliac crest. The third passage of MSCs were induced with H-DMEM containing TGF-beta3, IGF-I, Dexamethasone and VitC. The shape and ultrastructure of cells were observed, toluidine blue stain for GAG and immunohistochemistry for type II collagen were applied for chondrogenic phenotype identification. After 14 days of induction, MSCs changed from a spindle-like appearance to a polynal shape, a large amount of endoplasmic reticulum, Golgi complex and mitochondria were observed, and the differentiation of MSCs chondrogenic phenotype was verified by positive staining of toluidine blue and immunohistochemistry. MSCs derived from bone marrow can differentiate to chondrogenic phenotype when induced in vitro and can be used as optimal seed cells for cartilage tissue engineering.
Subject(s)
Animals , Bone Marrow Cells , Cell Biology , Cell Differentiation , Cell Separation , Cells, Cultured , Chondrocytes , Cell Biology , Chondrogenesis , Mesenchymal Stem Cells , Cell Biology , Phenotype , Sheep , Tissue EngineeringABSTRACT
<p><b>OBJECTIVE</b>To evaluate the feasibility of growing tissue-engineered cartilage using chondrocytes seeded onto a biodegradable porous bioceramic, the beta-tricalcium phosphate (beta-TCP).</p><p><b>METHODS</b>A porous bioceramic template of beta-TCP was created in the shape of a disc. Chondrocytes isolated from rabbit articular cartilage were seeded on the beta-TCP template and then kept in rotatory cell culture system (RCCS) for 1 week prior to subcutaneous transplantation into athymic mice. The three-dimensional structure was well-maintained 16 weeks after implantation. After 4, 8, 16 weeks, the specimens were harvested and examined macroscopically, histologically and immunohistochemically.</p><p><b>RESULTS</b>Gross morphological and histological analysis of the specimens from the chondrocyte-beta-TCP complex demonstrated new cartilage construction. The overall configuration of the experimental specimens closely resembled the structure of beta-TCP template.</p><p><b>CONCLUSION</b>These findings suggest that porous bioceramic (beta-TCP) is a good "matrix" for chondrocyte, and can be used for cartilage engineering.</p>
Subject(s)
Animals , Female , Mice , Calcium Phosphates , Pharmacology , Cartilage , Transplantation , DNA , Glycosaminoglycans , Immunohistochemistry , Mice, Nude , Tissue EngineeringABSTRACT
AIM: To set up a method of inducing mouse embryonic stem cells (mESC) to differentiate into cardiomyocyte after treatment with 5-azacytidine. METHODS: Cytotoxicity of 5-azacytidine was measured by MTT assay. Treatment of mESC with conditioned culture mediums, which were composed of 5-azacytidine alone or combined with retinoic acid, induced the cell differentiation to cardiomyocytes. The cells induced were identified by detecting the expression of cardiac proteins (myosin, desmin, ?-actin and ?-actinin). Gene MLC-2v, a specific gene of ventricular-like cardiomyocyte, was also detected by RT-PCR. RESULTS: The non-cytotoxic dose of 5-azacytidine was 8 ?mol/L, which was able to induce mESC to differentiate into cardiac syncytiums. Cells induced expressed many cardiac proteins and MLC-2v mRNA. However, combined with retinoic acid inhibited mESC differentiation into cardiomyocyte. CONCLUSION: 5-azacytidine is able to promote mESC differentiation into cardiomyocytes. A method of inducing mESC to differentiate into cardiomyocytes in vitro has been established.
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Objective The purpose of this study was to investigate the effect of different level of sex steroids (?-estradiol and progesterone) on the proliferation of uterus endometrial epithelial cells. Methods Rabbit uterus endometrial epithelial cells were isolated by digestion of trypsin and centrifugation. The specificity and homogeneity of the endometrial epithelial cells cultured in media containing ?-estradiol and progesterone were determined by immunofluorescence staining of cytokeratin monoclonal antibody and the flow cytometric analysis. The different concentration of progesterone (P4) and ?-estradiol (E2) were added in media, the proliferative capability of the endometrial epithelial cells cells in vitro was determined indirectly by MTT assay. Results High purity endometrial epithelial cells (96%) were isolated using centrifugation method. The endometrial epithelial cells were examined by immunofluorescence staining. The endometrial epithelial cells were positive stained by anti-cytokeratin antibody. Immunofluorescence staining demonstrated that cytokeratin was detectable in the cytoplasm of epithelial cells. The cell growth curve showed that the proliferation of endometrial epithelial cells was not significantly changed by the treatment of E2 (100nmol/L) or P4 (100nmol/L) individually, but a combination of 100nmol/L E2 and 10nmol/L P4 could promote its proliferation greatly (P