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1.
Journal of Southern Medical University ; (12): 49-55, 2007.
Artículo en Chino | WPRIM | ID: wpr-298246

RESUMEN

<p><b>OBJECTIVE</b>To study the effect of superparamgnetic iron oxides (ferumoxides) on the survival and proliferation of neural stem cells (NSCs) and determine the optimal ferumoxides concentration for labeling.</p><p><b>METHODS</b>Bone marrow stromal cells (BMSCs) were obtained from rat femoral marrow and cultured in vitro to induce their differentiation into NSCs. Ferumoxides labeling of the NSCs was performed with different final concentrations of ferumoxides, and the labeling efficiency and viability of the labeled NSCs were evaluated by Prussian blue staining, MTT assay, flow cytometry and transmission electron microscope.</p><p><b>RESULTS</b>The NSCs could be effectively labeled with ferumoxides with a labeling efficiency of around 90%. Prussian blue staining showed numerous fine granules with blue staining in the cytoplasm of the labeled NSCs, and the intensity of the blue staining was in positive correlation with the ferumoxide concentration for labeling. Transmission electron microscopy of the labeled NSCs revealed the presence of numerous vesicles spreading in the cytoplasm and filled with electron-dense magnetic iron particles. The ferumoxides vesicles increased with the labeling concentration of ferumoxides, and at the final concentration exceeding 25 microg/ml, ferumoxides vesicles in the NSCs gave rise to conglomeration which hampered observation of the cellular ultrastructure by transmission electron microscope. The results of flow cytometry and MTT assay demonstrated that the cell viability, proliferation, differentiation and apoptosis of the labeled cells were affected by ferumoxides at the concentration above 25 microg/ml, but such effects could be minimal at lower concentrations.</p><p><b>CONCLUSION</b>Ferumoxides might be feasible for in vitro labeling of the NSCs with the optimal concentration of 25 microg/ml.</p>


Asunto(s)
Animales , Masculino , Ratas , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Dextranos , Óxido Ferrosoférrico , Hierro , Farmacología , Nanopartículas de Magnetita , Microscopía Electrónica de Transmisión , Neuronas , Biología Celular , Óxidos , Farmacología , Células Madre , Biología Celular
2.
Journal of Southern Medical University ; (12): 611-615, 2007.
Artículo en Chino | WPRIM | ID: wpr-268067

RESUMEN

<p><b>OBJECTIVE</b>To label rat neural stem cells (NSCs) with the complex of Sinerem, the ultrasmall superparamagnetic iron oxide (USPIO), and poly-L-lysine (PLL), and evaluate the feasibility of tracking the labeled cells with magnetic resonance imaging (MRI) in vitro and in vivo.</p><p><b>METHODS</b>Sinerem was incubated with PLL to obtain the complex of Sinerem-PLL. The mesenchymal stem cells (MSCs) isolated from the bone marrow of SD rats were cultured and induced to differentiate into the neural stem cells. The second-passage cells were cultured overnight with the Sinerem-PLL complex, after which Prussian blue staining and transmission electron microscopy were performed to observe the nanoparticles in the cytoplasm. Cell apoptosis assay was performed to assess the cell viability 1 day, 1 week, and 2 weeks after the labeling. Cell tracking with 4.7 MR system was carried out in vivo and in vitro using T(2)WI and T(2)*WI sequences.</p><p><b>RESULTS</b>The NSCs could be effectively labeled with Sinerem-PLL complex with the labeling efficiency exceeding 95%. Prussian blue staining showed numerous blue iron particles in the cytoplasm, and under transmission electron microscope, these particles accumulated in the endosomes/lysosomes. The labeling did not significantly affect the cell viability and proliferation. Remarkable low signal density changes of the labeled cells was seen on T(2)WI and T(2)*WI in vivo and in vitro.</p><p><b>CONCLUSION</b>NSCs can be effectively labeled with Sinerem-PLL complex, and MRI can be used to track the labeled cells in vivo and in vitro.</p>


Asunto(s)
Animales , Masculino , Ratas , Diferenciación Celular , Células Cultivadas , Dextranos , Metabolismo , Endosomas , Metabolismo , Óxido Ferrosoférrico , Metabolismo , Lisosomas , Metabolismo , Imagen por Resonancia Magnética , Métodos , Nanopartículas de Magnetita , Células Madre Mesenquimatosas , Biología Celular , Microscopía Electrónica de Transmisión , Neuronas , Biología Celular , Metabolismo , Polilisina , Metabolismo , Ratas Sprague-Dawley , Células Madre , Biología Celular , Metabolismo , Factores de Tiempo
3.
Chinese Journal of Oncology ; (12): 543-546, 2004.
Artículo en Chino | WPRIM | ID: wpr-254305

RESUMEN

<p><b>OBJECTIVE</b>To detect HPV 58, a common type of human papillomavirus (HPV), clone and express its E7 gene from biopsy specimens of cervical cancer.</p><p><b>METHODS</b>HPV 58 from 58 biopsy tissues of cervical cancer was detected by GP5+/GP6+ PCR followed by template-directed dye-terminator incorporation assay with fluorescence polarization detection (TDI-FP). HPV 58 E7 gene was amplified from one HPV 58-positive sample, and then cloned into pGEM-T Easy vector. The recombinant plasmid, HPV58E7-pGEM-T was confirmed by sequencing. Subsequently, E7 gene was cloned into prokaryotic expression vector pRSET-A. The constructed pRSET-58E7 plasmids were transfected into BL21(DE3) cells, and induced to express 58 E7 protein by IPTG.</p><p><b>RESULTS</b>Among the 58 biopsy tissues of cervical cancer, 10 were HPV 58-positive, accounting for 19.2% of 52 HPV-positive cases. HPV 58 E7 gene was amplified from one HPV 58-positive sample. The constructed plasmids were identified containing HPV58 E7 gene by restriction enzyme analysis and sequencing. SDS-PAGE analysis showed that HPV58 E7 His6 fusion protein of M(r) 16 x 10(3) was expressed by pRSET-58E7 after induction by IPTG. The fusion protein accounted for 30% of total bacterial proteins.</p><p><b>CONCLUSION</b>HPV 58 is not uncommon in Chinese women with cervical cancer in Shaanxi province. Constructed HPV58 E7 recombinant plasmids can be effectively expressed in E.coli, which may provide a tool in diagnosis and vaccine design for HPV of HPV58-associated tumors.</p>


Asunto(s)
Adulto , Femenino , Humanos , Persona de Mediana Edad , Clonación Molecular , Escherichia coli , Metabolismo , Genes Virales , Papillomaviridae , Genética , Proteínas E7 de Papillomavirus , Genética , Infecciones por Papillomavirus , Genética , Plásmidos , Proteínas Recombinantes , Genética , Transformación Genética , Neoplasias del Cuello Uterino , Genética , Virología
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