Optimización de unidades formadoras de colonias y detección del virus de papiloma humano en sangre de cordón umbilical / Colony-forming units optimization and human papillomavirus detection in umbilical cord blood

Resumen Introducción: Se requiere analizar diversos parámetros para el control de calidad adecuado de las unidades de sangre de cordón umbilical (USCU) cuando se utilizan con fines terapéuticos. Objetivo: Optimizar las unidades formadoras de colonias (UFC) de cultivos clonogénicos y detectar el genoma del virus del papiloma humano (VPH) en USCU. Métodos: Se incluyeron 141 muestras de sangre de cordón umbilical (SCU), de segmento y de UFC de cultivos clonogénicos de USCU. Se realizó extracción de ADN, cuantificación y amplificación por PCR del gen endógeno GAPDH. Se detectó el gen L1 del VPH con los oligonucleótidos MY09/MY11 y GP5/GP6+; los productos de PCR se migraron en electroforesis de agarosa. El ADN purificado de las UFC se analizó mediante electroforesis de agarosa y algunos ADN, con la técnica sequence specific priming. Resultados: La concentración de ADN extraído de UFC fue superior comparada con la de SCU (p = 0.0041) y la de segmento (p < 0.0001); así como la de SCU comparada con la de segmento (p < 0.0001). Todas las muestras fueron positivas para la amplificación de GAPDH y negativas para MY09/MY11 y GP5/GP6+. Conclusiones: Las USCU criopreservadas fueron VPH netativas; además, es factible obtener ADN en altas concentraciones y con alta pureza a partir de UFC de los cultivos clonogénicos.

Abstract Introduction: Analysis of several markers is required for adequate quality control in umbilical cord blood units (UCBU) when are used for therapeutic purposes. Objective: To optimize colony-forming units (CFU) from clonogenic cultures and to detect the human papillomavirus (HPV) genome in UCBU. Methods: One hundred and forty-one umbilical cord blood (UCB), segment or CFU samples from UCBU clonogenic cultures were included. DNA extraction, quantification and endogenous GAPDH gene PCR amplification were carried out. Subsequently, HPV L1 gene was detected using the MY09/MY11 and GP5/GP6+ oligonucleotides. PCR products were analyzed with electrophoresis in agarose gel. CFU-extracted purified DNA was analyzed by electrophoresis in agarose gel, as well as some DNAs, using the SSP technique. Results: CFU-extracted DNA concentration was higher in comparison with that of UCB (p = 0.0041) and that of the segment (p < 0.0001), as well as that of UCB in comparison with that of the segment (p < 0.0001). All samples were positive for GAPDH amplification and negative for MY09/MY11 and GP5/GP6+. Conclusions: Cryopreserved UCBUs were HPV-negative. Obtaining CFU DNA from clonogenic cultures with high concentrations and purity is feasible.

Involvement of inhibition of nucleus GAPDH over-expression in erythropoietin's reduction of neuronal apoptosis induced by brain ischemia/reperfusion in rats / 生理学报

To study whether recombinant human erythropoietin (rhEPO) reduces neuronal apoptosis through inhibiting over-expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in nucleus induced by brain ischemia/reperfusion in rats, 48 adult Sprague-Dawley rats were randomly divided into 3 groups: sham, saline and EPO groups. Animal models of brain ischemia/reperfusion were established by middle cerebral artery occlusion in rats. The effects of EPO on the sizes of ischemia tissue were observed by TTC staining. The over-expression of GAPDH in nucleus was detected by Hoechst-33258 and anti-GAPDH antibody double staining. The neuronal apoptosis in penumbral was detected by Nissl's staining and Hoechst-33258 immunofluorescence, respectively. The results showed that rhEPO treatment (3 000 U/kg, three times daily, i.p.) apparently reduced the sizes of infarct brain tissue in ischemia/reperfusion rats. rhEPO inhibited over-expression of GAPDH in nucleus of apoptotic neurons. In the meantime rhEPO decreased the number of apoptotic neurons in ischemia/reperfusion rats. These results suggest that rhEPO may induced reduction of neuronal apoptosis in penumbra may be through inhibiting over-expression of GAPDH in nucleus of apoptotic neurons induced by ischemia/reperfusion. Reduction of GAPDH over-expression in nucleus may play a pivotal role in EPO inhibiting neuronal apoptosis in cerebral ischemia/reperfusion rats, providing experimental evidence for EPO neuro-protecting effects against ischemia/reperfusion.

Enhanced tyrosine hydroxylase expression in PC12 cells co-cultured with feline mesenchymal stem cells

Mesenchymal stem cells (MSCs) secrete a variety of neuroregulatory molecules, such as nerve growth factor, brain-derived neurotrophic factor, and glial cell-derived neurotrophic factor, which upregulate tyrosine hydroxylase (TH) gene expression in PC12 cells. Enhancing TH gene expression is a critical step for treatment of Parkinson's disease (PD). The objective of this study was to assess the effects of co-culturing PC12 cells with MSCs from feline bone marrow on TH protein expression. We divided the study into three groups: an MSC group, a PC12 cell group, and the combined MSC + PC12 cell group (the co-culture group). All cells were cultured in DMEM-HG medium supplemented with 10% fetal bovine serum for three days. Thereafter, the cells were examined using western blot analysis and immunocytochemistry. In western blots, the co-culture group demonstrated a stronger signal at 60 kDa than the PC12 cell group (p < 0.001). TH was not expressed in the MSC group, either in western blot or immunocytochemistry. Thus, the MSCs of feline bone marrow can up-regulate TH expression in PC12 cells. This implies a new role for MSCs in the neurodegenerative disease process.