Modulação da diferenciação neural de células troco embrionárias por transientes de cálcio intracelulares: papéis dos receptores purinérgicos e de canais de cálcio voltagem-dependentes / Modulation of neural embryonic stem cell differentiation by intracellular Ca2+ oscillations. Roles of purinergic receptors and voltage gated Ca2+ channels

Receptores purinérgicos e canais de cálcio voltagem-dependentes estão envolvidos em diversos processos biológicos como na gastrulação, durante o desenvolvimento embrionário, e na diferenciação neural. Quando ativados, canais de cálcio voltagem-dependentes e receptores purinérgicos do tipo P2, ativados por nucleotídeos, desencadeiam transientes de cálcio intracelulares controlando diversos processos biológicos. Neste trabalho, nós estudamos a participação de canais de cálcio voltagem-dependentes e receptores do tipo P2 na geração de transientes de cálcio espontâneos e sua regulação na expressão de fatores de transcrição relacionados com a neurogênese utilizando como modelo células tronco (CTE) induzidas à diferenciação em células tronco neurais (NSC) com ácido retinóico. Descrevemos que CTE indiferenciadas podem ter a proliferação acelerada pela ativação de receptores P2X7, enquanto que a expressão e a atividade desse receptor precisam ser inibidas para o progresso da diferenciação em neuroblasto. Além disso, ao longo da diferenciação neural, por análise em tempo real dos níveis de cálcio intracelular livre identificamos 3 padrões de oscilações espontâneas de cálcio (onda, pico e unique), e mostramos que ondas e picos tiveram a frequência e amplitude aumentadas conforme o andamento da diferenciação. Células tratadas com o inibidor do receptor de inositol 1,4,5-trifosfato (IP3R), Xestospongin C, apresentaram picos mas não ondas, indicando que ondas dependem exclusivamente de cálcio oriundo do retículo endoplasmático pela ativação de IP3R. NSC de telencéfalo de embrião de camundongos transgênicos ou pré-diferenciadas de CTE tratadas com Bz-ATP, o agonista do receptor P2X7, e com 2SUTP, agonista de P2Y2 e P2Y4, aumentaram a frequência e a amplitude das oscilações espontâneas de cálcio do tipo pico. Dados, obtidos por microscopia de luminescência, da expressão em tempo real de gene repórter luciferase fusionado à Mash1 e Ngn2 revelou que a ativação dos receptores P2Y2/P2Y4 aumentou a expressão estável de Mash1 enquanto que ativação do receptor P2X7 levou ao aumento de Ngn2. Além disso, células na presença do quelante de cálcio extracelular (EGTA) ou do depletor dos estoques intracelulares de cálcio do retículo endoplasmático (thapsigargin) apresentaram redução na expressão de Mash1 e Ngn2, indicando que ambos são regulados pela sinalização de cálcio. A investigação dos canais de cálcio voltagem-dependentes demonstrou que o influxo de cálcio gerado por despolarização da membrana de NSC diferenciadas de CTE é decorrente da ativação de canais de cálcio voltagem-dependentes do tipo L. Além disso, esse influxo pode controlar o destino celular por estabilizar expressão de Mash1 e induzir a diferenciação neuronal por fosforilação e translocação do fator de transcrição CREB. Esses dados sugerem que os receptores P2X7, P2Y2, P2Y4 e canais de cálcio voltagem-dependentes do tipo L podem modular as oscilações espontâneas de cálcio durante a diferenciação neural e consequentemente alteram o padrão de expressão de Mash1 e Ngn2 favorecendo a decisão do destino celular neuronal

Purinergic receptors and voltage gated Ca2+ channels have been attributed with developmental functions including gastrulation and neural differentiation. Upon activation, nucleotide-activated P2 purinergic receptor and voltage-gated Ca2+ channel subtypes trigger intracellular calcium transients controlling cellular processes. Here, we studied the participation of voltage-gated calcium channels and P2 receptor activity in spontaneous calcium transients and consequent regulation expression of transcription factors related to retinoic acid-induced neurogenesis of mouse neural stem and embryonic stem cells (ESC). In embryonic pluripotent stem cells, proliferation is accelerated by P2X7 receptor activation, while receptor expression / activity needs to be down-regulated for the progress of neuroblast differentiation. Moreover, along neural differentiation time lapse imaging with means of a cytosolic calcium-sensitive fluorescent probe provided different patterns of spontaneous calcium transients (waves and spikes) showing that both, frequency and amplitude increased along differentiation. Cells treated with the inositol 1,4,5-trisphosphate receptor (IP3R) inhibitor Xestospongin C showed spikes but not waves, indicating that waves exclusively depended on calcium release from endoplasmic reticulum by IP3R activation. Cells treated with the P2X7 receptor subtype agonist Bz-ATP and the P2Y2 and P2Y4 receptor 2-S-UTP increased frequency and amplitudes of calcium transients, mainly spikes, in embryonic telencephalon neural stem cells (NSC) and NSC pre-differentiated from ESC. Data obtained by luminescence time lapse imaging of stable transfected cells with Mash1 or Ngn2 promoter-protein fusion to luciferase reporter construct revealed increased Mash1 expression due to activation of P2Y2/P2Y4 receptor subtypes, while increased expression of Ngn2 was observed following P2X7 receptor activation. In addition, cells imaged in presence of the extracellular calcium chelator EGTA or following endoplasmic reticulum calcium store depletion by thapsigargin showed a decrease in Mash1 and Ngn2 expression, indicating that both are regulated by calcium signaling. Investigation of the roles of voltage gated Ca2+ channels in neural differentiation showed that Ca2+ influx in NSC pre-differentiated from ESC is due to membrane depolarization and L-type voltage gated Ca2+ channel activation, thereby controlling cell fate decision, by stabilizing the expression of MASH1 and inducing differentiation, by phosphorylation of the transcription factor CREB. Altogether these data suggest that P2X7, P2Y2, P2Y4 receptors and L-type voltage gated Ca2+ channels can modulate spontaneous calcium oscillations during neural differentiation and consequently change the Mash1 and Ngn2 expression patterns, thus favoring the cell fate decision to the neuronal phenotype

Estudo citofotométrico da expressão dos marcadores tumorais Ki-67 e CD34 no adenocarcinoma de próstata / Cytophotometric expression of tumor antigen markers Ki-67 and CD-34 in prostate adenocarcinoma

OBJETIVO: Quantificar a porcentagem da imunomarcação no índice de marcagem e densidade óptica do Ki-67 e CD34 no adenocarcinoma de próstata e compará-las entre si. MÉTODOS: Foram estudados, através de imunoistoquímica, o Ki-67 e o CD34 em 34 casos de adenocarcinoma de próstata provenientes de prostatectomia radical no período de 2000 a 2005 realizado no Hospital Regional do Gama em Brasília. Estes marcadores foram quantificados através do software SAMBA 4000 ® Sistema de Análise Microscópica de Busca Automática e do software IMMUNO® para análise das variáveis índice de marcagem e densidade óptica. Para avaliação da associação entre as expressões do marcador, foi estimado o coeficiente de correlação de Spearman. Para a comparação do tipo de lesão, foi usado o teste t de Student em amostras pareadas e não paramétrico de Wilcoxon. RESULTADOS: Dos 34 blocos que foram para leitura dos marcadores tumorais, 15 marcaram expressão com Ki-67, 34 com CD34 e 14 com ambos os marcadores. O índice de marcagem do CD34 teve valor mediano de 72,72 por cento, valor mínimo 5,14 por cento e valor máximo 88,81 por cento. O índice de marcagem do Ki-67 teve mediana de 73,78 por cento, mínimo de 16,87 por cento e máximo de 87,47 por cento. A densidade óptica do CD34 teve mediana de 48,33, mínimo de 35,65 e máximo de 85,86. Na densidade óptica do Ki-67 o valor da mediana foi 40,03 sendo a mínima de 21,53 e a máxima de 52,43. CONCLUSÃO: A expressão citofotométrica do Ki-67 teve índice médio de marcação de 64,04 por cento e o CD34 de 61,64 por cento. A expressão citofotométrica da densidade óptica média do Ki-67 foi de 39,49 e no CD34 de 53,69. Há diferença significativa entre a imunomarcação do Ki-67 e CD34 em relação à densidade óptica (p=0,025), não havendo diferença significativa no índice de marcagem (p=0,470).

OBJECTIVE: To quantify the percentage of immunostaining through the labeling index as well as the optical density of Ki-67 and CD34 in prostate adenocarcinoma and compare the results between markers. METHODS: Markers Ki-67 and CD34 were studied using immunohistochemistry in 34 cases of prostate adenocarcinoma from radical prostatectomies performed at the Hospital Regional do Gama in Brasilia, Brazil from 2000 through 2005. Those markers were quantified using the SAMBA 4000® software - Automated Scanning Microscopic Analysis System - and the IMMUNO® software in the analysis of the variables labeling index and optical density. Spearman's correlation coefficient was estimated in order to evaluate the association between the expression levels of the markers. For the comparison of lesion types, Student's paired t-test and the nonparametric Wilcoxon test were used. RESULTS: Of the 34 blocks referred for the study of the tumor markers, 15 were positive for Ki-67, 34 showed CD34 expression, and 14 were positive for both markers. The median value for the labeling index of CD34 was 72.72 percent; the minimum was 5.14 percent and the maximum, 88.81 percent. The median for the Ki-67 labeling index was 73.78 percent, while the minimum was 16.87 percent, and the maximum, 87.47 percent. The median value for the optical density of CD34 was 48.33, the minimum was 35.65 and the maximum, 85.86. For the optical density of Ki-67, the median was 40.03, while the minimum and maximum values were 21.53 and 52.43, respectively. CONCLUSION: The cytophotometric expression of Ki-67 had a mean labeling index of 64.04 percent, and the mean CD34 labeling index was 61.64 percent. The cytophotometric expression of the mean optical density of Ki-67 was 39.49, while for CD34 it was 53.69. There was a significant difference between Ki-67 and CD34 immunostaining with respect to optical density (P=0.025); no significant difference occurred regarding labeling index (P=0.470).

Study on relationship between changes of geometry parameters in human spleen nuclei and the postmortem interval / 法医学杂志

OBJECTIVE@#Using computer image-analyze technique (CIAT) to study changes of geometry parameters in human spleen nuclei and seek a new experimental method to deduce the estimation the postmortem interval (PMI).@*METHODS@#31 cadavers that known accurate PMI, sampled and smeared respectively every hour within the first 36 hours after death, fixed with cold Carony fixation, stained by Feulgen-van's method, and measured 5 geometry parameters using the image-analyze instrument including Area (A), Mean-Dia (MD), Average Diameter (AD), perimeter (P), Index of density (ID).@*RESULTS@#A, MD, AD and P in the human spleen nuclei have no correlation with the PMI. But ID rose regularly with the prolongation of PMI in 36 hours. There was a definite correlation between ID and the PMI, r=0.983, linear regression equation with PMI (hours) as the dependent variable was calculated for ID.@*CONCLUSION@#Geometry parameter ID was proved to be preferable indexes for estimation of PMI in 36 hours.

DNA-feulgen cytophotometry in hyperplasia and adenocarcinoma of the endometrium

The present study was carried out on 35 endometrial hyperplasia, 30 endometrial carcinoma and 5 normal proliferative phase endometrium. Cases were stained with hematoxylin and eosin and Feulgen stain for cytophotometry. The majority of cells in the normal and the hyperplastic groups, including atypical hyperplasia were within the diploid range, whereas more than 65% of the cells in the carcinoma group, were aneuploid with increase in number of aneuploid cells as the grade of the tumor increased. The number of aneuploid cells was also proportionate to the nuclear area measurements. Small nuclear areas were usually diploid, while larger ones were associated with more DNA and were aneuploid content. Such results indicated that cytophotometry may be a helpful adjuvant in routine histolopathological diagnosis, increasing the diagnostic accuracy and shedding light on the biological potential of the malignant cells

Study of the effect of feeding different vegetable oils on liver parenchyma of hypercholesterolemic rats using cytophotometry and AgNORs enumeration

This study was carried out to evaluate the effect of continuous feeding of hypercholasterolemic rats on high fat diet [containing either corn oil,cotton oil,Canadian rapeseed oil or Egyptian rapeseed oil] on morphology, DNA content and nucleolar organizer regions [NORs] of liver cells. Hypercholesterolemia was induced in 50% adult male albino rats using high fat diet containing cholesterol, cholic acid and 20% palm stearin for 8 weeks. This diet caused fatty infiltration and mild periportal fibrosis of the liver. Feeding hypercholesterolemic rats on high fat diet containing 20% of either Egyptian or Canadian rapeseed oil for 5 weeks aggravated these changes and also caused mild to moderate dysplasia. These histopathological changes were confirmed by an increase in DNA content of the liver cells and increase number of AgNORs