Marilda Osti Spinelli

Protocolo da extração de DNA de material parafinado para análise de microssatélites em leiomioma

The standardization of the protocol of the DNA extraction from paraffin material in order to analyze the instability of microsatellites in genes related to the uterine leiomyoma, using 30 to 40mg of tissue removed from paraffin blocks is presented. Desparaffin throught xilol baths to 65oC, rehydrated with solutions of decreasing concentrations of ETOH and deionisation water. The extraction of DNA was obtained through the stages of cellular lysis, with Promega solution, digestion of proteins by Proteinase K, precipitation proteins with Promega solution and precipitation of DNA with ETOH 70% colded and ressuspended with Promega solution. This protocol results in a viable DNA to be use in the Polimerase Chain Reaction (PCR).

Inbred F344 rats as a biologic model of intra-abdominal sepsis

Aiming to design a biologic model of acute intra-abdominal sepsis for experimental studies, conventional, isogenic rats F344 were infected with the bacteria Escherichia coli (E.coli), strain 11775, serotype H7:01:K1. Six hours after inoculation with E. coli, the animals - males and females - showed the following symptoms: piloerection, hyperpnea and decreased motor activity. The lethal dosis (LD50), was 6 x 105 CFU/ml, analyzed in 32 males and 32 females. The highest mortality rate was observed on the first 24 hours. Liver dysfunction, common in intra-abdominal sepsis, was evaluated at 0, 24, 48 and 168 hours after inoculation, by means of serum enzyme activities. Study of the migration of polymorphonuclear-neutrophil cells (PMN) and mononuclear-macrophage ones (MN) showed a significant increase of PMN in E. coli inoculated males (z > 4.7; P 6.2; P < 0.0003), when compared to control groups. As for MN cells, there were no differences between inoculated and control groups, in males (z = 2.3; P = 0.0107) and in females (z = 1.8; P = 0.0359) as well. In conclusion, these results show that inbred F344 rats are adequate biologic models for studies of acute, intra-abdominal sepsis.