Studies on erythroid cell maturation

Burgos, Hugo (1971) Studies on erythroid cell maturation. PhD thesis, University of Glasgow.

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Abstract

Studies on erythroid cell maturation were carried out by means of two different experimental approaches leading to the characterization of (a) the haemoglobin type synthesized by spleen colonies produced by transplantation of rat haemopoietic tissue into heavily irradiated mice, and (b) the daughter cells derived from mitoses of earlier haemopoietic precursors, and factors influencing their maturation and proliferation. Optimal experimental conditions for spleen recolonization by heterologous (rat) transplants were investigated and established through a series of experiments, and methods for preparation and analysis of the type of haemoglobin synthesized by the re colonizing cells were developed. Investigation of the behaviour of normal and experimental haemoglobins in starch gel electrophoresis and development of assay for their differential characterization were undertaken. Supralethally irradiated mice (Porton strain) were transplanted with foetal liver and adult bone marrow cells from Wistar rats. After a suitable post-transplantation period, the exogenously recolonized spleens were obtained. The confluent spleen colonies were isolated and disaggregated, and cell cultures prepared and incubated with 59FeCl3, Haemoglobins were extracted, purified by CMC column chromatography, and fractionated by starch gel electrophoresis. The incorporation of 59Fe into haemoglobin was determined by cutting the gel strip containing the haemoglobin components into 1mm or 1 mm thick slices, which were then individuaJ.ly hydrolysed and counted by liquid scintillation system. It was possible to demonstrate the heterogeneity of rat haemoglobin, and the presence of haemoglobin aggregates. Synthesis of catalase and other non-haemoglobin, haem-containing proteins, along with synthesis of haemoglobin was also demonstrated. This makes the interpretation of assays based on iron incorporation difficult for haemoglobin synthesis studies. Although recolonization of mouse spleens by rat haemopoietic tissues was achieved in spite of the antigenic disparity between donor and host, the analysis of the type of haemoglobin synthesized by the spleen re colonizing cells was obscured, by the presence of endogenous haemoglobin and aggregates. However, there was some indication that the recolonizing cells continued to produce the same type of haemoglobin. For the in vitro study of behavioural and morphological characteristics of erythroid cells, and factors influencing their maturation and proliferation, 13-day foetal mouse livers were isolated and disaggregated. Cell cultures were prepared in plasma and fibrin clots, and observed with time-lapse cinemicrography during mitosis. The events recorded on the film were analysed and correlated with the fixed and stained cells. The effects of different plasma, and erythropoietin stimulation on mitosis of erythroid cells were studied by measuring differences in frequency distribution of mitoses and mitotic indices, and also by autoradiography. The results demonstrated an increased, number of mitoses in erythropoietin-stimulated cultures. Erythropoietin appeared to permit the continuation of the cell cycle and to maintain mitotic activity throughout the period of incubation. The results also suggested that erythropoietin produced shortening of G2 and G1, i.e. precipitated the initiation of mitosis as well as synthesis. By the use of plasma from mice made polycythaemic by hypertransfusion, and stimulation with erythropoietin, the presence of an erythropoietic mitotic inhibitor was demonstrated.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: John Paul
Keywords: Cellular biology
Date of Award: 1971
Depositing User: Enlighten Team
Unique ID: glathesis:1971-73080
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 14 Jun 2019 08:56
Last Modified: 14 Jun 2019 08:56
URI: http://theses.gla.ac.uk/id/eprint/73080

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