Módulo magnetismo y electro magnetismo

RESULTS.

Having investigated the aromatase expression in homogenised adipose tissue obtained from patients undergoing reduction mammoplasty, subsequent studies investigated the aromatase expression in adipocyte and stromal cell fractions of this tissue. Cell fractions were obtained by collagenase digestion and differential centrifugation into a "stromal" cell pellet and floating adipocyte layer. The stromal cell population was characterized by Giemsa staining and immunocytochemistry. Fig 3.2.1 a-d demonstrate the populations of cells seen in the pelleted fraction. The majority of the cells in the pelleted fraction had the histological characteristics of stromal cells.

RNA was extracted from the cell populations obtained, 20pg of which was utilised in the

competitive PCR reaction, along with lOpg rat cRNA. Fig 3.2.2 iliustrates the results of

amplification of four samples of mRNA from adipocytes and stromal cells derived from the breast tissue of three patients. The relative concentration of aromatase mRNA in the stromal cell fraction was 3.5 to 19 fold higher than in the adipocytes.

Figure 32.1.a. Giemsa stained pelleted cells.

e

*

!*•

" IE- %

"Stromal" cells extracted from one sample of adipose tissue, collagenase digested and separated were stained and representative photos from the samples are shown.

Cytochemical and immunological staining was performed on the pelleted cells after spinning onto poly-L-lysine coated slides.

Stained samples revealed predominantly medium and large diameter mononuclear cells of varying staining intensities. Some binucleate cells were seen as well as the occasional cell clusters and short tubular structures.

Figure 32.1b. Cytokeratin stained pelleted cells.

Cells were stained for cytokeratin using a monoclonal antibody (anti-human cytokeratin, M821). The antiborly is highly cross reactive to a variety of human keratins. Appropriate staining was verified using human breast epithelial cells.

Epithelial cells (cytokeratin positive) comprised only 0.3% and 1.7% of the total count and were often seen as clusters of small cells.

Figure 3.21c. Wmenlin stained pelleted cells.

#

3

Vimentin staining with murine vimentin monoclonal antibody (antiswine vimentin, M725). This has a broad interspecies cross reactivity including human.

Samples were also stained for vimentin intermediate filaments, characteristic of cells of mesenchymal origin, including stromal cells, endothelial cells and macrophages the latter of which show only weak vimentin staining. The majority of the cells were vimentin positive (81 % and 92%).

Figure 32.1d. Macrophage marker C D IIc (KB90) stained cells. [

#

The monocyte/macrophage staining was carried out using the KB90 murine monoclonal antibody and visualised using an alkaline phosphatase-anti-alkaline-phosphatase technique. Optimal dilutions were obtained using peripheral blood mononuclear cells.

Cells expressing macrophage markers comprised 8% and 18% of the cells, while those identified

as endothelial cells by Factor VIII staining were even fewer in number (6.3% and 7.6%) of the total cells.

Figure 3% 2. Relative levels of mRNA encoding aromatase in adipocytes and stromal cells. ADIPOCYTES PELLETED CELLS HUMAN ONLY RAT ONLY NO RNA O 50 T (/) Û 4 0 - > 3 0 - Ü! 1 0 " u 2 0- A P A P A P A P

HUMAN PATIENT 1 PATIENT 2 PATIENT 3 RAT

Legend:

A adipocytes.

P pelleted (stromal) cells.

Comparison of aromatase mRNA in breast adipocytes and pelleted "stromal" cells. 20^g of breast RNA was co-amplified with 10/xg rat cRNA. Results of 4 experiments using 3 patients are shown with repres<?^Vaû*«lot blot autoradiographs. Product yields are expressed as corrected percent total volume density (mean±SEM). The control amplifications contained only rat aromatase cRNA, breast RNA or no RNA.

DISCUSSION.

Adipose tissue is composed primarily of lipid-laden mature adipoctyes and stromal cells. Stromal cells are capable of further differentiation into mature adipocytes and are regarded as pre­ adipocytes. There are two populations of cells which can be separated from the adipose tissue; a stromal cell population, consisting of mainly stromal cells of mesenchymal origin but also containing some epithelial and endothelial cells. The adipocyte fraction consists of mature adipocytes alone. The low percentage of epithelial cells in the pelleted fraction is probably due to the method of preparation. Since breast parenchymal tissue is less susceptible to collagenase digestion than adipose tissue, the technique selects for adipose tissue cell types.

The levels of mRNA encoding aromatase in these two cell populations differed significantly, the majority of mRNA being expressed in the stromal cell fraction. There are three possible interpretations of this result. Firstly, that the stromal cells contain more mRNA for aromatase than the adipocytes. Alternatively, the minority of the cell types in the pelleted fraction, such as the epithelial cells, macrophages and endothelial cells, might contain such high levels of aromatase mRNA that the overall pelleted cell fraction has a high mRNA content, even when the stromal cell contribution to the total mRNA level is low. To date, aromatase activities in these particular breast cells have not been evaluated. However, isolated alveolar macrophages and culture

umbilical endothelial cells have undetectable aromatase activities (Milewich et a/., 1983 and

1987). It is likely, therefore that the aromatase activity resides in the stromal cells and not in the minority cell types. The third possible explanation is that the half life of the mRNA in these two cell populations differs such that the stromal cell mRNA has a longer half life than the adipocyte mRNA.

The observation that the majority of the aromatase mRNA is in the stromal cell fraction of adipose tissue is supportive of previous studies in which abdominal adipose tissue was separated into the same two fractions and the relative levels of aromatase activity assessed in

the two populations of cells. The adipocyte fraction was shown to contain 13% of the aromatase

activity, while the stromal cell pellet contained 87% (Ackerman etal., 1981). However, membrane

fractions of the two cell populations contained comparable aromatase activities, leading to the conclusion that the low level of aromatase activity in the intact adipocytes was due to the sequestration of the substrate into the lipid droplet of the adipocytes, leaving it unavailable for aromatisation. While this may be true, the difference in mRNA levels between the cell types will inevitably contribute to the differences in aromatase activity in intact cells.

3. DETERMINATION OF THE LEVELS OF mRNA ENCODING AROMATASE IN HUMAN