| dc.description.abstract | Pituitary-dependent hypercortisolism (PDH) or Cushing’s disease is a common endocrinopathy is dogs, caused by an ACTH-producing pituitary tumor. Clinical signs of PDH include polyuria/polydipsia, muscular atrophy , abdominal enlargement, alopecia and lethargy. Treatment consists of eliminating the glucocorticoid excess with a medical treatment or by eliminating the pituitary lesion via transsphenoidal hypophysectomy . With the removal of the tumor both endocrine and neurological problems are eliminated. Although postoperative remission rate is high, long term recurrence of the disease is common (around 23%).
It is hypothesized that Cancer Stem Cells (CSC) are a subpopulation of cells that are responsible for growth, maintenance and progression of tumors. Hypophysectomy and the availability of pituitary tumor tissue enable the isolation and characterization of these stem cells. In this way, new prognostic markers can be identified and ultimately, they could be used as new therapeutic targets, making therapy less invasive for the patient and more efficient against the disease. So far, no specific markers are identified to isolate stem or progenitor cells in the canine pituitary gland.
By means of a technique called Fluorescence Activated Cell Sorting (FACS), a small cell fraction (side population, SP) can be separated from the majority of cells (main population, MP). SP cells have been isolated from different tissues, and many times, the SP appeared to be enriched in stem cells. Previously, the SP from canine healthy pituitaries was successfully isolated.
In the present study, the cell digestion protocol and FACS technique was optimized for canine healthy pituitary and pituitary adenomas. Two different FACS instruments were used and several methods were tested to collect the cells after sorting, in order to obtain high quality RNA. There were high variations in percentage life cells and total life cell count among the samples. These variations may have influenced the SP appearance and with that, the proportion of sorted SP cells. When sorting bone marrow cells, RIN values of >6.5 were obtained from samples collected in RLT-buffer + β-mercaptoethanol (β -ME), empty tubes or in 1 ml RNA Later®, whereas RIN values were much lower in sorted pituitary gland samples . However, fresh samples sorted on the BD Influx showed a more stable expression of 3 reference genes compared with frozen samples sorted on the FACSAria II (SORP).
Next to the FACS study, the role of Pax7 and PC2 in canine pituitary adenomas was investigated. In mice, Pax7 expression is restricted to the melanotroph cells of the intermediate lobe. In canine, Pax7 expression was found in one healthy pituitary, and not in all intermediate lobe adenomas. Therefore, expression of the melanotrophe protein PC2 was investigated, but no expression was found in healthy pituitaries or pituitary adenomas. Besides, decreased gene expression of Pax7, Drd2, PC2, Sox2 and Thbs2 was found in canine pituitary adenomas, compared with healthy pituitaries. These results cannot support in an explanation for the Pax7 expression found in canine samples, but evoked new research questions about variations in expression of the investigated genes between the different species. | |
