Détection et Quantification du Parathyroid Hormone-Related Peptide (PTHrP) par RT-PCR compétitive

  • Ingo Beyer ((PhD) Student)
  • Pierre Bergmann (Promotor)
  • R Karmali (Co-promotor)

Scriptie/masterproef: Master-after-master

Uittreksel

A. Hypercalcemia of malignancy has been described for the first time in 1924 and has been ascribed to two mechanisms: local bone resorption from multiple and rapidly growing osteolytic bone metastases and production by the tumor of a humoral factor. In many cases of humoral hypercalcemia of malignancy (HHM) this factor is parathyroid hormone-related peptide (PTHrP), which has been identified in 1987. PTHrP has been detected and identified due to its endocrine effects that occur with massive production by certain tumors, but PTHrP exists as an intracrine or paracrine physiological factor in humans in utero and in adult life. Its role in enchondral bone formation has been demonstrated in knock-out mice. PTHrP to date has been demonstrated in most human tissues and its actions as a cytokine can be categorized into three major groups: 1) regulation of cellular proliferation and differentiation, 2) transepithelial calcium transport, and 3) relaxation of smooth muscle. The efects of PTHrP on bone resorption and on the osteoblast suggest that this factor, which is expressed by bone cells themeselves, might play a physiological role in th eregulation of bone turnover.
B. We have worked out a reverse transcription - polymerase chain reaction (RT-PCR) for quantification of PTHrP. A clonal cDNA has been modified by internal deletion of a 174 base pairs (bp) in its median section and has been cloned to serve as an internal standard (DeltaE). Two primers upstream and downstream from the deletion allow the coamplification of a 321 bp sequence of PTHrP¨and of a 144 bp sequence of DeltaE respectively. After competitive amplification of a given amount of PTHrP and of increasing amounts of the internal standard DeltaE the products are revealed by ethidium bromide and gel electrophoresis. The ratio of the photonic absorption at 260nm and at 280nm for both products is compared to the ratios for known quantities of DeltaE to allow quantification of PTHrP contained in the tissue. The method is validated by the quantification of a known amount of a synthetic PTHrP mRNA. The amounts of PTHrP mRNA present in a microgram of total mRNA have been measured to be 0,98 attomoles in gravid rat uterus and 0,065 attomoles in fetal rat long bones respectively. This method will allow to the study of PTHrP gene expressino in fetal rat long bones in vitro in a time effective manner end using only a small number of bones.
Datum Prijs30 sep 1999
TaalFrench
BegeleiderPierre Bergmann (Promotor) & R Karmali (Co-promotor)

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