Induction of single nucleotide polymorphisms in the purinergic P2X7 receptor and subsequent investigation of changes in function and gene expression of specific bone markers

Jan Rune Larsen

Studenteropgave: Speciale

Abstrakt

The investigation of bone is an interesting field in research involving crosstalk between several cells of different origins including several pathways of which many are still to be discovered and characterised. Through this system, a constant remodelling of the skeleton throughout the lifespan of a human is administered. If this system is no longer in homeostasis, it can lead to osteoporosis – a very invasive disease of which many elderly people suffers, in particular post-menopausal women. Involvement of the purinergic P2X7 receptor in regulation of bone formation has been reported. In addition, a subsequent identification of several single nucleotide polymorphisms (SNPs) within its gene has also been documented. This project has its basis in indicative findings of the negative and positive effect from SNPs in the P2X7 receptor gene has on the development of osteoporosis. As a result, it is the aim of this report to investigate the cellular effect of three selected SNPs transfected into an osteoblastic cell line in order to determine functional changes in the receptor followed by an analysis of the pattern of expression in selected bone markers. This was approached by inducing a single point mutation using site directed mutagenesis in the P2X7 receptor gene whilst it sits in a plasmid. This is followed by transfection of the plasmid into ROS 17/2.8 cells using Lipofectamine 2000 and a subsequent examination of change in receptor function by looking at changes in intracellular calcium concentration using. In addition, seven bone markers are tested for change in gene expression by RT-qPCR. A functional change of the P2X7 receptor observed by a change in intracellular calcium concentration was found for two SNPs according to their gain- or loss-of-function character when compared to WT. In contrast, the third SNP induced no significant change in function of the P2X7 receptor when compared to WT. For the seven bone markers, although significant differences between SNPs and WT was observed, no tangible patterns of increase or decrease in boneformation seen as a result of change in bone markers was observed.

UddannelserMolekylærbiologi, (Bachelor/kandidatuddannelse) KandidatMedicinalbiologi, (Bachelor/kandidatuddannelse) Kandidat
SprogEngelsk
Udgivelsesdato1 apr. 2014
VejledereSusanne Syberg & Ole Vang

Emneord

  • Osteoporosis
  • Bone
  • P2X7 receptor
  • Purinergig