Doktorsavhandling från Oxford

I en doktorsavhandling utförd av doktoranden Sumayya Nafisa Khan vid anrika Oxford University har man använt sig av PHI´s HoloMonitor M4 samt programvaran Hstudio 2.7.

Doktorsavhandlingen är omfattande milt uttryckt.Efter genomläsning och kommen till sid 100 av 228 blev undertecknad matt,såpass att jag inte vågar mig på en sammanfattning.Materialet är alltför omfattande.
Bloggen tror att detta handlar om avancerad stamcellsforskning.
Av vad jag kan förstå återuppbyggnad av brosk vid knäskador. 
Att med stamceller som bas bygga upp ny(a) broskstrukturer.

Thesis

Fibroblast Growth Factor 2 (FGF2) in cartilage injury and repair

Abstract:

Fibroblast Growth Factors (FGFs) are important pleiotropic growth factors with a proven role in joint development and postnatal homeostasis. Previously our group has shown that FGF2 is released upon cartilage injury and Fgf2 knockout (KO) mice develop accelerated osteoarthritis (OA), suggesting it is chondroprotective in vivo. Work from others has shown that the accelerated OA phenotype in the Fgf2 KOs is phenocopied by deletion of Fgfr3 but not Fgfr1, suggesting that the FGFR may determine FGF2’s action in the joint.
In the work presented here, I investigate the role of FGF2 in promoting enhanced cartilage regeneration. I first explore the control and function of different FGF receptors (FGFRs) and ligands; then perform an in vivo model of focal cartilage injury in the Fgf2 KO mouse.
FGFR3 mRNA expression levels were rapidly down-regulated by injury in an FGF2-dependent manner, but recovered after chondrocytes were isolated and cultured in vitro. I did not observe significant differences in intracellular signalling or expression of a panel of FGF-dependent genes using receptor selective mutant ligands. In vivo, Fgf2 KO mice failed to repair focal cartilage defects compared with wild-type (WT) controls. Mesenchymal stem cells (MSCs) were activated by FGF2; leading to enhanced scratch assay closure and keeping cells in a more motile state but not in promoting cell adhesion to damaged tissue. FGF2 suppressed chondrogenesis of MSCs in vitro. Taken together, I identified a primary role for FGF2 in promoting intrinsic cartilage repair, which may be due to local activation of MSCs. These results provide mechanistic insights into how cartilage repairs and may have important clinical implications.

Ur den 228 sidiga texten klipper jag som vanligt in enbart PHI-relaterat.

Tillägg med forskarens egna kommentarer (fetningarna är dock bloggens egna):

One advantage of using time-lapse video is the ability to observe real-time cell division.
I counted cell division over the 24-hour period in each of the treatment groups.
Time-lapse video also allows one to examine the morphology of the cells with different treatments over time.
In the first hour after scratch injury, cells had a more rounded appearance and appeared to clump together.
In order to track the velocity and course of individual cells, the assay was repeated with GFP murine MSCs (passage 11) on a holographic phase microscope that focused on one side of the scratch front.
Time-lapse images were taken every 20 minutes for 36 hours (videos available on enclosed USB stick, in PowerPoint presentation, slide 5).
10 (randomly chosen) cells were tracked for each treatment group over this time period.
Hstudio 2.7 software was used to calculate the velocity of each cell.


Figure 5.13A, B and C show the speed of 10 172 tracked cells for untreated (control) cells, 20ng/ml FGF2 treated cells and 250nM FGFRi treated cells respectively.
The mean speed of 10 tracked cells for each treatment group (Figure 5.13D) shows that the speed of control cells varied across the time period from 20-80μm/h,whereas FGF2-treated cells maintained a more constant speed of 20-30 μm/h.FGFRi-treated cells had reduced speed at around 10 μm/h.
Interestingly we also observed an apparent 4-hourly periodicity in the control cells that was lost with FGF2 treatment.In this small sample size, the significance of this is unclear.
Control cells also showed a peak in speed at around 13-17 hours post-scratch. 

Materials and Methods

Time-lapsed images of the scratch were carried out in an incubator at

37°C, 5% CO2 on either a JuLi stage microscope, or an ibidi holographic phase microscope (HoloMonitor M4) with images taken every 20 min for 24 h or 36 h respectively. 

Figure 5.13:Speed of tracked murine GFP MSCs post-scratch

(C) The plate was placed in a holographic phase microscope in an incubator at 37° C, 5% CO2.The microscope was set up to image one side of the scratch and take images every 20 min for 36h.Hstudio 2.7

software was used to track 10 cells per group and calculate their speed.

 Figure 5.14:Migration of tracked murine GFP MSCs post-scratch

(C) The plate was placed in a holographic phase microscope in an incubator at 37° C, 5% CO2.The microscope was set up to image one side of the scratch and take images every 20 min for 36h.Hstudio 2.7

software was used to track 10 cells per group and calculate the migration of the cells.

Figure 5.15:Trajectory map of individual murine GFP MSCs post-scratch

(C).The plate was placed in a holographic phase microscope in an incubator at 37°C, 5% CO2. The microscope was set up to image one side of the scratch and take images every 20 min for 36h.

Hstudio 2.7 software was used to track the relative position of 10 cells per group and their trajectories plotted as an axis from initial coordinates 0,0(x,y).n=1.

     Min kommentar

       Denna doktorsavhandling från anrika Oxford får nog anses som en av de främre   

       eller kanske främsta, av doktorsavhandlingar PHI´s instrument medverkat i. 

                                       Mvh the99