Changes in gene expression in pressure ulcers debrided by different approaches - a pilot study.

Pressure ulcers (PUs), also known as pressure injuries, are chronic wounds that represent potential lifelong complications. Pressure ulcers of a deep category (III and IV) are often indicated for surgical treatment - debridement and surgical reconstruction. Sharp surgical debridement is widely used in the debridement of PUs; however, the Versajet® hydrosurgery system is becoming an increasingly popular tool for tangential excision in surgery due to its numerous advantages. This work focused on the expression of selected genes, especially those associated with oxidative stress, in PUs debrided by two approaches - sharp surgical debridement and debridement using Versajet® hydrosurgery system. Expression of following genes was evaluated: NFE2L2, ACTA2, NFKB1, VEGFA, MKI67, HMOX1, HMOX2, HIF1A, and SOD2. ACTB and PSMB were used as housekeeping genes. So far, five patients have been enrolled in the study. Preliminary results suggest no significant difference in gene expression with different pressure ulcer treatment approaches except NFE2L2, despite the macroscopic differences. However, the results revealed correlations between the expression of some genes, namely HIF1A and SOD2, VEGFA and SOD2 and VEGFA and HIF1A. These results may indicate a connection between hypoxia, oxidative stress, pressure ulcer healing processes and angiogenesis.

Appropriate reference gene selection for real-time PCR data normalization during rat mesenchymal stem cell differentiation.

Reverse transcription quantitative PCR (RT—qPCR) is one of the best methods for the study of mesenchymal stem cell (MSC) differentiation by gene expression analysis. This technique needs appropriate reference or housekeeping genes (HKGs) to normalize the expression of the genes of interest. In the present study the expression stability of six widely used HKGs including Actb, Btub, Hprt, B2m, Gusb and Tfrc was investigated during rat MSC differentiation into osteocytes, adipocytes and chondrocytes lineages using geNorm and NormFinder software. RT—qPCR data analyzed by geNorm revealed the different sets of suitable reference genes for each cell type. NormFinder also showed similar results. Analysis of the combined data of MSCs with each differentiated cell type revealed the considerable shift in expression of some reference genes during differentiation; for example Gusb and B2m were among the least stable genes in MSCs but the most stable in chondrocytes. Normalization of specific genes for each lineage by different reference genes showed considerable difference in their expression fold change. In conclusion, for the appropriate analysis of gene expression during rat MSC differentiation and also for monitoring differentiation procedures, it is better to consider precisely the reference gene stability and select suitable reference genes for each purpose.