Next, we studied the effects of leukemia cells on BMSCs co cultured in direct get in touch with. BMSCs from 3 healthy donors have been co cultured with the three distinct leukemia cell lines in direct speak to. The cells have been har vested at four h, ten h and 24 h and total RNA was ex tracted. The total RNA from BMSC mono cultures was mixed with the total RNA from TF 1, TF 1 or K562 cell mono cultures and also the resulting three mixed total RNA samples had been applied as a mono culture handle inside the gene expression profiling evaluation. The RNA from BMSCs co cultured using the TF 1, TF 1a and K562 cells had been ex tracted along with the gene expression profiles were analyzed by microarrays. The analysis of microarray data working with Partek Genomic Suite revealed that 544 genes have been differentially expressed involving co cultured and mono cultured handle cells.
Hierarchical clustering analysis of these genes clearly separated PI3K delta inhibitor the samples into two groups, co cultures and mono cultures. The results were equivalent for the analysis of BMSCs co cultured in transwells with all the leukemia cells. We located that CXCL1, CXCL6, TEP1, IL8, CCL2 and PTGS2 genes were by far the most up regulated genes in BMSCs co cultured within the direct speak to with leukemia cells. Ingenuity Path way Analysis in the differentially expressed genes revealed that the major canonical pathways involved were the gluco corticoid receptor signaling, IL 17 signaling and acute phase response signaling. Gene expression analysis of BMSCs co cultured with CD34 cells revealed alterations in metabolism related genes To evaluate no matter if the observed BMSC gene induction was specifically induced by leukemia cells, BMSCs had been co cultured in transwells with CD34 cells from healthy donors.
The BMSCs had been harvested at four h, ten h and 24 h and total RNA was extracted. The gene expres sion profiles of BMSC mono cultures and co cultured with the CD34 cells have been analyzed by microarrays. Analysis of your microarray data revealed that 4904 genes were differentially expressed between the two groups. Hierarchical clustering analysis of these genes separated the BMSCs into two Mocetinostat structure groups however the separation between co cultured and mono cultured cells was not perfect. 1 group consisted of eight co cultured samples and two mono cultures, the sec ond group consisted of 7 mono cultured samples and 1 co cultured sample.
We found that probably the most up regulated genes in BMSCs co cultured with CD34 cells compared with BMSC mono cultures have been SERPINB2, IL1B, RTP3, CCL7 and IL8. Ingenuity pathway analysis revealed that the best ca nonical pathways involved had been the purine metabolism, mTOR signaling and EIF2 signaling. To valid ate the microarrays data, we performed a quantitative RT PCR evaluation which confirmed the higher expression of IL8 in BMSCs co cultured with CD34 cells compared with BMSC mono cultures.