Screening And Identification Of Key Genes For Activation Of Islet Stellate Cell

Part1:A transcriptional sequencing analysis of islet stellate cell and pancreatic stellate cellBackground and aims: Recent studies have shown that islet fibrosis is an important ingredient in the progression of type 2 diabetes whether in patients or in animals.Our previous studies have shown that islet stellate cell(ISC),similar to pancreatic stellate cell(PSC)in phenotype and biological characters,may be responsible for the islet fibrosis in type 2 diabetes.However,it is still not clear about ISCs' origin.Previous studies have demonstrated that the two cells are similar but not identical from the biological phenotype.To further identify the differences between PSC and ISC,we performed genome-wide transcriptional analysis on the PSC and ISC of Wistar Rats.Method: PSCs and ISCs were isolated from 10-week-old healthy male Wistar Rats' pancreatic tissue and islets.Genome-wide transcriptional sequence of stellate cells was generated using Hiseq3000 platform.Then the KEGG biological pathway enrichment and GO gene function enrichment analysis were performed on the differentially expressed genes.The identified differentially expressed genes were validated using quantitative RT-PCR.Results: A total of 21,901 genes were finally detected from the samples,and 32 significant differentially expressed genes(adjusted-P Value<0.05)between PSC and ISC were identified.Among these genes,14 of them were up-regulated and 18 were down-regulated in ISC group.Quantitative RT-PCR further confirmed the m RNA levels of these genes.Conclusions: Our study identified and validated the differences between PSC and ISC in genome-wide transcriptional scale,confirming the assumption that ISC and PSC are similar other than identical.They are two different kinds of cells,but probably from the same origin.The stellate cells in the fibrotic islets are the ISCs and are not migrated from the PSC.Part2:Screening and identification of key genes for activation of islet stellate cellBackground and aims: Previous studies found that ISCs in diabetic animals have strong activity and proliferative capacity,and express large amounts of extracellular matrix(ECM)components.Therefore,it is very important to identify the key molecules which can cause ISC activation.So we can find a way to inhibit ISC activation and prevent islet fibrosis.In this experiment,ISCs of GK rats and Wistar rats were subjected to transcriptional sequencing to screen out the key genes that may cause ISC activation.Method: Ten-week-old GK rats and Wistar rats were selected.Islets were isolated from fresh pancreatic issue.And ISCs were extracted from the surrounding of cultured islets and then subjected to transcriptome sequencing analysis.Then the KEGG biological pathway enrichment and GO gene function enrichment analysis were performed on the differentially expressed genes.The identified differentially expressed genes were validated using quantitative RT-PCR.We validated the protein expression level of the gene which may results in different biological phenotypes of two kinds of ISCs by Western Blot and immunofluorescence.Results: A total of 35362 genes were finally detected from the samples,and 204 significant differentially expressed genes(adjusted-P Value<0.05)between GK Rats' ISCs and Wistar Rats' ISCs were identified.Among these genes,73 of them were up-regulated and 131 were down-regulated in GK Rats' ISCs group compared to Wistar.Quantitative RT-PCR further confirmed the m RNA levels of these genes.GO gene function enrichment analysis showed that Pdpn gene is most likely to cause ISC activation in diabetic rats.The high expression of Pdpn in ISCs of GK rat was confirmed by Western Blot and immunofluorescence.Conclusions: There are significant differences in genes between GK rats' and Wistar rats' ISCs.Pdpn may be a key gene for accelerated activation of ISCs in diabetes.It is still necessary to determine its biological effects through gene silencing or other methods.