The Role Of Rat Betacellulin In Pancreatic Stem Cell's Transdifferentiation

The current version does not support copying Cyrillic text to the Clipboard. You should activate the programless differentiated, and less restricted phenotype that can serve as a multipotent progenitor or functional stem cell. External stimuli can then direct the differentiation of these cells to endocrine, acinar or mature duct phenotypes. It has been reported that pancreatic ductal epithelial cells isolated from adult non-obese diabetic mice can be grown in long term cultures and induced to produce functioning islets. These generated islets were capable of lowering blood glucose concentrations to near normal when implanted in diabetic non-obese mice. The mice remained normoglyacemic for three month's duration of the study. Some success has been achieved in generating islets in culture from human pancreatic ductal cells remaining after islet isolation from adult cadaveric pancreas. These duct cells were grown in culture and were then coaxed into forming three-dimensional structures, called cultivated human islets buds, from which sprout islets containing β cells and the other islet cell types. These results show that the duct cells may be functional stem cells of the pancreas. In order to obtain a large number of insulin producing islet cells from adult pancreatic duct epithelial cells, the ways of expansion and transdifferentiation of pancreatic duct cells have been pursued.Up to now, the perfect method has not been found in culturing pancreatic duct cells. Growth factors may play an important role in the differentiation of stem cells. Betacellulin (BTC) is a member of the epidermal growth factor (EGF) family originally identified from the contioned medium of a mouse pancreatic beta tumor cell line. Human BTC cDNA was cloned from a human breast cancer cell line, MCF-7. BTC is proteolytically processed from a larger membrane-anchored precursor and is a potent mitogen for a wide variety of cell types. It binds and activates ErbB-1 and ErbB-4 homodimers. BTC is widely expressed in most tissues and various body fluids, induing milk. Expression is particularly high in the pancreas. Northern blot and immunohistochemical analyses showed that BTC is mainly localized in pancreatic islets. Furthermore, the human BTC can convert rat pancreatic amylase-secreting cells(AR42J) into insulin-secreting cells. These data strongly suggest the importance of BTC in the differentiation and growth of β cells inpancreatic islets. It has not been reported that whether BTC can promote the pancreatic duct cells replication and differentiation into islet cells.Mitogen-activated protein kinases (MAPKs) are a family of protein kinases playing a central role in signal transduction and thought to mediate diverse processes ranging from transcription of protooncogenes to programmed cell death (PCD). MAPKs include several serine-threonine kinases in three interrelated signal transduction cascades, and can be activated by stimuli such as growth factors, stress and inflammation. ERK pathway engages in regulating the proliferations and differentiations of cells. It is still not clear that whether MAPKs participate in the process of pancreatic stem cell's proliferation by rBTC stimuli.In this study, the aim is to obtain a large amount of rat betacellulin protein with good bioactivity by gene engineering; to establish stable methods for culturing rat pancreatic stem cell in vitro; to observe the biological roles of BTC in the expansion and transdifferentiation of rat pancreatic duct epithelial cells, and investigate its mechanism.Molecular cloning of rat betacellulin: the complete sequence of rat betacellulin gene was amplified by reverse transcription PCR from rat kidney. The amplified fragment was cloned into pMD18-T vector and identified by digestion. The positive clone was sequenced by shenggong Corporation. BLAST analysis indicated that the cDNA sequence of rat betacellulin was identical with that reported in Genebank. In this study, we constructed prokaryotic expression plasmids of pET28a(+)-rBTC, and no mutations were found after sequencing. The recombinant plasmid was transformed into E.coli BL-21(DE3), and the rat betacellulin protein with Mr20,000 was expressed under IPTG induction. The expressed betacellulin was detected by SDS-PAGE and western blot. The expressed protein was purified by Ni²⁺ affinity chromatography and then renatured by dialysis. It's purity could reach over 96%. The effect of the renatured protein on proliferation of NIH3T3 cells was detected by MTT colorimetry. The results show that the the recombinant ratbetacellulin could significantly stimulate the proliferation of BALB/ 3T3 cells. We obtained the rat betacellulin with good biological activity and made a foundation for our following experiments.Isolation culture and identification of rat pancreatic stem cells:The rat pancreas were digested with collagenase V, followed by incontinuous density gradient to separate islets from the acinar and ductal tissue. Duct epithelial cells were cultivated in CMRL1066 and then in serum-free DMEM/F12 medium with the addition of growth factors including rat betacellulin for 30 days. Samples were taken at different time points for light microscopic examination and for immunocytochenical study with antibodies against transdifferentiation gene PDX-1 and protein CK-19, which are the main marker of the adult pancreatic stem cells. Insulin contents in the medium were assayed. In our study, a large number of rat pancreatic duct epithelial cells were harvested after the isolation of islets. Some duct epithelial cells were PDX-1 and CK-19 positive at day one and duct epithelial cells proliferated and expanded rapidly and then transdifferentiated into stem cells and finally 3D islets.The Role of rat betacellulin in pancreatic stem cell'stransdifferentiation: In our study, the pancreatic stem cells were treated in different dose of rBTC. At dose 20ug/ml of rBTC, the number of islet clone transdifferentiated from pancreatic duct epithelial cells were higher than that in control group, and the insulin content are also higher than that in control group (p<0.01). The results show that rat betacellulin can significantly promote the pancreatic stem cells transdifferentiating into islet. Futhermore, the molecular mechanism of rBTC in expansion and transdiiferentiation of rat pancreatic duct epithelial cells were investigated. We found that rat betacellulin can increase the DNA systhesis of pancreatic duct epithelial cells. By western blot analysis, the signal transduction pathway of rBTC in rat pancreatic stem cells transdiiferentiating into islet maybe through ERK pathway.Conclusion: In this study, we have successfully obtained the rat betacellulin...