Studies of Human Adult Liver Stem Cell Transdifferention into Pancreatic Exocrine Cells

• Main Authors: Sin-Yu Lin, 林心榆
• Other Authors: 陳百薰
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/98384823853706479747

碩士 === 高雄醫學大學 === 醫學研究所碩士班 === 94 === Exogenous insulin therapy alone can not give a promise of resolving glucose homeostasis and prevent the occurrence of variety of chronic complications in type 1 diabetes mellitus (T1DM) patients. The transplantation of islet grafts or insulin-secreting tissue gives us the exciting expectancy of blood glucose control. The availability of islet grafts as a source for islet transplantation is severely limited, owing to the scarcity of donor pancrease. Some researches are, therefore, now oriented towards the use of glucose-responsive insulin-producing cells as the source of β cell-replacement therapy. These kinds of studies represent the main aspect for TIDM therapy. The successfully cultivating pluripotent stem cell and some promising results in generating insulin-producing cells obtained with embryonic stem cells（ES cells）of human origin give the hope for a permanent cure of diabetes. However, potential use of ES cells for the treatment of disease in human is beclouded in controversy because of the ethical issues. The recently successful research in adult stem cells has sparked that pancreatic ducts, islets and even hepatic oval cell could be the origin of adult pancreatic stem/progenitor cells, instead of embryo-derived stem cells, for the treatment of diabetes. We isolated human liver stem/progenitor cells by traditional glass-ring method. These oval cells we got expressed specific markers, such as vimentin, α-fetoprotein, cytokeratin peptide 19 and Thy 1.1, but not albumin. Additionally, these cells with highly proliferation potential ability, the cumulative population doubling level (cpdl), anchorage independent growth and the colony-forming efficiency were 49, 5.5 and 10.7%, respectively. We switched the culture medium to the Neurobasal medium with N2 and B27 supplement, adding 0.1 μM exendin-4, 10 mM nicotinamide, and 5 μM LY294002. The result indicating this oval cells monolayer with round cell cluster would change their morphology to duct-like structure and islet-like structure. Harvesting these cells conducted the protocols of RT-PCR for insulin, glucose transporter 2, somatostatin, glucagon and α-amylase. Only α-amylase was positive in RT-PCR and immunostaining. Additionally, in the period of stimulation, we used the 1 nM betacellulin and 2 nM activin A for 7 days trial, or replenished 50 μM β-mercaptoethanol in the 23 mM glucose KNC medium with 10 % FBS. The results revealed that these two methods did not induce insulin secreting, merely producing α-amylase. The outcome of our study revealed that high glucose condition in KNC medium with exendin-4 and 10% FBS could induce the Pancreatic duodenal homeobox-1 protein ( PDX-1 ) expression, but the nitches for adult liver oval cells to transdifferentiate into insulin-secreting were still unknown. While, most of the studies about organ-specific stem cells possess plasticity that permitted differentiation along new lineage were about mammalian but homo sapiens. In our study, we initiated these liver oval cells with pluripotent marker Oct-4 in high glucose condition（23 mM）, 10 mM nicotinamide, 10% FBS and 0.1 ?嵱 exendin-4 in K-NAC medium. After this stimulation, there were many round cells appeared above the monolayer of oval cells. These round cells were PDX-1 positive for immunostaining. When we switched the culture medium to the neurobasal medium with N2 and B27 supplement, adding 0.1?嵱 exendin-4, 10mM nicotinamide, and 5 ?嵱 LY294002, these oval cells monolayer with round cells clusters would change their morphology to duct-like structure and islet-like structures. After harvesting these cells, we conducted the protocols of immunostaining and RT-PCR. We observed that these cells were ??-amylase positive in both detecting methods. In summary, our study confirmed that these cells with stem cell phenotype could overstretch the lineage commitment transdifferentiating into exocrine producing cells. With this potential ability, we could confirm that these liver oval cells are multipotent stem/progenitor cells.