Isolating Culture And Identification Of Pancreatic Stem Cell Of The Neonatal Pig

with development of standard's enhancement,diabetes' prevalence rate advances day by day,has become after the cardiovascular disease,the tumor third big succeeding in the imperial civil service examinations chronic illness.Fast,effective,supplemented for a long time organism insulin treats diabetes' basic plan.At present,three method are used for supplementing hypoinsulinism the best one depends on transplanting the function pancreatic gland stem because stem cell multiplication ability is strong,quantity is sufficient,after the transplant,the rejection reaction is low,and organism may also according to the need to adjust many kinds of cell proportion,and avoid the complication occurrence.But the donor cell is seldom it has limited its widespread application,therefore the people start to seek for the heterogeneous animal pancreatic gland stem cell for donor cell origin in the research,we discover that Pancreatic stem cells of the Neonatal Pig may take the best heterogeneous animal pancreatic gland stem cell's donor cell,therefore it brings new hope for treating diabetes。This experiment makes two aspects study,Isolating culture and identification, establishes the cell's best isolated culture and identification system,in the research,we discover that Pancreatic stem cells of the Neonatal Pig may take the best heterogeneous animal pancreatic gland stem cell's donor cell,therefore it brings new hope for treating diabetes。This experiment makes two aspects study, Isolating culture and identification,establishes the cell's best isolated culture and identification system1.The establishment of the optimal method for isolating piglet pancreatic cells and the culture system.The fresh pancreatic tissue,collected surgically from the living piglets,was chipped into small fragments after all the lipid tissue and vascular tissue were clipped on the ice,and digested by 0.1%collagease typeⅤand 0.25%trypsin into small cell-clusters and single cell.Then filtered and centrifuged three times at lower speed or via density gradient by 5%bovine serum albumin(BSA),through which the pancreatic cells were collected,and cultured in the Petri dish or flask coated with coated by laminin or gelatin,using the lower-glucose DMEM supplemented with 15%neonatal bovine serum(NBS) and 10 mmol/L nicotinamide as basic medium.4 days later,the none adhered(floating) cells were removed,the lower-glucose DMEM supplemented 10 ng/mL Epidermal growth factor(EGF) instead of nicotinamide was used as the medium to enhance the proliferation of the pancreas stem/progenitor cells.The results showed that Neonatal Pig has many pancreatic stem cells2.Identification and form characteristics of pancreatic stem cells from piglets.Primary cells adhered to dishes in form of single cell or cell mass.During the culture,there are some cells which departed from the cell mass continually.About 8days,there appeared the multisynaptic-endocrine progenitor cells increase among the remained cells and cell mass,developing into clone-like,and quickly into jointed reticular form.The number of small cells increased thereafter,while the Epithelium-like stem cells proliferated much slowly.The data observed show that the characteristic of multisynaptic cells is long synapse about 50～80μm,polarization,with big nucleo-cytoplasmic ratio and the immature organelle.The characteristic of small cells is 10～12μm diameter,positive CK7.The epithelium-like cells are round,at big nucleo-cytoplasmic ratio,the progenitor with organelles and petty secretory granules.On the whole,Four kinds of different cells with stem cell properties were isolated successfully.The immunohistochemical staining showed that the isolated cells express not only the markers of stem cell including SSEA-1 and Oct3/4,but the pancreatic cells including PDX-1,CK7 and nestin as well,which indicated that the cells isolated possess the properties of pancreas stem cells.The growth curve of pancreatic stem cell in neonatal piglets has been performed.The isolated cells after 10—13 days culture in vitro made passage.And they were frozen and thawed successfully and the viability of the thawed cells was more than 90%.For the first time,with monoclonal and microenvironment together,multipotent capability of pancreatic stem cells in vitro was certificated,studying in vitro initially show that reduplication Potentiality of Pancreatic stem cells of the Neonatal Pig is influenced by different factors.