Research Of The Nestin Positive Cell Origined From Liver Induction And Differentiation For Pancreatic Island β Cell Treat Type 1 Diabetes

There are lots of progenitive and differentiated stem cells in the embryo liver ofanimals. We have applied technology of immune-histological chemistry, cell culture,molecular cloning, reflex immune, to solve the hot problems of stem cells: (1) stemcells can induce differentiation of pancreas secretory cell; (2) transplantation of stemcells. This research emphasized induction and differentiation of nestin positive cellfrom liver of rats embryos. Firstly, we fractionally cultivated the nestin positive cellsin vitro; secondly, we made them differentiate to pancreas secretory cells that cansecret insulin, which can prove them stem cells.The full text altogether divides three parts.Part 1:(1)embryo liver of sd rats stem cell separation and raise.The embryo liver stem cell separation method uses the mechanicalpartingmethod union which we originally creates hanging drops to cultivatethe method ofpreserving health, fixedly is pregnant the mouse fourlimbs, cuts open the abdominalcavity, the exposition assumes "Y" theshape womb, after the embryo puts in togetherwith the womb contains75% ethyl alcohol to clean 1 minute, takes unfavorable balance oftrade only. Rips open with the tweezers fetal membrane takes out theembryo, the one byone rips open the skin to take out the embryo liver, then will put in4℃D-Hanks balance fluid in the blue bottle, thickly will cut,after again will rinse 3time, will blot the unnecessary bloodcorpuscle and the liquid. Adds by drops 2-3 dropof high sugar DMEM thin to cut the 1 mm3 size the organization, joins the D-Hanksbalancefluid collection single cell to hang the fluid, after settles 20-30minto abandonthe upper formation blood corpuscle, by 700rpm speedgentrifugalism 5min, except onserum, collection precipitation cell. Hangs the cell with the D-hanks fluid to move inagain the culturedish, joins precipitation cell two time of about the pancreasenzyme-EDTA digestion enzyme, after mixes uniform observes the cellularformunder the microscope, when the cell changes the circle, has when thewhite cottonwool shape precipitation explained the digestion isappropriate, joins the little D-hanksfluid termination digestion,will digest after the cell gentrifugalism, by 30ul/ drop(0.5×10⁵/ml) the density vaccination in the 100mm³ culture dish top head, inthe non-blood serum increase growth factor high sugar DMEM culturemedium, 37℃, 5%CO2, under the saturated humidity condition willraise, after 1 day will trade the fluid,mainly will be eliminates theblood corpuscle and so on not to paste the wall cell, 2days later (inculture dish will join little PBS) on the inversion aerosol raise,every daywill take out half quantity to trade the fluid, will waitthe cytomixis to achieve 4/5o'clock, will digest with the trypsin,according to 1:2-1:3 proportions hand downfrom generation togeneration. May obtain the shape function good massive embryosliverstem cell. Simplified the stem cell separation step, reduced thepollutionopportunity, is advantageous to the further raise.(2) the nest protein (nestin) expands in the embryo liver stem cellincreases.This topic tried to find out set of better inductions methods,increase nicotinicacid amide 10mmol/L, insulin 1 mg/L, the N₂ chemicaladditive, the alkalinity becomes the ciliary cell growth factor (basicfibroblast growth factor, bFGF), the epidermisgrowth factor(epidermal growth factor, EGF), the stem cell factor (stem cellfactor,SCF), the leukemia inhibiting factor (leukemia inhibitoryfactor, LIF) each 20 ug/L,the experiment proved organizes inenvironment through the partial simulationsembryo liver, then thepromotion embryo liver stem cell massive increments,massively expandfor in vitro increase provide the reliable experimental basis. Twosulfur hydrazone are one kind of zinc ion mastiff mixture, can specificity with insulinthe cell in zinc ion union, cause the cell to assume red,through to clones the nestinmasculine cell which forms to carry ontwo sulfur hydrazone dyeing under the highsugar environment, also hadproven the nestin masculine cell has splits up into insulinthe cellpotential, has the stem cell the characteristic.Part two: Diabetes mouse model establishment.The discussion chain urea assists the fungus element (streptozocin, STZ) sendsthe big mouse 1 diabetes model establishment the method. This research adopts onthe international popular abdominal cavity injection chain urea to assist the funguselement to destroy the island function, unifies the high glucose and the fat diet,through the improvement, adjusts the chain urea to assist the fungus element thedosage and the density, can enable the diabetes big mouse through the stochasticblood sugar examination prompt to maintain the stable high blood sugar condition,the experiment proved the diabetes big mouse makes the mold success ratio above90%, is the diabetes research provides an appropriate animal model.Part three:stem cell transplant treats diabetes.The stem cell totipotency opened the new way for the research diabetes celltreatment. This research adopts from 12-15 day-long embryo mouse liver separates,raises the embryo liver stem cell, the crosswise differentiation can express the nestin antibody under the high sugar in vitro cell culture, gathers the island of langerhanstype cell group, and can express and the island oflangerhans cell differentiationrelated duplication factor and the island of langerhans unique hormone, when receivesthe glucose stimulation, can synthesize the secretion insulin under the nicotinic acidamide function, theoritically speaking may reverse diabetes big mouse'shyperglycemia condition.Above research explanation, the niacinamide and under the highly concentratedglucose function induction, the oval cell may to the pancreatic gland internalsecretion cell direction differentiation. And can express the insulin.We believed that,the oval cell direction detection possibly has two kind of situation, to the insulinsecretory cell induction differentiation. (1), liver oval cell itself can express nestin,itself also has to the insulin secretory cell differentiation potential. In normalcondition also has little the partial livers oval cell to the insulin secretory celltransformation.(2), the nicotinic acid amide and the highly concentrated glucose addon each kind of growth factor again, can start the liver egg round cell PDX-1 gene theexpression, in the enormous degree has simulated the embryo time pancreatic glandstem cell growth and the growth environment, causes it to the insulin secretory celldirection differentiation.In the oval cell the nestin masculine cell can the directional inductiondifferentiation insulin secretory cell be a result which these two factors affectstogether. Finally, after we induce the cell which obtains to the diabetes big mousemodel to carry on the cell transplant, can completely cure the diabetes, but also needsour scientific worker long-term, the difficult endeavor.