CN102899288A - Method for constructing human islet-derived pancreatic stem cell line and method for differentiation of human islet-derived pancreatic stem cell line into insulin-producing cells - Google Patents
Method for constructing human islet-derived pancreatic stem cell line and method for differentiation of human islet-derived pancreatic stem cell line into insulin-producing cells Download PDFInfo
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Abstract
The invention discloses a method for constructing a human islet-derived pancreatic stem cell line and a method for differentiation of the human islet-derived pancreatic stem cell line into insulin-producing cells. The pancreatic stem cell line is obtained by amplifying pancreatic stem cells in human pancreatic clusters, and is a potential source for multidirectional differentiation. When induced, the pancreatic stem cell line can differentiate to form neuron-like cells, adipose-like cells, osteoblast-like cells and functional islet-like cell clusters. By transplanting the functional islet-like cell clusters into a body of a diabetic nude mouse model, the blood glucose concentration of the nude mouse model can be reduced and reach a normal level within 24 days.
Description
Technical field
The invention belongs to the cell engineering field, particularly the pancreatic stem cells system in a kind of people's pancreas islet source makes up and to the method for insulin secretory cell differentiation.
Background technology
Diabetes are the chronic diseases the third-largest occurred frequently after cardiovascular disorder and cancer, and human life and health in its serious harm.At present, although the treatment of diabetes there are a lot of methods, such as the orally-taken blood sugar reducing medicine, injection of exogenous Regular Insulin, and in conjunction with the method for dietary restrictions and physical activity, all can not tackle the problem at its root.Organ transplantation is a kind of reasonable solution, but the application of the method is subject to the restriction of donor deficiency and immunological rejection problem.And stem cells hyperplasia power is strong, abundant amount, graft-rejection are relatively low.Therefore, people place hope on the problem that the stem cell alternative medicine can solve donor deficiency and immunological rejection.
Although the use of stem cell can solve donorcells quantity problem, before application, also need first it to be induced to differentiate into functional insulin secretory cell.Pancreatic stem cells (Pancreatic stem cells) is that a class is present in the adult stem cell in fetus and the adult pancreatic tissue, has self, and multi-lineage potential at first is divided into the various cells in the pancreatic tissue in the natural differentiation process.Because this cell derived in pancreatic tissue, therefore, has larger potentiality to aspect the insulin secretory cell differentiation.Increasing evidence shows and all exists the stem cell with multi-lineage potential in pancreatic duct, pancreas islet and the acinar tissue.There is at present viewpoint to think, the mesenchymal stem cells that is obtained by islet tissue may derive from the β cell (Russ after dedifferenting, H.A., Bar, Y., Ravassard, P., Efrat, In vitroproliferation of cells derived from adult human beta-cells revealed bycell-lineage tracing.Diabetes S.2008,57:1575～1583).And these cells external repeatedly go down to posterity after its insulin promoter still be in active state, therefore have huge treating diabetes potentiality.
In-vitro separation, cultivating pancreatic stem cells as seed cell, and be the functional islets cell with its Induction of committed differentiation, is the effective way that solves the shortage of pancreas islet donor.Existing a plurality of research groups have carried out certain element task in the research of pancreatic stem cells.Xibei Univ. of Agricultural ﹠ Forest Science ﹠ Technology effect plum (foundation [J] of monoclonal human pancreatic stem cells separation and Culture system. molecular cytobiology, 2008,41 (6): 450-456.), Zhao Ting (the human pancreas stem cell is induced the effect [J] of insulin-like cell group and treatment rat diabetes thereof. Chinese Tissue Engineering Study and clinical rehabilitation, 2007,11 (7): 1259-1262.), auspicious (the Isolation and culture of human pancreas stem cell of the Yao Zhong of Third Military Medical University, identify and differentiation characteristic [J]. Third Military Medical University's journal, 2003,25 (1): 23-25.) Jilin University etc. is successively in the separation and purification of pancreatic stem cells, the screening of substratum, and cytobiology, a large amount of work has been done in the molecular biology identification aspect; The pancreatic stem cells that is separated to forms insulin-like cell group after the directional induction in vivo, and detects the secretion of Regular Insulin and C-peptide, but differentiation efficiency is all very low, and is not remarkable to the result for the treatment of of diabetes after transplanting.
Summary of the invention
The technical problem that the present invention solves is that it is the method for external structure that the pancreatic stem cells in a kind of people's pancreas islet source is provided, this stem cell line Differentiation Induction in vitro is become the method for insulin-like cell group, and the pancreatic stem cells system and the insulin-like cell group that obtain with aforesaid method.Can improve its glucose level after described insulin-like cell group migrated to the diabetes model animal.
The pancreatic stem cells that the invention provides a kind of people's pancreas islet source is the method for external structure, the method comprises the steps: to digest Human Pancreas, choose islet cells group, further digestion forms single cell suspension, goes down to posterity to cultivate the rear mesenchyme sample pancreatic stem cells that forms.
Described pancreatic stem cells system, its cell is for becoming fiber-like, how triangular in shape and short shuttle shape, monocyte has two or more kernels, and there is contact inhibition in iuntercellular.
Described pancreatic stem cells is, can be differentiated to form insulin-like cell group through inducing.
A first aspect of the present invention provides the construction process of the pancreatic stem cells system in a kind of people's pancreas islet source, may further comprise the steps:
1) will cultivate 2-3 days among human pancreas's cell adding nutrient solution A;
2) choose islet cells group in the above-mentioned culture, become unicellular with protease digestion, be inoculated among the nutrient solution A and cultivate;
When 3) treating that Growth of Cells merges to 80-90%, go down to posterity, add nutrient solution B and cultivate, obtain human pancreas's stem cell line (hPSC) in islet cells source;
Wherein said nutrient solution A is the RPMI1640 that contains 10-25% FBS, 8-12mM nicotinamide, 18-22ng/mL EGF and 18-22ng/mL bFGF; Described nutrient solution B is the Low-DMEM that contains 10-25%FBS, 18-22ng/mLbFGF.
In one embodiment, described nutrient solution A also contains 0.8-1.2mM Sodium.alpha.-ketopropionate, 1.8-2.2mM glutaminase, 0.05-0.15mM beta-mercaptoethanol and 90-110mg/mL penicillin/streptomycin; Described nutrient solution B also contains 0.05-0.15mM beta-mercaptoethanol, 1.8-2.2mM glutamine and 90-110mg/mL penicillin/streptomycin.
In a preferred embodiment, aforesaid method may further comprise the steps:
1) in human pancreas's tissue block, adds 0.05-0.2% (mass/volume) collagenase digesting, stop filtering after the digestion, centrifugal and clean and obtain unicellularly, add described nutrient solution A and cultivated 2-3 days;
2) choose islet cells group at microscopically, 0.25% (mass/volume) proteolytic enzyme further is digested to it unicellular, is inoculated in the porous plate, adds described nutrient solution A and cultivates;
When 3) treating that Growth of Cells merges to 80-90%, with 0.25% protease digestion, with 1: 3-1: 5 ratios go down to posterity, and add described nutrient solution B and cultivate; Cultivate above the human pancreas's stem cell line that obtains the islet cells source after 50 generations.
A second aspect of the present invention provides a kind of human pancreas's of making stem cell line to induce and is divided into the method that insulin-like cell is rolled into a ball, may further comprise the steps:
1) the human pancreas stem cell is inoculated in the RPMI1640/B27 nutrient solution that contains 0.8-1.2% BSA, 0.8-1.2mM Sodium propanecarboxylate, 80-120ng/mL ActivinA, 40-60mM LY294002,8-12mM nicotinamide, 0.8-1.2 * ITS, in adherent ware, cultivated 3-4 days; 0.25% protease digestion, suspension culture forms EB (embryoid), changes above-mentioned nutrient solution is removed Sodium propanecarboxylate, ActivinA and LY294002 and adds 0.8-1.2 * 10
-6The nutrient solution of M RA (vitamin A acid), 18-22ng/mLEGF and 18-22ng/mL bFGF continues to carry out preliminary induction 7-9 days in the cultivation of cellar culture condition low suspension; In a preferred embodiment, described inoculation culture liquid also contains 1.8-2.2mM L-glutaminate, 0.05-0.15mM beta-mercaptoethanol and 0.8-1.2mM Sodium.alpha.-ketopropionate;
2) with the cell transfer behind the preliminary induction to the RPMI1640/B27 nutrient solution that contains 0.8-1.2% BSA, 1.8-2.2mM glutamine, 0.8-1.2mM Sodium.alpha.-ketopropionate, 22-28 μ M zinc acetate, 0.8-1.2 * ITS, 90-110mM nicotine, 8-12ng/mL exendin-4 and 8-12ng/mL beta cell element, under the cellar culture condition, continue suspension culture, obtain insulin-like cell group; In a preferred embodiment, described nutrient solution also contains 1.8-2.2mM L-glutaminate, 0.8-1.2mM beta-mercaptoethanol and 0.8-1.2mM Sodium.alpha.-ketopropionate.
In a preferred embodiment, employed human pancreas's stem cell line is the pancreatic stem cells system in people's pancreas islet source of making up according to the method for first aspect present invention.
A third aspect of the present invention provides that a kind of to make the pancreatic stem cells in people's pancreas islet source of setting up according to the method for first aspect present invention be the method that Differentiation Induction in vitro becomes the class neurocyte, the method may further comprise the steps: the cell that the pancreatic stem cells that described people's pancreas islet is originated is is inoculated in and discards nutrient solution after nutrient solution B cultivates 20-30h, after adding the pre-induced liquid processing of neurocyte 20-30h, change neurocyte induced liquid continuation effect 8-12h into; The pre-induced liquid of described neurocyte is to add the 1mmol/L beta-mercaptoethanol in the Low-DMEM basic culture solution; Described neurocyte induced liquid is to add the 5mmol/L beta-mercaptoethanol in the Low-DMEM basic culture solution.
A fourth aspect of the present invention provides a kind of pancreatic stem cells system that makes people's pancreas islet source of setting up according to the method for first aspect present invention to induce into the method for class adipocyte, the method may further comprise the steps: the cell that the pancreatic stem cells that described people's pancreas islet is originated is is inoculated in above-mentioned nutrient solution B and cultivates, treat that the cytogamy degree reaches about 80-90%, the replacing inducing culture (
Adipogenesis Differentiation Kit test kit (GIBCO)) continues to cultivate, continue to cultivate 14～21 days.
A fifth aspect of the present invention provides a kind of pancreatic stem cells system that makes people's pancreas islet source of setting up according to the method for first aspect present invention to induce into the method for class osteocyte, the method may further comprise the steps: the cell that the pancreatic stem cells that described people's pancreas islet is originated is is inoculated in above-mentioned nutrient solution B and cultivates, treat that the cytogamy degree reaches about 80-90%, namely change inducing culture (
Osteogenesis Differentiation Kit test kit (GIBCO)) continues to cultivate, continue to cultivate 21～28 days.
In one embodiment, the class osteocyte that obtains by aforesaid method is that Alizarin red staining is positive.
A sixth aspect of the present invention provides human pancreas's stem cell line of setting up according to the method for first aspect present invention.
A seventh aspect of the present invention provides the insulin-like cell group that sets up according to the method for second aspect present invention.
A eighth aspect of the present invention provides medicine, reagent, composition, the test kit that comprises the described human pancreas's stem cell line of sixth aspect present invention or the described insulin-like cell of seventh aspect present invention group.
A ninth aspect of the present invention provides the described human pancreas's stem cell line of sixth aspect present invention or the described insulin-like cell of seventh aspect present invention group for the preparation of the purposes in the medicine of lowering blood glucose or treatment diabetes, reagent, composition, the test kit.
Compared with prior art, the present invention has following useful technique effect:
1) the pancreatic stem cells system in people's pancreas islet source of the present invention's acquisition, its upgrowth situation is good, its population doubling time shortens to 24.75 ± 1.34h by 50.48 original ± 1.56h, and the survival rate of cell than before greatly raising, be convenient to the application of mass-producing; Still have preferably vigor at subculture in vitro separately more than 50 generations, still keep division growth, be difficult for occuring aging and apoptosis.
2) the pancreatic stem cells system in people's pancreas islet source of the present invention's acquisition, its nutritional requirement is lower, the various nutritional factor such as the foetal calf serum of the cultivation needs 20% of the islet cells that original separation obtains, EGF, bFGF, nicotinamide, and only require now about 10% foetal calf serum and bFGF, greatly reduce the cost of cultivation.
3) human pancreas's stem cell line of the present invention's acquisition derives from dedifferenting of people's beta Cell of islet, adding behind the inducible factor it is differentiated to form functional islets like cell group efficient and significantly improves, can excreting insulin and C-peptide after high sugar stimulates, it is transplanted in the body of the nude mice model of suffering from diabetes, can lowering blood glucose concentration even can return to normal glucose level, and in 40 days, can keep lower glucose level.
4) because the insulin-like cell group that the present invention obtains is the people source, therefore can expect that its compatibility when being used for the treatment of the patient who suffers from diabetes is better than the insulin-like cell group in other sources.
Description of drawings
The behave pancreatic stem cells morphological feature demonstration figure in pancreas islet source of Fig. 1;
The behave pancreatic stem cells characteristic sign evaluation figure in pancreas islet source of Fig. 2;
The cell proliferation of the pancreatic stem cells in people's pancreas islet source that Fig. 3 Brdu detects is figure as a result;
The behave cell growth curve figure of pancreatic stem cells system in pancreas islet source of Fig. 4, wherein X-coordinate is time (fate), ordinate zou is cell count (10
4);
The behave pancreatic stem cells in pancreas islet source of Fig. 5 is induced to differentiate into the characteristic sign evaluation figure of class neurocyte;
Fig. 6 characteristic sign evaluation figure that the pancreatic stem cells in pancreas islet source becomes fat to induce that behaves;
The behave characteristic sign evaluation figure of pancreatic stem cells osteogenic induction in pancreas islet source of Fig. 7;
The behave pancreatic stem cells in pancreas islet source of Fig. 8 is induced to differentiate into the characteristic sign evaluation figure of insulin-like cell group;
Fig. 9 is the blood sugar concentration change curve after the nude mice diabetes model is transplanted by insulin-like cell group, and wherein X-coordinate is time (fate), and ordinate zou is blood sugar concentration.
Embodiment
The structure that the pancreatic stem cells that the invention provides a kind of people's pancreas islet source is and the method for differentiation.This clone still has preferably vigor at subculture in vitro separately more than 50 generations, keeps division growth, is difficult for occuring aging and apoptosis, can be differentiated to form functional islets like cell group through after inducing.Below by to gene and the Immunological Identification of the structure of this clone, cell morphological characteristic, mark, induce differentiation, high low sugar stimulates Regular Insulin and the secretion of C-peptide and transplants the interior change of blood sugar of diabetes model body and test to elaborate, and the explanation of the invention is not limited.
FBS in this specification sheets refers to foetal calf serum, about the percentage ratio of FBS, if no special instructions, all refers to percent by volume.BSA in this specification sheets refers to bovine serum albumin, about the percentage ratio of FBS, if no special instructions, all refers to mass percent.Other percentage ratios that use in this specification sheets if no special instructions, are then comply with the usual implication of using in this area.
The nutrient solution of mentioning in this specification sheets is all available from Gibico company, and reagent all available from Sigma company originally.
Cellar culture liquid described in the specification sheets, cellar culture condition refer to that those skilled in the art can be according to nutrient solution and the culture condition of the definite described cell of suitable cultivation of prior art.
Embodiment
The structure of the pancreatic stem cells system in embodiment 1, people's pancreas islet source
The structure of the pancreatic stem cells system in people's pancreas islet source specifically may further comprise the steps:
1) in the situation that the patient agrees, carry out the Human Pancreas that aseptic collection is excised in the pancreatectomy surgical procedure of patients with acute pancreatitis of part pancreatectomy treatment at needs, place the Hank ' s flushing of precooling and remove the blood of periphery and cut off around fat, blood vessel, organize tunicle and reticular tissue, then be cut into 1mm
3About tissue block, add the 0.1%IV Collagenase Type, in 37 ℃ of digestion 20-30min.During this time frequently vibration, rear 80 eye mesh screens of crossing of termination digestion that contain 10% FBS Hank ' s, centrifugal (500r/min) 3min, and clean 2 times, add the RPMI1640 nutrient solution that contains 20% FBS, 1mM Sodium.alpha.-ketopropionate, 10mM nicotinamide, 0.1mM beta-mercaptoethanol, 2mM glutamine, 20ng/mL EGF, 20ng/mL bFGF and 100mg/mL penicillin/streptomycin and cultivate 48h.
2) choose by hand islet cells group at microscopically, 0.25% trypsinase further digests it and forms single cell suspension, stop being inoculated in 12 orifice plates after the digestion, add the RPMI1640 nutrient solution contain 20%FBS, 1mM Sodium.alpha.-ketopropionate, 10mM nicotinamide, 0.1mM beta-mercaptoethanol, 2mM glutamine, 20ng/mL EGF, 20ng/mL bFGF and 100mg/mL penicillin/streptomycin, place 37 ℃, the incubator of 5% CO2 to cultivate.
When 3) treating that Growth of Cells to 90% merges, with 0.25% tryptic digestion, go down to posterity with 1: 3 ratio, cell is inoculated in the Low-DMEM nutrient solution that contains 10%FBS, 0.1mM beta-mercaptoethanol, 2mM glutamine, 20ng/mL bFGF and 100mg/mL penicillin/streptomycin cultivates, per two and half amounts are changed liquid.
The evaluation of the pancreatic stem cells system in embodiment 2, people's pancreas islet source
2.1 morphocytology feature
The metamorphosis of the pancreatic stem cells in observer's pancreas islet source under phase microscope.Concrete outcome as shown in Figure 1, wherein, figure A is the human pancreatic island cell group that separates in former generation, figure B is the pancreatic stem cells (hPSC) in the people's pancreas islet source after the enlarged culturing, compares with islet cells, and cell is for becoming fiber-like, many triangular in shape and short shuttle shapes, network-like arrangement, there is contact inhibition in iuntercellular, and figure C is for to carry out Giemsa staining to the human pancreas stem cell, cell is monokaryon, examine apparent in viewly, the kernel more than 2 or 2 is arranged, can see obvious cell fission phase.This cell still has preferably vigor at subculture in vitro separately more than 50 generations, still keeps division growth, is difficult for occuring aging and apoptosis.
2.2 the evaluation of pancreatic stem cells mark of correlation
2.2.1 immunofluorescence detects
After 4% Paraformaldehyde 96 was fixing, immunofluorescence dyeing was identified pancreatic stem cells characteristic sign with the pancreatic stem cells in people's pancreas islet source.
The immunofluorescence dyeing step is:
A. cell is through the fixing 10min of 4% Paraformaldehyde 96, with PBS washing 2-3 time;
B. add 0.1% TritionX-100 room temperature effect 10min, PBS washes 2-3 time, acts on 5min at every turn;
C. add the PBS effect 30min that contains 1%BSA;
D. add primary antibodie, 4 ℃ are spent the night;
E.PBS washes 2-3 time, acts on 5min at every turn, adds fluorescence two and resists, 37 ℃ of effect 1h;
F.PBS washes 2-3 time, the fluorescence microscopy Microscopic observation.
Immunofluorescence detects employed primary antibodie and mainly contains: vimentin (Vim), PDX1, Ngn3, glucose transcription factor (Glut2) and CK19.Shown in Fig. 2 A, the result shows: this pancreatic stem cells is albumen in wave shape (Vim), PDX1, Ngn3 and glucose transcription factor (Glut2) positive to the immunofluorescence dyeing result, and does not express epithelial cell mark CK19 respectively.
2.2.2PCR detect the expression of pancreatic stem cells marker gene
According to the reverse transcriptase gene reference sequences of having delivered among the GenBank, design and synthetic specific detection primer, relevant primer sequence, length, and the Tm value see Table 1.
Table 1. pancreatic stem cells Research of predicting markers and corresponding pcr amplification primer thereof
The extraction of cell total rna: the pancreatic stem cells in the people's pancreas islet source after collecting 0.25% tryptic digestion and going down to posterity and induce after insulin-like cell group in centrifuge tube, PBS washing 2-3 time; Then add 1mL Trizol concussion mixing, leave standstill 5min under the room temperature and carry out lysis, add the 0.2mL chloroform in centrifuge tube, concussion is left standstill 15min under the room temperature, and 4 ℃, the centrifugal 15min of 10000r/min; Get top section after centrifugal and move into another centrifuge tube, add the 0.2mL Virahol, turn upside down, mixing leaves standstill 30min under the room temperature gently, and 4 ℃, the centrifugal 15min of 10000r/min; Abandon supernatant, add the vibration of 1mL 75% ethanol, the centrifugal 5min of 7500r/min; To precipitate seasoning (placing about 10min under the room temperature), with the resuspended RNA of 30 μ L DEPC water.
Pcr amplification carries out the synthetic of cDNA:
The pcr amplification system is: 5 * PrimeScript Buffer, 2 μ L, Random 6 mers 2 μ L, Oliqo (dT) Primer 0.5 μ L, reversed transcriptive enzyme (PrimeScript RT EnzymeMix) 0.5 μ L, total RNA of 500ng, RNase Free H2O polishing to 10 μ L; Mixing is hatched 30min for 37 ℃, and then 85 ℃ are extended 5s, obtain cDNA.
The pcr amplification double-stranded DNA, reaction system is as follows: 10 * PCR Buffer1.5 μ L, MgCl2 1.5 μ L, each 0.3 μ L of upstream and downstream primer, Taq enzyme 0.1 μ L, dNTP Mixture1.2 μ L, cDNA 1.0 μ g add two pure water polishing to 15.0 μ L.
The pcr amplification program is: 94 ℃ of denaturation 5min; 94 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 40s, totally 35 circulations; 72 ℃ are extended 10min again, 4 ℃ of preservations.
Get 5 μ L PCR products and carry out 2% agarose gel electrophoresis analysis, the expression of pancreatic stem cells mark of correlation gene is shown in Fig. 2 B, and this strain clone is all expressed Vim, PDX1, Ngn3 and Mafa gene, illustrates that it has the feature of pancreatic stem cells.
2.2.3PCR detect the pancreatic stem cells telomerase activation
According to
Telomerase Detection Kit S7700 (CHEMICON) test kit operation instructions detects the pancreatic stem cells telomerase activation, and the result shows that this clone has very strong telomerase activation (Fig. 2 C), external very strong amplification ability is arranged
2.3BrdU detect the cell proliferation of the pancreatic stem cells system in people's pancreas islet source
The pancreatic stem cells that people's pancreas islet is originated is as treating test sample, cell sample to be measured is made single cell suspension, be inoculated in 48 orifice plates, add nutrient solution B and cultivate 3d, behind the nutrient solution processing 4～6h that contains 30 μ g/mLBrdU, fixing 15min under methanol/acetone stationary liquid (methyl alcohol: acetone is 1: 1) room temperature, PBS buffer solution for cleaning 2～3 times, 2mol/L hydrochloric acid effect 45min, PBS buffer solution for cleaning 2～3 times, the Sodium Tetraborate effect 15min of adding PH8.5, PBS buffer solution for cleaning 2～3 times, add more than 4 ℃ of effects of mouse-anti BrdU (1: 100, Santa Cruz) 16h PBS buffer solution for cleaning 2～3 times, adding two resists, Hochest33342 effect 2～5min, PBS buffer solution for cleaning 2～3 times, fluorescence microscopy Microscopic observation.The result is as shown in Fig. 3, and through counting, this pancreatic stem cells is that the positive rate of BrdU is 40.68 ± 1.56%, illustrates that this clone has a very strong amplification ability external.
2.4 the mensuration of growth curve
Get the pancreatic stem cells (the 5th generation and 15 generation cell) in people's pancreas islet source of going down to posterity, with 1 * 10
4The cell density in individual/hole is inoculated on 24 well culture plates, gets 3 holes every day, the tryptic digestion suspension cell, and the basic culture solution neutralization, cell counting, averaging is denoted as cell count on the same day, continuous counter 10 days.With cell count (10
4) be ordinate zou, cultivated days (d) is X-coordinate, draws growth curve, the result is as shown in Figure 4.The 5th generation after relatively going down to posterity and 15 generation cell growth curve, wherein 1～3 day is latent period, 4～7 days is increased logarithmic phase always, illustrates that cell has very strong competence for added value.
The calculating of population doubling time (PDT): calculation formula is PDT=(T-T0) lg2/ (lgNt-lgN0).T0 wherein: the time of origin of cell cultures; T: cell arrives the time of plateau; N0: the quantity of cell when cultivating beginning; Nt: the quantity of cell when arriving phase plateau.The result shows: the population doubling time of the pancreatic stem cells in people's pancreas islet source is 24.75 ± 1.34h.
The function assessment feature of the pancreatic stem cells system in embodiment 3, people's pancreas islet source
Pancreatic stem cells is to be present in pancreatic tissue, has self, the early stage cell of the growth of multi-lineage potential.In-vitro separation, clone's pancreatic stem cells, and directional induction its be divided into the functional islets cell transplantation for diabetes, be the effective way that solves the shortage of pancreas islet donor.
The present invention induces the pancreatic stem cells system in people's pancreas islet source, make its directed differentiation become insulin-like cell group, be used for treating the nude mice diabetes model, stimulate insulin release test and transplanting nude mice diabetes model to detect its glucose level by associated molecule and immunofluorescence detection, high sugar.
3.1 inducing of class neurocyte broken up and evaluation
With 0.25% tryptic digestion, cell is inoculated in 48 orifice plates, add and discard nutrient solution after nutrient solution is cultivated 24h, after adding the pre-induced liquid of neurocyte (adding the 1mmol/L beta-mercaptoethanol in the Low-DMEM basic culture solution) processing 24h, change neurocyte induced liquid (adding the 5mmol/L beta-mercaptoethanol in the Low-DMEM basic culture solution) continuation effect 8h into.
Operation instructions according to the universal SP kit of SP-9000 is carried out immunohistochemical staining to the cell after inducing.Detecting employed primary antibodie mainly contains: nidogen (Nestin), neuronspecific enolase (NSE) and neuronspecific enolase (GFAP).The immunofluorescence dyeing result as shown in Figure 5, the result shows: cell form after inducing occurs significantly to change, a plurality of cynapses that cell is protruding are similar to the aixs cylinder of neurocyte.Coloration result shows that the class neurocyte after inducing is neurocyte sign Nestin, NSE, GFAP are positive.
3.2 inducing of class adipocyte broken up and evaluation
Cell is inoculated in 12 orifice plates, adds nutrient solution and cultivate, treat that the cytogamy degree reaches about 80-90%, namely change inducing culture (
Adipogenesis DifferentiationKit (GIBCO) test kit) continue to cultivate, per 2～3 days replaced mediums continue to cultivate 14-21 days.
After inducing 14-21 days, discard substratum, PBS washs once, added 4% Paraformaldehyde 96 fixing about 1 hour, and inhaled and abandon stationary liquid, PBS washing one time, every hole adds an amount of about 1ml oil red O stain liquid (preparation, storage liquid: Virahol is configured to 0.5% solution, working fluid: storage liquid and PBS were with dilution in 3: 2, and filtration can be used), dyeed 15 minutes, staining fluid is abandoned in suction, PBS washing twice, microscopy.Coloration result as shown in Figure 6.The result shows: occur a large amount of fat in the cell and drip, oil red O stain takes on a red color.
3.3 inducing of class osteocyte broken up and evaluation
Cell is inoculated in 12 orifice plates, adds nutrient solution and cultivate, treat that the cytogamy degree reaches about 80-90%, namely change inducing culture (
Osteogenesis DifferentiationKit test kit (GIBCO)) continue to cultivate, per 2～3 days replaced mediums continue to cultivate 21～28 days.
After inducing about 21～28 days, discard substratum, the PBS washing once added 4% Paraformaldehyde 96 fixing about 1 hour, inhaled and abandoned stationary liquid, physiological saline washing one time, every hole adds an amount of about 1ml1% Alizarin red staining liquid oil red O stain liquid (pH4.1～4.3), and lucifuge dyeed 15 minutes, inhales and abandons staining fluid, distilled water wash twice, microscopy.Coloration result as shown in Figure 7.The result shows: the visible positive induces sample to be dyed to redness, and sees that the calcium of reddish black tubercle is arranged.
3.4 inducing of insulin-like cell group broken up and evaluation
3.4.1 insulin-like cell group induces differentiation
1) 0.25% tryptic digestion pancreatic stem cells is inoculated in the adherent ware of the RPMI1640/B27 nutrient solution that contains 1% BSA, 2mM L-glutaminate, 0.1mM beta-mercaptoethanol, 1mM Sodium propanecarboxylate, 100ng/mL ActivinA, 50mM LY294002,10mM nicotinamide, 1mM Sodium.alpha.-ketopropionate, 1 * ITS and cultivated 3-4 days; 0.25% tryptic digestion, suspension culture forms EB, changes above-mentioned nutrient solution is removed Sodium propanecarboxylate, ActivinA and LY294002 and adds 1.0 * 10
-6The nutrient solution of M RA, 20ng/mL EGF, 20ng/mL bFGF continues to cultivate and to carry out preliminary induction 7-9 days at 37 ℃, the condition low suspension of 5%CO2;
2) with the cell transfer behind the preliminary induction to the RPMI1640/B27 nutrient solution of the BSA that contains massfraction 1%, 2mM glutamine, 1mM beta-mercaptoethanol, 1mM Sodium.alpha.-ketopropionate, 25 μ M zinc acetates, 1 * ITS, 100mM nicotine, 10ng/mL exendin-4 and 10ng/mL beta cell element, under 37 ℃, the condition of 5%CO2, continue suspension culture, obtain insulin-like cell group.
3.4.2 the evaluation of islet cells group
To induce the insulin-like cell group that is differentiated to form to be inoculated in 48 orifice plates or carry out adherent culture in 6 orifice plates.Insulin-like cell group in 48 orifice plates is used for the detection of immunofluorescence, and the cell mass in 6 orifice plates is then analyzed the secretion situation of the post-stimulatory Regular Insulin of high low sugar.
48 orifice plates are cultivated the result that the immunofluorescence behind the 24h detects, as shown in Figure 8, and DTZ positive (Fig. 8 B), and PDX1, C-peptide, Regular Insulin positive (Fig. 8 C).
Respectively with 5.6 and the glucose of 25mmol/L stimulate Regular Insulin to discharge liquid to stimulate insulin-like cell group after inducing, the Regular Insulin and the C-peptide amount that discharge are analyzed.6 orifice plates are cultivated sucking-off inducing culture liquid behind the 24h, and the stimulation fluid that after PBS (-) flushing 3 times, add 0.4mL/ hole serum-free, contains 5.6mmol/L (low sugar) and 25mmol/L (high sugar) glucose concn stimulates Regular Insulin release.Collecting after 2 hours stimulates rear nutrient solution, and-20 ℃ of refrigerated storage are to be measured.After the off-test, send benevolence Ji hospital laboratory test department radioimmunoassay, measure Regular Insulin and C-peptide content in the stimulation fluid, the result is as being shown in Table 2.Can find out insulin-like cell after the stimulation that is subject to the glucose stimulation fluid, external can uelralante and C-peptide, and induce the effect in 2 weeks will be significantly better than the effect of inducing for 1 week.
Table 2. glucose stimulates the release test of insulin-like cell group
A: compare the burst size of stimulating group Regular Insulin and C-peptide poor and extremely significantly (P＜0.01) with control group
C: compare with the low sugar stimulating group, the burst size difference of high sugared stimulating group Regular Insulin and C-peptide is (P＜0.01) extremely significantly
3.4.3 interior detection of blood sugar body behind the diabetes mice model transplanted by the insulin-like cell that is induced to differentiate group
At random choose 18 in the male nude mouse 24 6～10 ages in week, sets up diabetes animal model by injecting 0.2% streptozotocin (STZ), remains 6 injection citrate buffers as negative control.Nude mice is fasting 12h before molding, with 100mg/kg (body weight) dosage abdominal injection STZ, every day 1 time, inject continuously 2d, measure on an empty stomach the blood glucose value of molding nude mice behind the 3d, measure once every 2d, the fasting blood sugar of continuous 2 rating model nude mices is higher than 16.7mmol/L, judges that then the diabetes nude mice is successfully prepared.With normal nude mice in contrast, model is divided into the insulin-like cell group group of transplanting nutrient solution group, the groups of cells of not inducing and inducing, every group each 6.The nude mice of normally feeding after abdominal injection is transplanted, per 3 days interior blood sugar concentrations of detection nude mouse after transplanting, the blood sugar concentration change curve of drawing according to detected result as shown in Figure 9, wherein, X-coordinate is the time (fate after transplanting, d), ordinate zou is the blood sugar concentration (mmol/L) of nude mice.Can be found out by blood sugar concentration change curve shown in Figure 9, the blood sugar concentration of nude mice namely begins to descend after islet transplantation like cell group, to the level of transplanting rear 10 days blood sugar concentrations and can reach normal nude mice, in after this 24 days, can keep normal blood sugar concentration, the concentration of blood sugar begins again to rise subsequently, and blood sugar concentration is suitable with the model nude mice blood sugar concentration of transplanting nutrient solution after the 46th day.And the Transplanted cells group of not inducing begins to be down to behind the blood sugar concentration 3w of nude mice below the 10mmol/L, and has kept about 40 days always, and then the blood sugar of model nude mice continues again to rise.This shows that the insulin-like cell group that induces has definite physiological function, can reduce the concentration of diabetes model blood sugar in vivo.And the pancreatic stem cells of transplanting to tie up in the nude mouse can Spontaneous Differentiation be beta Cell of islet under the high sugared condition, play the effect of control blood sugar, but its hypoglycemic short run effect to slightly be worse than the insulin-like cell group after inducing.
Claims (12)
1. the construction process of the pancreatic stem cells system in people's pancreas islet source, the method may further comprise the steps:
1) will cultivate 2-3 days among human pancreas's cell adding nutrient solution A;
2) choose islet cells group in the above-mentioned culture, become unicellular with protease digestion, be inoculated among the nutrient solution A and cultivate;
When 3) treating that Growth of Cells merges to 80-90%, go down to posterity, add nutrient solution B and cultivate, obtain human pancreas's stem cell line (hPSC) in islet cells source;
Wherein said nutrient solution A is the RPMI1640 that contains 10-25% FBS, 8-12mM nicotinamide, 18-22ng/mLEGF and 18-22ng/mL bFGF; Described nutrient solution B is the Low-DMEM that contains 10-25%FBS, 18-22ng/mLbFGF.
2. the process of claim 1 wherein that described nutrient solution A also contains 0.8-1.2mM Sodium.alpha.-ketopropionate, 1.8-2.2mM glutaminase, 0.05-0.15mM beta-mercaptoethanol and 90-110mg/mL penicillin/streptomycin; Described nutrient solution B also contains 0.05-0.15mM beta-mercaptoethanol, 1.8-2.2mM glutamine and 90-110mg/mL penicillin/streptomycin.
3. claim 1 or 2 method, the method may further comprise the steps:
1) in human pancreas's tissue block, adds 0.05-0.2% (mass/volume) collagenase digesting, stop filtering after the digestion, centrifugal and clean and obtain unicellularly, add described nutrient solution A and cultivated 2-3 days;
2) choose islet cells group at microscopically, 0.25% (mass/volume) proteolytic enzyme further is digested to it unicellular, is inoculated in the porous plate, adds described nutrient solution A and cultivates;
When 3) treating that Growth of Cells merges to 80-90%, with 0.25% protease digestion, with 1: 3-1: 5 ratios go down to posterity, and add described nutrient solution B and cultivate; Cultivate above the human pancreas's stem cell line that obtains the islet cells source after 50 generations.
4. method that makes human pancreas's stem cell line Differentiation Induction in vitro become insulin-like cell group, the method may further comprise the steps:
1) the human pancreas stem cell is inoculated in the RPMI1640/B27 nutrient solution that contains 0.8-1.2% BSA, 0.8-1.2mM Sodium propanecarboxylate, 80-120ng/mL ActivinA, 40-60mM LY294002,8-12mM nicotinamide, 0.8-1.2 * ITS, in adherent ware, cultivated 3-4 days; 0.25% protease digestion, suspension culture forms EB, changes above-mentioned nutrient solution is removed Sodium propanecarboxylate, ActivinA and LY294002 and adds 0.8-1.2 * 10
-6The nutrient solution of M RA, 18-22ng/mL EGF and 18-22ng/mL bFGF continues to carry out preliminary induction 7-9 days in the cultivation of cellar culture condition low suspension; In a preferred embodiment, described inoculation culture liquid also contains 1.8-2.2mM L-glutaminate, 0.05-0.15mM beta-mercaptoethanol and 0.8-1.2mM Sodium.alpha.-ketopropionate;
2) with the cell transfer behind the preliminary induction to the RPMI1640/B27 nutrient solution that contains 0.8-1.2% BSA, 1.8-2.2mM glutamine, 0.8-1.2mM Sodium.alpha.-ketopropionate, 22-28 μ M zinc acetate, 0.8-1.2 * ITS, 90-110mM nicotine, 8-12ng/mL exendin-4 and 8-12ng/mL beta cell element, under the cellar culture condition, continue suspension culture, obtain insulin-like cell group; In a preferred embodiment, described nutrient solution also contains 1.8-2.2mM L-glutaminate, 0.8-1.2mM beta-mercaptoethanol and 0.8-1.2mM Sodium.alpha.-ketopropionate.
5. the method for claim 4, wherein said human pancreas's stem cell line are the pancreatic stem cells systems in people's pancreas islet source of setting up according to each method of claim 1-3.
6. one kind makes the pancreatic stem cells system in people's pancreas islet source of setting up according to each method of claim 1-3 induce the method that is divided into the class neurocyte, the method may further comprise the steps: the cell that the pancreatic stem cells that described people's pancreas islet is originated is is inoculated in and discards nutrient solution after described nutrient solution B cultivates 20-30h, after adding the pre-induced liquid processing of neurocyte 20-30h, change neurocyte induced liquid continuation effect 8-12h into; The pre-induced liquid of described neurocyte is to add the 1mmol/L beta-mercaptoethanol in the Low-DMEM basic culture solution; Described neurocyte induced liquid is to add the 5mmol/L beta-mercaptoethanol in the Low-DMEM basic culture solution.
7. a pancreatic stem cells system that makes people's pancreas islet source of setting up according to each method of claim 1-3 induces into the method for class adipocyte, the method may further comprise the steps: the cell that the pancreatic stem cells that described people's pancreas islet is originated is is inoculated in above-mentioned nutrient solution B and cultivates, treat that the cytogamy degree reaches 80-90%, change inducing culture and continue to cultivate, continue to cultivate 14～21 days; Wherein said inducing culture is
Adipogenesis Differentiation Kit test kit (GIBCO).
8. a pancreatic stem cells system that makes people's pancreas islet source of setting up according to each method of claim 1-3 induces into the method for class osteocyte, the method may further comprise the steps: the cell that the pancreatic stem cells that described people's pancreas islet is originated is is inoculated in above-mentioned nutrient solution B and cultivates, treat that the cytogamy degree reaches 80-90%, change inducing culture and continue to cultivate, continue to cultivate 21～28 days; Wherein said inducing culture is
Osteogenesis Differentiation Kit test kit (GIBCO).
9. human pancreas's stem cell line of setting up according to each method of claim 1-3.
10. the insulin-like cell group that sets up according to the method for claim 4 or 5.
11. comprise medicine, reagent, composition or the test kit of human pancreas's stem cell line claimed in claim 9 or insulin-like cell claimed in claim 10 group.
12. human pancreas's stem cell line claimed in claim 9 or insulin-like cell claimed in claim 10 group is for the preparation of the purposes in medicine, reagent, composition or the test kit of lowering blood glucose or treatment diabetes.
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