CN102433300A - Method for constructing pancreatic stem cell line from human insulin and differentiating to insulin secretion cell - Google Patents
Method for constructing pancreatic stem cell line from human insulin and differentiating to insulin secretion cell Download PDFInfo
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Abstract
The invention discloses a method for constructing and differentiating a pancreatic stem cell line from human insulin. The pancreatic stem cell line is a pancreatic stem cell amplified from human insulin mass and has a multi-lineage differentiation potential. The induced pancreatic stem cell line can be differentiated to form nerve-like cells, fat-like cells, bone-like cells and functional insulin-like cell mass. The functional insulin-like cell mass is transplanted to a body of a nude mouse model suffered from diabetes, so that the blood sugar concentration of the nude mouse model can be reduced and normal blood sugar level can be maintained within one month.
Description
Technical field
The invention belongs to the cell engineering field, the pancreatic stem cells system structure in particularly a kind of people's pancreas islet source and the method for breaking up to insulin secretory cell.
Background technology
Mellitus 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, though treatment of diabetes is had a lot of methods, like the orally-taken blood sugar reducing medicine, injection of exogenous Regular Insulin, and the method for combination 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 this method receives 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.
Though the use of stem cell can solve donorcells quantity problem, before application, also need earlier it to be induced to differentiate into functional insulin secretory cell.Pancreatic stem cells (Pancreatic stem cells) is one type of adult stem cell that is present in fetus and the adult pancreatic tissue, has self, and multidirectional differentiation potential at first is divided into the various cells in the pancreatic tissue in natural atomization.Because this cell derives from pancreatic tissue, therefore, to having bigger potentiality aspect the insulin secretory cell differentiation.More and more evidences shows and all exists the stem cell with multidirectional differentiation potential in pancreatic duct, pancreas islet and the acinar tissue.Have viewpoint to think at present, the mesenchyme appearance stem cell that is obtained by islet tissue possibly derive from β cell (Russ, H.A. after dedifferenting; Bar, Y., Ravassard; P.; Efrat, In vitro proliferation of cells derived from adult human beta-cells revealed by cell-lineage tracing.Diabetes S.2008,57:1575～1583).And these cells still are in active state at external its insulin promoter of back that repeatedly goes down to posterity, and therefore have huge treating diabetes potentiality.
In-vitro separation, cultivating pancreatic stem cells as seed cell, and its directional induction is divided into the functional islets cell, is the effective way that solves the shortage of pancreas islet donor.Existing a plurality of research groups have carried out certain basis work 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.), the Yao Zhong of Third Military Medical University auspicious (in-vitro separation cultivation, evaluation and the differentiation characteristic [J] of human pancreas stem cell. 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 treatment of diabetes effect 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; With the external evoked method that is divided into insulin-like cell group of this stem cell line, and the pancreatic stem cells system and the insulin-like cell group that obtain with aforesaid method.Can improve its glucose level after said insulin-like cell group migrated to the diabetes model animal.
It is the method for external structure that the present invention provides the pancreatic stem cells in a kind of people's pancreas islet source; This method comprises the steps: to digest Human Pancreas; Choose islet cells group, further digestion forms single cell suspension, and the cultivation back of going down to posterity forms mesenchyme appearance pancreatic stem cells.
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.
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) with cultivating 2-3 days among human pancreas's cell adding nutrient solution A;
2) choose islet cells group in the above-mentioned culture, become unicellular, be inoculated among the nutrient solution A and cultivate with protease digestion;
When 3) treating that cell grows to the 80-90% fusion, go down to posterity, add nutrient solution B and cultivate, obtain human pancreas's stem cell line (pPSC) 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; Said nutrient solution B is the Low-DMEM that contains 10-25%FBS, 18-22ng/mLbFGF.
In one embodiment, said 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; Said nutrient solution B also contains 0.05-0.15mM beta-mercaptoethanol, 1.8-2.2mM Stimulina 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 digestion after-filtration, centrifugal and clean and obtain unicellularly, add said 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 said nutrient solution A and cultivates;
When 3) treating that cell grows to the 80-90% fusion, with 0.25% protease digestion, with 1: 3-1: 5 ratios go down to posterity, and add said nutrient solution B and cultivate; Cultivate above the human pancreas's stem cell line that obtains the islet cells source after 50 generations.
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 conventional culture condition low suspension cultivation; In a preferred embodiment, said 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 containing on the plain RPMI1640/B27 nutrient solution of 0.8-1.2%BSA, 1.8-2.2mM Stimulina, 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; Under conventional culture condition, continue suspension culture, obtain insulin-like cell group; In a preferred embodiment, said 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.
The third aspect of the 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 for external evoked type of being divided into neurocyte; This method may further comprise the steps: the cell inoculation that the pancreatic stem cells that said people's pancreas islet is originated is discards nutrient solution after nutrient solution B cultivates 20-30h; After adding the preparatory induced liquid processing of neurocyte 20-30h, change neurocyte induced liquid continuation effect 8-12h into; The preparatory induced liquid of said neurocyte is to add the 1mmol/L beta-mercaptoethanol in the Low-DMEM basic culture solution; Said neurocyte induced liquid is to add the 5mmol/L beta-mercaptoethanol in the Low-DMEM basic culture solution.
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 the method for type of one-tenth adipocyte; This method may further comprise the steps: the cell inoculation that the pancreatic stem cells that said people's pancreas islet is originated is is cultivated in above-mentioned nutrient solution B; Treat that the cytogamy degree reaches about 80-90%; Change inducing culture (
Adipogenesis Differentiation Kit test kit (GIBCO)) and continue to cultivate, continue to cultivate 14～21 days.
The 5th 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 the method for type of one-tenth osteocyte; This method may further comprise the steps: the cell inoculation that the pancreatic stem cells that said people's pancreas islet is originated is is cultivated in above-mentioned nutrient solution B; Treat that the cytogamy degree reaches about 80-90%; Promptly change inducing culture (
Osteogenesis Differentiation Kit test kit (GIBCO)) and continue to cultivate, continue to cultivate 21～28 days.
In one embodiment, the class osteocyte that obtains through aforesaid method is the sodium alizarinsulfonate stained positive.
The 6th aspect of the present invention provides human pancreas's stem cell line of setting up according to the method for first aspect present invention.
The 7th aspect of the present invention provides the insulin-like cell group that sets up according to the method for second aspect present invention.
Eight aspect of the present invention provides medicine, reagent, compsn, the test kit that comprises described human pancreas's stem cell line of sixth aspect present invention or the described insulin-like cell of seventh aspect present invention group.
The 9th 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 to be used for medicine, reagent, the compsn of lowering blood glucose or treatment mellitus, the purposes of test kit in preparation.
Compared with prior art, the present invention has following beneficial technical effects:
1) the pancreatic stem cells system in people's pancreas islet source of obtaining of the present invention, its upgrowth situation is good, its population doubling time shortens to 22.36 ± 1.06h by 45.24 original ± 1.28h, and the survival rate of cell than before raising greatly, be convenient to the application of mass-producing; Still have vigor preferably at subculture in vitro separately more than 50 generations, still keep division growth, be difficult for taking place 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; 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 about 10% foetal calf serum and bFGF now, 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 mellitus, can lowering blood glucose concentration even can return to normal glucose level, and in 1 month, 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 to treat the patient who suffers from mellitus is superior to 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;
Fig. 3 is the figure as a result of the cell cycle of the pancreatic stem cells in people's pancreas islet source of flow cytometer detection, and wherein X-coordinate is a dna content, and ordinate zou is a cell count;
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 a cell count (10
4);
The behave characteristic sign evaluation figure of pancreatic stem cells type of being induced to differentiate into neurocyte in pancreas islet source of Fig. 5;
The behave pancreatic stem cells in pancreas islet source of Fig. 6 becomes fat inductive characteristic sign evaluation figure;
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 transplants the blood sugar concentration change curve behind the nude mice diabetes model for insulin-like cell group, and wherein X-coordinate is time (fate), and ordinate zou is a blood sugar concentration.
Embodiment
The structure that the pancreatic stem cells that the present invention provides a kind of people's pancreas islet to originate is and the method for differentiation.This clone still has vigor preferably at subculture in vitro separately more than 50 generations, keeps division growth, is difficult for taking place aging and apoptosis, can be differentiated to form functional islets like cell group through after inducing.Below through to the gene and the Immunological Identification of the structure of this clone, cell morphological characteristic, mark, induce differentiation; High low sugar stimulation Regular Insulin and the secretion of C-peptide and the intravital change of blood sugar of transplanting diabetes model are tested and are elaborated, and said is to explanation of the present invention rather than qualification.
FBS in this specification sheets is meant foetal calf serum, about the percentage ratio of FBS, if no special instructions, all is meant percent by volume.BSA in this specification sheets is meant bovine serum albumin, about the percentage ratio of FBS, if no special instructions, all is meant 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.
Conventional nutrient solution described in the specification sheets, conventional culture condition are meant that those skilled in the art can be according to the nutrient solution and the culture condition of the definite said 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) aseptic collection aborted fetus pancreas; The blood that places the Hank ' s flushing of precooling and remove periphery with cut off around fat, blood vessel, organize tunicle and reticular tissue; Be cut into the tissue block about 1mm3 then, add 0.1%IV Collagen Type VI enzyme, in 37 ℃ of digestion 20-30min.Vibration frequently during this time; 80 eye mesh screens are crossed in the termination digestion back of containing 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 Stimulina, 20ng/mLEGF, 20ng/mL bFGF and 100mg/mL penicillin/streptomycin and cultivate 48h.
2) choose islet cells group by hand 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 Stimulina, 20ng/mL EGF, 20ng/mL bFGF and 100mg/mL penicillin/streptomycin, place 37 ℃, the incubator of 5%CO2 to cultivate.
When 3) treating that cell grows to 90% fusion; With 0.25% tryptic digestion; Go down to posterity with 1: 3 ratio; Cell inoculation is cultivated in the Low-DMEM nutrient solution that contains 10%FBS, 0.1mM beta-mercaptoethanol, 2mM Stimulina, 20ng/mL bFGF and 100mg/mL penicillin/streptomycin, and 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 characteristic
The metamorphosis of the pancreatic stem cells in observer's pancreas islet source under phase microscope.Concrete outcome is as shown in Figure 1, and wherein, figure A is the isolating human pancreatic island cell group of former generation, and figure B is the pancreatic stem cells (pPSC) in the people's pancreas islet source after the enlarged culturing; Compare with islet cells, cell is for becoming fiber-like, how triangular in shape and short shuttle shape, 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, and cell is a monokaryon; Examine apparent in viewly, the kernel more than 2 or 2 is arranged, can see tangible cell fission phase.This cell still has vigor preferably at subculture in vitro separately more than 50 generations, still keeps division growth, is difficult for taking place aging and apoptosis.
2.2 the evaluation of pancreatic stem cells mark of correlation
2.2.1 immunofluorescence detects
After 4% Paraformaldehyde 96 was fixed, 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 washs 2-3 time with PBS through the fixing 10min of 4% Paraformaldehyde 96;
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 one and resist, 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, and fluorescent microscope is observed down.
Immunofluorescence detects employed one anti-mainly containing: vimentin (Vim), PDX1, Ngn3, glucose transcription factor (Glut2), hTERT and CK19.Shown in Fig. 2 A, the result shows the immunofluorescence dyeing result respectively: this pancreatic stem cells is that albumen in wave shape (Vim), PDX1, Ngn3, glucose transcription factor (Glut2) and hTERT are positive, and does not express epithelial cell mark CK19.
2.2.2PCR detect pancreatic stem cells, the expression of the islet cells group marker gene after inducing
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 correlating markings thing 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 with induce after insulin-like cell group in centrifuge tube, PBS washing 2-3 time; Add 1mL Trizol concussion mixing then, leave standstill 5min under the room temperature and carry out lysis, in centrifuge tube, add the 0.2mL chloroform, 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 (about room temperature held 10min), 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 6mers 2 μ L, Oliqo (dT) Primer 0.5 μ L, reversed transcriptive enzyme (PrimeScript RT Enzyme Mix) 0.5 μ L; Total RNA of 500ng, RNase Free H2O polishing to 10 μ L; Mixing is hatched 30min for 37 ℃, and 85 ℃ are extended 5s then, obtain cDNA.
The pcr amplification double-stranded DNA, reaction system is following: 10 * PCR Buffer, 1.5 μ L, MgCl
21.5 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 preparatory sex change 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, pancreatic stem cells mark of correlation expression of gene situation is shown in Fig. 2 B, and this strain clone is all expressed Vim, PDX1, Ngn3 and Mafa gene, explains that it has the characteristic of pancreatic stem cells.And after inducing, the expression of Vim is constant basically, and PDX1, Ngn3, the expression of Mafa and Insulin (Regular Insulin) all strengthens, and explains that pancreatic stem cells is to islet cells differentiation (Fig. 8 C).
2.3 flow cytometer detects the cell cycle of the pancreatic stem cells system in people's pancreas islet source
Pancreatic stem cells with people's pancreas islet source is for treating test sample, and cell sample to be measured is processed single cell suspension, and the centrifugal 5min of 1000r/min abandons supernatant; Fix with 4 ℃ of precooled ethanol then, at least fixedly 18h is to be checked;
Adjusting cell concn again is 10
6Individual/mL, get the 1mL cell suspension, PBS washes in the PI dye liquor that is resuspended in 1mL 400 μ g/mL for 2-3 time, hatches 30min for 37 ℃, carries out its cell cycle of flow cytometry analysis.
The detected result of flow cytometer is as shown in Figure 3, and Mean G1 is that G1 phase dna content MV is 64.7; %G1=60 be G1 phase (pre-synthesis phase of DNA) cell count account for the sum 60%; %S=32.3 be S phase (DNA synthesis phase) cell count account for the sum 32.3%, %G2=7.66 be G2 phase (DNA post-synthesis phase) cell count account for the sum 7.66%.
2.4 the mensuration of growth curve
Get the pancreatic stem cells (the 5th generation and 25 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, the note of averaging is made cell count on the same day, continuous counter 10 days.With cell count (10
4) be ordinate zou, cultivating fate (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 25 generation cell growth curve, wherein 1～3 day is latent period, 4～7 days is increased logarithmic phase always, explains 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 22.36 ± 1.06h.
The function assessment characteristic 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 multidirectional differentiation potential.In-vitro separation, clone's pancreatic stem cells, and directional induction its be divided into functional islets transplantation treatment mellitus, 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 transplant its glucose level of nude mice diabetes model detection through associated molecule and immunofluorescence detection, high sugar.
3.1 inducing of type neurocyte broken up and evaluation
With 0.25% tryptic digestion; With cell inoculation in 48 orifice plates; Add and discard nutrient solution after nutrient solution is cultivated 24h; After adding the preparatory 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.Detect employed one anti-mainly containing: nidogen (Nestin), neuronspecific enolase (GFAP) and 'beta '-tubulin (β-III Tubulin).The immunofluorescence dyeing result is as shown in Figure 5, and the result shows: cell form after inducing takes place significantly to change, and a plurality of cynapses that cell is protruding are similar with the aixs cylinder of neurocyte.Coloration result shows that the class neurocyte after inducing is neurocyte sign Nestin, NSE, β-III tubulin positive.
3.2 inducing of type adipocyte broken up and evaluation
With cell inoculation in 12 orifice plates; Adding nutrient solution cultivates; Treat that the cytogamy degree reaches about 80-90%; Promptly changing inducing culture (
Adipogenesis Differentiation Kit (GIBCO) test kit) continues to cultivate; Changed substratum in per 2～3 days, continue to cultivate 14-21 days.
After inducing 14-21 days, discard substratum, PBS washs once, adds 4% Paraformaldehyde 96 fixing about 1 hour; Stationary liquid is abandoned in suction, PBS washing one time, and every hole adds an amount of about 1ml oil red O stain liquid, and (prepare, 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 is 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 type osteocyte broken up and evaluation
With cell inoculation in 12 orifice plates; Adding nutrient solution cultivates; Treat that the cytogamy degree reaches about 80-90%; Promptly changing inducing culture (
Osteogenesis Differentiation Kit test kit (GIBCO)) continues to cultivate; Changed substratum in per 2～3 days, continue to cultivate 21～28 days.
After inducing about 21～28 days, discard substratum, PBS washs once; Added 4% Paraformaldehyde 96 fixing about 1 hour, and inhaled and abandon stationary liquid, saline water washing one time; Every hole adds an amount of about 1ml1% sodium alizarinsulfonate staining fluid 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 is as shown in Figure 7.The result shows: the visible positive induces sample to be dyed 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 plain RPMI1640/B27 nutrient solution of the BSA that contains massfraction 1%, 2mM Stimulina, 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; Under 37 ℃, the condition of 5%CO2, continue suspension culture, obtain insulin-like cell group.
3.4.2 the evaluation of islet cells group
With inducing the insulin-like cell group that is differentiated to form to be inoculated in 48 orifice plates or carrying 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 post-stimulatory secretion of insulin situation of high low sugar.
48 orifice plates are cultivated the result that the immunofluorescence behind the 24h detects, and are as shown in Figure 8, DTZ positive (Fig. 8 B), and PDX1, C-peptide, Regular Insulin positive (Fig. 8 C).
Stimulating Regular Insulin to discharge liquid with 5.6 with the glucose of 25mmol/L respectively stimulates the insulin-like cell group after inducing, the Regular Insulin and the C-peptide amount that discharge is 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 the back nutrient solution, and-20 ℃ of refrigerated storage are to be measured.After the off-test, send chemical examination section of benevolence Ji hospital to use radioimmunoassay, measure Regular Insulin and C-peptide content in the stimulation fluid, the result is as seeing shown in the table 2.Can find out insulin-like cell after the stimulation that receives the glucose stimulation fluid, external can uelralante and C-peptide, and induce the effects in 2 weeks will obviously be better than the effect of inducing for 1 week.
Table 2. glucose stimulates the release test of insulin-like cell group
A: compare with control group, the burst size difference of stimulating group Regular Insulin and C-peptide is (P＜0.01) extremely significantly
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
Detected 3.4.3 induce in the blood sugar body after the mouse diabetes model is transplanted by the insulin-like cell group of differentiation
Picked at random is 18 in 24 6～10 week age male nude mouses, sets up diabetes animal model through injecting 0.2% streptozotocin (STZ), remain 6 and injects 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 2d continuously; Measure the blood glucose value of molding nude mice behind the 3d on an empty stomach; Measure once at a distance from 2d, the fasting blood sugar of continuous 2 rating model nude mices is higher than 16.7mmol/L, judges that then the mellitus nude mice prepares successfully., model is divided into transplants nutrient solution group, not inductive groups of cells and inductive insulin-like cell group group, every group each 6 as contrast with normal nude mice.The nude mice of normally feeding after abdominal injection is transplanted is transplanted the back and detected the intravital blood sugar concentration of nude mice in per 3 days, and is as shown in Figure 9 according to the blood sugar concentration change curve that detected result is drawn; Wherein, (fate, d), ordinate zou is the blood sugar concentration (mmol/L) of nude mice to X-coordinate for the time after transplanting.Blood sugar concentration change curve by shown in Figure 9 can be found out; Blood sugar concentration islet transplantation like cell group back nude mice promptly begins to descend; Can reach the level of normal nude mice to transplanting back 7 days blood sugar concentrations; In after this 1 month, 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 40th day.And inductive Transplanted cells group not begins to reduce to behind the blood sugar concentration 2w of nude mice below the 10mmol/L, and has kept about 45 days always, and then the blood sugar of model nude mice continues again to rise.This shows that inductive insulin-like cell group has definite physiological function, can reduce the concentration of diabetes model blood sugar in vivo.And the pancreatic stem cells of transplanting ties up to and can spontaneously be divided into beta Cell of islet in the nude mouse under the high sugared condition, play the effect of controlling blood sugar, but its hypoglycemic short run effect will be worse than the insulin-like cell group after inducing slightly.
Claims (12)
1. the construction process of the pancreatic stem cells system in people's pancreas islet source, this method may further comprise the steps:
1) with cultivating 2-3 days among human pancreas's cell adding nutrient solution A;
2) choose islet cells group in the above-mentioned culture, become unicellular, be inoculated among the nutrient solution A and cultivate with protease digestion;
When 3) treating that cell grows to the 80-90% fusion, go down to posterity, add nutrient solution B and cultivate, obtain human pancreas's stem cell line (pPSC) 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; Said nutrient solution B is the Low-DMEM that contains 10-25%FBS, 18-22ng/mLbFGF.
2. the process of claim 1 wherein that said 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; Said nutrient solution B also contains 0.05-0.15mM beta-mercaptoethanol, 1.8-2.2mM Stimulina and 90-110mg/mL penicillin/streptomycin.
3. claim 1 or 2 method, this method may further comprise the steps:
1) in human pancreas's tissue block, adds 0.05-0.2% (mass/volume) collagenase digesting, stop digestion after-filtration, centrifugal and clean and obtain unicellularly, add said 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 said nutrient solution A and cultivates;
When 3) treating that cell grows to the 80-90% fusion, with 0.25% protease digestion, with 1: 3-1: 5 ratios go down to posterity, and add said nutrient solution B and cultivate; Cultivate above the human pancreas's stem cell line that obtains the islet cells source after 50 generations.
4. one kind makes the external evoked method that is divided into insulin-like cell group of human pancreas's stem cell line, and this 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 conventional culture condition low suspension cultivation; In a preferred embodiment, said 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 containing on the plain RPMI1640/B27 nutrient solution of 0.8-1.2%BSA, 1.8-2.2mM Stimulina, 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; Under conventional culture condition, continue suspension culture, obtain insulin-like cell group; In a preferred embodiment, said 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. a pancreatic stem cells system that makes people's pancreas islet source of setting up according to each method of claim 1-3 induces the method for type of being divided into neurocyte; This method may further comprise the steps: the cell inoculation that the pancreatic stem cells that said people's pancreas islet is originated is discards nutrient solution after said nutrient solution B cultivates 20-30h; After adding the preparatory induced liquid processing of neurocyte 20-30h, change neurocyte induced liquid continuation effect 8-12h into; The preparatory induced liquid of said neurocyte is to add the 1mmol/L beta-mercaptoethanol in the Low-DMEM basic culture solution; Said 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 the method for type of one-tenth adipocyte; This method may further comprise the steps: the cell inoculation that the pancreatic stem cells that said people's pancreas islet is originated is is cultivated in above-mentioned nutrient solution B; 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 the method for type of one-tenth osteocyte; This method may further comprise the steps: the cell inoculation that the pancreatic stem cells that said people's pancreas islet is originated is is cultivated in above-mentioned nutrient solution B; 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, compsn or the test kit of the described insulin-like cell of described human pancreas's stem cell line of claim 9 or claim 10 group.
12. the described insulin-like cell of described human pancreas's stem cell line of claim 9 or claim 10 group is used for the purposes of medicine, reagent, compsn or the test kit of lowering blood glucose or treatment mellitus in preparation.
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