CN103881961A - Rat pancreas islet epithelioid stem cell system - Google Patents
Rat pancreas islet epithelioid stem cell system Download PDFInfo
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- CN103881961A CN103881961A CN201410138844.2A CN201410138844A CN103881961A CN 103881961 A CN103881961 A CN 103881961A CN 201410138844 A CN201410138844 A CN 201410138844A CN 103881961 A CN103881961 A CN 103881961A
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Abstract
The invention relates to a rat pancreas islet epithelioid stem cell system and belongs to the field of animal cell (tissue) engineering. The rat pancreas islet epithelioid stem cell is from a normal adult rat pancreas islet tissue, cultured in vitro, and can grow in a clonal manner; moreover, the cell has a plurality of differentiation potentials, specifically cell adherent culture is polygonal and epithelioid, cell nucleus is oval, and nucleolus is a single nucleolus or amphinucleolus; in cell clonal growth, cells grow slowly on the first day after being inoculated, enter a logarithm growth period on the second day to the fourth day and enter a platform period on the fifth day to the sixth day, and multiplication capacity is reduced on the seventh day; the cells are normal diploid cells with a chromosome number 2n equal to 42; an octamer transcription factor 4, a paired-gene cassette 4, paired-gene cassette 6, a neurogenic factor 3 and nidogen are expressed on protein level. The rat pancreas islet epithelioid stem cell system has the plurality of differentiation potentials, is directionally induced in vitro to differentiate and form nerve cells, fat cells and osteoblast, and is free of oncogenicity.
Description
Technical field
The present invention relates to the mankind and other Mammals pancreatic stem cell system and feature thereof, particularly the feature such as a routine rat Langerhans islet Epithelial stem cell line and form, growth, expression and differentiation, belongs to zooblast (tissue) engineering field.
Background technology
Diabetes (Diabetes mellitus, DM) be one of the major disease of harm humans health, whole world diabetic subject has reached 3.46 hundred million (Am J Stem Cells.2012 at present according to statistics, 1 (3): 196-204.), and traditional medicine and insulin substitution therapy can not fundamentally be cured this disease.Nearly ten years, along with the development of implantation technique, pancreatic islets transplantation treatment diabetes have obtained certain curative effect (N Engl J Med.2000,343 (4): 230-238. Diabetes.2005,54 (7): 2060-2069. Am J Transplant.2008,8 (11): 2463-2470. Immune Netw. 2013,13 (6): 235-239.).But due to donor source shortage, the application of pancreatic islets transplantation treatment diabetes technology is restricted.Pancreatic stem cell is to be present in pancreatic endocrine to organize the adult stem cell in pancreas islet, and energy self also has many differentiation potentials.In-vitro separation clone pancreatic stem cell, induce it to be differentiated to form functional islets, and cell transplantation for diabetes is one of approach effectively solving donor shortage.At present, set up pancreatic stem cell system, research pancreatic stem cell is differentiated to form functional islets cell transplantation for diabetes has become focus.Abroad, (the Diabetes.2001 such as Zulewski, 50 (3): 521-533.Endocrinology.2002,143 (8): 3152-3161.StemCells. 2004,22 (7): 1134-1141.) first report that collagenase digesting rat or adult pancreas separate tissue obtain individual cells and pancreas islet, RPMI1640 nutrient solution suspension culture, after 96h, choose suspension pancreas islet, in nutrient solution, add again 20ng/mLbFGF, 20ng/mLEGF adherent culture, the growth of Epithelial pancreatic stem cell clone property.Wherein, rat Langerhans islet stem cell has been increased 10 times for 8.5 months, and people's pancreatic stem cell has increased 7 times for 8 months.Immuno-chemical reaction shows pancreatic stem cell express nestin (nestin), does not express insulin, glucagon, somatostatin and CK19.Directional induction in vitro, pancreatic stem cell is differentiated to form pancreatic duct cell, expresses CK19; Be differentiated to form islet cells, express insulin, glucagon etc.; Be differentiated to form liver cell, express α-fetoprotein; Be differentiated to form neurocyte, express Neuron-specific adhesion molecule.Be implanted in mouse liver, humanized's pancreatic stem cell is differentiated to form people's liver cell.(the J Endocrinol.2004 such as Maria-Engler, 183 (3): 455-467.) collagenase HI perfusion digestion adult pancreas main pancreatic duct, separate and obtain cell and cell mass, density gradient centrifugation, dithizone (dithizon, DTZ) active coloring detects, and filters out diameter and is greater than 150 μ m pancreas islet, and CMRL1066+10%FCS cultivates.Confocal fluorescent microscopic examination and RT-PCR detect, and cultivate 24h, in islet cells group, only have a small amount of cell expressing nestin; Cultivate 60d, nestin
+expression amount significantly increases.Adopt containing 10%FBS nutrient solution and cultivate nestin
+cell 30d, expresses insulin; Cultivate nestin and adopt containing 1%FBS nutrient solution
+cell 30d, expresses insulin, glucagon and somatostatin.Therefore, author thinks external long-term cultivation, the nestin in people's pancreas islet
+cell has stem cell characteristic.(Diabetes. 2003,52 (10): 2519-2525.) the early stage pancreatic tissue of collagenase digesting human fetal obtains islet cells group, adherent culture, epithelioid cell's formation individual layer that increases such as Humphrey.Build nestin-GFP plasmid, Adenovirus Transfection, 418 screenings, are purified into nestin
+cell.RT-PCR detects nestin
+cell coexpression vimentin.Adopt the directional induction in vitro such as niacinamide, HGF, nestin
+cell but can not be differentiated to form the β cell of expressing Pdx1, insulin, is implanted in athymic mouse body and can not be differentiated to form functional β cell, secretion insulin.This shows that nestin is not the specific marker thing of β cell progenitor cell.Sugiyama etc. (Proc Natl Acad Sci USA.2007,104 (1): 175-180.) tryptic digestion mouse fetal pancreatic tissue, separates and obtains individual cells.Flow cytometry sorting, the pancreatic stem cell of acquisition coexpression Ngn3, CD133 and CD45f.Directional induction in vitro, pancreatic stem cell is differentiated to form β cell, secretion insulin.(the Stem Cells.2006 such as Ta, 24 (7): 1738-1749.) research shows to add fibroblast growth factor (basic fibroblast growth facter in nutrient solution, bFGF), leukocyte inhibitory factor (leukemia inhibitory factor, and bone morphogenetic protein 4 (bone morphogenetic protein-4 LIF), BMP4), all can promote the amplification of adult mice pancreatic stem cell.(the Proc Natl Acad Sci U S A. 2013 such as Jin, 110 (10): 3907-3912.) the adult mice pancreatic tissue of Gelatinase B+DNase Digestive system digestion obtains individual cells, CD133 mark, flow cytometry sorting, conditioned medium is cultivated, after 3w, cell clonal formation cell mass, RT-PCR detects clone cell and expresses CD133.In nutrient solution, add RSPO1 directional induction, CD133
+cytodifferentiation forms pancreatic stem cell and the acinous cell of expressing Ngn3.Flow cytometry further sub-elects Ngn3
+pancreatic stem cell, directional induction in vitro, is differentiated to form β cell.Glucose stimulates, secretion insulin.Domestic, (Chinese science C collects effect plums etc.: life science .2008, 38 (8): 699-707. Science in China Series C:Life Science.2008, 51 (9): 779-788. molecular cytobiology is reported .2008.41 (6): 450-457. molecular cytobiology is reported .2008, 41 (6): 457-464. dissects journal .2009, 40 (2): 55-58. China animal doctor journal .2009, 29 (2): 191-195.) report type Ⅳ collagen enzymic digestion human fetal pancreatic tissue, collect individual cells and cell mass, low sugar DMEM nutrient solution is cultivated.After primary Epithelial pancreatic stem cells grows, 0.25% tryptic digestive juice digestion, goes down to posterity.Clone's ring grizzly is selected monoclonal human pancreatic stem cells, adds 10ng/mLEGF amplification cultivation in nutrient solution, sets up human foetus pancreatic stem cell line.Immuno-chemical reaction and RT-PCR detect this stem cell and express Pdx1, glucagon, nestin and CK19 albumen, do not express insulin, CD34, CD44 and CD45.Beta-mercaptoethanol induction, is differentiated to form neurocyte, expresses NF albumen.Niacinamide induction, is differentiated to form DTZ stained positive, the functional class pancreas islet of secretion insulin and C peptide.In the diabetes rat scrotum that the pancreatic islets transplantation of induction class is prepared at STZ, can reduce blood glucose in diabetic rats level, prolongs life.The aseptic human fetal pancreatic tissue of getting of Yan Zhifeng etc. (Chinese Tissue Engineering Study and clinical .2008,12 (3): 485-489.), the digestion of V Collagenase Type, separates and obtains insulin-like cell group.Choose suspension insulin-like cell group adherent culture, Epithelial stem cell clone property grows to individual layer, goes down to posterity.Growth curve shows that the human fetal pancreas islet Epithelial stem cell 3d that goes down to posterity enters logarithmic phase, and 6d enters plateau, and cell growth curve is " S " shape.Fluorescence immune reaction detects primary Epithelial stem cell and expresses PCNA, Pdx1, nestin, CK19, insulin, glucagon and CD29, and after going down to posterity, stem cell is not expressed insulin, glucagon and CD29.Daytime magnitude (the Chinese magazine .2004 of general surgery, 13 (10): 746-749) collagenase digesting neonate rat pancreatic tissue, fragment of tissue adopts serum-free RPMI1640 nutrient solution to cultivate.Cultivate 36h, nestin
+cell attachment growth.Add bFGF, EGF, N
2, nestin
+cell rapid growth.Cultivate 18～24d, nestin
+cytodifferentiation forms class insulin-like cell group, and Regular Insulin release experiment detects, secretion insulin.Feng Ruopeng etc. (Scientia Agricultura Sinica .2007, (3) .582-587. Journal of Agricultural Biotechnology, 2011.19 (1): 9-17.) collagenase digesting tire pig pancreatic tissue separates and obtains pancreas islet, adherent culture, pancreatic stem cell fast breeding, passed for 18 generations, built to be.Immuno-chemical reaction shows pancreatic stem cell expression CK19 and α-actin, does not express nestin.Serum-free induction, after 2w, pancreatic stem cell is differentiated to form the insulin-like cell group of DTZ stained positive, expresses the relevant albumen of insulin secretory cell function such as insulin and Glut2.Glucose stimulates, secretion insulin and C-peptide.Inducing cell is transplanted in diabetes nude mice abdominal cavity, can be alleviated its hyperglycemia situation.At present, the domestic report of setting up rat Langerhans islet Epithelial stem cell line that there is no.The present invention one routine rat Langerhans islet Epithelial stem cell line and feature thereof, will be for the research mankind and other Mammals pancreatic stem cell provide foundation, this for the further research mankind and other mammalian pancreas grow, generation of diabetes and reproduce pancreas islet miniorgan and treat diabetes and have great importance.
Summary of the invention
The object of this invention is to provide routine rat Langerhans islet Epithelial stem cell line and a feature thereof, for the research mankind and other Mammals pancreatic stem cell system provide foundation, this for the further research mankind and other mammalian pancreas grow, generation of diabetes and reproduce pancreas islet miniorgan and treat diabetes and have great importance.
In order to address the above problem, the technical solution adopted in the present invention is:
One routine rat Langerhans islet Epithelial stem cell line, preserving number is C201460, preservation date on April 2nd, 2014, depositary institution: Chinese Typical Representative culture collection center, address: Wuhan University's Chinese Typical Representative culture collection center.
Comprise following feature:
1. rat Langerhans islet Epithelial stem cell morphological specificity
HE dyeing, after rat Langerhans islet Epithelial stem cell is completely adherent, is polygon Epithelial (Fig. 1); Giemsa dyeing, nucleus is oval, kernel mostly is nucleolus or double-core benevolence (Fig. 2);
2. rat Langerhans islet Epithelial stem cell growth characteristic
The growth of rat Langerhans islet Epithelial stem cell clone property; Growth chart clear-cells is inoculated 1d poor growth, and 2nd～4d enters logarithmic phase, and 5th～6d is plateau, 7d multiplication capacity decline (Fig. 3);
3. rat Langerhans islet Epithelial stem cell chromosome number
Chromosome specimen shows that rat Langerhans islet Epithelial stem cell is normal diploid cell, chromosome number 2n=42(Fig. 4);
4. rat Langerhans islet Epithelial stem cell expression characteristic
Flow cytometry and fluorescence immunoassay chemical reaction show that rat Langerhans islet Epithelial stem cell is at protein expression octamer biading factor 4 (Octamer-binding transcription factor 4, Oct4, Fig. 5), pairing box gene 4(Paired box 4, Pax4, Fig. 6), pairing box gene 6(Paired box 6, Pax6, Fig. 7), neurogenic factor 3(Neurogenin 3, Ngn3, Fig. 9) and nidogen (nestin, Figure 10), do not express Pdx1, MafA, Ptf1a, CK19, NeuroD2, PCNA, Nanog, Sox2, CD117, CD15, CD34, CD45, insulin, glucagon, ghrelin, somatostatin and secretin, Fig. 8 is Flow cytometry negative control, and Figure 11 is fluorescence immunoassay chemical reaction mouse fetal inoblast negative control,
5. rat Langerhans islet Epithelial differentiation of stem cells potential
Rat Langerhans islet Epithelial stem cell has many differentiation potentials, and directional induction in vitro is differentiated to form neurocyte, adipocyte and scleroblast;
5.1 are differentiated to form neurocyte: adopt DMEM+10%NBS+5mg/mL rat cerebral tissue extracting solution directional induction in vitro, induction 6d, approximately there is 50% rat Langerhans islet polygon Epithelial differentiation of stem cells to be formed with the neurocyte (Figure 12) of cynapse, fluorescence immunoassay chemical reaction is expressed neuronspecific enolase (Neuron specific enolase, NSE) (Figure 13);
5.2 are differentiated to form adipocyte: adopt DMEM+10%NBS+1umol/L dexamethasone+1ug/Linsulin+ 0.5mmol/LIBMX induced liquid directional induction in vitro, induction 7d, occurs in rat Langerhans islet polygon Epithelial stem cell that little and few fat drips; 14d, cell lactones drips and increases, and size is evenly; 21d, approximately 80% polygon Epithelial stem cell becomes round cell, and cell lactones drips and continues to increase, not of uniform size; 27d, round cell lactones drips showed increased, increase, the oil red O stain positive (Figure 14);
5.3 are differentiated to form scleroblast: adopt RPMI1640+10%NBS+0.1umol/L dexamethasone+10mmol/L β-phospho-glycerol+50ug/mL ascorbate salt induced liquid directional induction in vitro, induction 7d, there is shrinkage in approximately 30% rat Langerhans islet polygon Epithelial stem cell; 14d, pyknotic cells increases to 50%, part necrocytosis; 21d, emiocytosis increases, and starts to occur mineralising tubercle; 28d, the mineralising tubercle Alizarin red staining positive (Figure 15), Vonkossa dyeing mineralising tubercle presents black (Figure 16);
6. rat Langerhans islet Epithelial stem cell tumorigenicity
Rat Langerhans islet Epithelial stem cell is without tumorigenicity; Rat Langerhans islet Epithelial stem cell is seeded in the subcutaneous 30d in nude mice back, analyses, and observes and has no knob.
7. the rat Langerhans islet Epithelial stem cell cultivation of going down to posterity
Rat Langerhans islet Epithelial stem cell is by 1 × 10
6individual/mL cell density is cultivated, when stem cell growth to 80% fusion, 0.25% trypsinase+0.04%EDTA Digestive system had digestive transfer culture, the cell of collection is seeded in the culture dish that is covered with 0.1% gelatin, RPMI1640+10%NBS+10ng/mLbFGF nutrient solution amplification cultivation; This example rat Langerhans islet Epithelial stem cell line passed for 50 generations;
8. the freezing preservation of rat Langerhans islet Epithelial stem cell
Freezing: 80%DMEM+10%DMSO+10%NBS cell freezing liquid dilution rat Langerhans islet Epithelial stem cell to 1 × 10
6individual/mL, is sub-packed in freeze pipe; 4 ℃ of balance 30min, the 2h that suspends on liquid nitrogen surface, drops into liquid nitrogen long-term frozen and preserves;
Thaw: from liquid nitrogen container, take out rat Langerhans islet Epithelial stem cell freeze pipe, drop into rapidly in 37 ℃ of waters and thaw, RPMI1640+10%NBS+10ng/mLbFGF nutrient solution adherent culture; Trypan Blue detects, and the rat Langerhans islet Epithelial stem cell surviving rate of thawing is about 93%.
The invention has the beneficial effects as follows: routine rat Langerhans islet Epithelial stem cell line and a feature thereof is provided, will be for the research mankind and other Mammals pancreatic stem cell provide foundation, this for the further research mankind and other mammalian pancreas grow, generation of diabetes and reproduce pancreas islet miniorgan and treat diabetes and have great importance.
Accompanying drawing explanation
This specification sheets has 16 width accompanying drawings, wherein:
Fig. 1 rat Langerhans islet Epithelial stem cell HE dyeing morphological specificity (× 200);
Fig. 2 rat Langerhans islet Epithelial stem cell Giemsa dyeing morphological specificity (× 400);
Fig. 3 rat Langerhans islet Epithelial stem cell growth curve;
Fig. 4 rat Langerhans islet Epithelial stem cell chromosome number 2n=42;
Fig. 5 Flow cytometry, rat Langerhans islet Epithelial stem cell is expressed Oct4;
Fig. 6 Flow cytometry, rat Langerhans islet Epithelial stem cell is expressed Pax4;
Fig. 7 Flow cytometry, rat Langerhans islet Epithelial stem cell is expressed Pax6;
Fig. 8 Flow cytometry negative control;
Fig. 9 fluorescence immunoassay chemical reaction, rat Langerhans islet Epithelial stem cell is expressed Ngn3(× 400);
Figure 10 fluorescence immunoassay chemical reaction, rat Langerhans islet Epithelial stem cell is expressed nestin(× 400);
Figure 11 fluorescence immunoassay chemical reaction mouse fetal inoblast negative control (× 200);
Figure 12 directional induction in vitro, rat Langerhans islet Epithelial differentiation of stem cells is formed with the neurocyte (× 200) of cynapse;
Figure 13 fluorescence immunoassay chemical reaction, the neurocyte that rat Langerhans islet Epithelial differentiation of stem cells forms is expressed NSE(× 200);
Figure 14 directional induction in vitro, rat Langerhans islet Epithelial differentiation of stem cells forms adipocyte, the oil red O stain positive (× 200);
Figure 15 directional induction in vitro, rat Langerhans islet Epithelial differentiation of stem cells is formed into osteocyte, the Alizarin red staining positive (× 100);
Figure 16 rat Langerhans islet Epithelial differentiation of stem cells is formed into osteocyte, and Vonkossa dyeing mineralising tubercle presents black (× 100).
Embodiment
Below by example, the present invention is described in further details, these examples are only used for illustrating the present invention, do not limit the scope of the invention.
Embodiment 1
1. rat Langerhans islet Epithelial stem cell morphological specificity
HE dyeing: after rat Langerhans islet Epithelial stem cell is completely adherent, be polygon Epithelial (Fig. 1).Dyeing process is with reference to Si Tuzhenqiang, Wu Junzheng. cell cultures [M]. and the .2007. of world book publishing company is slightly;
Giemsa dyeing: rat Langerhans islet Epithelial stem cell core is oval, and kernel mostly is nucleolus or double-core benevolence (Fig. 2).Dyeing process is with reference to R.I. Fu Leixieni. animal cell culture-basic fundamental guide [M]. and the .2008. of Science Press is slightly;
2. rat Langerhans islet Epithelial stem cell growth characteristic
The growth of rat Langerhans islet Epithelial stem cell clone property; Growth chart clear-cells is inoculated 1d poor growth, and 2nd～4 d enter logarithmic phase, and 5th～6d is plateau, 7d multiplication capacity decline (Fig. 3).
Growth curve measuring method: resurrection the 20th, 30, the 40 generation rat Langerhans islet Epithelial stem cell of thawing, RPMI1640+ 10%NBS+10ng/mLbFGF nutrient solution is diluting cells to 1 × 10 respectively
4individual/mL.Get 7 of 96 orifice plates, inoculate respectively the cell of 3 kinds of different algebraically on each plate, in per generation, is established 6 holes, and every hole adds 100 μ L cell suspensions, and establishes negative control (basic culture solution, acellular), is placed in 37 ℃, 5%CO
2, cultivate in saturated humidity incubator.Now be designated as 0d.Every 24h takes out a culture plate, adopts CCK-8 method to measure cell absorbance (OD value), surveys altogether 7d.When mensuration, every hole adds after 10 μ LCCK-8, is placed in 37 ℃, 5%CO
2, act on 2h in saturated humidity incubator, in microplate reader, measure the OD of 450nm place value.Take the time (d) as X-coordinate, absorbance (OD) is ordinate zou, draws rat Langerhans islet Epithelial stem cell growth graphic representation.
3. rat Langerhans islet Epithelial stem cell chromosome number
Chromosome specimen shows that rat Langerhans islet Epithelial stem cell is normal diploid cell, chromosome number 2n=42(Fig. 4).
Rat Langerhans islet Epithelial stem cell is by 1 × 10
5individual/mL cell density is inoculated in the culture dish that is covered with 0.1% gelatin, adherent culture.While being expanded to 80% fusion, adding final concentration is 0.1 μ g/mL colchicine, continues to cultivate 5h, makes most cell chromosomes be stopped at metacinesis phase.0.25% trypsinase+0.04%EDTA Digestive system room temperature digestion 3min, RPMI1640+10%NBS nutrient solution stops digestion, centrifugal.Abandon supernatant, add 0.4%KCl, and be placed in 37 ℃ of waters, hypotonic 30min is centrifugal.Abandon supernatant, add methyl alcohol-Glacial acetic acid stationary liquid (methyl alcohol: Glacial acetic acid=3:1, matching while using), mix, leave standstill fixing 20min, centrifugal.So repeatedly fix 2～3 times.Abandon supernatant, add 0.5mL stationary liquid, mix, make karyomit(e) suspension.Get freezing slide glass, highly drip sheet in about 80cm, flame is dry.Drip Giemsa staining fluid, dyeing 10min.The micro-Microscopic observation chromosome specimen of General Biology, takes a picture.
4. rat Langerhans islet Epithelial stem cell expression characteristic
Flow cytometry and fluorescence immunoassay chemical reaction show that rat Langerhans islet Epithelial stem cell is at protein expression octamer biading factor 4 (Octamer-binding transcription factor 4, Oct4, Fig. 5), pairing box gene 4(Paired box 4, Pax4, Fig. 6), pairing box gene 6(Paired box 6, Pax6, Fig. 7), neurogenic factor 3(Neurogenin 3, Ngn3, Fig. 9) and nidogen (nestin, Figure 10), do not express Pdx1, MafA, Ptf1a, CK19, NeuroD2, PCNA, Nanog, Sox2, CD117, CD15, CD34, CD45, insulin, glucagon, ghrelin, somatostatin and secretin.Fig. 8 is Flow cytometry negative control, and Figure 11 is fluorescence immunoassay chemical reaction mouse fetal inoblast negative control.
4.1 Flow cytometry
Whether be with fluorescent mark according to purchased specific antibody, adopt direct immunofluorescence labelling method or indirect immunofluorescence labelling method to detect.
4.1.1 direct immunofluorescence labelling method
Direct immunofluorescence labelling method detects rat Langerhans islet Epithelial stem cell and does not express MafA, PCNA, CD117, CD15, CD34 and CD45.
PBS(-) washing rat Langerhans islet Epithelial stem cell 2 times, diluting cells to 1 × 10
6individual/mL.Get 7 of EP pipes, add respectively 1mL cell suspension, centrifugal (1000rpm, 5min).Abandon supernatant, add respectively 200uL to contain the PBS(-of 1%BSA) fluorescein labelled antibody (the anti-mouse MafA of the Golden Hamster fluorescence antibody of 0.25ug/100uL dilution of dilution, the mouse anti human PCNA fluorescence antibody of 5uL/100uL dilution, the mouse anti human CD117 fluorescence antibody of 5uL/100uL dilution, the mouse anti human CD15 fluorescence antibody of 5uL/100uL dilution, the mouse anti human CD45 fluorescence antibody of the mouse anti human CD34 fluorescence antibody of 5uL/100uL dilution or 5uL/100uL dilution, all purchased from eBioscience company), negative control adds PBS(-), mix, hatch 30min for 4 ℃, centrifugal (1000rpm, 5min).Abandon supernatant, PBS(-) washed cell 2 times, centrifugal (1000rpm, 5min), to remove unconjugated unnecessary antibody.Every pipe adds respectively 0.5mLPBS(-) re-suspended cell, flow cytometer (Accuri C6, BD, the U.S.) detects positive cell ratio.Experiment repeats 3 times.
4.1.2 indirect immunofluorescence labelling method
Indirect immunofluorescence labelling method detects rat Langerhans islet Epithelial stem cell and expresses octamer biading factor 4 (Oct4), pairing box gene 4(Paired box 4, Pax4) and pairing box gene 6(Paired box 6, Pax6).
PBS(-) washing rat Langerhans islet Epithelial stem cell 2 times, diluting cells to 1 × 10
6individual/mL.Get 4 of EP pipes, add respectively 1mL cell suspension, centrifugal (1000rpm, 5min).Abandon supernatant, add respectively 200uL to contain the PBS(-of 1%BSA) dilution without fluorescein-labeled primary antibodie, (the mouse Pax6 of the rabbit Chinese People's Anti-Japanese Military and Political College of the anti-human Oct4 of rabbit of 1:100 dilution or 1:500 dilution, purchased from Epitomics company; The anti-human Pax4 of 1:50 rabbit, purchased from Abgent company), negative control adds PBS(-), mix, hatch 30min for 4 ℃, centrifugal (1000rpm, 5min).Abandon supernatant, PBS(-) washed cell 2 times, centrifugal (1000rpm, 5min), to remove unconjugated unnecessary antibody.Abandon supernatant, add 200uL to contain the PBS(-of 1 %BSA) fluorescein-labelled two anti-(the goat-anti rabbit fluorescence two of 1:100 dilution is anti-, purchased from Epitomics company) of dilution, negative control adds PBS(-), mix, hatch 30min for 4 ℃, centrifugal (1000rpm, 5min).Abandon supernatant, PBS(-) washed cell 2 times, centrifugal (1000rpm, 5min), to remove unconjugated unnecessary antibody.Every pipe adds respectively 0.5mLPBS(-) re-suspended cell, flow cytometer (Accuri C6, BD, the U.S.) detects positive cell ratio.Experiment repeats 3 times.
4.2 fluorescence immunoassay chemical reactions
For the antibody of buying less than Flow cytometry, buy the antibody test of fluorescence immunoassay chemical reaction.
Fluorescence immunoassay chemical reaction shows rat Langerhans islet Epithelial stem cell expression neurogenic factor 3(Neurogenin 3, Ngn3) and nidogen (nestin), do not express Pdx1, Ptf1a, CK19, Nanog, NeuroD2, Sox2, insulin, glucagon, ghrelin, somatostatin and secretin.
In the culture dish that is covered with 0.1 % gelatin, put into cover glass, rat Langerhans islet Epithelial stem cell is by 1 × 10
5individual/mL cell density is seeded in the culture dish that is placed with cover glass.When Growth of Cells to 80% fusion, 4% paraformaldehyde room temperature fixed cell creep plate 15min.PBS(-) after washed cell creep plate 2 times, the 1%Triton-X100 30min that bores a hole, 1%BSA seals 45min.Wash unnecessary confining liquid off, adding the PBS(-containing 1%BSA) (the mouse anti human Ngn3 of 1:100 dilution, the mouse anti human Ptf1a of 3 μ g/mL dilutions, the mouse anti human ghrelin of 5 μ g/mL dilutions or the anti-human secretin of rabbit of 1:100 dilution, all purchased from Abcam company for the primary antibodie of dilution; The mouse nestin of the mouse Chinese People's Anti-Japanese Military and Political College of 1:300 dilution, purchased from Novus company; The mouse anti human CK19 of the mouse anti human Pdx1 of 20 μ g/mL dilutions or 20 μ g/mL dilution, purchased from eBioscience company; The anti-human Sox2 of rabbit of the mouse NeuroD2 of the rabbit Chinese People's Anti-Japanese Military and Political College, the 1:100 dilution of the anti-human Nanog of rabbit, the 1:300 dilution of 1:100 dilution or the anti-human somatostatin of rabbit of 1:100 dilution, all purchased from Epitomics company; The mouse glucagon of the rabbit Chinese People's Anti-Japanese Military and Political College of the mouse insulin of the mouse Chinese People's Anti-Japanese Military and Political College of 1:500 dilution or 1:500 dilution, purchased from Boster company; ), negative control adds PBS (-), and 4 ℃ are spent the night.PBS(-) washing 3 times, each 3min.Adding the PBS(-containing 1%BSA) anti-(the goat-anti mouse fluorescence two of 1:1000 dilution is anti-for the fluorescence two of dilution, goat-anti rabbit fluorescence two purchased from Novu company or 1:100 dilution is anti-, purchased from Epitomics company), negative control adds PBS (-), hatches 1h for 37 ℃.PBS(-) washing 3 times, each 3min, glycerine mounting.Fluorescence microscopy Microscopic observation, takes a picture.Negative control cell is mouse fetal inoblast.
5. rat Langerhans islet Epithelial differentiation of stem cells potential
Rat Langerhans islet Epithelial stem cell has many differentiation potentials, and directional induction in vitro is differentiated to form neurocyte, adipocyte and scleroblast.
5.1 are differentiated to form neurocyte
Be differentiated to form neurocyte: adopt DMEM+10%NBS+5mg/mL rat cerebral tissue extracting solution directional induction in vitro, induction 6d, approximately there is 50% rat Langerhans islet polygon Epithelial differentiation of stem cells to be formed with the neurocyte (Figure 12) of cynapse, fluorescence immunoassay chemical reaction is expressed neuronspecific enolase (Neuron specific enolase, NSE) (Figure 13).
Rat Langerhans islet Epithelial stem cell is by 1 × 10
5individual/mL cell density inoculation, RPMI1640+10%NBS+10ng/mL bFGF nutrient solution amplification cultivation.In the time of Growth of Cells to 80% fusion, change the extracting solution directional induction of DMEM+10%NBS+5mg/mL rat cerebral tissue and cultivate, change every other day liquid.Induction 6d, approximately has 50% rat Langerhans islet polygon Epithelial differentiation of stem cells to be formed with the neurocyte of cynapse, and fluorescence immunoassay chemical reaction is expressed NSE.
5.2 are differentiated to form adipocyte
Be differentiated to form adipocyte: adopt DMEM+10%NBS+1umol/L dexamethasone+1ug/Linsulin+0.5mmol/L IBMX induced liquid directional induction in vitro, induction 7d, occurs in rat Langerhans islet polygon Epithelial stem cell that little and few fat drips; 14d, cell lactones drips and increases, and size is evenly; 21d, approximately 80% polygon Epithelial stem cell becomes round cell, and cell lactones drips and continues to increase, not of uniform size; 27d, round cell lactones drips showed increased, increase, the oil red O stain positive (Figure 14).
Rat Langerhans islet Epithelial stem cell is by 1 × 10
5individual/mL cell density inoculation, RPMI1640+10%NBS+10ng/mL bFGF nutrient solution amplification cultivation.In the time of Growth of Cells to 80% fusion, change the directional induction of DMEM+10%NBS+1umol/L dexamethasone+1ug/Linsulin+0.5mmol/LIBMX induced liquid and cultivate, change every other day liquid.Induction 7d, occurs in rat Langerhans islet polygon Epithelial stem cell that little and few fat drips; 14d, cell lactones drips and increases, and size is evenly; 21d, approximately 80% polygon Epithelial stem cell becomes round cell, and cell lactones drips and continues to increase, not of uniform size; 27d, round cell lactones drips showed increased, increase, the oil red O stain positive.
5.3 are differentiated to form scleroblast
Be differentiated to form scleroblast: adopt RPMI1640+10%NBS+0.1umol/L dexamethasone+10mmol/L β-phospho-glycerol+50ug/mL ascorbate salt induced liquid directional induction in vitro, induction 7d, there is shrinkage in approximately 30% rat Langerhans islet polygon Epithelial stem cell; 14d, pyknotic cells increases to 50%, part necrocytosis; 21d, emiocytosis increases, and starts to occur mineralising tubercle; 28d, the mineralising tubercle Alizarin red staining positive (Figure 15), Vonkossa dyeing mineralising tubercle presents black (Figure 16).
Rat Langerhans islet Epithelial stem cell is by 1 × 10
5individual/mL cell density inoculation, RPMI1640+10%NBS+10ng/mL bFGF nutrient solution amplification cultivation.In the time of Growth of Cells to 80% fusion, change the directional induction of RPMI1640+10%NBS+0.1umol/L dexamethasone+10mmol/L β-phospho-glycerol+50ug/mL ascorbate salt induced liquid and cultivate, change every other day liquid.Induction 7d, there is obvious shrinkage in approximately 30% rat Langerhans islet polygon Epithelial stem cell; 14d, pyknotic cells increases to 50%, part necrocytosis; 21d, emiocytosis increases, and starts to occur mineralising tubercle; 28d, the mineralising tubercle Alizarin red staining positive, Vonkossa dyeing mineralising tubercle presents black.
6. rat Langerhans islet Epithelial stem cell tumorigenicity
Rat Langerhans islet Epithelial stem cell is without tumorigenicity.Rat Langerhans islet Epithelial stem cell is seeded in the subcutaneous 30d in nude mice back, dissects, and observes and has no knob.
Subcutaneously at 6 nude mice backs inject respectively rat Langerhans islet Epithelial stem cell, dosage is 1 × 10
6individual/0.2mL/site, 3 nude mice back subcutaneous injection 0.2mL cell culture fluid RPMI1640 in contrast, normal feeding and management 30d.As a result, 6 experiment nude mices and 3 contrast nude mices all survive to experiment end, and anatomic observation, is showed no knob.
7. the rat Langerhans islet Epithelial stem cell cultivation of going down to posterity
In the time of the fusion of rat Langerhans islet Epithelial stem cell growth to 80%, 0.25% trypsinase+0.04%EDTA Digestive system digestion 3min, RPMI1640+10%NBS nutrient solution stops digestion, centrifugal (1000rpm, 5min).The cell of collecting is by 1 × 10
6individual/mL cell density is seeded in the culture dish that is covered with 0.1% gelatin, RPMI1640+10%NBS+10ng/mLbFGF nutrient solution amplification cultivation.This example rat Langerhans islet Epithelial stem cell line passed for 50 generations.
8. the freezing preservation of rat Langerhans islet Epithelial stem cell
Freezing: when the fusion of rat Langerhans islet Epithelial stem cell growth to 80%, PBS(-) clean, the digestion of 0.25% trypsinase+0.04%EDTA Digestive system, RPMI1640+10%NBS nutrient solution stops digestion, centrifugal (1000rpm, 5min).80%RPMI1640+10%DMSO+10%NBS refrigerating fulid diluting cells to 1 × 10
6individual/mL, is sub-packed in freeze pipe.4 ℃ of balance 30min, the 2h that suspends on liquid nitrogen surface, drops into liquid nitrogen long-term frozen and preserves.
Thaw: from liquid nitrogen container, take out rat Langerhans islet Epithelial stem cell freeze pipe, drop into rapidly in 37 ℃ of waters and thaw, RPMI1640+10%NBS nutrient solution cleans the cell 2～3 times of thawing, RPMI1640+10%NBS+10ng/mLbFGF nutrient solution adherent culture.The cell that takes a morsel, Trypan Blue detects, and cell thawing surviving rate is about 93%.
Adopt this freezing and storing method, this example rat Langerhans islet Epithelial stem cell 50 of freezing preservation is managed, and approximately 0.5 × 10
8individual cell.
Claims (9)
1. a routine rat Langerhans islet Epithelial stem cell line, it is characterized in that: rat Langerhans islet Epithelial source of human stem cell is in Normal Adult Rat islet tissue, vitro culture can clone property be grown, and there are many differentiation potentials, preserving number is C201460, preservation date on April 2nd, 2014, depositary institution: Chinese Typical Representative culture collection center.
2. a routine rat Langerhans islet Epithelial stem cell line according to claim 1, it is characterized in that: rat Langerhans islet Epithelial stem cell morphological specificity is: after rat Langerhans islet Epithelial stem cell is completely adherent, be polygon Epithelial, nucleus is oval, and kernel mostly is nucleolus or double-core benevolence.
3. a routine rat Langerhans islet Epithelial stem cell line according to claim 1, is characterized in that: rat Langerhans islet Epithelial stem cell growth characteristic is: the growth of rat Langerhans islet Epithelial stem cell clone property; Cell is inoculated 1d poor growth, and 2nd～4d enters logarithmic phase, and 5th～6d is plateau, and 7d multiplication capacity declines.
4. a routine rat Langerhans islet Epithelial stem cell line according to claim 1, is characterized in that: rat Langerhans islet Epithelial stem cell chromosome number is: rat Langerhans islet Epithelial stem cell is normal diploid cell, chromosome number 2n=42.
5. a routine rat Langerhans islet Epithelial stem cell line according to claim 1, it is characterized in that: rat Langerhans islet Epithelial stem cell expression characteristic is: rat Langerhans islet Epithelial stem cell is at protein expression octamer biading factor 4 (Octamer-binding transcription factor 4, Oct4), pairing box gene 4(Paired box 4, Pax4), pairing box gene 6(Paired box 6, Pax6), neurogenic factor 3(Neurogenin 3, and nidogen (nestin) Ngn3), do not express Pdx1, MafA, Ptf1a, CK19, NeuroD2, PCNA, Nanog, Sox2, CD117, CD15, CD34, CD45, insulin, glucagon, ghrelin, somatostatin and secretin.
6. a routine rat Langerhans islet Epithelial stem cell line according to claim 1, it is characterized in that: rat Langerhans islet Epithelial differentiation of stem cells potential is: rat Langerhans islet Epithelial stem cell has many differentiation potentials, directional induction in vitro, rat Langerhans islet Epithelial differentiation of stem cells is formed with the neurocyte of cynapse, expresses NSE; Be differentiated to form and contain the adipocyte that fat drips, the oil red O stain positive; Be differentiated to form scleroblast, produce mineralising tubercle, the Alizarin red staining positive, Vonkossa dyeing mineralising tubercle presents black.
7. a routine rat Langerhans islet Epithelial stem cell line according to claim 1, is characterized in that: rat Langerhans islet Epithelial stem cell tumorigenicity: rat Langerhans islet Epithelial stem cell is without tumorigenicity.
8. a routine rat Langerhans islet Epithelial stem cell line according to claim 1, it is characterized in that: the rat Langerhans islet Epithelial stem cell cultivation of going down to posterity: in the time of the fusion of rat Langerhans islet Epithelial stem cell growth to 80%, 0.25% trypsinase+0.04%EDTA Digestive system had digestive transfer culture, the cell of collection is by 1 × 10
6individual/mL cell density is seeded in the culture dish that is covered with 0.1% gelatin, RPMI1640+ 10%NBS+10ng/mLbFGF nutrient solution amplification cultivation; This stem cell line passed for 50 generations.
9. a routine rat Langerhans islet Epithelial stem cell line according to claim 1, is characterized in that: the freezing preservation of rat Langerhans islet Epithelial stem cell:
Freezing: 80%DMEM+10%DMSO+10%NBS cell freezing liquid dilution rat Langerhans islet Epithelial stem cell to 1 × 10
6individual/mL, is sub-packed in freeze pipe; 4 ℃ of balance 30min, the 2h that suspends on liquid nitrogen surface, drops into liquid nitrogen long-term frozen and preserves;
Thaw: from liquid nitrogen container, take out rat Langerhans islet Epithelial stem cell freeze pipe, drop into rapidly in 37 ℃ of waters and thaw, RPMI1640+10%NBS+10ng/mLbFGF nutrient solution adherent culture; Trypan Blue detects, and the rat Langerhans islet Epithelial stem cell surviving rate of thawing is about 93%.
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| WO2004011621A2 (en) * | 2002-07-29 | 2004-02-05 | Es Cell International Pte Ltd. | Multi-step method for the differentiation of insulin positive, glucose |
| WO2006045105A2 (en) * | 2004-10-20 | 2006-04-27 | Vitro Diagnostics, Inc. | Generation and differentiation of adult stem cell lines |
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