CN109486766A - The cultivating system and cultural method of a kind of lachrymal gland stem cell, lachrymal gland stem cell - Google Patents

The cultivating system and cultural method of a kind of lachrymal gland stem cell, lachrymal gland stem cell Download PDF

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CN109486766A
CN109486766A CN201811415998.6A CN201811415998A CN109486766A CN 109486766 A CN109486766 A CN 109486766A CN 201811415998 A CN201811415998 A CN 201811415998A CN 109486766 A CN109486766 A CN 109486766A
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stem cell
lachrymal gland
gland stem
culture
lachrymal
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CN109486766B (en
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张雁
肖洒
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Sun Yat Sen University
National Sun Yat Sen University
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Abstract

The present invention relates to the cultivating systems and cultural method of a kind of lachrymal gland stem cell, lachrymal gland stem cell, are related to cell engineering field.The cultivating system of lachrymal gland stem cell of the invention is basic culture medium with DMEM/F12, add following components and constitute culture solution: cell culture additive, nonessential amino acid, Ala-Gln, blocking effect of mitogen activated protein kinases/extracellular signal-regulated kinase signal path ligand, fibroblast growth factor, Wnt signal path ligand and ROCK signal pathway inhibitor composition, and using matrigel as the bracket of stereoscopic culture.Cultural method of the invention is, by mouse lacrimal tissue be digested to it is unicellular be inoculated into matrigel, after its solidification, be added in above-mentioned lachrymal gland stem cell medium and carry out originally culture.Using the mouse lachrymal gland stem cell of method culture of the invention, quantity is more, enough to stablize lasting passage, and has the effect for restoring lacrimal secretion function, can effectively treat xerophthalmia.

Description

The cultivating system and cultural method of a kind of lachrymal gland stem cell, lachrymal gland stem cell
Technical field
The present invention relates to cell engineering fields, more particularly to a kind of 3 D stereo cultivating system and use this cultivating system The mouse lachrymal gland stem cell (LGSC) of culture.
Background technique
Lachrymal gland is eye vitals, is mainly made of secretion acinus and secretor.Lachrymal gland is by sympathetic nerve and secondary friendship Feel nerve modulation, it is partially adrenergic nerve that wherein most, which is cholinergic nerve,.Sympathetic nerve is originated from neck epineural Section, parasympathetic nerve are then connected with pterygopalatine ganglion and ciliary ganglion, and certain sensory nerves of lachrymal gland and trident mind Through being connected.There are many acetylcholinergic receptor in lachrymal gland, including muscarinic receptor, I type and II type vip receptor and go Methylepinephrine receptor alpha 1, β.Nerve modulation glandula lacrimalis inferior can secrete lysozyme, lactotransferrin, and lipocalin protein is immunized Globulin A etc..
Lacrimal secretion tears form tear film, so that eyes is kept wet, to protect eyes.Once lachrymal gland lesion is damaged Wound, tears hyposecretion, people will suffer from xerophthalmia (Dry eye Disease, DED).The aging of people, self immune system Disorder, the radiotherapy of eye and the reduction of Serum Testosterone Levels can all cause lacrimal gland function to be lacked of proper care.Shape secreted by patients with dry eye At tear film it is higher than normal tear film osmotic pressure, so as to cause Corneal inflammation, lead to that keratocyte is impaired, forms ulcer, finally lead Patient's vision decline is caused, its quality of life is seriously affected.
For patients with dry eye, the eyedrops that current therapeutic scheme mainly has drop isotonic or hypotonic, inhibit because Eye conjunctiva caused by xerophthalmia and the inflammation of cornea occur, and obstruction drain hole increases tear film thickness etc..But these treatments can only be alleviated The symptom of xerophthalmia makes eyes keep wet as much as possible, keratocyte is avoided to be damaged.Moreover, using medicament for the eyes for a long time Water and anti-inflammatory medicaments can also kill keratocyte and cause the inflammatory reaction for being more difficult to cure.Therefore, drug therapy can not It fundamentally solves the problems, such as lacrimal secretion functional disturbance, cannot thoroughly cure the lachrymal gland of aging or lesion.
In order to more effectively more thoroughly treat xerophthalmia, researchers have carried out a series of researchs, it is intended to restore lachrymal gland Secreting function.To restore the secreting function of lachrymal gland completely, it is necessary to obtain new healthy cell or organ and carry out transplantation treatment. The lachrymal gland cell for transplanting, researcher just begin trying Isolation and culture lachrymal gland stem cell in order to obtain.By nearly 30 The effort in year, the in vitro culture of lachrymal gland stem cell has breakthrough research, however, existing lachrymal gland Stem cells cultured in vitro side The efficiency that method sorts progenitor cells is very low, quantity is few, and cannot continue secondary culture, greatly limits its clinical application.Cause This, needs more excellent separation method and cultivating system to separate and cultivate lachrymal gland stem cell, provides well for clinical application Technology platform.
Summary of the invention
Based on this, it is necessary to for existing lachrymal gland stem cell cultivating system and cultural method sorting progenitor cells low efficiency, The few problem of quantity provides the cultivating system and cultural method of a kind of lachrymal gland stem cell, dry thin to obtain greater number of lachrymal gland Born of the same parents, and the lachrymal gland stem cell is able to carry out stable secondary culture.
A kind of cultivating system of lachrymal gland stem cell, is basic culture medium with DMEM/F12, and addition following components constitutes culture Liquid: cell culture additive, nonessential amino acid, Ala-Gln, blocking effect of mitogen activated protein kinases/extracellular Signal regulated kinase signal path ligand, fibroblast growth factor, Wnt signal path ligand and ROCK signal path inhibit The composition of agent, and using matrigel as the bracket of stereoscopic culture.
Further, the cell culture additive is the mixture of N2 and B27.
The cell culture additive can also select 9F, by beta -mercaptoethanol, 2- ethylaminoethanol, sodium selenite, heparin, Transferrins, vitamin C, insulin, bovine serum albumin(BSA)-oleic acid and fibroblast growth factor-2 (FGF2) composition.
Further, the blocking effect of mitogen activated protein kinases/extracellular signal-regulated kinase signal path (MAPK/ERK letter Number access) ligand is EGF, the fibroblast growth factor is FGF10, and the Wnt signal path ligand is Wnt3A, institute Stating ROCK signal pathway inhibitor is Y-27632.
Further, it is 2% that N2 concentration, which is 1%, B27 concentration, in the cell culture additive；The non-essential amino Acid concentration is 1%, and the Ala-Gln concentration is 1%.
Further, each component concentration in the cell culture additive 9F are as follows: 5-10 μM of beta -mercaptoethanol, 2- amino second 5-10 μM of alcohol, 10-20 μM of sodium selenite, heparin 50-150ng/ml, transferrins 2-5ng/ml, vitamin C 5-10ng/ml, Insulin 5-10ng/ml, bovine serum albumin(BSA)-oleic acid 6.8-9.4ng/ml and fibroblast growth factor-2 5-10ng/ ml；The nonessential amino acid 1%, the Ala-Gln 1%.
Further, the blocking effect of mitogen activated protein kinases/extracellular signal-regulated kinase signal path ligand is EGF, The fibroblast growth factor is FGF10, and the Wnt signal path ligand is Wnt3A, and the ROCK signal path inhibits Agent is Y-27632.
Further, EGF concentration is 10-50ng/ml in the cell culture additive, and FGF10 concentration is 10-100ng/ Ml, Wnt3A concentration are 5-10ng/ml, and Y-27632 concentration is 5-10mM.
Further, the matrigel is Matrigel.Matrigel is the three dimensional matrix with biological activity, simulation Structure, composition, physical characteristic and the function of internal cell basilar memebrane, are conducive to Cultured Mouse tear as matrigel using it The culture and differentiation of gland stem cell.
Further, nonessential amino acid uses NEAA, wherein containing Gly, l-Alanine, altheine, L- asparagus fern 7 kinds of nonessential amino acid such as propylhomoserin, Pidolidone, L-PROLINE, Serine can effectively improve cell culture medium proportion.
A kind of cultural method of lachrymal gland stem cell, by mouse lacrimal tissue be digested to it is unicellular be inoculated into matrigel, to After it is solidified, it is added in above-mentioned lachrymal gland stem cell medium, and in 37 DEG C, 5%~10%CO2Originally culture is carried out under environment.
A kind of lachrymal gland stem cell, is prepared using above-mentioned cultivating system and cultural method.
Compared with prior art, the invention has the following advantages:
One, it cannot effectively cultivate to obtain the defect of lachrymal gland stem cell the present invention overcomes traditional plane cultural method, The method for constructing 3 D stereo culture carries out Cultured Mouse lachrymal gland stem cell, dry by the mouse lachrymal gland of the method culture Cell quantity is more, and sufficient cell material is provided for clinical research；
Two, the present invention carries out 3 D stereo culture lachrymal gland stem cell using matrigel, and stem cell can be certainly as time goes by I replicates, and a part will do it differentiation, forms micro-organs approximate with the organ structure or that cell turnover process is similar, i.e., " organoid body ", stem cell interact in organoid body with the cell of differentiation, and signaling molecule influences each other, and form one Metastable stem cells hyperplasia microenvironment enables stem cell to continue to pass on, and keeps certain tissue characteristics and gene Type stability；
Three, the mouse lachrymal gland stem cell obtained using the culture system in vitro that the present invention establishes, the mouse tear after transplanting Secretory volume dramatically increases, and not only has the lachrymal gland for repairing damage, is also equipped with the effect for restoring lacrimal secretion function, to study dry eyes The stem-cell therapy of disease provides good basis.
Detailed description of the invention
Fig. 1 is the state change map of difference culture generation when LGSCM conditional mouse lachrymal gland stem cell is persistently passed on, ratio Ruler: 100 μm；
Fig. 2 is the total number of cells statistics of difference culture generation when LGSCM conditional mouse lachrymal gland stem cell is persistently passed on；
Fig. 3 is LGSCM conditional mouse lachrymal gland stem cell with the increased state change map of incubation time, scale bar: 100 μ m；
Fig. 4 is the state diagram of originally culture lachrymal gland stem cell, a, LGSCM, b, 9F-LGSCM, scale bar: 100 μm；
Fig. 5 is the expression figure of the stemness gene for the lachrymal gland stem cell that RT-PCR detects different algebra；
Fig. 6 is individually to remove the 7th day each growth factor, ligand and inhibitor originally culture lachrymal gland stem cell (P0 generation) shape State figure, scale bar: 400 μm；
Fig. 7 is individually to remove the size after each growth factor, ligand and inhibitor P0 are cultivated 7 days for lachrymal gland stem cell and thin Born of the same parents' sum figure, * * *, P < 0.01；
Fig. 8 is individually to remove each growth factor, ligand and inhibitor P2 for the state diagram in the 7th day of lachrymal gland stem cell, ratio Ruler: 400 μm；
Fig. 9 is individually to remove the size after each growth factor, ligand and inhibitor P2 are cultivated 7 days for lachrymal gland stem cell and thin Born of the same parents' sum, * * *, P < 0.01；
Figure 10 is the IF detection figure for cultivating 14 days lachrymal gland stem cells, scale bar: 100 μm；
Figure 11 is that the IF of mature mouse lachrymal gland detects figure, scale bar: 100 μm；
Figure 12 is IF detection of the culture more than 14 days lachrymal gland stem cells, scale bar: 200 μm；
Figure 13 is the pretherapy and post-treatment eye symptom figure of xerophthalmia mouse；
Figure 14 is that the IHC that lachrymal gland is sliced after xerophthalmia mouse is transplanted lachrymal gland stem cell 8 weeks detects figure, scale bar: 100 μm；
Figure 15 is the secretory volume that xerophthalmia mouse transplants 8 weeks tears of lachrymal gland stem cell, *, P < 0.05；* *, P < 0.01.
Specific embodiment
To facilitate the understanding of the present invention, a more comprehensive description of the invention is given in the following sections with reference to the relevant attached drawings.In attached drawing Give presently preferred embodiments of the present invention.But the invention can be realized in many different forms, however it is not limited to this paper institute The embodiment of description.On the contrary, purpose of providing these embodiments is keeps the understanding to the disclosure more thorough Comprehensively.
Unless otherwise defined, all technical and scientific terms used herein and belong to technical field of the invention The normally understood meaning of technical staff is identical.Term as used herein in the specification of the present invention is intended merely to description tool The purpose of the embodiment of body, it is not intended that in the limitation present invention.
Material explanation:
Mouse is purchased from Guangdong Medical Lab Animal Center in present invention experiment；N2, B27, Glutamax, NEAA are purchased It is purchased from Pepro Tech from Gibco, EGF, FGF10, Wnt3A, Y-27632 is purchased from Sigma.
Main solution and formula:
(1) solution or reagent used in cell culture experiments
1) NaCl 8g, KCl 0.2g, Na the configuration of PBS Buffer: are weighed2HPO41.155g KH2PO40.2g adds Enter the ultrapure water of about 800ml, after completely dissolution, pH value is adjusted to 7.4, constant volume 1L, after autoclave sterilization, 4 DEG C of preservations.
2) configuration of 10 × Trypsin-EDTA (TE): weighing 1.25g trypsin, and 1.00g EDTA is dissolved in 250ml PBS in, filtration sterilization, -20 DEG C preservation.1 × TE is diluted to for vitellophag with addition PBS using preceding.
3) when being directly used in originally culture from BD company purchase neutral enzymatic (Dispase), digestion lacrimal tissue is used.
4) lachrymal gland stem cell medium: LGSCM, 9F-LGSCM.
5) DMEM/F12 (Sigma) culture medium constituent is as shown in the table:
(2) solution or reagent used in nucleic acid electrophoresis experiment
1) 242g Tris, 18.6g 50 × TAE electrophoresis Buffer (pH8.5): are added in the vial of 1L Na2EDTA·2H2Then O is added the second level water of about 800ml, dissolution is sufficiently stirred, adds the acetic acid of 57.1ml, sufficiently stir It mixes, second level water is added to be settled to 1L, room temperature preservation.50 times are diluted when use.
2) ethidium bromide (EB): storing liquid concentration is 10mg/ml, and working concentration is 0.5 μ g/ml.
3) 1% Normal Agarose Gel: weighing 1g agarose (agarose), and 1 × TAE of 100ml buffer is added, adds Heat, which is boiled to agarose, to be completely dissolved, and is cooled to 50 DEG C or so, and 5 μ l EB storing liquids are added, makes final concentration of 0.5 μ g/ml, shakes up Casting of gels plate afterwards.
(3) solution or reagent used in immunofluorescence experiment
1) 4%PFA:20g paraformaldehyde is dissolved in 1 × PBS of 400ml, and 65 DEG C dissolve by heating overnight (about 12h).The bottom of to When portion's residue precipitates on a small quantity, 40 μ l 1N NaOH dissolutions, and constant volume 500ml, 4 DEG C of preservations are added.
2) 30% sucrose solution: 3g sucrose powder is dissolved in 4%PFA, constant volume 10ml, 4 DEG C of preservations.
3) confining liquid: with the nonimmune lowlenthal serum of 1 × PBS dilution to 10%, ready-to-use preservation.
4) primary antibody: Krt14 (Abcam, ab181595), Krt19 (Abcam, ab52625), AQP5 (Abcam, Ab104751), E-cadherin (Abcam, ab11512), mCherry (Abcam, ab167453)
5) secondary antibody: purchase is in life company.
Embodiment 1:
A kind of cultivating system of lachrymal gland stem cell is basic culture medium with DMEM/F12, adds following components and concentration structure At culture solution: 1%N2,2%B27,1%NEAA, 1%Glutamax, 10-50ng/ml EGF, 10-100ng/ml FGF10,5- 10ng/ml Wnt3A, 5-10mM Y-27632, and using Matrigel as the bracket of stereoscopic culture.Culture solution herein is known as LGSCM。
Embodiment 2:
A kind of cultivating system of lachrymal gland stem cell is basic culture medium with DMEM/F12, adds following components and concentration structure At culture solution: 5-10 μM of beta -mercaptoethanol, 5-10 μM of 2- ethylaminoethanol, 10-20nM sodium selenite, 50-150ng/ml heparin, 2-5ng/ml transferrins, 5-10ng/ml vitamin C, 5-10 μ g/ml insulin, 6.8-9.4 μ g/ml bovine serum albumin(BSA)-oil Acid, 5-10ng/ml FGF2,1%NEAA, 1%Glutamax, 10-50ng/ml EGF, 10-100ng/ml FGF10,5- 10ng/ml Wnt3A, 5-10mM Y-27632, and using Matrigel as the bracket of stereoscopic culture.Culture solution herein is known as 9F-LGSCM
Embodiment 3:
A kind of cultural method of lachrymal gland stem cell, comprising the following steps:
A) unicellular using enzyme digestion separation acquisition mouse lachrymal gland；
B) according to 1 × 104The ratio kind of cells/well enters Matrigel Medium Culture, and deployed lachrymal gland stem cell training is added It is cultivated in nutrient solution LGSCM, culture environment is 37 DEG C, 5%~10%CO2, just have sacculus shape population of stem cells later within culture 7 days Body is formed, and uses 1 × 104The ratio of cells/well passes on.
Experimental example 4:
A kind of cultural method of lachrymal gland stem cell, comprising the following steps:
A) unicellular using enzyme digestion separation acquisition mouse lachrymal gland；
B) according to 1 × 104The ratio kind of cells/well enters Matrigel Medium Culture, and deployed lachrymal gland stem cell training is added It is cultivated in nutrient solution 9F-LGSCM, culture environment is 37 DEG C, 5%~10%CO2, culture just has sacculus shape to do after 7 days thin Born of the same parents group is formed, and uses 1 × 104The ratio of cells/well passes on.
Embodiment 5:
The lachrymal gland stem cell obtained using the cultural method of embodiment 3 or embodiment 4.
In order to support above-described embodiment, the present invention has carried out the originally culture of mouse lachrymal gland stem cell, secondary culture, lachrymal gland The verifying of stem cell medium ingredient, the identification of mouse lachrymal gland stem cell, treatment xerophthalmia of mouse lachrymal gland stem cell etc. are a series of Experiment, for example following experimental example of the specific experiment step respectively tested:
Experimental example 1: mouse lachrymal gland stem cell primary culture
1) mouse is put to death, dissection obtains mouse lachrymal gland, and 1 × PBS soaking and washing 1 minute, 75% ethyl alcohol impregnated 15s, and immediately 1 × PBS is cleaned 2 times；
2) tissue is shredded as far as possible, is transferred in 15ml centrifuge tube, 500 μ l Dispase, 37 DEG C of digestion 1h are added；
3) blow and beat postdigestive tissue with 1ml micropipette rifle, it made to scatter, it is unicellular to obtain, using pre-cooling 1 × PBS is cleaned twice；
4) cell after 40 μm of membrane filtrations is collected, is counted, according to 1 × 104The ratio of cells/well is resuspended in 40 μ l culture It after being mixed in liquid with 40 μ l Matrigel, plants in 24 orifice plates, is placed in 37 DEG C of incubators, solidifies Matrigel, 20 minutes The stem cell medium prepared in advance is added afterwards to be cultivated.
Under the conditions of mouse lachrymal gland stem cell LGSCM, state change such as Fig. 1 institute that is primary and persistently passing on different culture generations Show；For the state of originally culture lachrymal gland stem cell as shown in figure 4, wherein a is using LGSCM as culture solution, b is using 9F-LGSCM as training Nutrient solution, the lachrymal gland stem cell group diameter of 9F-LGSCM culture are slightly less than the lachrymal gland stem cell group of LGSCM culture.
Experimental example 2: lachrymal gland stem cell secondary culture
1) 100 μ l1 × PBS and 20 μ lDispase are added in every hole in 24 orifice plates, and Matrigel and cell mixture are smash It is broken, it is placed in 37 DEG C of incubators and digests 30 minutes, Matrigel is digested to liquid form,
2) the lachrymal gland stem cell of culture is collected using 15ml centrifuge tube, 500rpm is centrifuged 4 minutes, collects precipitating；
3) 1 × TE vitellophag is added, 37 DEG C are incubated for 10 minutes；
4) TE is terminated, by the piping and druming of lachrymal gland stem cell at unicellular, 1200rpm is centrifuged 4 minutes, cell is collected, according to 1 × 104The ratio of cells/well is resuspended in mixed in 40 μ l culture solutions with 40 μ l Matrigel after, plant in 24 orifice plates, be placed in 37 DEG C In incubator, Matrigel is solidified, the stem cell medium LGSCM or 9F-LGSCM prepared in advance is added after twenty minutes and carries out Culture.
Using the mouse lachrymal gland stem cell of this method culture, under conditions of stem cell medium, stabilization was passaged to for 40 generations More than, continuing the secondary culture time is more than 1 year or more.Mouse lachrymal gland stem cell persistently passes on the state change of different culture generations As shown in Figure 1；The statistics that mouse lachrymal gland stem cell persistently passes on the total number of cells of different culture generation cultures 7 days is as shown in Figure 2； Mouse lachrymal gland stem cell is as shown in Figure 3 with the increased state change of incubation time.
Interpretation of result: the culture of mouse lachrymal gland stem cell is generally 7 days, and when originally culture, mouse lachrymal gland stem cell is presented It sprouts branched, and after passing on, form then keeps solid sacculus shape, and with the increase of passage number, shape State is more stable, such as Fig. 1.Count the total number of cells discovery of different generations, proliferative capacity of lachrymal gland stem cell under the conditions of LGSCM It is relatively strong, 1 × 104After a cell culture 7 days, 4 × 10 can be amplified5It is more than a cell.And with the increase of passage number, Lachrymal gland stem cell gradually adapts to the environment of in vitro culture, and proliferative capacity gradually increases, and finally tends towards stability, such as Fig. 2.? Different incubation times, the state of lachrymal gland stem cell are varied under the conditions of LGSCM, and the sacculus cell mass of formation can be increasing, Such as Fig. 3.Similarly, 9F-LGSCM condition can also obtain similar sacculus shape lachrymal gland stem cell, and property is also and under the conditions of LGSCM Lachrymal gland stem cell is similar, such as Fig. 4.Since under the conditions of 9F-LGSCM, the proliferation of lachrymal gland stem cell is relatively slow, therefore, connecing In the various confirmatory experiments to get off, we use the lachrymal gland stem cell under the conditions of LGSCM.
Experimental example 3: lachrymal gland stem cell medium ingredient verifying
In order to verify the necessity of main growth factor in culture medium, ligand and inhibitor, using experimental example 1 and in fact The cultural method of example 2 is tested, each growth factor, ligand and inhibitor are individually removed, prepares corresponding culture solution, respectively Lachrymal gland stem cell is cultivated, its growth conditions and growth rate are then compared.
Individually remove Wnt3A, FGF10, Y-27632, EGF, the 7th day shape of originally culture lachrymal gland stem cell (P0 generation) State is as shown in Figure 6；
Wnt3A, FGF10, Y-27632, EGF are individually removed, P0 is for the size after the culture of lachrymal gland stem cell 7 days and carefully Born of the same parents' sum is as shown in Figure 7；
Wnt3A, FGF10 are individually removed, P2 is as shown in Figure 8 for the 7th day state of lachrymal gland stem cell；
Wnt3A, FGF10 are individually removed, P2 is for the size and total number of cells such as Fig. 9 after the culture of lachrymal gland stem cell 7 days It is shown.
Interpretation of result: it removes in LGSCM after each essential growth factors, it has been found that EGF, Y-27632 are removed, FGF10 has a significant impact the originally culture of lachrymal gland stem cell, and the balloon diameter and total number of cells of lachrymal gland stem cell are all aobvious Writing reduces.After passage 2 times, removing for Wnt3A can also be had a significant impact.Thus it could be speculated that in the cultivating system that we establish Each growth factor, ligand and the inhibitor of addition all play an important role to the growth of lachrymal gland stem cell and proliferation.
Experimental example 4: the detection of each gene expression mRNA level in-site of lachrymal gland stem cell
I. the extraction of cell RNA
1) cell for collecting 14 days cultivated days different with same generation of different algebra cultures, is added 1ml Trizol, whirlpool Rotation oscillation is complete to cell cracking；
2) 0.2ml chloroform is added, vortex oscillation 30 seconds, is stored at room temperature 10 minutes, until liquid layered, 4 DEG C of 12000rpm Centrifugation 15 minutes；
3) after being centrifuged, it can remove and observe that liquid is divided into three layers in centrifuge tube, the careful transparent aqueous phase for drawing top layer turns New 1.5ml is moved to without in RNA enzyme centrifuge tube；
4) isometric isopropanol is added, mixes gently, is stored at room temperature 10 minutes, 12000rpm4 DEG C is centrifuged 15 minutes；
5) supernatant is abandoned, 75% ethyl alcohol 1ml is added, overturns cleaning, 4 DEG C of 12000rpm are centrifuged 5 minutes, abandon supernatant；
6) centrifuge tube is placed in superclean bench, dries up, becomes the translucent nothing that 20 μ l can be added to white precipitate RNA enzyme water dissolution precipitating；
Ii.RT-PCR reverse transcription reaction (TOYOBO ReverTra Ace kit)
1) it is sequentially added into PCR reaction tube:
2 μ l (1000ng) of total serum IgE
65 DEG C act on 5 minutes；
2) it behind RNA ice bath 2 minutes after being denaturalized, is added:
37 DEG C act on 5 minutes；
3) it is placed on ice, 5 × RT Master Mix II is added；
4) reverse transcription program be 37 DEG C 15 minutes, 50 DEG C 5 minutes, 98 DEG C 5 minutes, 4 DEG C ice bath 5 minutes, complete reverse transcription CDNA sample be placed in -20 DEG C and save backup.
Iii.PCR program
1) in the PCR system that total volume is 20 μ l are as follows:
2) different primers are arranged according to various annealing temperatures, recurring number, extension of time etc.；
3) nucleic acid electrophoresis testing result.
Interpretation of result: nucleic acid electrophoresis testing result as shown in figure 5, cultivate resulting lachrymal gland stem cell expression Epcam, The marker gene of the adult stem cells such as Krt5, Krt14, Pax6, P63, Nestin, also, pass through the tear to different culture generations The detection of gland stem cell, it is found that each stem cell marker genes can stablize expression.
Experimental example 5: the differentiation of mouse lachrymal gland stem cell in vitro and mouse lachrymal gland IF detection
1) resulting mouse lachrymal gland stem cell will be cultivated, cultivate 14 days and be added serum in vitro or reduces matrigel Hardness can all be such that it breaks up,
2) the lachrymal gland stem cell or mouse lachrymal gland 4%PFA solution after breaking up are fixed overnight；
3) be dehydrated: 70% ethyl alcohol 2 hours, 80% ethyl alcohol 2 hours, 95% ethyl alcohol 30 minutes, 95% ethyl alcohol 30 minutes are anhydrous Ethyl alcohol 30 minutes, dehydrated alcohol 2 hours, dehydrated alcohol+TO (1:1) 30 minutes, TO 30 minutes, TO 2 hours, TO+ paraffin (1: 1) 30 minutes, paraffin 3 hours, paraffin 2 hours.Totally 12 barrels, 16 hours；
4) paraffin embedding；
5) paraffin section, slice thickness are 4 μm；
6) staining procedure is as follows:
1. 60 DEG C of paraffin section 10 minutes, dimethylbenzene 10 minutes, dehydrated alcohol 3 minutes, 90% ethyl alcohol 3 minutes, 80% second Alcohol 3 minutes, 70% ethyl alcohol 3 minutes, distillation washing 3 times 3 minutes every time；
2. antigen retrieval 17 minutes；
3. being washed on 1 × PBS shaking table 3 times, every time 5 minutes；
4. after 10% lowlenthal serum is closed 30 minutes, primary antibody, 4 DEG C of overnight incubations are added；
5. removing primary antibody, cleaned 3 times, every time 5 minutes with 1 × PBS；
6. secondary antibody is added, it is incubated at room temperature 1 hour；
7. removing secondary antibody, cleaned 3 times, every time 5 minutes with 1 × PBS；
8. DAPI dyeing is added, room temperature 5 minutes；
9. removing DAPI, cleaned 3 times, every time 5 minutes with 1 × PBS；
10. using fluorescence mountant mounting and taking pictures.
It reduces the IF testing result of 14 days lachrymal gland stem cells of matrigel hardness culture and 14 days lachrymal glands of serum free culture system is added and do The IF testing result difference of cell is as shown in fig. 10 and fig. 12.The IF testing result of lacrimal tissue is as shown in figure 11.Figure 10 a and figure Lachrymal gland stem cell is displayed in red fluorescence in 11a, and mature lumen cell is displayed in red fluorescence in Figure 10 b and Figure 11 b.A is in Figure 12 Acinus like cell shows that green fluorescence, b are that lachrymal gland stem cell is displayed in red fluorescence, and c is that nucleus is displayed in blue fluorescence.
Interpretation of result: finding by Immunofluorescence test, cultivates in 14 days lachrymal gland stem cell groups, only part cell table Up to Krt14, outer layer is focused primarily upon, illustrates that these cells not yet break up, its stem cell properties can be maintained；And another part cell Then expressing K rt19 focuses primarily upon inside, and illustrating that this part cell has broken up becomes the cell with lumen characteristic.Comparison The localization and expression of two class cells in lacrimal tissue lumen, the Distribution and localization and the positioning class in lachrymal gland lumen for finding two kinds of cells Seemingly.As seen from Figure 12, serum free culture system 14 days lachrymal gland stem cells, which are added, can also differentiate expression AQP5, and it is very low to obtain nucleocytoplasmic ratio Acinus like cell, illustrates that cultivate resulting lachrymal gland stem cell cultivates in vitro, has the potential to lumen and acinar differentiation.
Experimental example 6: damage lachrymal gland is repaired in lachrymal gland stem cell body
I. lachrymal gland stem cell transplantation
1) the lachrymal gland stem cell of included red fluorescence (td-Tomato) mouse is cultivated by the 7th day；
2) lachrymal gland stem cell is digested to unicellular, is resuspended, is made with the matrix of Matrigel and DMEM/F12 mixed in equal amounts Cell becomes 2 × 106The concentration of cells/ml；
3) after the xerophthalmia model mouse of anesthesia lachrymal gland damage, dissection exposes lachrymal gland；
4) 20000 each cells are injected in left side, and the matrix of cell is resuspended as control in right side injection；
5) it after injecting, sews up a wound, after raising 8 weeks, detects repairing effect.
Ii. the therapeutic effect after stem cell transplantation is detected
1) anesthetized mice shoots mice eye symptom；
2) using the amount for making phenol red cotton thread measurement mouse lacrimal secretion by oneself, phenol red cotton thread is placed in mouse canthus about 10 Second, then measure the length of color change portion；
3) mouse is dissected, lachrymal gland is taken out, it is fixed, after slice, carry out IHC detection.Slice and colouring method and 5 phase of experimental example Together.
As shown in figure 13, xerophthalmia mouse transplants lachrymal gland stem cell 8 to the pretherapy and post-treatment eye symptom comparison of xerophthalmia mouse As shown in figure 14, xerophthalmia mouse transplants secretory volume such as Figure 15 of 8 weeks tears of lachrymal gland stem cell for the IHC detection that lachrymal gland is sliced after week It is shown.
Interpretation of result: after injection lachrymal gland stem cell, arriving for the eye dry eye symptoms of mouse inhibits and alleviates the (left side Figure 13 Side), and inject matrix side mice eye and obviously seriously fester (on the right side of Figure 13)；It can be detected in lachrymal gland slice after dissection mouse From the lachrymal gland stem cell of transplanting, and break up the acinar cells for becoming lachrymal gland and lumen cell (on the left of Figure 14), and control group (on the right side of Figure 14) has no the newborn acinus of differentiation and lumen cell, illustrates that lachrymal gland stem cell has the ability in mouse differentiation in vivo, The lachrymal gland of damage can be repaired.(LGSC), control group (Vehicle) mouse tear after comparison normal (Normal), lesions treatment Secretory volume (Figure 15), the later mouse lacrimal secretion of lachrymal gland stem cell transplantation is slightly below normal level, but relative to not moving Its lacrimal secretion of the group of plant stem cell dramatically increases, and the lachrymal gland of damage can be repaired by illustrating lachrymal gland stem cell transplantation not only, It is also equipped with the effect for restoring its secreting function.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.

Claims (10)

1. a kind of cultivating system of lachrymal gland stem cell, which is characterized in that with DMEM/F12 be basic culture medium, add following components Constitute culture solution: cell culture additive, nonessential amino acid, Ala-Gln, mitogen-activated protein swash Enzyme/extracellular signal-regulated kinase signal path ligand, fibroblast growth factor, Wnt signal path ligand and ROCK letter The composition of number pathway inhibitor, and using matrigel as the bracket of stereoscopic culture.
2. the cultivating system of lachrymal gland stem cell according to claim 1, which is characterized in that the cell culture additive is The mixture of N2 and B27.
3. the cultivating system of lachrymal gland stem cell according to claim 1, which is characterized in that the cell culture additive is 9F, the 9F are beta -mercaptoethanol, 2- ethylaminoethanol, sodium selenite, heparin, transferrins, vitamin C, insulin, cow's serum The mixture of albumin-oleic acid and fibroblast growth factor-2.
4. the cultivating system of lachrymal gland stem cell according to claim 2, which is characterized in that in the cell culture additive N2 volumetric concentration is 0.5%-1%, and B27 volumetric concentration is 1%-2%；The nonessential amino acid volumetric concentration is 1%, described Ala-Gln volumetric concentration is 1%.
5. the cultivating system of lachrymal gland stem cell according to claim 3, which is characterized in that the cell culture additive 9F Middle each component concentration are as follows: 5-10 μM of beta -mercaptoethanol, 5-10 μM of 2- ethylaminoethanol, 10-20 μM of sodium selenite, heparin 50- 150ng/ml, transferrins 2-5ng/ml, vitamin C 5-10ng/ml, insulin 5-10ng/ml, bovine serum albumin(BSA)-oleic acid 6.8-9.4ng/ml with fibroblast growth factor-2 5-10ng/ml；The nonessential amino acid 1%, third ammonia of L- Acyl-L-Glutamine 1%.
6. -5 any lachrymal gland stem cell cultivating system according to claim 1, which is characterized in that the mitogen activates egg White kinases/extracellular signal-regulated kinase signal path ligand is EGF, and the fibroblast growth factor is FGF10, described Wnt signal path ligand is Wnt3A, and the ROCK signal pathway inhibitor is Y-27632.
7. the cultivating system of lachrymal gland stem cell according to claim 6, which is characterized in that in the cell culture additive EGF concentration is 10-50ng/ml, and FGF10 concentration is 10-100ng/ml, and Wnt3A concentration is 5-10ng/ml, and Y-27632 concentration is 5-10mM。
8. the cultivating system of lachrymal gland stem cell according to claim 1, which is characterized in that the matrigel is Matrigel。
9. a kind of cultural method of lachrymal gland stem cell, which is characterized in that be digested to mouse lacrimal tissue and unicellular be inoculated into base In matter glue, after its solidification, be added as described in claim any one of 1-8 in lachrymal gland stem cell medium, 37 DEG C, 5%~ 10%CO2It is cultivated under environment.
10. a kind of lachrymal gland stem cell obtained using method for claim 9 culture.
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