CN106591220A - Human normal skin epithelial cell and use thereof - Google Patents
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Abstract
The invention discloses a human normal skin epithelial cell and use thereof. The human normal skin epithelial cell is named as human normal eyelid epithelial cell HNEEC/HL-014 and HNEEC/HL-015 with preservation numbers respectively of CCTCC NO: C2015180 and C2015181. A separation culture method of the human normal skin epithelial cell comprises the following steps: removing fat in a normal eyelid skin sample cut in an eye operation, adding dispase and DNase I for acting after digestion, collecting cells by virtue of filtering and centrifuging, re-suspending the cells with an HL culture medium, and performing inoculated culture. A subculturing method comprises the following steps: proliferating the cells until the abundance is 70 to 90 percent, digesting the cells with pancreatin-EDTA (Ethylene Diamine Tetraacetic Acid), and neutralizing with DMEM (Dulbecco Modified Eagle Medium); and centrifuging and collecting the cells, re-suspending the cells with the HL culture medium, and performing the inoculated culture. The cell can be used for physiologically studying the human normal cell, studying and detecting drug toxicity of in vitro normal cells and evaluating pathogenesis of skin-related diseases, skin regeneration and cosmetics safety.
Description
Technical field
The invention belongs to cell biology, be related to a kind of people's normal skin epithelial cell and its primary separation and Culture and
Secondary Culture method and purposes.
Background technology
The epithelial cell that human body vitals such as skin, lung, kidney, liver and pancreas are all broken up by organ specificity is constituted.This
The epithelial cell of a little specific differentiations is directly related with the specific function of Different Organs, the gas exchange function, kidney such as lung
Filtering function, the removing toxic substances of liver and neutralization function, pancreatic cell generation insulin, skin have protects body from external environment
Injury.And these vitals will threaten the health of the mankind once there are pathological changes or degeneration, because these vitals are very
What difficulty was replaced, the specific cell of Different Organs can not mutually replace the cell of other organs.These specific differentiations
Cell is all to be difficult regeneration, original cuiture propagation is carried out in vitro and is always a bottleneck.So greatly limit people couple
The understanding of organism normal cell function and application, currently carry out much to come from the cell life of scientific and technical literature even textbook
The description that thing is gained knowledge is inaccurate even mistake.Real the reason for is that the Biological Knowledge of most normal cells is derived from
" cancerous cell " result of study of In vitro culture.Although various countries scientist constantly attempts all the time culture and proliferation of human body weight wants device
Functional epithelial cell of official, but, for the primary epithelial cells for being isolated from people and mammal In vitro culture still
It is highly difficult.Using current serum-free medium, original cuiture (the survival a couple of days, such as skin that can only carry out short-term having
With the epithelial cell such as pancreas), what is had can only carry out limited Secondary Culture (on 1-3 generations, such as trachea/bronchus and prostate
Chrotoplast), what is had is even unable to In vitro culture (such as liver, colon, prostate epithelial cell) at all, only comes from human body skin
The superficial cell of skin with or so generation of Secondary Culture 10, but can be still not able to meet the requirement of researcher.And from every
In individual animal or biopsy sample obtain epithelial cell yield it is still very low, including cell quantity, be directly separated or short
The cell purity of phase culture is all very low, and these are primary and pass on the growth rate of epithelial cell and be also extremely limited.
For the culture for carrying out epithelial cell in vitro, attempt at present by genetic manipulation, such as proceed to virus (SV40T or
HPV16E6E7) or cellular oncogene, the algebraically of external cell survival can be extended.But the disadvantage of genetic manipulation is meeting
Change genetic background and the phenotype of these cells so that allow these normal epithelium cells to lose its normal physiological function, such as p53 and
PRB signal paths are usually suppressed.And, these genetically modified cells can not possibly be transplanted again into body again.But
It is the technology due to lacking effective In vitro culture epithelial cell at present, medical science and life of above-mentioned these cells in the world today
Still favor is enjoyed in scientific research.In non-cancer research field, they just represent the tissue of " function " property in initial source
Or organ.In cancer research field, they are also frequently used to be compareed as " normal cell ", and their market price is also
It is very expensive.At this stage, " normal cell " is also had no precedent both at home and abroad can apply to basis and clinic study.
Skin is the maximum organ of human body, it is estimated that the weight of Normal adult human dermal is 5 kilograms, and surface area is about 2 squares
Rice.Because it is directly exposed in environment, it makes various in vivo organizing with organ from physical property, mechanicalness, chemical and disease
The invasion and attack of pathogenic microorganism, carry protection body, perspire, feel cold and hot and pressure function.Skin include three layers, i.e. epidermis,
Corium and subcutaneous tissue.Skin has the barrier action of two aspects：On the one hand internal moisture content, electrolyte, other materials are prevented
Lose；On the other hand the intrusion of extraneous harmful substance is prevented.Skin remains stablizing for human internal environment, while skin is also assisted in
The metabolic process of human body.Epidermis is that skin is outmost one layer, and average thickness is 0.2 millimeter, according to the different development ranks of cell
Section and Morphological Features, ecto-entad can be divided into 5 layers, i.e. horny layer, clear layer, granular layer, prickle cell layer and basal layer.Grind at present
The skin keratinocytes studied carefully and use using on are usually primary separation, using serum-free culture, by various effort in vitro
It is most long to pass on more than ten times, cell senescence and death will occur.Immortalized cell line is built on this basis, and such as HaCaT is thin
Born of the same parents, although can Long Term Passages, but its chromosome is distorted, and between normal horn cell only 30% base
It is similar because expressing, it is impossible to represent normal cell truly.Therefore, if the normal skin of people that stabilization in vitro is passed on can be obtained
Skin epithelial cell, by the normal function for alloing people more fully to study cell, disease pathogenesis, tissue organ function's weight
Build or reproduce, and determine the toxicity of agents on normal cells, these all there will be weight to basis and clinic study with application
Big meaning.
The content of the invention
The primary and foremost purpose of the present invention is the shortcoming and deficiency for overcoming prior art, there is provided people's normal skin epithelial cell,
Name as people normal eyes eyelid epithelial cell HNEEC/HL-014 and HNEEC/HL-015, in being preserved on January 13rd, 2016
State's Type Tissue Collection, deposit number is CCTCC NO:C2015180 and CCTCC NO:C2015181.
The purpose of the present invention is achieved through the following technical solutions：
A kind of people's normal skin epithelial cell, name as people's normal eyes eyelid epithelial cell HNEEC/HL-014 (male) and
HNEEC/HL-015 (women), deposit number is respectively CCTCC NO：C2015180 and C2015181.
The cell derived is in the Chinese normal skin epithelial cell of sex, it is characterised in that：Chromosome is two
Times body, 22 tandem repeat locis of str locus type/allele length representing, respectively：AMEL/X/Y,
D3S1358/15, D13S317/8/11, D7S820/10/11, D16S539/11/13, Penta E/5/21, D2S441/10/11,
TPOX/8/11, TH01/9/9.3, D2S1338/17/23, CSF1PO/11/12, Penta D/12/13, D10S1248/13/15,
D19S433/14/15, vWA/14/17, D21S11/28/31.2, D18S51/14/21, D6S1043/11/13, D8S1179/10/
11, D5S818/11/12, D12S391/17/18, FGA/24 and AMEL/X, D3S1358/16/17, D13S317/11, D7S820/
11/12, D16S539/11/12, Penta E/14/16, D2S441/10/11, TPOX/8/11, TH01/6/9, D2S1338/19/
20, CSF1PO/10/12, Penta D/8/9, D10S1248/13, D19S433/13, vWA/18, D21S11/29/32.2,
D18S51/14/21, D6S1043/12/19, D8S1179/13/15, D5S818/9/13, D12S391/19/21, FGA/22/25.
The condition of culture of described people's normal skin epithelial cell is preferably and is based on 37 DEG C, 5%CO with HL cultures2Culture；
Described HL culture medium is：DMEM and serum-free medium SFM by volume 1:3 mixing, while adding the FBS of 5% (v/v)
(hyclone), and 0.4 μ g/mL hydrocortisones (hydrocortisone), 5 μ g/mL insulins (insulin), 8.4ng/mL
Cholera toxin (cholera toxin), 10ng/mL epidermal growth factors (epithelial growth factor (EGF)), 24
μ g/mL adenine (adenine), 100U/mL penicillins (penicillin), 100 μ g/mL streptomycins (streptomycin),
0.25 μ g/mL Amphotericin B (Fungizone), 30 μM of fasudils (Fasudil), above-mentioned culture medium needs 0.22 μm of aperture of Jing
Membrane filtration.
The primary isolated culture method of above-mentioned people's normal skin epithelial cell, comprises the steps：
(1) in the case of patient or patient care people's informed consent, collect sex operated eye and cut off just
Normal skin tissue sample.
(2) detached tissue sample is washed with the ethanol of 95～100% (v/v), then is washed with PBS (0.01M, pH 7.4), so
Tissue sample is put in the sterile petri dish containing pre-cooling PBS afterwards, under anatomic microscope, with dissection tweezers and shears removal group
The fat remained in tissue samples.
(3) tissue sample is digested with Digestive system；Preferably, described Digestive system is the trainings of the HL containing collagenase and Bacillus polymyxa Neutral proteinase
Foster base.
(4) supernatant is removed in postdigestive tissue centrifugation, and cell precipitation is resuspended in into 0.25% (mass volume ratio) pancreas enzyme -EDTA
Middle digestion.
(5) the DMEM culture medium containing 10% (v/v) FBS, centrifugation is added to remove supernatant.
(6) Bacillus polymyxa Neutral proteinase and DNase I of tepidarium are added, with pipette tips sample is blown and beaten repeatedly.
(7) the DMEM culture medium containing 10% (v/v) FBS is added, is hanged with the filter filtration cell in 40～70 μm of apertures
Liquid, collects the cell suspension after filtering, and supernatant is removed in centrifugation.
(8) re-suspended cell is deposited in HL culture medium, is inoculated in culture bottle culture, obtains people's normal skin epithelial cell.
Specifically, the primary isolated culture method of people's normal skin epithelial cell
Pre-cooling described in step (2) is preferably in pre-cooling on ice.
The consumption of the Digestive system described in step (3) is preferably 10 times of tissue sample volumes.
The condition of the digestion described in step (3) is preferably 37 DEG C and digests 1～3 hour.
The concentration of collagenase and Bacillus polymyxa Neutral proteinase described in step (3) is preferably and is 0.2mg/mL.
Digestion described in step (4) preferably digests 1 hour on ice or room temperature digests 10 minutes.
Tepidarium described in step (6) is preferably 37 DEG C of tepidarium.
Step (4), (5), the centrifugation described in (7) are preferably 1000rpm and are centrifuged 5 minutes.
The condition of the culture described in step (8) is preferably 37 DEG C, 5%CO2。
The Secondary Culture method of above-mentioned people's normal skin epithelial cell, comprises the steps：
(1) when people's normal skin epithelial cell proliferation is to 70～90% abundance, washed with 1 × PBS (0.01M, pH 7.4)
Cell is washed, then cell monolayer is digested with 0.05% (mass volume ratio) pancreas enzyme -EDTA.
(2) add in DMEM and digestion reaction；Supernatant is removed in centrifugation, is precipitated with HL culture medium re-suspended cell, is inoculated in culture
Bottle culture.
The time of the digestion described in step (1) is preferably 2～5 minutes.
Centrifugation described in step (2) is preferably 1000rpm and is centrifuged 5 minutes.
The condition of the culture described in step (2) is preferably 37 DEG C, 5%CO2。
Above-mentioned people's normal skin epithelial cell can be used for the Physiologic Studies of human normal cell line, the medicine of external normal cell
The research such as toxicity research and detection, safety evaluation of cosmetics and skin related disease.
The present invention has the advantage that and effect relative to prior art：
(1) present invention provide people's normal skin epithelial cell, primary separation and Culture from Chinese normal skin tissue,
The cell does not import any exogenous gene, and the normal diploid cell for people is identified in Jing karyotypings.Jing str locus typing reflects
It is fixed, it is a kind of normal cell system of the people for never registering both at home and abroad.
(2) people's normal skin epithelial cell that the present invention is provided, the form of basis of microscopic observation cell is tight, thin for arrangement
Born of the same parents' distinct, third dimension are strong, the epithelial cell of multiangular.This cellular morphology maintains to pass on by 57 and 58 days always, remains to
Keep vegetative state normal growth.
(3) people's normal skin epithelial cell that the present invention is provided, can be used for the Physiologic Studies of human normal cell line, in vitro just
Often application in the drug toxicity research and detection, skin regeneration and safety evaluation of cosmetics of cell etc..
Description of the drawings
Fig. 1 is the cellular morphology figure of people's normal skin epithelial cell.
Fig. 2 is the growth curve chart of people's normal skin epithelial cell.
Fig. 3 is the chromosome karyotype analysis figure of people's normal skin epithelial cell.
Fig. 4 is the str locus typing figure of people's normal skin epithelial cell.
Fig. 5 is the drug toxicity testing result figure of people's normal skin epithelial cell.
Specific embodiment
By combination accompanying drawing described further below it will be further appreciated that the features and advantages of the invention.The enforcement for being provided
Example is only the explanation to the inventive method, and limits remaining content of present invention announcement never in any form.
【Embodiment 1】The primary separation and Culture of primary people's normal skin epithelial cell
(1) in the case of patient or patient care people's informed consent, collect what is cut off during masculinity and femininity operated eye
Part normal eyes skin tissue.
(2) preparation of Digestive system：HL culture medium containing collagenase and the equal 0.2mg/mL of Bacillus polymyxa Neutral proteinase；Wherein, HL culture medium is：
DMEM (GIBCO#11965-092) and serum-free medium SFM (GIBCO#10744-019) by volume 1:3 mixing, while adding
Plus 5% (v/v) hyclone, and 0.4 μ g/mL hydrocortisones (hydrocortisone), 5 μ g/mL insulins
(insulin), 8.4ng/mL cholera toxins (cholera toxin), 10ng/mL epidermal growth factor (epithelial
Growth factor (EGF)), 24 μ g/mL adenine (adenine), 100U/mL penicillins (penicillin), 100 μ g/mL
Streptomycin (streptomycin), 0.25 μ g/mL Amphotericin B (Fungizone), 30 μM of fasudils (Fasudil) are above-mentioned
Culture medium needs 0.22 μm of aperture membrane filtration of Jing).
(3) detached tissue sample 1 time is washed with the ethanol of 95～100% (v/v), then 2 is washed with PBS (0.01M, pH 7.4)
It is secondary, during then tissue is put into containing the sterile petri dish of pre-cooling PBS on ice, under anatomic microscope, with dissection tweezers and shears
Remove the fat remained in tissue.
(4) by 1～2cm of tissue sample3In being put into 14mL the or 50mL centrifuge tubes of the 10mL Digestive systems of (2), 37 DEG C of digestion 1
～3 hours.
(5) organize low-speed centrifugal (1000rpm) 5 minutes by postdigestive, remove supernatant.
(6) cell precipitation is resuspended in 0.25% (mass volume ratio) pancreas enzyme -EDTA of 2～5mL, is placed in 1 little on ice
When or room temperature 10 minutes.
(7) DMEM culture medium of the 10mL containing 10% (v/v) FBS is subsequently adding, low speed 1000rmp is centrifuged 5 minutes；As far as possible will
Supernatant removes clean.
(8) the 5mg/mL Bacillus polymyxa Neutral proteinases of 2mL tepidariums (37 DEG C) and the 1mg/mL DNase I of 200 μ L are added, with aseptic
P1000 disposable plastics pipette tips blow and beat sample 1 minute repeatedly.
(9) DMEMs of the 10mL containing 10% (v/v) FBS is added, with the filter filtration cell suspension in 40～70 μm of apertures, is received
Cell suspension after collection filtration, low speed 1000rmp is centrifuged 5 minutes, removes supernatant.
(10) re-suspended cell is deposited in HL culture medium, is inoculated in the culture bottle culture of T25 or T75, and condition of culture is 37
DEG C, 5%CO2。
The successful primary people's normal skin epithelial cell of separation and Culture according to the method described above, the shape of basis of microscopic observation cell
State such as Fig. 1 (arrangement is closely, cell boundary is clear, third dimension is strong, multiangular epithelial cell).The cell is named as that " people is normal
Eyelid epithelia cell HNBEC/HL-014 " and " the normal eyelid epithelial cell HNBEC/HL-015 of people ", on January 13rd, 2015
It is preserved in China typical culture collection center (address：China. Wuhan. Wuhan University), deposit number is CCTCC NO：
C2015180 and C2015181.
【Embodiment 2】The Secondary Culture of people's normal skin epithelial cell
(1) when the people's normal skin epithelial cell proliferation cultivated in the culture bottle in T25 or T75 is to 70～90% abundance
When, then monolayer is digested with 0.05% (mass volume ratio) pancreas enzyme -EDTA with 1 × PBS (0.01M, pH 7.4) washed cell twice
Cell 2～5 minutes.
(2) add in the complete DMEM of 10mL and 1～2 point of kind of digestion reaction.
(3) 1000rmp is centrifuged 5 minutes, goes supernatant, re-suspended cell to be deposited in 10mL HL inoculation of medium cultures.
(4) if necessary can be by 1 × 106Epithelial cell be resuspended in 1～2mL cells frozen storing liquid (90% hyclone and
10%DMSO, v/v) in, it is stored in standby in liquid nitrogen.
According to the method described above Secondary Culture people normal skin epithelial cell, cell growth curve such as Fig. 2 of culture, connect
Culture 57 and 58 days are resumed, people's normal skin epithelial cell of the present invention remains to keep vegetative state normal growth.
【Embodiment 3】The karyotyping identification of people's normal skin epithelial cell
(1) when people's normal skin epithelial cell (1 × 106) in exponential phase of growth when, plus Colchicine is final concentration of
0.08 μ g/mL, continue to cultivate 3 hours.
(2) blowing and beating cell repeatedly makes it come off, and 1000rpm is centrifuged 5 minutes harvestings.
(3) supernatant is abandoned, the 0.075mol/L KCl solution 8mL of 37 DEG C of pre-temperatures are added, cell mass is gently blown and beaten and is mixed, put
37 DEG C of Hypotonic treatments 28 minutes.
(4) fixative (methanol for adding 2mL newly to prepare:Glacial acetic acid=3:1, v/v), careful piping and druming, mixing, 1000rpm
Centrifugation 10 minutes.
(5) supernatant is abandoned, adds 8mL fixatives, piping and druming to make after cell suspension, 20 minutes are fixed under room temperature.
(6) 1000rpm is centrifuged 10 minutes, abandons supernatant, repeats to fix once.
(7) supernatant is abandoned, leaves and takes about 300 μ L fixatives and gently blow and beat and make cell suspension, taken 2～3 hanging drops and soak in frozen water
On the microscope slide for steeping.
(8) microscope slide is put and do in 70 DEG C of baking boxs roasting 2.5 hours, natural cooling.
(9) 0.025% (mass volume ratio) trypsin solution (pH6.8～7.2) 5mL are processed 8～15 seconds.
The normal saline rinsing of (10) 37 DEG C of pre-temperatures, Giemsa is dyeed 5～10 minutes, does the aobvious band analyses of G.
(11) basis of microscopic observation cell caryogram and photograph, carry out karyotyping；At least observation analysis have silk for more than 20
Metaphase of cell division cell.Representational cell karyotyping result is shown in Fig. 3, and people's normal skin epithelial cell is normal diploid, 46
Bar chromosome no abnormality seen is arranged.
【Embodiment 4】The Genotyping analysis identification of people's normal skin epithelial cell
(1) people's normal skin epithelial cell (1 × 10 of adherent growth6), with 1 × PBS washed cells twice, 0.05% pancreas
Enzyme-EDTA digestion cell monolayers 2～5 minutes, in the complete DMEM of 10mL and digestion reaction.
(2) 10000rpm is centrifuged 1 minute, supernatant, plus 200 μ L buffer GA (cell/tissue extracting genome DNA to the greatest extent
Test kit DP304, Tiangeng company), vibrate to thoroughly suspension.
(3) 20 μ L Proteinase K solution are added, is mixed.
(4) 200 μ L buffer GB (cell/tissue genome DNA extracting reagent kit DP304, Tiangeng company) are added, fully
It is reverse to mix, 70 DEG C of placement 10min, brief centrifugation.
(5) the μ L dehydrated alcohol of people 200, fully vibration is added to mix 15 seconds, brief centrifugation.
(6) resulting solution and flocculent deposit are all added into (cell/tissue extracting genome DNA reagent in an adsorption column
Box DP304, Tiangeng company), 12000rpm is centrifuged 30 seconds, removes waste liquid.
(7) 500 μ L buffer GD (cell/tissue genome DNA extracting reagent kit DP304, Tiangengs are added in adsorption column
Company), 12000rpm is centrifuged 30 seconds, removes waste liquid.
(8) 600 μ L rinsing liquid PW (cell/tissue genome DNA extracting reagent kit DP304, Tiangengs are added in adsorption column
Company), 12000rpm is centrifuged 30 seconds, removes waste liquid.
(9) adsorption column is proceeded in another centrifuge tube, to the μ L elution buffers of middle part Deca 50～200 of adsorbed film
TE (cell/tissue genome DNA extracting reagent kit DP304, Tiangeng company), room temperature 2～5min of placement, 12000rpm (～
13400 × g) it is centrifuged 2 minutes, the DNA solution of extraction is collected in centrifuge tube.
(10) 22 locus (21 STR bit points and 1 sex are carried out using ABI 9700PCR amplification instruments (ABI companies)
Site) DNA composite amplifications.
(11) detection of amplified fragments is carried out using ABI 3130XL types genetic analyzers.
(12) using GenoMapper3.2 software analysis sample datas, automatic gene typing, STR genotyping result figures are carried out
4,22 str locus sites are detected, represented with " str locus seat/allele length "：AMEL/X/Y, D3S1358/15,
D13S317/8/11, D7S820/10/11, D16S539/11/13, Penta E/5/21, D2S441/10/11, TPOX/8/11,
TH01/9/9.3, D2S1338/17/23, CSF1PO/11/12, Penta D/12/13, D10S1248/13/15, D19S433/
14/15, vWA/14/17, D21S11/28/31.2, D18S51/14/21, D6S1043/11/13, D8S1179/10/11,
D5S818/11/12, D12S391/17/18, FGA/24 and AMEL/X, D3S1358/16/17, D13S317/11, D7S820/11/
12, D16S539/11/12, Penta E/14/16, D2S441/10/11, TPOX/8/11, TH01/6/9, D2S1338/19/20,
CSF1PO/10/12, Penta D/8/9, D10S1248/13, D19S433/13, vWA/18, D21S11/29/32.2, D18S51/
14/21, D6S1043/12/19, D8S1179/13/15, D5S818/9/13, D12S391/19/21, FGA/22/25.
People's normal skin epithelial cell Jing str locus Classification Identifications of the present invention, are never to register both at home and abroad
A kind of normal cell system of people.
【Embodiment 5】The drug toxicity detection of people's normal skin epithelial cell
(1) people's normal skin epithelial cell is digested with 0.05% pancreatin, is prepared into single cell suspension, be inoculated in 96 holes
Plate, every μ L of hole inoculating cell suspension 200, is about 8000 cells, in 37 DEG C, 5%CO per hole2Incubator culture.
(2) dosing (paclitaxel and cisplatin) after being inoculated with 4～6 hours is processed, and variable concentrations medicine, drug level are added per hole
Gradient (μM) is 100,33.3,10,3.33,1,0.333,0.1, and each gradient of every kind of medicine arranges 6 multiple holes, while arranging
Cell controls group (inoculating cell not agent-feeding treatment) and only plus HL culture medium blank control group, 6 multiple holes of per group of setting.
(3) drug treating (37 DEG C, 5%CO2Incubator culture) after 48 hours, solution in hole is sucked, 100 μ L are added per hole
CKK-8 detectable (Dojindo, Japan), i.e. 10 μ L CCK-8+100 μ L HL culture medium.
(4) in 37 DEG C, 5%CO2Continue to be incubated 0.5～2 hour in cell culture incubator, incubation time is with the more of cell concentration
Few correlation, the concrete time determines (can primarily determine that according to liquid color change) according to preliminary experiment, non-medicine feeding hole absorbance model
Contain system best between 1.0～1.5.
(5) absorbance at 450nm is determined with microplate reader.
People's normal skin epithelial cell to sensitivity (toxicity) testing result of two kinds of cancer therapy drugs, cisplatin and paclitaxel such as
Fig. 5, cisplatin affects not substantially in low concentration on cytoactive, and paclitaxel just can be killed effectively carefully in low concentration level
Born of the same parents, the result that two plants of cells are reflected is similar to, and reactions of the C2015180 to two kinds of medicines is more sensitive than C2015181.Show
People's normal skin epithelial cell is different with discrimination to the sensitivity of different pharmaceutical toxicity detection, and different cells are to same medicine
Reaction is also different.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention not by above-described embodiment
Limit, other any spirit without departing from the present invention and the change, modification, replacement made under principle, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (7)
1. a kind of people's normal skin epithelial cell, it is characterised in that：The normal eyelid epithelial cell HNEEC/HL- of Classification And Nomenclature behaviour
014 and HNEEC/HL-015, China typical culture collection center is preserved in, deposit number is respectively CCTCC NO：
C2015180 and C2015181.
2. people's normal skin epithelial cell according to claim 1, it is characterised in that：Deposit number is CCTCC NO：
In male, deposit number is CCTCC NO to people's normal skin epithelial cell origin of C2015180：The normal skin of people of C2015181
Skin epithelial cell origin is in women.
3. application of the people's normal skin epithelial cell described in claim 1 in the Physiologic Studies of human normal cell line.
4. the people's normal skin epithelial cell described in claim 1 in vitro normal cell drug toxicity research and detect in
Using.
5. application of the people's normal skin epithelial cell described in claim 1 in the study of pathogenesis of skin related disease.
6. application of the people's normal skin epithelial cell described in claim 1 in skin regeneration research.
7. application of the people's normal skin epithelial cell described in claim 1 in safety evaluation of cosmetics.
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