CN103451148B - People's normal bronchial epithelial cell and primary separation and Culture thereof and Secondary Culture method and purposes - Google Patents
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Abstract
The invention discloses people's normal bronchial epithelial cell and primary separation and Culture thereof and Secondary Culture method and purposes.The name of this cell is people's normal bronchial epithelial cell HNBEC/HL-001, and deposit number is CCTCC NO:C201311.The method of its primary separation and Culture is: remove fat in cancer beside organism's sample of Operation for Lung Cancer excision, after digestion, add Dispase and DNase I effect, by filtration, centrifugal collecting cell, then use HL substratum re-suspended cell, inoculation culture.The method of its Secondary Culture is: during cell proliferation to 70 ~ 90% abundance, with pancreas enzyme-EDTA digestion, then neutralizes with DMEM; Centrifugal collecting cell, with HL substratum re-suspended cell, inoculation culture.Cell of the present invention can be used for the Physiologic Studies of human normal cell line, and external Normocellular drug toxicity research and detection, segmental bronchus and lung relative disease comprise the study of pathogenesis of lung bronchogenic carcinoma.
Description
Technical field
The invention belongs to cell biology, relate to a kind of people's normal bronchial epithelial cell and primary separation and Culture thereof and Secondary Culture method and purposes.
Background technology
The epithelial cell of human body vitals as lung, kidney, liver, pancreas and skin are all broken up by organ specificity is its moiety.The epithelial cell of these specific differentiation is directly related with the specific function of Different Organs, as the gaseous interchange function of lung, the filtering function of kidney, the removing toxic substances of liver and in and function, pancreatic cell produce Regular Insulin, skin has the injury of protection body from external environment.And these vitals are once pathology occur or degenerates to threaten the health of the mankind, because these vitals are difficult to be replaced, the specific cell of Different Organs can not replace the cell of other organ mutually.The cell of these specific differentiation is all be difficult to regeneration, lets alone to carry out cultivation in vitro and bred.Greatly limit the understanding of people to organism normal cell function like this, it is inaccurate or even wrong for much coming from the description that the scientific and technical literature even cell biological of textbook gain knowledge.Real reason is that most Normocellular Biological Knowledge derives from vitro culture " cancer cells " result of study.Although various countries scientist constantly attempts functional epithelial cell of cultivation and proliferation of human body vitals all the time, separation is remained very difficult from the vitro culture of people and mammiferous primary epithelial cells.Apply current serum free medium, original cuiture (the survive for days that can only carry out short-term had, as the epithelial cell such as lung and pancreas), what have can only carry out limited Secondary Culture (1-3 generation, as tracheae/epithelial cell such as segmental bronchus and prostate gland), what have even at all can not vitro culture (as epithelial cells such as liver, colon, prostate glands), and the superficial cell only coming from human body skin can about Secondary Culture 10 generation.And the epithelial cell output obtained from each animal or biopsy sample is still very low, it is all very low for comprising the quantity of cell, the directly cell purity of separation or Short-term Culture, and these are primary and the epithelial rate of propagation that goes down to posterity is also very limited.
In order to carry out epithelial cultivation in vitro, abroad attempt by genetic manipulation at present, as proceeded to virus (SV40 T or HPV16E6E7) or cellular oncogene, can the algebraically of the outer cell survival of extension body.But the disadvantage of genetic manipulation is genetic background and the phenotype that can change these cells, so that these normal epithelium cells are allowed to lose its normal physiological function, as p53 and pRB signal path is usually suppressed.And these genetically modified cells can not be transplanted into body again again.But owing to lacking the epithelial technology of effective vitro culture at present, these cells above-mentioned still enjoy favor in the medical science and life science of the world today.At non-cancer research field, they just represent tissue or the organ of " function " property in initial source.At cancer research field, contrast that they are also usually used as " normal cell ", and also their market value is also very expensive.Present stage, also have no precedent " normal cell " both at home and abroad and can be applied to basis and clinic study.
Normal human bronchial's epithelial cell major function: the epithelial cell of (1) bronchial surface constitutes substrate columnar structure, removes mucociliary.(2) form physical barriers by mixed cellularity groups such as cilium, fibre-less and energy cells slimy, can effectively prevent multiple toxic substance.(3) produce and secrete the host defense system of a large amount of chemical mediator and cytokine height of formation complexity.Human bronchial epithelial cell is relevant with main pathophysiological disease, as lung bronchogenic carcinoma (how referred to as lung cancer), chronic bronchitis, bronchiectasis, bronchostenosis.If people's normal bronchial epithelial cell that stabilization in vitro goes down to posterity can be obtained, by the normal function making people more fully can study cell, disease pathogenesis, tissue organ function rebuilds or reproduces, and measuring the toxicity of agents on normal cells, these all will be of great importance to basis and clinic study and application.
Summary of the invention
Primary and foremost purpose of the present invention is that the shortcoming overcoming prior art is with not enough, provides a kind of people's normal bronchial epithelial cell.This cellular segregation is cultivated from Chinese normal lung tissue, and this cell does not import any foreign gene, is normal diploid cell, and gene type is accredited as the normal cell system of a kind of people never registered both at home and abroad.
Another object of the present invention is to provide above-mentioned people's normal bronchial epithelial primary isolation cultivation method.
Another object of the present invention is to provide above-mentioned people's normal bronchial epithelial Secondary Culture method.
The present invention also aims to provide above-mentioned people's normal bronchial epithelial purposes.
Object of the present invention is achieved through the following technical solutions:
A kind of people's normal bronchial epithelial cell, Classification And Nomenclature behaviour normal bronchial epithelial cell HNBEC/HL-001, be preserved in China typical culture collection center, deposit number is CCTCC NO:C201311.This cell derived is in Chinese normal lung epithelial cell, and karyomit(e) is diploid, STR(STR) genotype represents with 16 " str locus seat/allelotrope length ":
aMEL/X/Y,
d3S1358/17,
tH01/7/8,
d21S11/31/32,
d18S51/20,
penta E/11,
d5S818/11/12,
d13S317/8/11,
d7S820/8/13,
d16S539/11,
cSF1PO/10/12,
penta D/7/13,
vWA/17,
d8S1179/15/16,
tPOX/8/11,
fGA/20/24.
The described epithelial culture condition of people's normal bronchial is preferably to be cultivated based on 37 DEG C, 5% CO with HL
2cultivate, described HL substratum is: DMEM and serum free medium SFM by volume 1
:3 mixing, add 5%(v/v simultaneously) FBS(foetal calf serum), and 0.4 μ g/mL hydrocortisone (hydrocortisone), 5 μ g/mL Regular Insulin (insulin), 8.4 ng/mL Toxins,exo-, choleras (cholera toxin), 10 ng/mL Urogastrons (epithelial growth factor (EGF)), 24 μ g/mL VITAMIN B4 (adenine), 100 U/mL penicillin (penicillin), 100 μ g/mL Streptomycin sulphates (streptomycin), 0.25 μ g/mL amphotericin B (Fungizone), 30 μMs of fasudils (Fasudil), above-mentioned substratum need through 0.22 μm of aperture membrane filtration.
The epithelial primary isolation cultivation method of above-mentioned people's normal bronchial, comprises the steps:
(1) when patient or patient care people informed consent, the Carcinoma side normal tissue sample of Operation for Lung Cancer excision is collected.
(2) with 95 ~ 100%(v/v) ethanol wash the tissue sample of separation, use PBS(0.01M again, pH 7.4) wash, then tissue sample is put into the sterile petri dish containing precooling PBS, under dissecting microscope, with dissecting fat residual in tweezers and scissors removal tissue sample.
(3) tissue sample Digestive system is digested; Preferably, described Digestive system is the HL substratum containing collagenase and Dispase.
(4) postdigestive organizing centrifugally removes supernatant, cell precipitation is resuspended in 0.25%(mass volume ratio) digest in pancreas enzyme-EDTA.
(5) add containing 10%(v/v) the DMEM substratum of FBS, centrifugally removes supernatant.
(6) add Dispase and the DNase I of warm water bath, repeatedly blow and beat sample with rifle head.
(7) add again containing 10%(v/v) the DMEM substratum of FBS, with the metre filter cell suspension in 40 ~ 70 μm of apertures, collect the cell suspension after filtering, centrifugally remove supernatant.
(8) re-suspended cell is deposited in HL substratum, is inoculated in culturing bottle and cultivates, obtain people's normal bronchial epithelial cell.
Precooling described in step (2) is preferably in precooling on ice.
The consumption of the Digestive system described in step (3) is preferably 10 times to tissue sample volume.
The condition optimization of the digestion described in step (3) is 37 DEG C of digestion 1 ~ 3 hour.
The concentration of the collagenase described in step (3) and Dispase is preferably 0.2 mg/mL.
Digestion described in step (4) is preferably digestion 1 hour or room temperature on ice and digests 10 minutes.
Warm water bath described in step (6) is preferably the warm water bath of 37 DEG C.
Centrifugal 5 minutes of centrifugal preferably 1000 rpm described in step (4), (5), (7).
The condition optimization of the cultivation described in step (8) is 37 DEG C, 5% CO
2.
The epithelial Secondary Culture method of above-mentioned people's normal bronchial, comprises the steps:
(1) when people's normal bronchial epithelial cell proliferation to 70 ~ 90% abundance, with 1 × PBS(0.01M, pH 7.4) washed cell, then use 0.05%(mass volume ratio) pancreas enzyme-EDTA digestion monolayer cell.
(2) add in DMEM and digestion reaction; Centrifugally remove supernatant, by HL substratum re-suspended cell precipitation, be inoculated in culturing bottle and cultivate.
The time of the digestion described in step (1) is preferably 2 ~ 5 minutes.
Centrifugal 5 minutes of centrifugal preferably 1000 rpm described in step (2).
The condition optimization of the cultivation described in step (2) is 37 DEG C, 5% CO
2.
Above-mentioned people's normal bronchial epithelial cell can be used for the Physiologic Studies of human normal cell line, and external Normocellular drug toxicity research and detection, segmental bronchus and lung relative disease comprise the study of pathogenesis of lung bronchogenic carcinoma.
The present invention has the following advantages and effect relative to prior art tool:
(1) people's normal bronchial epithelial cell provided by the invention, primary separation and Culture is from the normal lung tissue of people, and this cell does not import any foreign gene, identifies the normal diploid cell into people through karyotyping.
(2) people's normal bronchial epithelial cell provided by the invention, primary separation and Culture, from Chinese normal lung tissue, through str locus Classification Identification, is the normal cell system of a kind of people never registered both at home and abroad.
(3) people's normal bronchial epithelial cell provided by the invention, can be used for the Physiologic Studies of human normal cell line, and external Normocellular drug toxicity research and detection, segmental bronchus and lung relative disease comprise the study of pathogenesis of lung bronchogenic carcinoma.
Accompanying drawing explanation
Fig. 1 is the epithelial cellular form figure of people's normal bronchial.
Fig. 2 is the epithelial growth curve chart of people's normal bronchial.
Fig. 3 is the epithelial chromosome karyotype analysis figure of people's normal bronchial.
Fig. 4 is people's normal bronchial epithelial str locus somatotype figure.
Fig. 5 is the epithelial susceptibility detected result figure of people's normal bronchial.
Embodiment
Below in conjunction with embodiment and accompanying drawing, further detailed description is done to the present invention, but embodiments of the present invention are not limited thereto.
The epithelial primary separation and Culture of embodiment 1 primary people normal bronchial
(1) when patient or patient care people informed consent, the Carcinoma side normal tissue sample of Operation for Lung Cancer excision is collected.
(2) preparation of Digestive system: containing the HL substratum of collagenase and equal 0.2 mg/mL of Dispase, wherein, HL substratum is: DMEM(GIBCO # 11965-092) with serum free medium SFM (GIBCO # 10744-019) by volume 1
:3 mixing, add 5%(v/v simultaneously) foetal calf serum, and 0.4 μ g/mL hydrocortisone (hydrocortisone), 5 μ g/mL Regular Insulin (insulin), 8.4 ng/mL Toxins,exo-, choleras (cholera toxin), 10 ng/mL Urogastrons (epithelial growth factor (EGF)), 24 μ g/mL VITAMIN B4 (adenine), 100 U/mL penicillin (penicillin), 100 μ g/mL Streptomycin sulphates (streptomycin), 0.25 μ g/mL amphotericin B (Fungizone), 30 μMs of fasudils (Fasudil), above-mentioned substratum need through 0.22 μm of aperture membrane filtration).
(3) with 95 ~ 100%(v/v) ethanol wash the tissue sample 1 time of separation, use PBS(0.01M again, pH 7.4) wash 2 times, then tissue is put into the sterile petri dish containing precooling PBS on ice, under dissecting microscope, with dissecting fat residual in tweezers and scissors removal tissue.
(4) by tissue sample 1 ~ 2 cm
3put into 14 mL or the 50 mL centrifuge tubes of the 10 mL Digestive systems of (2), 37 DEG C digest 1 ~ 3 hour.
(5) organize low-speed centrifugal (1000 rpm) 5 minutes by postdigestive, remove supernatant.
(6) cell precipitation is resuspended in the 0.25%(mass volume ratio of 2 ~ 5 mL) in pancreas enzyme-EDTA, be placed in 1 hour or room temperature 10 minutes on ice.
(7) 10 mL are then added containing 10%(v/v) the DMEM substratum of FBS, centrifugal 5 minutes of low speed 1000 rmp; Supernatant is removed clean as far as possible.
(8) add 5 mg/mL Dispases of 2 mL warm water baths (37 DEG C) and the 1 mg/mL DNase I of 200 μ L, repeatedly blow and beat sample 1 minute with aseptic P1000 disposable plastic rifle head.
(9) 10 mL are added containing 10%(v/v) DMEM of FBS, with the metre filter cell suspension in 40 ~ 70 μm of apertures, collects the cell suspension after filtering, centrifugal 5 minutes of low speed 1000 rmp, removal supernatant.
(10) re-suspended cell is deposited in HL substratum, and the culturing bottle being inoculated in T25 or T75 is cultivated, and culture condition is 37 DEG C, 5% CO
2.
The successful primary people's normal bronchial epithelial cell of separation and Culture according to the method described above, the form of basis of microscopic observation cell is as Fig. 1 (arrangement closely, cell boundary is clear, stereoscopic sensation is strong, the epithelial cell of multiangular).This cell divide called after " people's normal bronchial epithelial cell HNBEC/HL-001 ", on April 10th, 2013 be preserved in China typical culture collection center (address: China. Wuhan. Wuhan University), deposit number is CCTCC NO:C201311.
The epithelial Secondary Culture of embodiment 2 people normal bronchial
(1) when people's normal bronchial epithelial cell proliferation to 70 ~ 90% abundance of cultivating in the culturing bottle at T25 or T75, with 1 × PBS(0.01M, pH 7.4) washed cell twice, then use 0.05%(mass volume ratio) pancreas enzyme-EDTA digestion monolayer cell 2 ~ 5 minutes.
(2) add in the complete DMEM of 10 mL and digestion reaction 1 ~ 2 point of kind.
Centrifugal 5 minutes of (3) 1000 rmp, remove supernatant, and re-suspended cell is deposited in 10 mL HL inoculation of mediums and cultivates.
(4) if desired can by 1 × 10
6epithelial cell is resuspended in the cells frozen storing liquid (90% foetal calf serum and 10% DMSO, v/v) of 1 ~ 2 mL, is stored in liquid nitrogen for subsequent use.
Secondary Culture people normal bronchial epithelial cell according to the method described above, the cell growth curve of culture is as Fig. 2, and continuous passage cultivates 55 days, and people's normal bronchial epithelial cell of the present invention still can keep vegetative state normal growth.
The epithelial karyotyping qualification of embodiment 3 people normal bronchial
(1) when people's normal bronchial epithelial cell (1 × 10
6) when being in exponential phase of growth, add colchicine, final concentration is 0.2 μ g/mL, continues cultivation 3.5 hours.
(2) repeatedly blowing and beating cell makes it come off, 2000 rpm centrifugal 5 minutes harvested cells.
(3) abandon supernatant liquor, add 0.075 mol/L KCl solution 8 mL of 37 DEG C of pre-temperature, blow and beat cell mass mixing gently, put 37 DEG C of Hypotonic treatment 25 minutes.
(4) fixing agent (methyl alcohol that 1 mL newly prepares is added
:glacial acetic acid=3
:1, v/v), careful piping and druming, mixing, centrifugal 5 minutes of 2000 rpm.
(5) abandon supernatant liquor, add 8 mL fixing agents, after cell suspension is made in piping and druming, under room temperature, fix 20 minutes.
Centrifugal 5 minutes of (6) 2000 rpm, abandon supernatant liquor, repeat to fix once.
(7) abandon supernatant liquor, add several fixing agents and make cell suspension, get on 2 ~ 3 slide glasss soaked in frozen water.
(8) slide glass is put dry in 70 DEG C of baking boxs baking 2 hours, naturally cooling.
(9) 2.5%(mass volume ratio) trypsin solution (pH6.8 ~ 7.2) 5 mL process 25 ~ 45 second.
The physiological saline rinsing of (10) 37 DEG C of pre-temperature, Giemsa dyeing 5 ~ 10 minutes, makees the aobvious band of G and analyzes.
(11) basis of microscopic observation cell caryogram photographing, carries out karyotyping; At least observation analysis more than 20 cell mitosis metaphase.Representational nucleus type analysis the results are shown in Figure 3, and people's normal bronchial epithelial cell is normal diploid, 46 karyomit(e) no abnormality seen arrangements.
The epithelial genotypic analyses qualification of embodiment 4 people normal bronchial
(1) people's normal bronchial epithelial cell (1 × 10 of adherent growth
6), with 1 × PBS washed cell twice, 0.05% pancreas enzyme-EDTA digestion monolayer cell 2 ~ 5 minutes, in the complete DMEM of 10 mL and digestion reaction.
Centrifugal 1 minute of (2) 10000 rpm, use up supernatant, add 200 μ L damping fluid GA(cell/tissue genome DNA extracting reagent kit DP304, Tian Gen company), vibration is to thoroughly suspending.
(3) 20 μ L Proteinase K solution are added, mixing.
(4) 200 μ L damping fluid GB(cell/tissue genome DNA extracting reagent kit DP304 are added, Tian Gen company), fully put upside down mixing, place 10 min, brief centrifugation for 70 DEG C.
(5) people 200 μ L dehydrated alcohol is added, fully vibration mixing 15 seconds, brief centrifugation.
(6) gained solution and flocks are all added (cell/tissue genome DNA extracting reagent kit DP304, Tian Gen company) in an adsorption column, centrifugal 30 seconds of 12000 rpm, remove waste liquid.
(7) in adsorption column, 500 μ L damping fluid GD(cell/tissue genome DNA extracting reagent kit DP304 are added, Tian Gen company), centrifugal 30 seconds of 12000 rpm, remove waste liquid.
(8) in adsorption column, 600 μ L rinsing liquid PW(cell/tissue genome DNA extracting reagent kit DP304 are added, Tian Gen company), centrifugal 30 seconds of 12000 rpm, remove waste liquid.
(9) adsorption column is proceeded in another centrifuge tube, middle part to adsorption film drips 50 ~ 200 μ L elution buffer TE(cell/tissue genome DNA extracting reagent kit DP304, Tian Gen company), room temperature places 2 ~ 5 min, 12000 rpm(~ 13400 × g) centrifugal 2 minutes, the DNA solution of extraction is collected in centrifuge tube.
(10) PowerPlex 16 HS system (DC2101, promega company) is utilized to carry out the DNA composite amplification in 16 locus (15 STR sites and 1 sex site).
(11) ABI PRISM 3100 type genetic analyzer (1.1 edition datas collect software) is used to carry out the detection of amplified fragments.
(12) use Genotyper and PowerTyperTM 16 Macro software analysis sampled data, carry out automatic gene somatotype, STR genotyping result Fig. 4, detect 16 str locus sites, represent with " str locus seat/allelotrope length ":
aMEL/X/Y,
d3S1358/17,
tH01/7/8,
d21S11/31/32,
d18S51/20,
penta E/11,
d5S818/11/12,
d13S317/8/11,
d7S820/8/13,
d16S539/11,
cSF1PO/10/12,
penta D/7/13,
vWA/17,
d8S1179/15/16,
tPOX/8/11,
fGA/20/24.
People's normal bronchial epithelial cell of the present invention, through str locus Classification Identification, is the normal cell system of a kind of people never registered both at home and abroad.
The epithelial susceptibility of embodiment 5 people normal bronchial detects
(1) by 0.05% trysinization of people's normal bronchial epithelial cell, be prepared into single cell suspension, be inoculated in 96 orifice plates, every hole inoculating cell suspension 100 μ L, every hole is about 5000 cells, in 37 DEG C, 5% CO
2incubator is cultivated.
Dosing (5-Fu(5-Fluracil) in (2) second days, Zorubicin, cis-platinum) process, every hole adds 10 μ L different concns medicines, drug concentration gradient (μM) is 0.25,0.5,2.5,5.0,25,50, each gradient of often kind of medicine arranges three multiple holes, arrange cell controls group (inoculating cell is agent-feeding treatment not) simultaneously and only add the blank group of HL substratum, often group arranges three multiple holes.
(3) drug treating (37 DEG C, 5% CO
2incubator is cultivated) after 24 hours, suck solution in hole, every hole adds 10 μ L CKK-8 detection reagent (the green skies, Shanghai), i.e. 10 μ L CCK-8+90 μ L HL substratum.
(4) at 37 DEG C, 5% CO
2continue in cell culture incubator to hatch 0.5 ~ 2 hour, incubation time with cell concentration number relevant, the concrete time determines (tentatively can determine according to liquid color change) according to preliminary experiment, and range of absorbency controls between 1.0 ~ 1.5 best.
(5) absorbancy at 450nm place is determined at by microplate reader.
People's normal bronchial epithelial cell to susceptibility (toxicity) detected result of three kinds of cancer therapy drug 5-Fu, Zorubicin, cis-platinum as Fig. 5,5-Fu affects not obvious at lower concentration and high density on cell survival rate, the concentration versus cell survival rate of cis-platinum below 5 μMs is little, and Zorubicin cell survival rate when 2.5 μMs of concentration is worth close to 0.Show that people's normal bronchial epithelial cell is to the susceptibility of different pharmaceutical toxicity detection and discrimination.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (6)
1. people's normal bronchial epithelial cell, is characterized in that: Classification And Nomenclature behaviour normal bronchial epithelial cell HNBEC/HL-001, deposit number is CCTCC NO:C201311.
2. people's normal bronchial epithelial cell according to claim 1, it is characterized in that: karyomit(e) is diploid, STR genotype represents with 16 tandem repeat loci/allelotrope length: AMEL/X/Y, D3S1358/17, TH01/7/8, D21S11/31/32, D18S51/20, Penta E/11, D5S818/11/12, D13S317/8/11, D7S820/8/13, D16S539/11, CSF1PO/10/12, Penta D/7/13, WA/17, D8S1179/15/16, TPOX/8/11, FGA/20/24.
3. people's normal bronchial epithelial cell according to claim 1, is characterized in that: cultivate based on 37 DEG C, 5%CO with HL
2cultivate; Described HL substratum is: DMEM and serum free medium SFM by volume 1:3 mix, add the foetal calf serum of 5% simultaneously, and 0.4 μ g/mL hydrocortisone, 5 μ g/mL Regular Insulin, 8.4ng/mL Toxins,exo-, cholera, 10ng/mL Urogastron, 24 μ g/mL VITAMIN B4,100U/mL penicillin, 100 μ g/mL Streptomycin sulphates, 0.25 μ g/mL amphotericin B, 30 μMs of fasudils.
4. the epithelial Secondary Culture method of the people's normal bronchial described in any one of claim 1-3, is characterized in that comprising the steps:
(1) when people's normal bronchial epithelial cell proliferation to 70 ~ 90% abundance described in any one of claim 1-3, PBS washed cell is used, then with 0.05% pancreas enzyme-EDTA digestion monolayer cell;
(2) add in DMEM and digestion reaction; Centrifugally remove supernatant, by HL substratum re-suspended cell precipitation, be inoculated in culturing bottle and cultivate.
5. the epithelial Secondary Culture method of people's normal bronchial according to claim 4, is characterized in that:
The time of the digestion described in step (1) is 2 ~ 5 minutes;
Centrifugal described in step (2) is centrifugal 5 minutes of 1000rpm, and the condition of described cultivation is 37 DEG C, 5%CO
2.
6. the Physiologic Studies of the human normal cell line of people's normal bronchial epithelial cell in the Clinics and Practices of non-diseases described in any one of claim 1-3, external Normocellular drug toxicity research and detect, application in the study of pathogenesis of segmental bronchus and lung relative disease.
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| CN104531607B (en) * | 2014-12-29 | 2017-11-07 | 南京农业大学 | The primary bronchiole epithelial cell of dog and its application in immortalized cells is prepared |
| CN104694460A (en) * | 2015-02-13 | 2015-06-10 | 武汉大学深圳研究院 | Culture medium for normal epithelial cell of human or mammal, culture methods, normal epithelial cell and application of normal epithelial cell |
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