CN103451148A - Human normal bronchial epithelial cell and preliminary isolated culture and subculture methods and purposes thereof - Google Patents
Human normal bronchial epithelial cell and preliminary isolated culture and subculture methods and purposes thereof Download PDFInfo
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Abstract
The invention discloses a human normal bronchial epithelial cell and preliminary isolated culture and subculture methods and purposes of the human normal bronchial epithelial cell. The cell is named as the human normal bronchial epithelial cell HNBEC/HL-001, the preservation number of the cell is CCTCCNO: C201311. The preliminary isolated culture method includes the steps that fat in a para-carcinoma tissue sample excised from a patient with lung cancer through an operation is removed, dispase and DNasel are added to the fat for action after the fat is digested, the cell is collected through filtering and centrifugal operations, and the cell is suspended again with an HL culture medium for inoculated culture. The subculture method includes the steps that when the cell proliferates to 70-90% abundance, pancreatin-EDTA is used for digestion, and then DMEM is used for neutralization; the cell is collected through a centrifugal operation, and the cell is suspended again with an HL culture medium for inoculated culture. The human normal bronchial epithelial cell can be used for physiological research of human normal cells, drug toxicity research and detection of external normal cells, and research on pathogenesis of bronchia and lung diseases including bronchogenic carcinoma.
Description
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Technical field
The invention belongs to the cytobiology field, relate to the normal bronchial epithelial cell of a kind of people and primary separation and Culture thereof and go down to posterity cultural method and purposes.
Background technology
The human body vitals are all its moiety by the epithelial cell of organ specificity differentiation as lung, kidney, liver, pancreas and skin.The epithelial cell of these specificitys differentiation is directly related with the specific function of Different Organs, as the removing toxic substances of the filtering function of the gaseous interchange function of lung, kidney, liver and in and function, pancreatic cell produce Regular Insulin, skin and have and protect body to avoid the injury of external environment.And the health that these vitals once pathologies occur or degenerate and will threaten 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 specificity differentiation is all to be difficult to regeneration, lets alone in vitro and cultivates and bred.So greatly limited the understanding of people to the organism normal cell function, it is inaccurate or even wrong much coming from the scientific and technical literature description that even cell biological of textbook is gained knowledge.Real reason is that most Normocellular Biological Knowledges derive from vitro culture " cancer cells " result of study.Although the various countries scientist is constantly attempting functional epithelial cell that cultivation and proliferation of human body weight are wanted organ all the time,, remain very difficult for separating from people and mammiferous primary epithelial vitro culture.Apply current serum free medium, the former culture that can only carry out short-term had (is survived a couple of days, as epithelial cells such as lung and pancreas), what have can only carry out the limited cultivation of going down to posterity (1-3 generation, as epithelial cells such as tracheae/segmental bronchus and prostate glands), what have even at all can not vitro culture (as epithelial cells such as liver, colon, prostate glands), and the superficial cell that only comes from human body skin can go down to posterity and cultivate about 10 generations.And the epithelial cell output obtained from each animal or biological tissue sample is still very low, comprise the quantity of cell, directly separate or the cell purity of Short-term Culture is all very low, these are primary and the epithelial rate of propagation that goes down to posterity is also very limited.
In order to carry out in vitro epithelial cultivation, current external the trial by genetic manipulation, as proceed to virus (SV40 T or HPV16E6E7) or cellular oncogene, algebraically that can the outer cell survival of extension body.Yet the disadvantage of genetic manipulation is genetic background and the phenotype that can change these cells, so that allows these normal epithelium cells lose its normal physiological function, as p53 and the pRB signal path usually suppressed.And these genetically modified cells can not be transplanted into body again again.But, owing to lacking at present the epithelial technology of effective vitro culture, above-mentioned these cells still enjoy favor in the medical science of the world today and life science.In non-cancer research field, they are just representing tissue or the organ of " function " property in initial source.In the cancer research field, they also usually are used as " normal cell " contrast, and 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 people's bronchial epithelial cell major function: the epithelial cell of (1) bronchial surface has formed the 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 mediators and cytokine height of formation complexity.Human bronchial epithelial cell is relevant with main pathology physiological maladies, as lung bronchogenic carcinoma (how referred to as lung cancer), chronic bronchitis, bronchiectasis, bronchostenosis.If can obtain the external stable normal bronchial epithelial cell of people gone down to posterity, to make people can more fully study the normal function of cell, disease pathogenesis, tissue organ function rebuilds or reproduces, and measure medicine to Normocellular toxicity, 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 that overcomes prior art, with not enough, provides a kind of people 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 a kind of people's who never registered normal cell system both at home and abroad.
Another object of the present invention is to provide the primary isolation cultivation method of the normal bronchial epithelial cell of above-mentioned people.
A further object of the present invention is to provide the cultural method that goes down to posterity of the normal bronchial epithelial cell of above-mentioned people.
The present invention also aims to provide the above-mentioned people purposes of normal bronchial epithelial cell.
Purpose of the present invention is achieved through the following technical solutions:
The normal bronchial epithelial cell of a kind of people, the Classification And Nomenclature normal bronchial epithelial cell HNBEC/HL-001 that behaves, be preserved in Chinese Typical Representative culture collection center, and deposit number is CCTCC NO:C201311.This cell derived is in Chinese normal lung epithelial cell, and karyomit(e) is diploid, the STR(STR) genotype means 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 culture condition of the normal bronchial epithelial cell of described people is preferably with HL and cultivates based on 37 ℃, 5% CO
2cultivate, described HL substratum is: DMEM and serum free medium SFM by volume 1
:3 mix, add 5%(v/v simultaneously) the 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-, cholera (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 μ M fasudils (Fasudil), above-mentioned substratum need be through 0.22 μ m aperture membrane filtration.
The primary isolation cultivation method of the normal bronchial epithelial cell of above-mentioned people, comprise the steps:
(1) in the situation that the other healthy tissues sample of cancer of Operation for Lung Cancer excision is collected in patient or patient care people informed consent.
(2) with 95～100%(v/v) ethanol wash the tissue sample of separation, use PBS(0.01M, pH 7.4 again) wash, then tissue sample is put into to the sterile petri dish containing precooling PBS, under dissecting microscope, with dissecting tweezers and scissors, remove residual fat in tissue sample.
(3) tissue sample is digested with Digestive system; Preferably, described Digestive system is the HL substratum containing collagenase and Dispase.
(4) postdigestively organize the centrifugal supernatant that goes, cell precipitation be resuspended in to the 0.25%(mass volume ratio) digest in pancreas enzyme-EDTA.
(5) add containing 10%(v/v) the DMEM substratum of FBS, the centrifugal supernatant that goes.
(6) add Dispase and the DNase I of warm water bath, with the rifle head, repeatedly blow and beat sample.
(7) add containing 10%(v/v again) the DMEM substratum of FBS, with the strainer filtration cell suspension in 40～70 μ m apertures, collect the cell suspension after filtering, the centrifugal supernatant that goes.
(8) re-suspended cell is deposited in the HL substratum, is inoculated in culturing bottle and cultivates, and obtains the normal bronchial epithelial cell of people.
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 the tissue sample volume.
The condition optimization of the digestion described in step (3) is 37 ℃ of digestion 1～3 hour.
The concentration of the collagenase described in step (3) and Dispase is preferably and is 0.2 mg/mL.
Digestion described in step (4) is preferably and digests on ice 1 hour or room temperature digestion 10 minutes.
Warm water bath described in step (6) is preferably the warm water bath of 37 ℃.
Centrifugal described in step (4), (5), (7) is preferably 1000 rpm centrifugal 5 minutes.
The condition optimization of the cultivation described in step (8) is 37 ℃, 5% CO
2.
The cultural method that goes down to posterity of the normal bronchial epithelial cell of above-mentioned people, comprise the steps:
(1) when the normal bronchial epithelial cell of people is bred to 70～90% abundance, with 1 * PBS(0.01M, pH 7.4) washed cell, then use the 0.05%(mass volume ratio) pancreas enzyme-EDTA digestion monolayer cell.
(2) add in DMEM and digestion reaction; The centrifugal supernatant that goes, 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 described in step (2) is preferably 1000 rpm centrifugal 5 minutes.
The condition optimization of the cultivation described in step (2) is 37 ℃, 5% CO
2.
The normal bronchial epithelial cell of above-mentioned people can be used for the Physiologic Studies of human normal cell line, external Normocellular drug toxicity research and detection, and segmental bronchus and lung relative disease comprise the study of pathogenesis of lung bronchogenic carcinoma.
The present invention has following advantage and effect with respect to prior art:
(1) the normal bronchial epithelial cell of people provided by the invention, primary separation and Culture is from people's normal lung tissue, and this cell does not import any foreign gene, through karyotyping, identifies the normal diploid cell into the people.
(2) the normal bronchial epithelial cell of people provided by the invention, primary separation and Culture, from Chinese normal lung tissue, through the str locus Classification Identification, is the domestic and international a kind of people's who never registered normal cell system.
(3) the normal bronchial epithelial cell of people provided by the invention, can be used for the Physiologic Studies of human normal cell line, external Normocellular drug toxicity research and detecting, and segmental bronchus and lung relative disease comprise the study of pathogenesis of lung bronchogenic carcinoma.
The accompanying drawing explanation
Fig. 1 is the cellular form figure of the normal bronchial epithelial cell of people.
Fig. 2 is the growth curve chart of the normal bronchial epithelial cell of people.
Fig. 3 is the chromosome karyotype analysis figure of the normal bronchial epithelial cell of people.
Fig. 4 is the str locus somatotype figure of the normal bronchial epithelial cell of people.
Fig. 5 is the susceptibility detected result figure of the normal bronchial epithelial cell of people.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is done to further detailed description, but embodiments of the present invention are not limited to this.
The primary separation and Culture of the normal bronchial epithelial cell of the primary people of embodiment 1
(1) in the situation that the other healthy tissues sample of cancer of Operation for Lung Cancer excision is collected in patient or patient care people informed consent.
(2) preparation of Digestive system: containing the HL substratum of collagenase and equal 0.2 mg/mL of Dispase, wherein, the HL substratum is: DMEM(GIBCO # 11965-092) with serum free medium SFM (GIBCO # 10744-019) by volume 1
:3 mix, 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-, cholera (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 μ M fasudils (Fasudil), above-mentioned substratum need be through 0.22 μ m aperture membrane filtration).
(3) with 95～100%(v/v) ethanol wash the tissue sample 1 time of separation, use PBS(0.01M, pH 7.4 again) wash 2 times, then tissue is put into and contained the sterile petri dish of precooling PBS on ice, under dissecting microscope, with dissecting tweezers and scissors, remove residual fat in 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 ℃ digest 1～3 hour.
(5) organize low-speed centrifugal (1000 rpm) 5 minutes by postdigestive, remove supernatant.
(6) cell precipitation is resuspended in to the 0.25%(mass volume ratio of 2～5 mL) in pancreas enzyme-EDTA, be placed on ice 1 hour or room temperature 10 minutes.
(7) then add 10 mL 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 ℃) and the 1 mg/mL DNase I of 200 μ L, with aseptic P1000 disposable plastic rifle head, repeatedly blow and beat sample 1 minute.
(9) add 10 mL containing 10%(v/v) DMEM of FBS, with the strainer filtration cell suspension in 40～70 μ m apertures, collect the cell suspension after filtering, centrifugal 5 minutes of low speed 1000 rmp, removal supernatant.
(10) re-suspended cell is deposited in the HL substratum, and the culturing bottle that is inoculated in T25 or T75 is cultivated, and culture condition is 37 ℃, 5% CO
2.
The normal bronchial epithelial cell of primary people of separation and Culture success according to the method described above, under microscope, the form of observation of cell is as Fig. 1 (arrange closely, cell boundary is clear, stereoscopic sensation is strong, the epithelial cell of multiangular).This cell divide called after " the normal bronchial epithelial cell HNBEC/HL-001 of people ", on April 10th, 2013 be preserved in Chinese Typical Representative culture collection center (address: China. Wuhan. Wuhan University), deposit number is CCTCC NO:C201311.
The cultivation of going down to posterity of the normal bronchial epithelial cell of embodiment 2 people
(1) when the normal bronchial epithelial cell of the people who cultivates in the culturing bottle at T25 or T75 is bred to 70～90% abundance, with 1 * PBS(0.01M, pH 7.4) washed cell twice, then use the 0.05%(mass volume ratio) pancreas enzyme-EDTA digestion monolayer cell 2～5 minutes.
(2) add in the complete DMEM of 10 mL and 1～2 minute kind of digestion reaction.
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) in case of necessity can be 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 standby.
The normal bronchial epithelial cell of the cultivator that goes down to posterity according to the method described above, the cell growth curve of culture is as Fig. 2, and continuous passage is cultivated 55 days, and the normal bronchial epithelial cell of people of the present invention still can keep the vegetative state normal growth.
The karyotyping of the normal bronchial epithelial cell of embodiment 3 people is identified
(1) when the normal bronchial epithelial cell (1 * 10 of people
6) when exponential phase of growth, adding colchicine, final concentration is 0.2 μ g/mL, continues to cultivate 3.5 hours.
(2) repeatedly blow and beat cell it is come off, centrifugal 5 minutes harvested cells of 2000 rpm.
(3) abandon supernatant liquor, add 0.075 mol/L KCl solution 8 mL of 37 ℃ of pre-temperature, blow and beat gently cell mass and mix, put 37 ℃ of hypotonic processing 25 minutes.
(4) add the fixing agent (methyl alcohol of the new preparation of 1 mL
:glacial acetic acid=3
:1, v/v), carefully blow and beat, mix, 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, repeats to fix once.
(7) abandon supernatant liquor, add several fixing agents to make cell suspension, get on 2～3 slide glasss that soaked in frozen water.
(8) slide glass is put to dry baking 2 hours in 70 ℃ of baking boxs, naturally cooling.
(9) 2.5%(mass volume ratio) pancreatin solution (pH6.8～7.2) 5 mL processed for 25～45 seconds.
The physiological saline rinsing of (10) 37 ℃ of pre-temperature, Giemsa dyeing 5～10 minutes, make the aobvious band of G and analyze.
(11) observation of cell caryogram photography under microscope, carry out karyotyping; 20 of observation analysis above mitosis metaphase of cell at least.Representational nucleus type analysis the results are shown in Figure 3, and the normal bronchial epithelial cell of people is normal diploid, and 46 karyomit(e) no abnormality seens are arranged.
The gene type Analysis and Identification of the normal bronchial epithelial cell of embodiment 4 people
(1) the normal bronchial epithelial cell (1 * 10 of the people 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, adds 200 μ L damping fluid GA(cell/tissue genome DNA extracting reagent kit DP304, day root company), vibration is to thoroughly suspension.
(3) add 20 μ L Proteinase K solution, mix.
(4) add 200 μ L damping fluid GB(cell/tissue genome DNA extracting reagent kit DP304, day root company), fully put upside down and mix, place 10 min for 70 ℃, briefly centrifugal.
(5) add people's 200 μ L dehydrated alcohols, fully vibration mixes 15 seconds, briefly centrifugal.
(6) gained solution and flocks are all added to (cell/tissue genome DNA extracting reagent kit DP304, day root company) in an adsorption column, centrifugal 30 seconds of 12000 rpm, remove waste liquid.
(7) add 500 μ L damping fluid GD(cell/tissue genome DNA extracting reagent kit DP304 in adsorption column, day root company), centrifugal 30 seconds of 12000 rpm, remove waste liquid.
(8) add 600 μ L rinsing liquid PW(cell/tissue genome DNA extracting reagent kit DP304 in adsorption column, day root 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, it root company), room temperature is placed 2～5 min, 12000 rpm(～13400 * g) centrifugal 2 minutes, the DNA solution of extraction is collected in centrifuge tube.
(10) utilize PowerPlex 16 HS systems (DC2101, promega company) to carry out the DNA composite amplification in 16 locus (15 STR sites and 1 sex site).
(11) use ABI PRISM 3100 type genetic analyzers (1.1 edition datas are collected software) to carry out the detection of amplified fragments.
(12) use Genotyper and PowerTyperTM 16 Macro software analysis sampled datas, carry out the automatic gene somatotype, the STR somatotype is Fig. 4 as a result, detects 16 str locus sites, with " str locus seat/allelotrope length ", means:
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 normal bronchial epithelial cell of people of the present invention, through the str locus Classification Identification, is the domestic and international a kind of people's who never registered normal cell system.
The susceptibility of the normal bronchial epithelial cell of embodiment 5 people detects
(1) by 0.05% trysinization of the normal bronchial epithelial cell of people, 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 ℃, 5% CO
2incubator is cultivated.
(2) second day dosing (5-Fu(5-Fluracil), Zorubicin, cis-platinum) process, every hole adds 10 μ L different concns medicines, drug level gradient (μ M) is 0.25,0.5,2.5,5.0,25,50, each gradient of every kind of medicine arranges three multiple holes, cell control group (not dosing of inoculating cell processing) is set simultaneously and reaches the blank group that only adds the HL substratum, every group arranges three multiple holes.
(3) drug treating (37 ℃, 5% CO
2the incubator cultivation) 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 ℃, 5% CO
2in cell culture incubator, continue to hatch 0.5～2 hour, incubation time with cell concentration the number relevant, the concrete time is determined (can change and tentatively determine according to liquid color) according to preliminary experiment, range of absorbency is best between being controlled at 1.0～1.5.
(5) be determined at the absorbancy at 450nm place by microplate reader.
The normal bronchial epithelial cell of people to susceptibility (toxicity) detected result of three kinds of cancer therapy drug 5-Fu, Zorubicin, cis-platinum as Fig. 5,5-Fu is not obvious on the cell survival rate impact at lower concentration and high density, the concentration of cis-platinum below 5 μ M is little on the cell survival rate impact, and Zorubicin cell survival rate when 2.5 μ M concentration has approached 0 value.Show susceptibility and discrimination that the normal bronchial epithelial cell of people detects different pharmaceutical toxicity.
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.
Claims (10)
1. the normal bronchial epithelial cell of people is characterized in that: the Classification And Nomenclature normal bronchial epithelial cell HNBEC/HL-001 that behaves, deposit number is CCTCC NO:C201311.
2. the normal bronchial epithelial cell of people according to claim 1, it is characterized in that: karyomit(e) is diploid, the STR genotype means with 16 STR locus/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. the normal bronchial epithelial cell of people according to claim 1, is characterized in that: with HL, cultivate based on 37 ℃, 5% CO
2cultivate; Described HL substratum is: DMEM and serum free medium SFM by volume 1
:3 mix, add 5% foetal calf serum simultaneously, and 0.4 μ g/mL hydrocortisone, 5 μ g/mL Regular Insulin, 8.4 ng/mL Toxins,exo-, choleras, 10 ng/mL Urogastrons, 24 μ g/mL VITAMIN B4,100 U/mL penicillin, 100 μ g/mL Streptomycin sulphates, 0.25 μ g/mL amphotericin B, 30 μ M fasudils.
4. the primary isolation cultivation method of the normal bronchial epithelial cell of the described people of claim 1-3 any one, is characterized in that comprising the steps:
(1) collect the other healthy tissues sample of cancer of Operation for Lung Cancer excision;
(2) wash the tissue sample of separation with 95～100% ethanol, then wash with PBS, then tissue sample is put into to the sterile petri dish containing precooling PBS, under dissecting microscope, with dissecting tweezers and scissors, remove residual fat in tissue sample;
(3) tissue sample is digested with Digestive system; Described Digestive system is the HL substratum containing collagenase and Dispase;
(4) postdigestively organize the centrifugal supernatant that goes, cell precipitation is resuspended in 0.25% pancreas enzyme-EDTA and digests;
(5) add the DMEM substratum containing 10% foetal calf serum, the centrifugal supernatant that goes;
(6) add Dispase and the DNase I of warm water bath, with the rifle head, repeatedly blow and beat sample;
(7) add again the DMEM substratum containing 10% foetal calf serum, with the strainer filtration cell suspension in 40～70 μ m apertures, collect the cell suspension after filtering, the centrifugal supernatant that goes;
(8) re-suspended cell is deposited in the HL substratum, is inoculated in culturing bottle and cultivates, and obtains the normal bronchial epithelial cell of people.
5. the primary isolation cultivation method of the normal bronchial epithelial cell of people according to claim 4 is characterized in that:
Precooling described in step (2) is in precooling on ice;
The consumption of the Digestive system described in step (3) be 10 times to the tissue sample volume, the condition of described digestion be 37 ℃ digestion 1～3 hour;
The concentration of the collagenase described in step (3) and Dispase is 0.2 mg/mL.
6. the primary isolation cultivation method of the normal bronchial epithelial cell of people according to claim 4 is characterized in that:
Digestion described in step (4) is for digesting on ice 1 hour or room temperature digestion 10 minutes;
The warm water bath that warm water bath described in step (6) is 37 ℃;
The condition of the cultivation described in step (8) is 37 ℃, 5% CO
2.
7. the primary isolation cultivation method of the normal bronchial epithelial cell of people according to claim 4 is characterized in that:
Centrifugal described in step (4), (5), (7) is centrifugal 5 minutes of 1000 rpm.
8. the cultural method that goes down to posterity of the normal bronchial epithelial cell of the described people of claim 1-3 any one, is characterized in that comprising the steps:
(1) when the normal bronchial epithelial cell of people is bred to 70～90% abundance, use the PBS washed cell, then digest monolayer cell with 0.05% pancreas enzyme-EDTA;
(2) add in DMEM and digestion reaction; The centrifugal supernatant that goes, by HL substratum re-suspended cell precipitation, be inoculated in culturing bottle and cultivate.
9. the cultural method that goes down to posterity of the normal bronchial epithelial cell of people according to claim 8 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 1000 rpm, and the condition of described cultivation is 37 ℃, 5% CO
2.
10. the normal bronchial epithelial cell of the described people of claim 1-3 any one comprises the application in the study of pathogenesis of lung bronchogenic carcinoma at the Physiologic Studies of human normal cell line, external Normocellular drug toxicity research and detection, segmental bronchus and lung relative disease.
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| CN104531607A (en) * | 2014-12-29 | 2015-04-22 | 南京农业大学 | Canine primary bronchial epithelial cell and application thereof in preparation of immortalized cells |
| 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 |
| CN106591220A (en) * | 2016-12-29 | 2017-04-26 | 深圳市永生原代细胞生物科技有限公司 | Human normal skin epithelial cell and use thereof |
| CN107151645A (en) * | 2017-05-16 | 2017-09-12 | 武汉大学深圳研究院 | A kind of method and culture medium that in vitro individuation drug test is provided for lung cancer |
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| CN106497866A (en) * | 2016-11-30 | 2017-03-15 | 武汉大学深圳研究院 | A kind of people's normal vagina epithelial cell and application thereof |
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| CN103881975A (en) * | 2014-04-04 | 2014-06-25 | 武汉大学 | Human prostate cancer cell as well as passage separation culture and subculturing culture method and application thereof |
| CN103881975B (en) * | 2014-04-04 | 2015-10-21 | 武汉大学 | Human Prostate Cancer Cells and primary separation and Culture thereof and Secondary Culture method and purposes |
| CN104531607A (en) * | 2014-12-29 | 2015-04-22 | 南京农业大学 | Canine primary bronchial epithelial cell and application thereof in preparation of immortalized cells |
| 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 |
| CN106591220A (en) * | 2016-12-29 | 2017-04-26 | 深圳市永生原代细胞生物科技有限公司 | Human normal skin epithelial cell and use thereof |
| CN107151645A (en) * | 2017-05-16 | 2017-09-12 | 武汉大学深圳研究院 | A kind of method and culture medium that in vitro individuation drug test is provided for lung cancer |
| CN110791471A (en) * | 2019-12-05 | 2020-02-14 | 苏州大学 | Separation method of mouse trachea-bronchus epithelial cells |
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