CN108034636B - Human breast cancer cell line and application - Google Patents
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- CN108034636B CN108034636B CN201711264316.1A CN201711264316A CN108034636B CN 108034636 B CN108034636 B CN 108034636B CN 201711264316 A CN201711264316 A CN 201711264316A CN 108034636 B CN108034636 B CN 108034636B
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Abstract
The invention belongs to technical field of microbe cell line, and in particular to human breast cancer cell line and application.Human breast cancer cell line, it include: to be respectively designated as human breast carcinoma Her2+/Basal cell line ZJU-0327, ZJU-0725 and ZJU-1127, and it is preserved in China typical culture collection center, preservation address are as follows: China, Wuhan, Wuhan University, deposit number are respectively as follows: the human breast cancer cell line of CCTCC NO:C2017171, CCTCC NO:C2017172 and CCTCC NO:C2017173.Human breast carcinoma Her2+/Basal cell line ZJU-0327, ZJU-0725 and ZJU-1127 of the invention is established from Chinese source, and building is that the time is shorter, biological heredity character is stablized, using the breast cancer cell line as research model, have very great help for understanding the pathogenesis of primary breast cancer Basal/HER2-positive phenotype of Chinese population.
Description
Technical field
The invention belongs to technical field of microbe cell line, and in particular to human breast cancer cell line and application.
Background technique
Breast cancer is one of the most common malignant tumors in women in the world.In China, the disease incidence of breast cancer has on gradually
The trend risen, in some coastal economy developed regions, the disease incidence of breast cancer has accounted for the first place of women's malignant tumour.Breast cancer is
One kind has the malignant tumour of height heterogeneity, by the development of the scientific research technology of many decades, by using the micro- battle array of gene
Column technology can be classified as 5 hypotypes: conduit A type, conduit Type B, Basaloid, HER2 overexpression type and normal breast sample
Type, the therapeutic effect for promoting clinical breast cancer patient significantly improve, and reduce what patient was born in treatment lysis
Body & mind pain.
Between in the past few decades, the proposition of several pathology and immunohistochemistry subclassification may advantageously facilitate the clinical water of differentiation
Flat hormone receptor and the range of triple negative breast cancer and heterogeneous feature.However, these classification are not related to clinically HER2+
Breast cancer classification.HER2+ refers to ER-, and PR- and HER2 are positive, accounts for 20%-25% of all breast cancer patients.And its
He compares type of mammary cancer, and the type histological grade is poor, and easy to recur and transfer, prognostic are poor.Although Trastuzumab drug is one
Determine to survive effectively to the patient of HER2+ in degree, but the early stage patient and 70% of about 15%HER2+ still occur or more to turn
It moves patient HER2+ and shows the tolerance to the drug, often pathological manifestations are substrate template in the breast cancer of the type tolerance
(Basal-like) breast cancer.
Summary of the invention
The first purpose of this invention is, for the current domestic Basal/HER2- for lacking Chinese population source
Positive breast carcinoma cell strain, and provide the type from Risk of Breast Cancer in Chinese Population cell line.
For this purpose, above-mentioned purpose of the invention is achieved through the following technical solutions:
Human breast cancer cell line, comprising: be respectively designated as human breast carcinoma Her2+/Basal cell line ZJU-0327, ZJU-
0725 and ZJU-1127, and it is preserved in China typical culture collection center, preservation address are as follows: China, Wuhan, Wuhan University,
Deposit number is respectively as follows: the human milk gland of CCTCC NO:C2017171, CCTCC NO:C2017172 and CCTCC NO:C2017173
Cancerous cell line.
While by adopting the above technical scheme, the present invention can also be used or be combined using technology further below
Scheme:
Preferably, the human breast cancer cell line further include: be respectively designated as human breast carcinoma Her2+/Basal cell line
ZJU-0327, ZJU-0725 and ZJU-1127, and it is preserved in China typical culture collection center, preservation address are as follows: China,
Wuhan, Wuhan University, deposit number are respectively as follows: CCTCC NO:C2017171, CCTCC NO:C2017172 and CCTCC NO:
The progeny cell system of the human breast cancer cell line of C2017173.
It preferably, is typical polygon epithelioid cell when the human breast cancer cell line is cultivated in vitro, under light microscopic,
It is typical Malignant Epithelium feature under Electronic Speculum；Karyotyping result is hypo-triploid caryogram, and mainstream chromosome number is 78-85.
Preferably, the human breast cancer cell line has infiltrative breast carcinoma characteristic, feature in breast cancer clinical diagnosis
Identify that phenotype is Basal/HER2-positive.
Preferably, the human breast cancer cell line is outer aobvious in the case where the 11 of TP53 mutated gene exon is sequenced
The polymorphism mutation of mononucleotide occurs for the 72nd coding of son 4, and it is prominent that missense occurs for the 126th coding of exon 5
Become.
Preferably, for the human breast cancer cell line when mycoplasma test reagent box detects, PCR result is feminine gender, not
Receive mycoplasma contamination.
Second object of the present invention is, for the deficiencies in the prior art, provides human breast cancer cell line
Using.
For this purpose, above-mentioned purpose of the invention is achieved through the following technical solutions:
According to human breast cancer cell line noted earlier in the cell model of the pathogenesis of breast carcinoma Mechanism Study of Her/Basal
Application.
A further object of the invention is, for the deficiencies in the prior art, provides a kind of human breast cancer cell
The purposes of system.
For this purpose, above-mentioned purpose of the invention is achieved through the following technical solutions:
A kind of purposes of human breast cancer cell line is used to prepare according to human breast cell noted earlier system and feeds in immune deficiency
The model of breast cancer, tumor formation rate 100% are generated in newborn animal.
While by adopting the above technical scheme, the present invention can also be used or be combined using technology further below
Scheme:
Preferably, the immune deficiency mammal is immune deficiency nude mouse.
Human breast cancer cell line ZJU-0725 and ZJU-1127 of the invention was from an example invasive ductal carcinoma breast cancer 65 years old
Female patient (ER+,PR—,Her+) and ZJU-0327 from 63 years old female patient (ER of another Infiltrating ductal breast cancer+,PR+,
Her+) the tumor specimen tissue after cutting off of performing the operation.After through originally culture and building and be tied to form function, it is respectively designated as human breast carcinoma Her2
+/Basal cell line ZJU-0327, ZJU-0725 and ZJU-1127.
Human breast carcinoma Her2+/Basal cell line ZJU-0327, ZJU-0725 and ZJU-1127 cell growth of the invention
Vigorous, cell is in flat irregular polygon adherent monolayers growth, Chang Kejian vacuole in endochylema more；CK-pan positive rate is respectively about
98.69%, 98.2% and 98.93；Cell still has good vigor after cryopreservation resuscitation；Above-mentioned cell line is at present
Or so 100 generations are passaged to, growth conditions are good, can immortalize.
Human breast carcinoma Her2+/Basal cell line ZJU-0327, ZJU-0725 and ZJU-1127 cell of the invention, cell
Doubling time is respectively 18.7h, 57.5h and 18.8h；There is exception in chromosome number and structure, and chromosome number is divided mostly
For cloth between 78-85, textural anomaly includes chromosome increase, missing and transposition etc.；Gimesa dyeing display nuclear division phase
Obvious active, karyon is big, and caryoplasm ratio increases, it is seen that megacaryocyte；Have typical epithelial character under Electronic Speculum, it is seen that desmosome, microfilament,
Abundant organelle (mitochondria, ribosomes and rough surfaced endoplasmic reticulum (RER)) and nucleocytoplasmic ratio are big.
The STR detection method that human breast cancer cell line of the invention uses detection method most authoritative at present to recommend for ATCC,
Testing result determines that three human breast cancer cell lines are source of people, and finds that ZJU-0725 and ZJU-1127 has identical knot
Fruit, and it is different from ZJU-0327；Believe with the cytogenetics in the foremost culture collection mechanism of ATCC and two whole world DSMZ
Breath is compared, and discovery is not affected by other cell contaminations.
Human breast cancer cell line of the invention detects TP53 tumor suppressor gene, and 4 He of exon occurs for discovery mutation
There is the point mutation of C → G in the 72nd coding of exon 4 in 5, tumor suppressor gene TP53 sequencing discovery, result in original
The proline (Pro) first encoded becomes arginine (Arg), is mutated for non-pathogenic, belongs to single nucleotide polymorphism；Exon 5
There is the point mutation of T → A in 126th coding, and resulting in the tyrosine (Try) originally encoded becomes asparagine (Asn),
For pathogenic missense mutation.
Human breast cancer cell line of the invention identifies breast cancer phenotype phase through Western Blot (Western Blot)
Albumen and the analysis of prognosis correlative protein expression are closed, is compared with existing established cell line, finds established cell line
Belong to Basal/HER2-positive classification.
The activity of radiotherapy assessment human breast cancer cell line: various dose irradiation is used for ZJU-0725 and ZJU-1127, application
ZJU-0725 is sensitive to radiotherapy with reported Sk-Br3 (Basal/HER2-positive) afterwards and ZJU-1127 is insensitive,
Wherein the cell model is two plants of cell lines of ZJU-0725 and ZJU-1127.
Chemotherapeutic handles ZJU-0725 and ZJU-1127: display only has docetaxel (Docetaxel) and hydrochloric acid after application
Epirubicin (Epirubicin HCl) can effectively inhibit ZJU-0725, and substantially invalid to ZJU-1127；And carboplatin
(Carboplatin), cyclophosphamide (Cyclophosphamide Monohydrate), taxol (Paclitaxel) and 5- fluorine
Uracil (5-Fu) then significantly inhibits effect to two plants of breast cancer cell lines nothing.
Human breast cancer cell line tumor formation experimental result discovery of the invention, the cell line established all have in nude mouse
Good external Tumor formation.By by a certain number of above-mentioned people's Primary breast cancerous cell line ZJU-0327, ZJU-0725 and
ZJU-1127 cell inoculation establishes the animal model of breast cancer in the subcutaneous of nude mouse.
The present invention has been provided with the progeny cell of the breast cancer cell line, and the progeny cell is basic or all protects
The characteristic of parental cell is stayed.
Invention further provides the human breast cancer cell lines in the cell model as mammary gland carcinogenesis mechanism research
In application.Due to human breast carcinoma Her2+/Basal cell line ZJU-0327, ZJU-0725 and ZJU-1127 of the invention be from
What Chinese source was established, and building is that the time is shorter, biological heredity character is stablized, using the breast cancer cell line as diagnostic cast
Type has very great help for understanding the pathogenesis of primary breast cancer Basal/HER2-positive phenotype of Chinese population.
In another aspect, the present invention provides a kind of mammary gland for establishing two kinds of different shapes from an example breast cancer sample tissue
The preparation method of cancerous cell line, comprising the following steps:
(1) method of collection of specimens and preservation: determining position of the lump in the breast tissue of excision, and tumor tissues are derived from
The surface part of knurl active proliferation.The acquisition of sample carries out under pathologists guidance, prevents from examining pathological replacement
It is disconnected to have an impact；It when sample wouldn't do subsequent operation, can save it in 4 DEG C of RPMI 1640, can generally place left for 24 hours
It is right.
(2) originally culture: three times through PBS washing, machinery shreds tissue, collagenase digesting, and centrifugation and washing are inoculated with and pass
It is commissioned to train and supports and observe culture.
(3) purifying cells: taking limiting dilution assay, specially after passing on several times, by cell according to 1:6 or more high magnification numbe
It is passaged in culture dish and cultivates, under the microscope and mark the cell mass of epithelial character, strike off its peripheral cell under aseptic condition,
PBS is washed three times, and new culture medium is added and continues culture to growing naked eyes visible plaques.
(4) expand culture: trypsin digestion cell group is seeded to 96 orifice plates after cell is collected by centrifugation, is then successively seeded to
In 48 orifice plates, 24 orifice plates, 12 orifice plates, 6 orifice plates and T-25 culture bottle.
Detailed description of the invention
Fig. 1 is that the optics of human breast carcinoma Her2+/Basal cell line ZJU-0328, ZJU-0725 and ZJU-1127 cell is aobvious
Micro mirror figure；
Fig. 2 is the transmission electricity of human breast carcinoma Her2+/Basal cell line ZJU-0328, ZJU-0725 and ZJU-1127 cell
Mirror figure；
Fig. 3 is that the streaming of human breast carcinoma Her2+/Basal cell line ZJU-0327, ZJU-0725 and ZJU-1127 cell is reflected
Determine result figure；
Fig. 4 is the cell week of human breast carcinoma Her2+/Basal cell line ZJU-0327, ZJU-0725 and ZJU-1127 cell
Phase distribution map (A-E), statistical chart (F) and growth curve chart (G-I)；
Fig. 5 is the phenotype point of human breast carcinoma Her2+/Basal cell line ZJU-0327, ZJU-0725 and ZJU-1127 cell
Analysis figure (A), detection of mycoplasma agarose electrophoresis figure (B), radiotherapy sensitivity detection statistics figure (C) and chemosensitivity statistical chart (D)；
Fig. 6 is that the tumor formation of human breast carcinoma Her2+/Basal cell line ZJU-0327, ZJU-0725 and ZJU-1127 cell tries
Test result；
Fig. 7 is that the TP53 of human breast carcinoma Her2+/Basal cell line ZJU-0327, ZJU-0725 and ZJU-1127 cell is prominent
Become testing result；
Fig. 8 is the representativeness of human breast carcinoma Her2+/Basal cell line ZJU-0327, ZJU-0725 and ZJU-1127 cell
Single celled karyotyping.
Specific embodiment
The present invention is described in further detail referring to the drawings and specific embodiments.
Embodiment 1
From 65 years old female patient (ER of an example invasive ductal carcinoma breast cancer+,PR-,Her+) and another invasive ductal carcinoma
63 years old (ER of breast cancer+,PR+,Her+) sample in situ tumor tissue after operation excision, necrosis and the tissue such as fat are rejected, is used
It is washed 3 times containing dual anti-physiology salt.By in tissue merging sterile petri dish, a little collagenase digesting liquid is added dropwise in tissue block surface,
It is sufficiently shredded with the small scissors of different Sterile ophthalmics to volume about 1-2mm3Size；The tumor tissues collagenase digesting that will be shredded
Liquid collection is placed in 15ml centrifuge tube, a certain amount of digestive juice is added, and be put into 37 DEG C of CO2It is digested overnight in incubator；Disappear
Change overnight sample tissue and cell suspension centrifugation (300 × g, 7min), discards supernatant, then be centrifuged 3 times with brine；
The dual anti-resuspension precipitating of RPMI 1640+10%FBS+ is placed in culture dish, is placed in 37 DEG C of CO2Incubator culture, at least 2 days not
Culture dish is moved, it is adherent to be conducive to primary tumor cell；6 10mm cultures are passaged to when cell is grown in ware to 70-80%
In ware, it is daily under the microscope, it is to be seen to cell under mirror be oval, sheet distributed cell group when, strike off around into fiber finer
Born of the same parents, physiology salt wash 3 times, new culture medium are added and continues to cultivate；When cell mass is long visible to naked eyes, pancreatin digestive juice disappears
Change cell to reach in 96 orifice plates, successively be reached in 48 orifice plates, 24 orifice plates, 12 orifice plates, 6 orifice plates and T-25 culture bottle later.It builds and is
After success, it is respectively designated as human breast carcinoma Her2+/Basal cell line ZJU-0327, ZJU-0725 and ZJU-1127, in 2017
Is preserved in China typical culture collection center (CCTCC), preservation address on September 30, are as follows: China, Wuhan, Wuhan University are protected
Hiding number is respectively as follows: CCTCC NO:C2017171, CCTCC NO:C2017172 and CCTCC NO:C2017173.
Embodiment 2
Breast cancer cell line ZJU-0327, ZJU-0725 and ZJU-1127 cell for taking secondary culture, in optical microscopy
Under (NIKO inverted microscope) observe cell growth status.
Giemsa shows band:
1. pretreatment: the metaphase chromosome specimen made being placed in 60-80 DEG C of oven and bakes piece 3h；
2. pancreatin digests: 0.025% trypsin solution that sample is placed in 37 DEG C (dilutes 0.25% trypsase with PBS
10 times) in digestion 1min after PBS wash slide；
3. Giemsa is dyed: sample being placed in Giemsa dye liquor and dyes 10min, tap water is rinsed well, is sealed after drying
Piece.
Microscopy: checking aobvious band sample under microscope high power ocular, if it is alternate to occur the clearly depth on chromosome
Banding pattern, as desirable sample, for camera shooting analysis.
As Fig. 1 (J-L) ZJU-0327, ZJU-0725 and ZJU-1127 nucleocytoplasmic ratio have greatly and often multinucleate giant cell appearance.
As shown in Figure 1, cytological appearance picture, cell refractiveness is good, and cell growth state is good, clear background, cell
It is in flat irregular polygon, Chang Kejian vacuole and apocyte in endochylema, nucleocytoplasmic ratio is big, no fibroblast pollution more.Fig. 1
Picture and Gimesa dyeing for ZJU-0327, under ZJU-0725 and ZJU-1127 cell phase contrast microscope.A-C is tumour in figure
Single cell suspension directly cultivates the attached cell after 48；D-I is the 72nd generation breast cancer primary cell form；J-L is Gimesa dye
Cellular morphology after color.
Embodiment 3
Preparation of samples: by 1 × 107It is received after ZJU-0327, ZJU-0725 and ZJU-1127 cell tryptase enzymic digestion of a culture
It combines in 15ml centrifuge tube, washing again is collected by centrifugation after cell and is collected by centrifugation；
It is fixed: the fixed 2h of 2.5% glutaraldehyde fixer to be added into cell, 0.1M phosphate buffer is washed 3 times, is added
1% starves sour fixer fixed 2h, and 0.1M phosphate buffer washs 3 times；
Dehydration: carried out in 4 DEG C of refrigerators respectively 50% ethyl alcohol → → 70% ethyl alcohol → → 90% ethyl alcohol → → 90% ethyl alcohol+
90% acetone (1:1) → → 90% acetone → → 100% acetone (3 times), every procedure maintain 15-20min；
Embedding: pure acetone+embedding liquid (2:1) room temperature 3-4h → → pure acetone+embedding liquid (1:2) ambient temperature overnight → → pure packet
Bury 37 DEG C of 2-3h of liquid；
Solidification: 37 DEG C of oven overnights → → 45 DEG C of baking oven 12h → → 60 DEG C of baking ovens are for 24 hours；
Ultra-thin section preparation: 50-60nm thickness slice is cut with ultramicrotome；
Dyeing: 3% acetic acid uranium-lead citrate double staining；
Observation film making: it is observed and is made film with transmission electron microscope (Hitachi HT7700).
As a result as shown in Fig. 2, visible Tonofibrils in endochylema, enrich organelle (mitochondria, ribosomes and asperities endoplasm
Net), cell link between visible desmosome and microvillus sample protrusion.Fig. 2 is ZJU-0327, ZJU-0725 and ZJU-1127 cell is saturating
Penetrate electromicroscopic photograph.(A-C) desmosome of Cell tracking；(D-F) nuclear membrane being recessed；(G-I) microfilament in cytoplasm.
Embodiment 4
(300 × g, 7min) is centrifuged after ZJU-0327, ZJU-0725 and ZJU-1127 cell tryptase enzymic digestion of culture to collect
Cell, PBS are washed 3 times；The fixed 10-15min of 4% enough paraformaldehyde fixer room temperature is added, cell PBS washes 3 after fixing
It is secondary；Cell fixed and permeabilized 40min in the PBS of 10% lowlenthal serum+0.03%Triton-X100；Centrifugation removal supernatant,
The pan-CK streaming antibody and cell room temperature of FITC fluorescent marker, which are protected from light, is incubated for 30min, and the Incubating Solution of no added antibody is as yin
Property control；PBS washing centrifugation 3 times；Upper machine testing after 30 μm of cell strainer filterings.As a result as shown in figure 3, ZJU-0725 purity about
It is 97%, ZJU-1127 purity 98% or so；ZJU-0328 purity is 99%.Fig. 3 is that streaming identifies ZJU-0327, ZJU-
0725 and ZJU-1127 cell purity.(A-C) Isotype control flow analysis chart；(D-F) CK-pan flow analysis chart；
(G-I) CK-pan positive rate statistical chart.
Embodiment 5
Collect about 1 × 106A ZJU-0327, ZJU-0725 and ZJU-1127 cell, MCF-7 and leaching in index division
The human lymphocyte of bar cell separating liquid separation；The piping and druming of 0.3ml PBS buffer solution is added into cell suspension, then is added dropwise -20
DEG C frost ethyl alcohol 0.7ml, side edged rock uniformly, by sample merging 4 DEG C of refrigerator overnights；Next day takes out PBS and washes 1 time；It is added
RNase A digests 30min in 37 DEG C of water-baths, adds 400 μ l PI mixing, 4 DEG C are protected from light 30min；30 μm of strainers are crossed to obtain
Upper machine testing after single cell suspension.
Fig. 4 is the cell week of human breast carcinoma Her2+/Basal cell line ZJU-0327, ZJU-0725 and ZJU-1127 cell
Phase distribution map (A-E), statistical chart (F) and growth curve chart (G-I).(A) human blood mononuclear cell's period profile figure；(B)MCF-7
Cell cycle distribution figure；(C) ZJU-0327 cell cycle distribution figure；(D) ZJU-0725 cell cycle distribution figure；(E)ZJU-
1127 cell cycle distribution figures；(F) cell cycle statistical chart；(G) ZJU-0327 cell multiplication figure；(H) ZJU-0725 cell times
Increase figure；(I) ZJU-1127 cell multiplication figure.
In Fig. 4 shown in (A-F), normal human lymphocytes are 2 times of bodies, and almost all cell is in cell cycle G0/G1
Phase；MCF-7 and ZJU-0328, ZJU-0725 cell are polyploid, and most cells are located at the G0/G1 phase, secondly when the S phase, most
It is less G2/M；Statistical analysis shows compared with MCF-7 cell, G0/G1 phase ratio: ZJU-0725 decline and ZJU-1127 and
ZJU-0327 cell increases；S phase ratio (SPF) and proliferation index (PI): ZJU-0725 cell increases and ZJU-1127 and ZJU-
The decline of 0328 cell；G2/M phase ratio declines.
Embodiment 6
After ZJU-0327, ZJU-0725 and ZJU-1127 cell tryptase enzymic digestion of culture, it is resuspended in RPMI 1640+
It in 10%FBS culture medium and is counted, final concentration of 2.5 × 10 is obtained after concentration adjusts4/ ml and 5 × 104/ ml's is thin
Born of the same parents' suspension；It is seeded in respectively in 96 orifice plates, every 200 μ l of hole, every group sets 6 multiple holes, and only plus the hole of culture medium is as blank
Control；Culture plate is put into 37 DEG C of incubators and is cultivated, CCK-8 incubation is added in 0,12,24,36,48,60 and 72h time point
Light absorption value is measured at 2.5h, subsequent 450nm.As a result as shown in Fig. 4 (G-I), population doubling time distinguishes: ZJU-0725 and is
57.5h；ZJU-1127 is 18.8h and ZJU-0327 is 18.7h, unrelated with initial inoculating cell density.
Embodiment 7
By close ZJU-0725, ZJU-1127, ZJU-0327, HBL-100, MCF-10A, MCF-7, the T- converged of growth
The cell of 47D, BT-549, MDA-MB-231, PBS are washed 3 times；It is added 1ml RIPA lysate (final concentration of 1nM PMSF),
With rifle piping and druming it is several under, collect lysate；4 DEG C of 12000g are centrifuged 5min, take supernatant, and packing saves.BCA method measures each lysate
Concentration, adjustment concentration is consistent, and 4 times of volume sample-loading buffers, boiling water boiling 5min is added.Preparation SDS-PAGE glue, 20 μ g of loading,
80V electrophoresis is to separation gel and concentration glue interface, and 110V electrophoresis to bromophenol blue is close to the bottom of glue；Transferring film instrument goes to PVDF film；
5% skimmed milk power (TBST) room temperature slowly shakes closing 2h；4 DEG C of antiantibody overnight incubations are added, TBST is washed 3 times,
5min/ times；Diluted secondary antibody incubation at room temperature 2h is added；ECL luminescent solution is incubated for, and the imaging of Western Blot Image analysis system is aobvious
Shadow.
In Fig. 5: (A) HBL-100, MCF-10A, MCF-7, T-47D, BT549, MDA-MB-231, ZJU-0327, ZJU-
The WB of 0725 and ZJU-1127 cell detects figure；(B) detection of mycoplasma agarose electrophoresis figure；(C) radiotherapy sensitivity detection statistics figure；
(D)Docetaxel、Carboplatin、Cyclophosphamide Monohydrate、Paclitaxel、Epirubicin
HCl and 5-Fu chemosensitivity detection statistics figure.
As shown in Fig. 5 A result, it is established that ZJU-0327, ZJU-0725 and the ZJU-1127 phenotype come are Basal/
HER2-positive, and pluripotency stemness is strong and prognostic is poor.
Embodiment 8
The detection of mycoplasma infection is needed while carrying out to cancer cell is grown, this research is sharp using PCR method
It, can be simultaneously with the mycoplasma PCR detection kit (HuaAn PCR Mycoplasma Test Kit) of Hangzhou Huaan biotech firm
48 common mycoplasma species are detected, steps are as follows:
1. kit forms:
1. 400 μ l of PCR reaction solution (20 μ l/ times, 20 secondary responses)
2. 20 μ l of Taq enzyme (1 μ l/ times, 20 secondary responses)
3. 1000 μ l of DNA release liquid (40 μ l/ times, 25 secondary responses)
4. DNA positive control 1 manages (freeze-dried powder)
5. 1000 μ l of atoleine
6. 1 part of operation instructions
2. sample collection and processing:
1. collecting cell: the cancer epithelial cell closely converged will be grown to and scraped from culture dish with cell scraper, cancer will be contained
The culture solution of cell moves in 15ml centrifuge tube；
2. centrifugation: the above cell suspension is centrifuged (12000rpm, 5-l0min)；
9. extracting DNA: removing supernatant, 40 μ l of DNA release liquid is added, move into 2ml EP pipe, sufficiently blow and beat, boil 5min；
4. being centrifuged (12000rpm, 5~l0min) again, take supernatant spare as PCR response sample template.
3. preparing PCR reaction reagent:
1. positive control prepares: DNA positive control powder bottle is first centrifuged (5000rpm, 5min) on centrifuge, then
It opens bottle cap and 25 μ l deionized waters is added, sufficiently dissolve, it is spare；
2. preparing PCR reaction solution: taking out PCR reaction solution from -20 DEG C of refrigerators, 3 repetition test samples are plus positive right
5 reaction solutions should be prepared altogether according to (positive DNA) and negative control (PBS), take PCR reaction solution l00 μ l, and 5 μ l Taq enzymes are added.One
Reaction system needed for secondary property is prepared keeps the homogeneity of PCR reaction.
3. mixing well PCR reaction solution and Taq enzyme, and dispensed with 21 μ l/ pipes into 5 PCR reaction tubes；
4. it is right that 4 μ l ready samples (3 repeating pipes), DNA positive control and PBS feminine gender are respectively added in 5 PCR reaction tubes
According to；
5. PCR reaction tube is centrifuged (10000rpm, l0s).
4.PCR reaction and parameter setting: being put into PCR instrument for PCR reaction tube, carries out PCR reaction by following parameter:
The electrophoresis detection of 5.PCR product:
1. 1 × TAE buffer: taking l ml 50 × TAE electrophoretic buffer that 49ml deionized water is added；
2. prepared by 2.0% Ago-Gel: weighing 1.09g agarose in conical beaker, 50ml l × TAE is added；
Electrophoretic buffer, micro-wave oven dissolve agarose, slightly 5 μ l GelRed nucleic acid dyes of cold rear addition, pour into gel mold
In, cooled and solidified is spare；
3. loading: each PCR pipe takes 8 μ l pcr amplification products, is added in the glue hole of agar offset plate, and amplified production includes bromine
Phenol indigo plant indicator；
4. electrophoresis: stopping electrophoresis according to bromophenol blue indicator after 110V electrophoresis 20min.
6. gel imaging analysis: the offset plate taking-up after electrophoresis being put into Bio-Rad gel image analyser and observes and claps
According to preservation, band identical with positive control occur is mycoplasma positive sample.As shown in B in Fig. 5, the ZJU- that is established
0327, ZJU-0725 and ZJU-1127 is not by mycoplasma contamination.
Embodiment 9
Cell prepares: being suspended in after Primary breast the cancer cell ZJU-0725 and ZJU-1127 of culture are digested with pancreatin
It in RPMI l640 culture solution and is counted, final concentration of 2.5 × 10 is obtained after concentration adjusts4The cell suspension of/ml；
Cell inoculation: the cell suspension of concentrations above is inoculated into respectively in 96 orifice plates, and every hole 0.2ml is (thin containing 5000
Born of the same parents), every group of 6 multiple holes, only plus the not celliferous hole of culture solution is as blank control；
For radiation exposure group: applying the x of 0,2,5,10 and 15Gy after cell to be seeded is adherent respectively to every group of cell
Line irradiation, then continue the cell merging incubator after irradiation to cultivate 72h；
Chemotherapeutic processing group: suitable chemotherapeutic docetaxel is separately added into every group after cell to be seeded is adherent
(Docetaxel), carboplatin (Carboplatin), cyclophosphamide (Cyclophosphamide Monohydrate), taxol
(Paclitaxel), various concentration drug is arranged in epirubicin hydrochloride (Epirubicin HCl) and 5 FU 5 fluorouracil (5-Fu),
It is put into incubator and continues to cultivate specific time；
CCK-8 detection: to above every group of cell, with CCK-8, (1:10 dilutes, and 37 DEG C incubate respectively after the specifically processing time
Change 2.5h) Activity determination is carried out, and every group of cell is recorded in the absorbance value of 450nm, utilize Gradpad Software on Drawing curve.
As shown in figure result 5C, Sk-Br3 and ZJU-0725 are sensitive to radiotherapy, and as dose of radiation increases, inhibiting rate rises, and
ZJU-1127 is insensitive to radiotherapy.As shown in D in Fig. 5, the chemotherapeutic of various concentration removes Docetaxel and Epirubicin HCl
Have outside partial inhibition to ZJU-0725, other chemotherapeutics do not have obvious inhibiting effect to ZJU-0725 and ZJU-1127.
Embodiment 10
1. cell prepares: after being centrifuged (800rpm, 5min) after three plants of breast cancer cell pancreatin of exponential phase of growth are digested
Centrifugation 3 times is washed with serum free medium；
2. cell is resuspended in PBS buffer solution, counting adjustment cell density is 5 × 107/ml；
3. the above cell suspension of 0.2ml (is contained about 1 × 107A cell) it is injected into 4-6 weeks BABL/c back of mice
Subcutaneously (n=3), mouse raising is in SPF grades of laminar flow；
4. observation 4 weeks palpation and measures Tumor diameter size weekly；
Mouse is euthanized after 5.4 weeks, knurl measurement is taken out and takes pictures, take part knurl to be soaked in 10% formalin molten
Liquid carries out routine paraffin wax embedding and microsection manufacture, spare.
Fig. 6 is ZJU-0327, nude mice (nu/nu, the BABL/C) mouse of ZJU-0725 and ZJU-1127 cell in athymia
Subcutaneous institute is at knurl and knurl growth curve.(A) tumour that ZJU-0327 cell inoculation is subcutaneously grown after two weeks, the lower left corner are
Knurl is substantially schemed.It is that HE dyes pathology figure above right side, prompting the tumour is poor differentiated carcinoma；(B) ZJU-0725 cell inoculation two
The tumour subcutaneously grown after week, the lower left corner are that knurl is substantially schemed.It is that HE dyes pathology figure above right side, it is low for prompting the tumour
Break up cancer；(C) tumour that ZJU-1127 cell inoculation is subcutaneously grown after two weeks, the lower left corner are that knurl is substantially schemed.Above right side
Pathology figure is dyed for HE, prompting the tumour is poor differentiated carcinoma.
As shown in fig. 6, ZJU-0327, ZJU-0725 and the ZJU-1127 equal tumor formation on nude mice, and with inoculation time
Extend, transplantable tumor tumor volume is gradually increased；Pathologic finding shows to be low differentiation tumor of breast.
Embodiment 11
Design of primers: primer reference literature report；
Extracting genome DNA: qiagen DNA kit extracts complete genome DNA；
PCR:PCR condition carries out PCR in PCR instrument referring to the Tm temperature of respective primer；
Exon sequencing: it after gel electrophoresis, cuts target gel band and Hangzhou Qing Xike biotech firm is transferred to complete to survey
Sequence work.
Fig. 7 is TP53 abrupt climatic change result figure.4 point mutation of (A, B) exon: single nucleotide polymorphism；(C, D) exon
5 point mutation: missense mutation.
Three plants of breast cancer tumour TP53 as shown in Fig. 7 result occur C in the 72nd bit codon of exon 4 and are mutated into G,
Amino acid becomes arginine (Arg) from proline (Pro), which belongs to single nucleotide polymorphism；Exon 5 the 126th close
There is T and is mutated into A in numeral, and amino acid becomes asparagine (Asn) from tyrosine (Try), belongs to missense mutation.
Embodiment 12
1. colchicine (final concentration of 0.05mg/ml) is added into three plants of breast cancer cell culture solutions of exponential phase of growth
Act on 6 h；
2. cell is collected in 15ml after washing and use collected by trypsinisation cell, PBS washing to be centrifuged after discarding culture solution
It is centrifuged bottom of the tube；
3. Hypotonic treatment: hypotonic KCl solution (0.075mol/L) 4- being fully warmed-up in 37 DEG C of water-baths is added
5ml acts on 20min, abundant turgid cell in 37 DEG C of water-baths；
4. pre-fixing: being directly added into the fixer (methanol: glacial acetic acid, 3:l, V of l ml Fresh in above-mentioned hypotonic medium
/ V), gently piping and druming uniformly, is centrifuged (1500rpm, 10min) after fixed 5min, abandons supernatant；
5. fixing again: the fixer of 6-8ml Fresh, the fixed 30min of room temperature is added；
6. blowing piece: 5-6 drop fixer will be added again after the above specimen centrifuge, examination takes 1 drop, it drips on 4 DEG C of distilled water slides,
It is toasted rapidly on alcolhol burner flame and blows piece；
7. Giemsa is dyed: appropriate Giemsa working solution is added dropwise in the above surface of glass slide blown and dyes 10-15min, from
Naturally dry obtains metaphase chromosome sample after water rinses slide.
Fig. 8 is ZJU-0327, ZJU-0725, the representative single celled karyotyping of ZJU-1127 cell.(A)ZJU-0327
35th generation unicellular representative caryogram；(B) the 25th generation of ZJU-0725 unicellular representative caryogram；(C) the 23rd ZJU-1127
For unicellular representative caryogram.
As shown in figure 8, caryogram map, which is shown in the three plants of cells established in chromosome number and structure, there is exception,
Chromosome number is hypo-triploid caryogram, is distributed between 78-85 mostly, and textural anomaly includes chromosome increase, missing and transposition
Deng.
Above-mentioned specific embodiment is used to illustrate the present invention, is merely a preferred embodiment of the present invention, rather than to this
Invention is limited, and within the spirit of the invention and the scope of protection of the claims, to any modification of the invention made, is equal
Replacement, improvement etc., both fall within protection scope of the present invention.
Claims (7)
1. human breast cancer cell line, which is characterized in that the human breast cancer cell line includes: to be respectively designated as human breast cancer cell
It is ZJU-0327, ZJU-0725 and ZJU-1127, and is preserved in China typical culture collection center, deposit number is respectively as follows:
The human breast cancer cell line of CCTCC NO:C2017171, CCTCC NO:C2017172 and CCTCC NO:C2017173.
2. human breast cancer cell line according to claim 1, which is characterized in that the human breast cancer cell line further include:
It is respectively designated as human breast cancer cell line ZJU-0327, ZJU-0725 and ZJU-1127, and is preserved in Chinese Typical Representative culture guarantor
Hiding center, deposit number are respectively as follows: CCTCC NO:C2017171, CCTCC NO:C2017172 and CCTCC NO:C2017173
Human breast cancer cell line progeny cell system.
3. human breast cancer cell line according to claim 1 or 2, which is characterized in that the human breast cancer cell line is in body
It is typical polygon epithelioid cell when outer culture, under light microscopic, is typical Malignant Epithelium feature under Electronic Speculum；Karyotyping knot
Fruit is hypo-triploid caryogram, and mainstream chromosome number is 78-85.
4. human breast cancer cell line according to claim 1 or 2, which is characterized in that the human breast cancer cell line is in cream
There is infiltrative breast carcinoma characteristic, characterization phenotype is Basal/HER2-positive when gland cancer clinical diagnosis.
5. human breast cancer cell line according to claim 1 or 2, which is characterized in that the human breast cancer cell line is right
11 exons of TP53 mutated gene are sequenced down, and the polymorphism of mononucleotide occurs for the 72nd coding of exon 4
Missense mutation occurs for the 126th coding of mutation, exon 5.
6. human breast cancer cell line according to claim 1 or 2, which is characterized in that the human breast cancer cell line is being propped up
When Mycoplasma Detection Reagent box detects, PCR result is feminine gender, is not affected by mycoplasma contamination.
7. a kind of human breast cancer cell line according to claim 1 or claim 2 is ground in the pathogenesis of breast carcinoma mechanism for preparing Her/Basal
The application in cell model studied carefully.
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| CN111019897B (en) * | 2019-02-14 | 2023-08-11 | 中山大学孙逸仙纪念医院 | Human benign phylliform tumor cell line GLK-1010 and application thereof |
| CN111019899B (en) * | 2019-02-14 | 2023-08-04 | 中山大学孙逸仙纪念医院 | Human malignant foliar tumor cell line LJ-0429 and application thereof |
| CN113088493B (en) * | 2021-03-17 | 2021-10-08 | 浙江大学 | Human breast cancer hydrothorax metastasis cell line and application thereof |
| CN114015655B (en) * | 2021-12-09 | 2023-09-05 | 北京和合医学诊断技术股份有限公司 | HBRCA-959 cell line and culture method and application thereof |
| CN117070462B (en) * | 2023-08-17 | 2024-02-23 | 镜像绮点(上海)细胞技术有限公司 | Isolation and purification of primary cells |
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