CN109370990A - Colon cancer cell line, its method for building up and application in people - Google Patents
Colon cancer cell line, its method for building up and application in people Download PDFInfo
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Abstract
The present invention relates to colon cancer cell line cc-006cpm8 and its method for building up and application in a kind of new people, the cell strain is deposited in China typical culture collection center, and deposit number is CCTCC NO:C2016153.Colon cancer cell line cc-006cpm8 can stablize passage in people of the invention and proliferation is very fast, and to study the generation of colorectal cancer, development is shifted, and then the pathogenesis for disclosing colorectal cancer provides certain research clue and experimental model.Meanwhile the platform that molecular biology, immunological investigation and the Comprehensive Clinical diagnosis and treatment etc. for establishing cc-006cpm8 cell strain not and be only colorectal cancer provide advantage, and provide convenience for screening prevention and treatment colorectal cancer drug.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to colon cancer cell line and its method for building up in a kind of people
And application.
Background technique
Colorectal cancer is common malignant tumour, including colon cancer and the carcinoma of the rectum.For site of pathological change, the morbidity of colorectal cancer
Rate is followed successively by rectum, sigmoid colon, caecum, colon ascendens, colon descendens and transverse colon from high to low, there is proximally (right half hitch in recent years
Intestines) development trend.Its morbidity is in close relations with life style, heredity, Colorectal Adenomas etc..Age of onset becomes astogeny, men and women
The ratio between be 1.65:1.
According to the literature, there are about 1,200,000 patients to be diagnosed as colorectal cancer in the annual whole world, wherein be more than 600,000 it is direct or
It connects and dies of the disease.In the U.S., there are about the colorectal cancer cases that 14.7 ten thousand newly make a definite diagnosis within 2009, and have 4.99 ten thousand deaths.
In China, the morbidity number and death toll of colorectal cancer in 2005 are respectively 17.2 ten thousand and 9.9 ten thousand, and new cases are with annual
4.2% speed increases, and far surpasses 2% world level.
Tumour cell in vitro culture is to study the preferable means of Carcinogenesis Mechanism and cellular biology of tumor characteristic, is trained in tissue
It is occupied an important position in supporting.In view of people at present to the limitation of Colorectal Cancer, Preventive mechanism understanding, body is established
The Human colorectal cancer cells strain of outer culture can further apparent colorectal cancer cell biological characteristics and occurrence and development mechanism.
On the other hand, tumor cell line/strain, which is established, also provides convenience and relatively inexpensive cell model for screening anticancer medicine etc..
Tumor cell line/the strain at the people's Colon and rectum position reported in world wide at present has tens of kinds, such as external SW480 established,
W403、Colo-320、LoVo、CW-2、HT-29、LIM l215、LS l74T、T84、Caco-2、HCT-8、Colo205、
SW620, HCTl16, HR-78, CoLo320MD etc..Most of these cell lines come from primary tumor site, and respective source is in abdomen
Water.China is the district occurred frequently of colorectal cancer, but the work for establishing colon or rectum cancer cell strain is started late.It is reported to be derived from
Colorectal cancer cell system/the strain in China only has THC8908, SIC-8101, HR-8348, Hce8693, HRC-99 and DXH-1.This
Wherein, most cell strains due to cell contamination or are saved improper and have not been available；Other cell strain is not due to submitting state
The generally acknowledged preservation mechanism in border is standardized identification, whether can be advantageously used in scientific research and industrial use is that there are queries
's.Mono- colorectal cancer cell system of the only DXH-1 announced in National Laboratory cellular resources shared platform at present.Therefore, more
The colon cancer cell line of Chinese's genetic background is established on ground, and carries out China's colorectal cancer biological characteristics and morbidity machine using it
The research of system is particularly important.
Since tumour has the characteristics that heterogeneous, complexity and by not agnate and region bring otherness, even if
It is the cell line/strain set up with the tumour originally culture of same area, correlative study result cannot may also reflect completely
Whole features of disease.In addition, the tumour cell of in vitro culture is in and internal entirely different living environment, same cell strain
The biological characteristics after passing on repeatedly cause the uncertainty of experimental result it can also happen that change.Based on this, selection is had been established
The research that the cell strain of the west ethnic origin of long period carries out China's colorectal cancer is unfavorable for accurately analyzing China's knot directly
Intestinal cancer pathogenesis and therapeutic strategy.Therefore, this field exists is carried out using the colorectal cancer from Chinese's genetic background
The demand of tumour cell originally culture research, establishing more colorectal cancer cell system/strains is very important.
Summary of the invention
For the deficient status of above-mentioned colorectal cancer cell lines with Chinese's genetic background/be, in a first aspect, this
Invention provides a kind of new middle colon cancer cell line cc-006cpm8 with Chinese's genetic background, the cell strain
It is deposited in China typical culture collection center (address: Wuhan University of wuchang, wuhan area of Hubei China province collection), protects
Hiding number is CCTCC NO:C2016153, the deposit date is on August 9th, 2016, classification naming are as follows: low differentiation colon cancer in people
Cell strain.
Cc-006cpm8 cell strain character of the invention is stablized, and can stablize passage more than 100 generations, tumor formation rate is high, homogeneity
It is good, a kind of experimental material with new clinical tumor biological characteristics is provided for China's colorectal cancer research.
Cell strain of the invention is derived from a nearly ileocecus with Right-sided Colon Cancer with 89 years old female patient of hepatic metastases
Operation excision sample (in profit ulcer type gland cancer is invaded), which was not used chemotherapeutics.For the colon cancer group of operation excision
Block is knitted, (every milliliter of DPBS contains 1000U benzyl penicillin, 1000 μ g streptomycin sulphates in three anti-(10 times) DPBS liquid at 4 DEG C first
With 2.5 μ g amphotericin Bs) in impregnated 1-2 hours, effectively overcome pollution.Then, by three anti-(10 times) of tissue
DPBS liquid is washed 2 times, then is shredded tissue and be seeded in the 6cm plate of RPMI1640 complete medium and cultivated.It is poor using pancreatin
Speed sticks method removal fibroblast, obtains purer Epithelial Tumors cell (cc-006cp cell line) after 5 times are handled repeatedly.
When separating cell strain, cc-006cp cell line is formed through soft agar culture first and is cloned, then the monoclonal in picking agar exists
Expand culture in Liquid Culture, to obtain the monoclonal cell of the high oncogenicity of high-purity, which is named as cc-
006cpm8。
The cell strain has following biological characteristics:
1, cell adherent growth, contactless inhibition, in superposition growth；
2, cell doubling time is close to 27h；
3 ﹑ cells, which can pass on, reached for > 100 generations, and cell quality still keeps stable；
4, cell strain has very strong clonality, and agar colony formation rate is 73.2%；Planting plate efficiency is
66.5%；
5, cellular immunity groupization is as the result is shown: this plant of cell is in keratin (+++), vimentin (-), EMA (-), MLH1
(-), MSH2 (2+), MSH6 (3+), PMS2 (-), CEA (3+), p53 (-), Ki-67 stained positive rate about 50%；
6, chromosome analysis is as the result is shown: this plant of cell is stable hypo-triploid (46 < chromosome number≤69) cell, dye
Colour solid mode is 50, and the cell between 50 ± 3 chromosomes accounts for 84.2%；
7, full exon genes detection display: this plant of cell is MMR mutant clone, KRAS, NRAS ﹑ BRAF ﹑ APC,
The gene mutations situation such as TP53, SMAD4 detailed in Example 2；
8, tumor formation rate is high: the whole tumor formations after being inoculated with this plant of cell of 5 nude mices；
The results are shown in Table 1 for the analysis of 9 ﹑ short tandem repeat.
1 short tandem repeat result of table
The result is carried out cell STR with ATCC ﹑ DSMZ ﹑ JCRB and National Laboratory resource cell shared platform to compare,
The cell homologous with cc-006cpm8 is not found, illustrates that the cell is unique.
In second aspect, the present invention provides a kind of methods for obtaining monoclonal cell strain, and the method includes walking as follows
It is rapid: 1, to prevent from polluting: since intestinal cancer specimens from pri is carried out under open condition, easily pollute, soaked using high concentration antibiotic
Bubble, can effectively overcome pollution；2, cell culture: sample tissue is shredded in the plate for being inoculated in 6cm, and culture solution is added, sets 37
DEG C, 5%CO2It is cultivated in incubator；3, it separates cell: sticking method removal fibroblast using pancreatin differential, through multiple
After processing, the higher Epithelial Tumors cell line of purity is obtained；4, soft-agar cloning monoclonal cell culture: is carried out to cell line
Separation, in 37 DEG C, 5%CO2Culture growth forms colony in incubator, periodically with micro- sem observation during Colony forming, really
Its fixed Clonal Origin, selects monoclonal；5, picking purpose clone inoculation in liquid medium, sets 37 DEG C, 5%CO2Incubator
Middle expansion culture, forms the monoclonal cell strain of sufficient amount.Being generally separated unicellular strain is using infinite dilution Fa ﹑ or plate gram
Grand method.And the present invention is to separate cell strain by soft-agar cloning.Since normal cell cloning efficiency is weak in soft agar
(0.05-5%, even zero), and tumor cell cloning efficiency is strong (> 10%), and in the prior art, soft-agar cloning is formed
Method is normally used for observing cell colony dependence and proliferative capacity the two important characters.And the present invention dexterously utilizes this
Feature more effectively separates the unicellular strain of tumour.Cc-006cp cell line is in soft agar in the monoclonal shape of dispersion, 12-15
It when grow it is vigorous.At this moment accurate monoclonal cell is carried out again to select, when picked clones without plus pancreatin, utmostly reduce
The damage of cell, the clone cell then chosen amplification cultivation in RPMI1640 complete medium.Pass through the above method, success
Ground establishes colon cancer cell line cc-006cpm8 in the people for the high oncogenicity of high-purity for capableing of continuous passage.
In the third aspect, the present invention provides the purposes of colon cancer cell line in cc-006cpm8 people.The use
Way includes but is not limited to: (animal) screening prevention and treatment colorectal cancer drug in vitro or in vivo；Study the genesis mechanism of colorectal cancer；It builds
The cell model that vertical colorectal cancer occurs, develops or shifts；Establish colorectal cancer animal (including but not limited to mouse, rat etc.
Common experimental animal) model；Establish research colorectal cancer differentiation mechanism, cellular morphology and dysfunction, tumor invasion and metastasis machine
Make and instruct the cell model of Comprehensive Clinical diagnosis and treatment.The method for building up and particular use of cell model be all it is known in the art,
It can be designed and be used according to specific situation.
Beneficial effects of the present invention are as follows: colon cancer cell line in the people by providing a kind of novel stabilising passage
Cc-006cpm8, to study the generation of colorectal cancer from now on, development is shifted, and then the pathogenesis for disclosing colorectal cancer provides one
Fixed research clue and experimental model.Meanwhile it establishing cc-006cpm8 cell strain and not being only colorectal cancer molecular biology, be immunized
It learns and the researchs such as Comprehensive Clinical diagnosis and treatment provides advantage, and put down for what screening prevention and treatment colorectal cancer drug provided convenience
Platform.
Detailed description of the invention
For above and other purpose, feature, advantage and embodiment of the invention can be clearer and more comprehensible, to saying for institute's attached drawing
It is bright as follows:
Fig. 1 is the figure (× 20) that cc-006cpm8 forms clone in agar, and scale bar shows 200 μm.
Fig. 2 is the morphological observation figure (× 10) of cc-006cpm8 under phase contrast microscope, and scale bar shows 200 μm.
The HE that Fig. 3 is cc-006cpm8 dyes aspect graph (× 40), and scale bar shows 40 μm.
Fig. 4 is cc-006cpm8 transmission electron microscope aspect graph (× 10000).
Fig. 5 shows that the keratin immunohistochemistry of cc-006cpm8 is positive (× 40), and scale bar shows 40 μm.
Fig. 6 shows that the vimentin immunohistochemistry of cc-006cpm8 is negative (× 40), and scale bar shows 40 μm.
Fig. 7 shows that the EMA immunohistochemistry of cc-006cpm8 is negative (× 40), and scale bar shows 40 μm.
Fig. 8 shows that the CEA immunohistochemistry of cc-006cpm8 is in strong positive (× 20), and scale bar shows 40 μm.
Fig. 9 shows that the p53 immunohistochemistry of cc-006cpm8 is negative (× 20), and scale bar shows 40 μm.
Figure 10 shows that the ki-67 immunohistochemistry positive rate about 50% (× 20) of cc-006cpm8, scale bar show 40 μm.
Figure 11 shows that the MLH1 immunohistochemistry of cc-006cpm8 is negative (× 40), and scale bar shows 40 μm.
Figure 12 shows that the MSH2 immunohistochemistry of cc-006cpm8 is positive (× 40), and scale bar shows 40 μm.
Figure 13 shows that the MSH6 immunohistochemistry of cc-006cpm8 is in strong positive (× 40), and scale bar shows 40 μm.
Figure 14 shows that the PMS2 immunohistochemistry of cc-006cpm8 is negative (× 40), and scale bar shows 40 μm.
Figure 15 shows the cell growth curve of cc-006cpm8.Abscissa is the time (h), and ordinate is degrees of fusion (%).
Figure 16 shows the kind plate efficiency violet staining figure of cc-006cpm8.
Figure 17 a and Figure 17 b show cc-006cpm8 chromosome karyotype analysis figure.
Figure 18 shows the internal Tumor formation experimental result of immunodeficient mouse inoculation cc-006cpm8.
Figure 19 shows the colon cancer tissue biopsy slice HE colored graph (× 40) of the patient in the source cc-006cpm8.
Specific embodiment
In order to keep the objectives, technical solutions, and advantages of the present invention clearer, below in conjunction with the drawings and the specific embodiments,
The present invention will be described in further detail.It should be appreciated that the specific embodiments described herein are only used to explain this hair
It is bright, it is not intended to limit the present invention.Experimental method described in following embodiments is unless otherwise specified conventional method.It is following
Experimental material used in embodiment, unless otherwise specified, conventional biochemical reagent are purchased from GIBCO company.
Reagent:
1.DPBS solution
2.RPMI1640 culture medium
3. three anti-(10 times) DPBS liquid: every milliliter of DPBS contains 1000U benzyl penicillin, 1000 μ g streptomycin sulphates and 2.5 μ g
Amphotericin B.
4.RPMI1640 complete medium: 10% inactivated fetal bovine serum, 100U/ml penicillin is added in RPMI1640 culture medium
G, 100 μ g/ml streptomycin sulphates and 0.25 μ g/ml amphotericin B.
5. pancreatin: 0.025w/v% trypsin solution.
6. cells frozen storing liquid: fetal calf serum: DMSO=9:1 (v/v) (is purchased from ScienCellTM Research
Laboratories)。
Specimen origin:
The attached Nanjing hospital Oncological Surgery Right-sided Colon Cancer companion Patients with Liver Metastasis of Nanjing Medical University, 89 years old, women.Art
Before solicit sufferers themselves agreement, endorsed informed consent form.Row Right-sided Colon Cancer radical correction.From primary cancerous tissue obtain sample into
Row cell culture.Postoperative pathological is diagnosed as right hemicolon and invades poorly differentiated adenocarcinoma in profit ulcer type.
Colon cancer cell line cc-006cpm8 is established in 1 people of embodiment
It establishes cell line: for the cancerous tissue in the colon cancer sample of operation excision, is immersed in three anti-DPBS liquid immediately,
It send to laboratory rapidly.It is rinsed twice with three anti-(10 times) DPBS liquid, cancerous tissue is soaked in three anti-(10 times) DPBS liquid at 4 DEG C
Middle 1-2h.It is rinsed twice with three anti-(10 times) DPBS liquid again, tissue is shredded, is inoculated in the disposable plastic plate of 6cm, adds
Enter RPMI1640 complete medium, is placed in 37 DEG C, 5%CO2It is cultivated in incubator.Changed according to culture solution pH, every 2-3 days
It carries out half amount and changes liquid.Stick method removal fibroblast using pancreatin differential, establishes Epithelial Tumors cell line cc-006cp.
It establishes cell strain: cc-006cp cell line being isolated into monoclonal cell strain using soft agar assay.Specifically, will
0.6% low melting-point agarose-RPMI1640 complete medium is poured into 6cm culture dish, and bottom-layer agar is made；1000 will be contained
Low melting point agar-RPMI1640 the complete medium of the 0.3% of a cc-006cp cell is prepared into top-layer agar, be placed in 37 DEG C,
5%CO212-15 days formation colonies of culture growth, directly observe light field at inverted phase contrast microscope (Olympus) in incubator
Take pictures (Fig. 1).
Picking monoclonal is added in RPMI1640 complete medium, is placed in 37 DEG C, 5%CO2Expand culture in incubator.It should
Method has been successfully set up colon cancer cell line cc- in the people for the high oncogenicity of high-purity that one plant is capable of continuous passage
006cpm8。
The biological characteristics detection of colon cancer cell line cc-006cpm8 in 2 people of embodiment
1, cytomorphology
(1) living cells is observed: directly being observed under phase contrast microscope (Olympus CKX41 type), is had taken cc-
The living cells (Fig. 2) in the 10th generation of 006cpm8.As can be seen that cell is grown in adherent overlapping, contactless suppression.
(2) HE is dyed: cell reached for the 10th generation, and cell number is up to 106-107When, carry out specimens paraffin embedding slices, HE dyeing and
Under the microscope.As shown in figure 3, cell is in pernicious class Epithelial form, nuclear proportion is big, and kernel is multiple, and cell differs in size
Core, be clear to mitosis figures.
(3) transmission electron microscope observing: cell reached for the 10th generation, and cell is in logarithmic growth phase, and cell number is up to 106-107When collect
Centrifugation, fixes inspection with 2.5% glutaraldehyde.It observes and takes pictures under JEM-1011 type transmission electron microscope.As shown in figure 4, cc-
006cpm8 nucleus is larger, oval or round, there is multiple kernels, and Distribution of chromatin is uniform, and euchromatin is relatively abundant；Clearly
There are the organelles such as ribosomes abundant, mitochondria, rough surfaced endoplasmic reticulum (RER), lysosome in clear visible cytoplasm；There are many micro- for cell surface
Villus, it is seen that intercellular tight connection.
2 ﹑ cellular immunity groups
Reached for the 10th generation in cell, cell number is up to 106-107When be enriched with, carry out specimens paraffin embedding slices, using Envision bis-
Footwork does immunohistochemistry.The keratin of cc-006cpm8 cell is expressed as positive (Fig. 5)；Vimentin is feminine gender, endochylema
Without brown chromatic colorant (Fig. 6), meet epithelial origin characteristic.In addition, EMA (-) (Fig. 7), CEA (3+) is presented in cc-006cpm8 cell
(Fig. 8), p53 (-) (Fig. 9) and the positive rate (Figure 10) of Ki-67 dyeing about 50%.These results further show the cell
Related neoplasms biological nature and heterogeneity.In addition, detecting DNA mismatch revision points (MMR) albumen table with ImmunohistochemistryMethods Methods
Up to situation, show that cc-006cpm8 cell is MLH1 (-) (Figure 11), MSH2 (2+) (Figure 12), MSH6 (3+) (Figure 13), PMS2
(-) (Figure 14), these show the cell, and there are two types of DNA mismatches to repair protein expression missing, illustrates cc-006cpm8 for MMR mutation
Cell strain.
3, cytogene abrupt climatic change
Using Integrated DNA Technologes (IDT) the full exon trapping of people (Exome
Research Panel) kit, the implementation exon group capture of illumina Hiseq microarray dataset.With high-throughput information collection
Technology carries out information collection to the DNA fragmentation of acquisition, and using PE150 sequencing strategy, average each sample generates 10Gb clean
data.The data obtained carries out SNVs and InDels annotation statistics.Detect that there are 22863 monokaryon glycosides in the cellular exons region
Sour Mutation (SNVs), wherein same sense mutation 11257, missense mutation 11539, initiation codon loss 37, termination are close
Code 30 in advance.Detecting exon region, there are 168 insertion/deletions (InDels), and wherein frameshift mutation 165, originate
Password loss 2, termination codon shifts to an earlier date 1.It is carried out with Chinese population genome, Hapmap database and international thousand human genomes
It compares, 95% the above are common mutations (MAF > 0.05 common mutation) in the cell, and 12 are mutated (low for low frequency
Frequency mutation, 0.05>MAF>0), 3 are rare mutation (rare mutation, 0<MAF<0.01).Described 3
A rare mutation is ENO1 (rs3737148), HIST1H1C (rs12111009) and HIST1H2BD (rs2298091).
Exon SNVs result of the colorectal cancer common mutations gene in this cell strain is as follows: KRAS (-), NRAS (-) ﹑
BRAF (p.Val600Glu) ﹑ APC (p.Val1822Asp), TP53 (p.Pro72Arg), SMAD4 (p.Arg420His),
PIK3CA(p.Glu545Gly)、DCC(p.Phe23Leu、p.Arg201Gly、p.Gln460His)、MCC(p.Lys380Arg)、
MLH1(-)、MLH3(p.Thr942Ile、p.Asn826Asp)、MSH2(p.Gln915Arg)、MSH3(p.Ile79Val、
p.Gln949Arg,p.Ala1045Thr),MSH6(-),PMS2(p.Lys541Glu,p.Thr485Lys).And KRAS, NRAS ﹑
The exon InDels inspection of BRAF ﹑ APC, TP53, SMAD4, PIK3CA, DCC, MLH1, MLH3, MSH2, MSH3, MSH6, PMS2
Surveying result is feminine gender；There is frameshift mutation (p.Ser15_Gly16insGly) in MCC.
4, cell growth curve measures
The 7th generation cell for being in logarithmic growth phase is obtained, is made 1 × 103/ ml single cell suspension, then according to 1000 μ l/
Hole is inoculated in 24 orifice plates, has done 5 multiple holes.It is placed on JuLi Stage real-time cell calculating instrument and carries out cell count, per hour
Record is primary, records 8 days altogether.The growth curve that cell fusion degree changes over time is drawn, as shown in figure 15.According to following formula
It calculates cell population doublings time (PDT): PDT=(t-t0)〔lg2/(lgNt-lgN0)), average times of cc-006cpm8 cell
Increasing the time is about 27h.
5, soft-fractrue rock mass is tested
Bottom-layer agar is prepared with -1640 complete medium of 0.6% low melting-point agarose covering 6cm culture dish, 0.5ml is added
- 1640 complete medium of 0.3% low melting point agar containing 1000 cc-006cpm8 cells is prepared into top-layer agar, set 37 DEG C,
5%CO2It is cultivated 10 days in incubator, the colony containing 50 or more cells is counted, calculate cell colony formation rate.Colony
Formation rate=clone's number/inoculating cell number × 100%.The results show that the colony-forming efficiency of cc-006cpm8 cell is 73.2%.
6, plate efficiency is planted
Finely dispersed 6th generation cell is inoculated with every 100 cells/6cm plate, 3 parallel controls are set, is trained
It supports 9 days.First fixed with methanol after culture, then plus violet staining, counted to containing the clones of 50 or more cells.Kind
Plate efficiency=formation colony number/inoculating cell number × 100%.As shown in figure 16, kind plate efficiency is approximately equal to 66.5%.
7, chromosome analysis
The generation cell of the 5th, 10 and 25 of logarithmic growth phase respectively, respectively does 20 karyotypings.Cell through colchicine at
It manages 2h and after 0.075mol/L KCl carries out Hypotonic treatment, is fixed with methanol-glacial acetic acid, ice humidity strip drop piece, after aged at room temperature
It is handled with pancreatin, then carries out the aobvious band analysis of Giemsa dyeing.As the result is shown: it is respectively similar for modal number, illustrate the cell
Strain chromosome number is more stable.This cell strain modal number is 50, accounts for 84.2% between the cell of 50 ± 3 chromosomes, belongs to
Hyperdiploid.Figure 17 a and Figure 17 b be 50 articles of chromosomes of the 10th generation cell karyotyping: xx ,+7 ,+9 ,+9 ,+12, -15 ,+
18.Be also observed other cell chromosome caryogram :+8 ,+10 ,+12 ,+13 ,+14 ,+16 ,+19 ,+21 and+x, but with+7 ,+8 ,+
9 ,+12 ,+18 frequencies of occurrences are height.As it can be seen that this cell strain has the feature of malignant cell.
8 ﹑ short tandem repeat (short tandem repeat, STR) analysis
Short tandem repeat is also known as microsatellite DNA, refers on chromosome by several base-pairs as core unit (2-6
A base-pair), (number of repetition is 10~60 times or higher to a kind of DNA sequence dna that tandem sequence repeats are formed, and genetic fragment is in 400 alkali
Base is to following)；Each duplicate number of core unit will appear individual difference, to form the different equipotential base of fragment length
Cause.Therefore, the number of repetition of one group of STR sequence is almost unique in Different Individual, is the gene identities feature of individual,
It is the main method that cell biology identifies cell identity and source.The STR cellular identification of this cell strain uses
Goldeneye20A kit carries out Capillary Electrophoresis using ABI3730xd instrument, and Gene Mapper4.0 software is analyzed and obtained
It arrives.The result of STR analysis is displayed in Table 1.
9, tumor formation is tested in immunodeficient mouse body
4 week old BALB/C mices (being purchased from Yangzhou University's comparative medicine center, quality certification number No.201701126) 5 are taken,
Mouse back subcutaneous injection is carried out to side shoulder and opposite side buttocks respectively, each site is with 5 × 107A cell/ml injects 100 μ l.
1 week i.e. visible transplantable tumor is grown after nude mice by subcutaneous inoculating cell.As shown in figure 18, after four weeks, 5 nude mice whole tumor formations, transplanting
Tumor is in nodositas, has adhesion with skin.
10, the pathological section of tumour
It takes patient's intestinal cancer tissue to carry out biopsy, carries out HE dyeing after the fixation of formalin routine, specimens paraffin embedding slices.Such as figure
Shown in 19, HE coloration result shows that Cell differentiation degree is lower, and nucleus differs in size, and nuclear staining is deep, it is seen that pathology division
Picture, glandular tube shape are imperfect.
The foregoing is merely the preferred embodiment of the present invention, are not intended to limit the invention, claimed
Range is limited by claims and its equivalent replacement mode, done within the spirit and principles of the present invention any
Modifications, equivalent substitutions and improvements etc., should all be included in the protection scope of the present invention.
Claims (4)
1. colon cancer cell line cc-006cpm8 in a kind of people, the cell strain are preserved in China typical culture collection
Center, deposit number are CCTCC NO:C2016153.
2. application of the cell strain as described in claim 1 in the cell model for occurring as human large intestine cancer, developing or shifting.
3. cell strain as described in claim 1 is in the application established in large intestine carcinoma animal model.
4. cell strain as described in claim 1 is in the genesis mechanism of research colorectal cancer and/or screening prevention and treatment large intestine cancer drug
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| CN105296430A (en) * | 2015-09-29 | 2016-02-03 | 浙江大学 | Human colon carcinoma cell line DXH-1 and application thereof |
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| CN114540392A (en) * | 2022-03-02 | 2022-05-27 | 南京市第一医院 | Preparation method and application of basic fibroblast growth factor |
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