CN106148409A - A kind of method for building up of the colorectal cancer animal model with inflammatory basis - Google Patents
A kind of method for building up of the colorectal cancer animal model with inflammatory basis Download PDFInfo
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Abstract
The present invention provides the method for building up of a kind of colorectal cancer animal model with inflammatory basis, and step is as follows: the first step, the structure of pBabe CMV EGFP puro plasmid；Second step, the foundation of the EGFP process LAN system of retrovirus-mediated method；3rd step, stably expresses the foundation of the colorectal cancer cell system of EGFP；4th step, the foundation of A group nude mice oxter colorectal cancer xenografts model；5th step, the foundation of B group nude mice inflammatory model: the 6th step, there is the foundation of the colorectal cancer animal model of inflammatory basis: extract the tumor of A group nude mice oxter, shred to diameter about 2mm tumor mass with Sterile ophthalmic, take a tumor mass, directly it is seamed on B group nude mice Colon and rectum intestinal wall, repeat step in the 5th step, continue to raise one week, again repeat step in the 5th step, continue to raise one week, obtain carcinoma animal model.The animal model of the foundation of the present invention is able to verify that the existence of tumor, and assesses the size of tumor.
Description
Technical field
The invention belongs to biological technical field, particularly relate to a kind of colorectal cancer animal model with inflammatory basis
Method for building up.
Background technology
Adult's tumor of epidemiology and clinical research confirmation about 25% is caused by chronic inflammatory disease.Such as, stomach, liver and
The slow virus of cervix uteri or antibacterial infect and lung is exposed to tobacco smoke or other environmental compound for a long time, all can substantially promote
The chronic tissue injury of these organs and tumor subsequently is made to occur.There is colorectal cancer in chronic ulcerative colitis (Crohn is sick)
Risk calibration ordinary person high 4～20 times；The hemorrhagic ulcer Colon and rectum inflammation risk that cancerates is bigger, and the course of disease, more than 10 years persons, has
50% develops into cancer；The application of anti-inflammatory preparation is relevant with the reduction of colorectal cancer, breast carcinoma and incidence gastric cancer rate.
Although the relation of inflammation and cancer is clearly, and is referred to as " the seventh-largest feature of cancer ", but chronic inflammatory disease exists
The potential mechanism played a role in cancer understands not yet, the molecule machine that chronic inflammatory disease promotes tumor cell migration, attacks and shift
System still needs to be studied.Promote that the research of colorectal cancer growth transfer still is limited to cell in vitro level about inflammation at present,
I.e. use various inflammatory factor to process colorectal cancer cell, observe the impact of cell growth transfer ability.But cell in vitro water
Flat research has a certain limitation, first, inflammatory factor huge number, single inflammatory factor stimulate or several inflammation because of
The combination of son is all difficult to reflect intravital truth；Secondly, vitro culture conditions is huge with internal truth difference,
In internal milieu, tumor cell is in three dimensional growth state, and by Interstitial cell, new vessels generation, inflammation cell
The impact of the factors such as infiltration.Therefore, the research inflammation impact on colorectal cancer growth transfer, it is necessary to set up the dynamic of live body
Object model, but there is no successful example at present.
Set up for studying the animal model that colorectal cancer evolution process is affected by chronic inflammatory disease, need to enter in terms of two
Hands, on the one hand, need to induce at the Colon and rectum wall tissue of laboratory animal or be implanted into colorectal cancer, sets up Colon and rectum original position
Tumor model；On the other hand, need in laboratory animal, induce Colon and rectum chronic inflammatory disease.Two aspects combine, and can set up
There is the colorectal cancer animal model of inflammatory basis.
Nude mice Colon and rectum Orthotopic Transplantation Model, is the most classical Colon and rectum situ tumor model.First by people
Colorectal cancer cell injects nude mice oxter, sets up Transplanted tumor model.After tumor length to 0.5～1cm, tumor ablation, and use nothing
Bacterium eye scissors cuts the tumor mass to diameter about 2mm.Take the nude mice of the most inoculated tumor of another, a 2mm tumor mass is stitched to this nude mice
On Colon and rectum intestinal wall, i.e. set up nude mice in-situ junction rectal cancer model.
That induction Colon and rectum chronic inflammatory disease is more common at present is 2,4,6-trinitro-benzene-sulfonic acid (2,4,6-
Trinitrobenzenesulfonic acid, TNBS) and the mixed liquor of ethanol.TNBS is a kind of hapten, can be with intestinal
Macromolecule histone combines, and is transformed into antigen.Induction body produces immunoreation, causes colitis.The inflammation of TNBS induction
Disease property enteropathy trends towards Crohn disease, causes the immunoreation based on Th1 type.
Summary of the invention
Solve the technical problem that: for there is no the animal model model being successfully established inflammation to colorectal cancer growth transfer at present
The shortcoming of example, the present invention provides the method for building up of a kind of colorectal cancer animal model with inflammatory basis.
Technical scheme: the method for building up of a kind of colorectal cancer animal model with inflammatory basis, step is as follows:
The first step, the structure of pBabe-CMV-EGFP-puro plasmid:
1) preparing pBabe-puro digestion products by following reaction system and condition, wherein DNA is pBabe-puro
Plasmid,
Reaction system:
DNA, 1 μ g
10 × NEbuffer3+BSA, 3 μ L
Restriction endonuclease BamHI, 1 unit
Restriction endonuclease EcoRI, 1 unit
Add sterilizing ultra-pure water to volume 30 μ L
Reaction condition: 37 DEG C of water-bath 2h
2) pBabe-puro digestion products is carried out agarose gel electrophoresis, cut glue and reclaim the DNA sheet containing 5100 bp sizes
Section, and use AxyPrep DNA gel recovery test kit to be purified, obtain pBabe-puro digestion products after purification,
3) preparing CMV-EGFP DNA fragmentation by following PCR reaction system and condition, wherein DNA profiling is pEGFP-
C1 plasmid,
PCR reaction system is as follows:
5 × PrimeSTAR Buffer(Mg2+ plus), 10 μ L
Each 2.5 mM of dNTP Mixture(), 4 μ L
Forward primer 1:5'CCGGATCCTAATAGTAATCAATTACGGG3'(10 μM), 1 μ L
Reverse primer 2:5'AAGAATTCTTACTTGTACAGCTCGTCCAT3'(10 μM), 1 μ L
DNA profiling, 100 ng
PrimeSTARHS DNA Polymerase(2.5 U/ μ l), 0.5 μ L
Add sterilizing ultra-pure water to volume 50 μ L
Reaction condition:
98℃ 10 sec
55℃ 15 sec
72℃ 1 min/kbp
30 Cycles
Product: 1400bp
4) CMV-EGFP DNA fragmentation employing AxyPrep PCR cleaning agents box is purified, obtains not yet enzyme action
CMV-EGFP DNA fragmentation,
5) preparing CMV-EGFP digestion products by following reaction system and condition, wherein DNA is the CMV-of not yet enzyme action
EGFP DNA fragmentation,
Reaction system:
DNA, 1 μ g
10 × NEbuffer3+BSA, 3 μ L
Restriction endonuclease BamHI, 1 unit
Restriction endonuclease EcoRI, 1 unit
Add sterilizing ultra-pure water to volume 30 μ L
Reaction condition: 37 DEG C of water-bath 2h
6) CMV-EGFP digestion products is carried out agarose gel electrophoresis, cuts glue and reclaim the DNA fragmentation containing 1400 bp sizes,
And use AxyPrep DNA gel recovery test kit to be purified, obtain CMV-EGFP digestion products after purification,
7) use T4 DNA ligase and rapid ligation kit, by CMV-EGFP digestion products after purification with after purification
PBabe-puro digestion products is attached, and obtains connecting product,
8) connection product is transformed into competence Bacillus coli cells, and carries out resistance screening, obtain the Dan Ke after resistance screening
Grand bacterium colony,
9) bacterium colony after resistance screening is carried out positive colony screening, obtain pBabe-CMV-EGFP-puro plasmid；
Second step, the foundation of the EGFP process LAN system of retrovirus-mediated method:
PBabe-CMV-EGFP-puro plasmid is used with auxiliary packaging original paper vector plasmid VSVG and pMDL g/p RRE
Lipofectamine 3000 transfection reagent transfection 293T cell, obtains the EGFP process LAN system of retrovirus-mediated method, its
The mass ratio of middle plasmid is as follows: pBabe-CMV-EGFP-puro:VSVG:pMDL g/p RRE=2:1:1；
3rd step, stably expresses the foundation of the colorectal cancer cell system of EGFP:
The EGFP process LAN system of retrovirus-mediated method is added in colorectal cancer cell culture fluid, infection cell, infected
The EGFP gene of cell can be integrated in cellular genome, form the cell stably expressing EGFP, fast with final concentration 4 μ g/mL
The cell of EGFP is stably expressed in the screening of purine mycin, sets up the colorectal cancer cell system obtaining stable expression EGFP；
4th step, the foundation of nude mice oxter colorectal cancer xenografts model:
1. by 1 × 107The individual colorectal cancer cell system stably expressing EGFP, is resuspended in DMEM culture fluid, and adjusting volume is 50
μ L, cell density is 1 × 107/ 50 μ L, then mix with 50 μ L Matrigel equal-volumes, obtain cell suspension, as the most standby
With,
2. use insulin syringe, 100 μ L pallium cell injections are entered A group nude mice oxter, after 2 weeks, i.e. obtain nude mice oxter knot
Rectal cancer Transplanted tumor model；
5th step, the foundation of inflammatory model:
1. B group nude mice fasting 24 hours,
2. refetch with TNBS according to B group nude mouse, add water and mend to 50 μ L, add 50 μ L dehydrated alcohol, be i.e. made into 100 μ L TNBS
Working solution, wherein every 1kg nude mice takes the TNBS of 100 mg,
3. B group nude mice is anesthetized with ether, and carries tail decorporation just, and gastric perfusion needle is inserted colon through mice anus by ventricumbent position gently,
To the distance anus 4cm of needle tubing top, make mice stand upside down, inject 100 μ L TNBS working solutions, make mice keep handstand attitude
30s, extracts gastric perfusion needle,
1.～3. 4. after one week, come again the 5th step, continue to raise one week, obtain inflammatory model；
6th step, has the foundation of the colorectal cancer animal model of inflammatory basis:
1. extract the tumor of the A group nude mice oxter of the 4th step, shred to diameter about 2mm tumor mass with Sterile ophthalmic,
2. take a tumor mass, be directly seamed on the B group nude mice Colon and rectum intestinal wall of the 5th step,
1.～3. 3. repeat the 5th step, continue to raise one week,
1.～3. the most again repeating the 5th step, continue to raise one week, i.e. in nude mouse, foundation has the Colon and rectum of inflammatory basis
Carcinoma animal model.
The 2 of the first step described above) the middle concrete steps using AxyPrep DNA gel recovery test kit to be purified
It is:
1. under uviol lamp, cut the agarose gel containing target DNA, exhaust gel surface liquid with napkin and shred, meter
Calculating gel weight, this weight is as a gel volume；
2. gel is put in Eppendorf pipe, then add the Buffer DE-A of 3 gel volumes toward Eppendorf pipe, mixed
In 75 DEG C of heating after closing uniformly, it is interrupted mixing until gel piece is completely melt；
3. add the Buffer DE-B of 0.5 gel volume, mix homogeneously, obtain mixed liquor；
4. aspiration step 3. in mixed liquor, transfer to DNA and prepare in pipe, be placed in preparing pipe in 2 mL centrifuge tubes, 12,
Under 000 × g centrifugal force, centrifugal 1 min, abandons filtrate；
5. put back into 2 mL centrifuge tubes by preparing pipe, add 500 μ L Buffer W1,12, under 000 × g centrifugal force, be centrifuged 30
S, abandons filtrate；
Put back into 2 mL centrifuge tubes by preparing pipe the most again, add 700 μ L Buffer W2,12, centrifugal under 000 × g centrifugal force
30 s, abandon filtrate, add 700 μ L Buffer W2, and 12, under 000 × g centrifugal force, centrifugal 1 min, abandons filtrate；
7. put back in 2mL centrifuge tube by preparing pipe, 12, centrifugal 1 min under 000 × g centrifugal force；
8. it is placed in preparing pipe in 1.5 mL centrifuge tubes of cleaning, adds 25-30 μ L Eluent preparing film central authorities or go
Ionized water, room temperature stands 1 min, and 12, under 000 × g centrifugal force, centrifugal 1min eluted dna, i.e. completes purification.
The 4 of the first step described above) the middle concrete steps using AxyPrep PCR cleaning agents box to be purified
It is:
1., in PCR, enzyme action, enzyme mark or sequencing reaction liquid, Buffer PCR-A, the Buffer PCR-A of 3 volumes is added, no
Add to 100 μ L during foot 100 μ L, then mix, transfer to prepare in pipe, be placed in preparing pipe in 2 mL centrifuge tubes, 12,000
× g is centrifuged 1 min, abandons filtrate；
2. putting back into 2 mL centrifuge tubes by preparing pipe, add 700 μ LBuffer W2,12,000 × g is centrifuged 1 min, abandons filtrate；
3. it is placed in preparing pipe in centrifuge tube, puts back into 2 mL centrifuge tubes by preparing pipe, add 400 μ L Buffer W2,12,
000 × g is centrifuged 1 min；
4. by prepare pipe be placed in cleaning 1.5 mL centrifuge tubes in, prepare periosteum central authorities add 25-30 μ LEluent or go from
Sub-water, room temperature stands 1 min, and 12,000 × g is centrifuged 1 min eluted dna.
The 7 of the first step described above) in by CMV-EGFP digestion products after purification and pBabe-puro after purification
What digestion products was attached comprises the concrete steps that:
1. by 50 ng pBabe-puro digestion products after purification and the CMV-EGFP digestion products after purification of 3 times of moles
Mixing, adding distilled water to cumulative volume is 10 l；
2. 10 l 2 × Quick Ligation Buffer are added, mixing；
3. 1 l Quick T4 DNA ligase is added, mixing；
The most at room temperature effect i.e. obtains connection product after 5 minutes, link product is stored in the environment of-20 DEG C.
The 8 of the first step described above) in resistance delete comprising the concrete steps that of choosing:
1. take a pipe competence Bacillus coli cells, add 3 μ L after dissolving on ice and connect product, mixing, ice bath 30min；
2. pipe is statically placed in 42 DEG C after water-bath 90s, is transferred quickly in ice, ice bath 1-2min；
3. in pipe, add 900 μ L LB, water-bath 10min in 37 DEG C；
4. pipe is placed in shaking table, 37 DEG C, shake bacterium 45min under rotating speed 210 rpm；
5. the cell that 100 μ L have converted is coated on ampicillin plate；
6. it is inverted flat board, cultivates 16-20h at 37 DEG C, i.e. complete resistance screening.
The 9 of the first step described above) the comprising the concrete steps that of positives colony screening:
(1) bacterium colony PCR screening:
Picking monoclonal bacterium colony, is placed in the 300 μ L LB containing ampicillin, 37 DEG C, shake bacterium 6-8h under rotating speed 280 rpm,
Obtaining bacterium solution, bacterium solution uses PrimerSTAR HS DNA polymerase to carry out PCR screening, obtains bacterium colony PCR result positive
Clone；
(2) enzyme action is identified:
Take the clone that bacterium colony PCR result is positive, use plasmid miniprep dna to extract test kit and extract plasmid, and use BamHI and
EcoRI restricted enzyme carries out enzyme action, and the enzyme action that is that can cut out 1400bp fragment identifies positive clone；
(3) order-checking is identified:
Taking enzyme action and identify positive clone, check order, sequencing result uses Clustalx software to compare with EGFP sequence,
Sequences match i.e. successfully constructs, and i.e. obtains pBabe-CMV-EGFP-puro plasmid.
Comprising the concrete steps that of (2) described above middle employing plasmid miniprep dna extraction test kit extraction plasmid:
1. take the bacterium solution of the positive clone of the bacterium colony PCR result of 1-4 mL overnight incubation in LB culture medium, 12,000 × g from
Under mental and physical efforts, centrifugal 1 min, abandons most supernatant；
2. add 250 μ L Buffer S1 suspended bacterial to precipitate；
3. add 250 μ L Buffer S2, spin upside down mixing 4-6 time, make thalline fully crack, until being formed bright molten
Liquid；
4. add 350 μ L Buffer S3, spin upside down mixing 6-8 time, 12, centrifugal 10 min under 000 × g centrifugal force；
5. aspiration step 4. in centrifuged supernatant and transfer to prepare in pipe, be placed in preparing pipe in 2 mL centrifuge tubes, 12,
Under 000 × g centrifugal force, centrifugal 1 min, abandons filtrate；
6. putting back into centrifuge tube by preparing pipe, add 500 μ L Buffer W1,12, under 000 × g centrifugal force, centrifugal 1 min, abandons
Filtrate；
7. putting back into centrifuge tube by preparing pipe, add 700 μ L Buffer W2,12, under 000 × g centrifugal force, centrifugal 1 min, abandons
Filtrate, adds 700 μ L Buffer W2, and 12, under 000 × g centrifugal force, centrifugal 1 min, abandons filtrate；
8. put back in 2 mL centrifuge tubes by preparing pipe, 12, centrifugal 1 min under 000 × g centrifugal force；
9. move in 1.5 mL centrifuge tubes by preparing pipe, add 60-80 μ L Eluent or deionized water, room preparing periosteum central authorities
Gentle and quiet putting 1 min, 12, under 000 × g centrifugal force, centrifugal 1 min, i.e. completes the extraction of plasmid.
What in second step described above, the EGFP process LAN system of retrovirus-mediated method was set up comprises the concrete steps that:
1. transfection when inoculation 293T cell to degree of converging is 70-90%；
2. serum-free medium dilution Lipofectamine 3000 reagent is used；
3. serum-free medium dilution pBabe-CMV-EGFP-puro plasmid, VSVG plasmid and pMDL g/p RRE plasmid are used,
Then add P3000 reagent, mixing, obtain premixed liquid；
4. in Lipofectamine 3000 reagent diluted, add the premixed liquid of equivalent, incubated at room 5min, obtain
DNA-liposome complex；
5. DNA-liposome complex is added in cell culture fluid；
6. transfection after cultivate 24,48,72h, collect the supernatant rich in retroviral particle respectively, supernatant passes through ultracentrifugation
Concentrating virus, i.e. obtains the EGFP process LAN system of retrovirus-mediated method.
Beneficial effect: the method for building up of a kind of colorectal cancer animal model with inflammatory basis that the present invention provides, should
The EGFP used in method for building up can be excited generation fluorescence by laser instrument, and its luminous intensity also can be (little by corresponding instrument equipment
Living animal imaging system, IVIS;Lumina II, PerkinElmer company) detect such that it is able to confirm depositing of tumor
, and assess the size of tumor.
Accompanying drawing explanation
Fig. 1 is to use small animal living body imaging system detection inflammation group and matched group that tumor growth is affected testing result
Figure.
Fig. 2 is the weighing results figure after the nude mice taking-up tumor putting to death inflammation group and matched group.
Detailed description of the invention
PBabe-puro plasmid used in following example is purchased from Addgene company, and endonuclease reaction uses NEB public
Department's restricted enzyme.
AxyPrep DNA gel reclaims test kit purchased from AXYGEN company.
PEGFP-C1 plasmid is purchased from Clontech company, and PCR reaction uses the PrimerSTAR HS DNA of TAKARA company
Polymerase, PrimerSTAR HS DNA polymerase is purchased from TAKARA company.
AxyPrep PCR cleaning agents box is purchased from AXYGEN company.
Not yet the CMV-EGFP DNA fragmentation of enzyme action is with pEGFP-C1 plasmid as template, makes by PCR reaction system.
T4 DNA ligase and rapid ligation kit are purchased from NEB company.
Competence Bacillus coli cells is purchased from Tiangeng company.
Plasmid miniprep dna extracts test kit purchased from AXYGEN company.
Plasmid VSVG and pMDL g/p RRE is purchased from Addgene company, and Lipofectamine 3000 transfection reagent is purchased from
Lifetechnologies company.
TNBS(2,4,6-trinitro-benzene-sulfonic acid, 2,4,6-trinitrobenzenesulfonic acid) purchased from Sigma
Company, (lot number: 1002020045), is 50 g/L aqueous solutions.
Embodiment 1
A kind of method for building up of the colorectal cancer animal model with inflammatory basis, step is as follows:
The first step, the structure of pBabe-CMV-EGFP-puro plasmid:
1) preparing pBabe-puro digestion products by following reaction system and condition, wherein DNA is pBabe-puro
Plasmid,
Reaction system:
DNA, 1 μ g
10 × NEbuffer3+BSA, 3 μ L
Restriction endonuclease BamHI, 1 unit
Restriction endonuclease EcoRI, 1 unit
Add sterilizing ultra-pure water to volume 30 μ L
Reaction condition: 37 DEG C of water-bath 2h
2) pBabe-puro digestion products is carried out agarose gel electrophoresis, cut glue and reclaim the DNA sheet containing 5100 bp sizes
Section, and use AxyPrep DNA gel recovery test kit to be purified, obtain pBabe-puro digestion products after purification, its
What middle employing AxyPrep DNA gel recovery test kit was purified comprises the concrete steps that:
1. under uviol lamp, cut the agarose gel containing target DNA, exhaust gel surface liquid with napkin and shred, meter
Calculating gel weight, this weight is as a gel volume；
2. gel is put in Eppendorf pipe, then add the Buffer DE-A of 3 gel volumes toward Eppendorf pipe, mixed
In 75 DEG C of heating after closing uniformly, it is interrupted mixing until gel piece is completely melt；
3. add the Buffer DE-B of 0.5 gel volume, mix homogeneously, obtain mixed liquor；
4. aspiration step 3. in mixed liquor, transfer to DNA and prepare in pipe, be placed in preparing pipe in 2 mL centrifuge tubes, 12,
Under 000 × g centrifugal force, centrifugal 1 min, abandons filtrate；
5. put back into 2 mL centrifuge tubes by preparing pipe, add 500 μ L Buffer W1,12, under 000 × g centrifugal force, be centrifuged 30
S, abandons filtrate；
Put back into 2 mL centrifuge tubes by preparing pipe the most again, add 700 μ L Buffer W2,12, centrifugal under 000 × g centrifugal force
30 s, abandon filtrate, add 700 μ L Buffer W2, and 12, under 000 × g centrifugal force, centrifugal 1 min, abandons filtrate；
7. put back in 2mL centrifuge tube by preparing pipe, 12, centrifugal 1 min under 000 × g centrifugal force；
8. it is placed in preparing pipe in 1.5 mL centrifuge tubes of cleaning, adds 25-30 μ L Eluent preparing film central authorities or go
Ionized water, room temperature stands 1 min, and 12, under 000 × g centrifugal force, centrifugal 1min eluted dna, i.e. completes purification；
3) preparing CMV-EGFP DNA fragmentation by following PCR reaction system and condition, wherein DNA profiling is pEGFP-
C1 plasmid,
PCR reaction system is as follows:
5 × PrimeSTAR Buffer(Mg2+ plus), 10 μ L
Each 2.5 mM of dNTP Mixture(), 4 μ L
Forward primer 1:5'CCGGATCCTAATAGTAATCAATTACGGG3'(10 μM), 1 μ L
Reverse primer 2:5'AAGAATTCTTACTTGTACAGCTCGTCCAT3'(10 μM), 1 μ L
DNA profiling, 100 ng
PrimeSTARHS DNA Polymerase(2.5 U/ μ l), 0.5 μ L
Add sterilizing ultra-pure water to volume 50 μ L
Reaction condition:
98℃ 10 sec
55℃ 15 sec
72℃ 1 min/kbp
30 Cycles
Product: 1400bp
4) CMV-EGFP DNA fragmentation employing AxyPrep PCR cleaning agents box is purified, obtains not yet enzyme action
CMV-EGFP DNA fragmentation, what wherein employing AxyPrep PCR cleaning agents box was purified comprises the concrete steps that:
1., in PCR, enzyme action, enzyme mark or sequencing reaction liquid, Buffer PCR-A, the Buffer PCR-A of 3 volumes is added, no
Add to 100 μ L during foot 100 μ L, then mix, transfer to prepare in pipe, be placed in preparing pipe in 2 mL centrifuge tubes, 12,000
× g is centrifuged 1 min, abandons filtrate；
2. putting back into 2 mL centrifuge tubes by preparing pipe, add 700 μ LBuffer W2,12,000 × g is centrifuged 1 min, abandons filtrate；
3. it is placed in preparing pipe in centrifuge tube, puts back into 2 mL centrifuge tubes by preparing pipe, add 400 μ L Buffer W2,12,
000 × g is centrifuged 1 min；
4. by prepare pipe be placed in cleaning 1.5 mL centrifuge tubes in, prepare periosteum central authorities add 25-30 μ LEluent or go from
Sub-water, room temperature stands 1 min, and 12,000 × g is centrifuged 1 min eluted dna；
5) preparing CMV-EGFP digestion products by following reaction system and condition, wherein DNA is the CMV-of not yet enzyme action
EGFP DNA fragmentation,
Reaction system:
DNA, 1 μ g
10 × NEbuffer3+BSA, 3 μ L
Restriction endonuclease BamHI, 1 unit
Restriction endonuclease EcoRI, 1 unit
Add sterilizing ultra-pure water to volume 30 μ L
Reaction condition: 37 DEG C of water-bath 2h
6) CMV-EGFP digestion products is carried out agarose gel electrophoresis, cuts glue and reclaim the DNA fragmentation containing 1400 bp sizes,
And use AxyPrep DNA gel recovery test kit to be purified, obtain CMV-EGFP digestion products after purification,
7) use T4 DNA ligase and rapid ligation kit, by CMV-EGFP digestion products after purification with after purification
PBabe-puro digestion products is attached, and obtains connecting product, wherein by CMV-EGFP digestion products after purification and purification
After comprising the concrete steps that of being attached of pBabe-puro digestion products:
1. by 50 ng pBabe-puro digestion products after purification and the CMV-EGFP digestion products after purification of 3 times of moles
Mixing, adding distilled water to cumulative volume is 10 l；
2. 10 l 2 × Quick Ligation Buffer are added, mixing；
3. 1 l Quick T4 DNA ligase is added, mixing；
The most at room temperature effect i.e. obtains connection product after 5 minutes, link product is stored in the environment of-20 DEG C；
8) connection product is transformed into competence Bacillus coli cells, and carries out resistance screening, obtain the Dan Ke after resistance screening
Grand bacterium colony, wherein resistance deletes comprising the concrete steps that of choosing:
1. take a pipe competence Bacillus coli cells, add 3 μ L after dissolving on ice and connect product, mixing, ice bath 30min；
2. pipe is statically placed in 42 DEG C after water-bath 90s, is transferred quickly in ice, ice bath 1-2min；
3. in pipe, add 900 μ L LB, water-bath 10min in 37 DEG C；
4. pipe is placed in shaking table, 37 DEG C, shake bacterium 45min under rotating speed 210 rpm；
5. the cell that 100 μ L have converted is coated on ampicillin plate；
6. it is inverted flat board, cultivates 16-20h at 37 DEG C, i.e. complete resistance screening；
9) bacterium colony after resistance screening is carried out positive colony screening, obtain pBabe-CMV-EGFP-puro plasmid, its middle-jiao yang, function of the spleen and stomach
Comprising the concrete steps that of sex clone screening:
(1) bacterium colony PCR screening:
Picking monoclonal bacterium colony, is placed in the 300 μ L LB containing ampicillin, 37 DEG C, shake bacterium 6-8h under rotating speed 280 rpm,
Obtaining bacterium solution, bacterium solution uses PrimerSTAR HS DNA polymerase to carry out PCR screening, obtains bacterium colony PCR result positive
Clone；
(2) enzyme action is identified:
Take the clone that bacterium colony PCR result is positive, use plasmid miniprep dna to extract test kit and extract plasmid, and use BamHI and
EcoRI restricted enzyme carries out enzyme action, and the enzyme action that is that can cut out 1400bp fragment identifies positive clone, uses plasmid little
Comprising the concrete steps that of amount DNA extraction kit extraction plasmid:
1. take the bacterium solution of the positive clone of the bacterium colony PCR result of 1-4 mL overnight incubation in LB culture medium, 12,000 × g from
Under mental and physical efforts, centrifugal 1 min, abandons most supernatant；
2. add 250 μ L Buffer S1 suspended bacterial to precipitate；
3. add 250 μ L Buffer S2, spin upside down mixing 4-6 time, make thalline fully crack, until being formed bright molten
Liquid；
4. add 350 μ L Buffer S3, spin upside down mixing 6-8 time, 12, centrifugal 10 min under 000 × g centrifugal force；
5. aspiration step 4. in centrifuged supernatant and transfer to prepare in pipe, be placed in preparing pipe in 2 mL centrifuge tubes, 12,
Under 000 × g centrifugal force, centrifugal 1 min, abandons filtrate；
6. putting back into centrifuge tube by preparing pipe, add 500 μ L Buffer W1,12, under 000 × g centrifugal force, centrifugal 1 min, abandons
Filtrate；
7. putting back into centrifuge tube by preparing pipe, add 700 μ L Buffer W2,12, under 000 × g centrifugal force, centrifugal 1 min, abandons
Filtrate, adds 700 μ L Buffer W2, and 12, under 000 × g centrifugal force, centrifugal 1 min, abandons filtrate；
8. put back in 2 mL centrifuge tubes by preparing pipe, 12, centrifugal 1 min under 000 × g centrifugal force；
9. move in 1.5 mL centrifuge tubes by preparing pipe, add 60-80 μ L Eluent or deionized water, room preparing periosteum central authorities
Gentle and quiet putting 1 min, 12, under 000 × g centrifugal force, centrifugal 1 min, i.e. completes the extraction of plasmid；
(3) order-checking is identified:
Taking enzyme action and identify positive clone, check order, sequencing result uses Clustalx software to compare with EGFP sequence,
Sequences match i.e. successfully constructs, and i.e. obtains pBabe-CMV-EGFP-puro plasmid.
Second step, the foundation of the EGFP process LAN system of retrovirus-mediated method:
1. transfection when inoculation 293T cell to degree of converging is 70-90%；
2. serum-free medium dilution Lipofectamine 3000 reagent is used；
3. serum-free medium dilution pBabe-CMV-EGFP-puro plasmid, VSVG plasmid and pMDL g/p RRE plasmid are used,
Then adding P3000 reagent, mixing, obtain premixed liquid, wherein the mass ratio of plasmid is as follows: pBabe-CMV-EGFP-puro:
VSVG:pMDL g/p RRE=2:1:1；
4. in Lipofectamine 3000 reagent diluted, add the premixed liquid of equivalent, incubated at room 5min, obtain
DNA-liposome complex；
5. DNA-liposome complex is added in cell culture fluid；
6. transfection after cultivate 24,48,72h, collect the supernatant rich in retroviral particle respectively, supernatant passes through ultracentrifugation
Concentrating virus, i.e. obtains the EGFP process LAN system of retrovirus-mediated method.
3rd step, stably expresses the foundation of the colorectal cancer cell system of EGFP:
The EGFP process LAN system of retrovirus-mediated method is added in rectum cancer cell culture fluid, infection cell, infected
The EGFP gene of cell can be integrated in cellular genome, forms the cell stably expressing EGFP, with final concentration 4 μ g/mL purine
The cell of EGFP is stably expressed in mycin screening, sets up the rectum cancer cell system obtaining stable expression EGFP；
4th step, the foundation of nude mice oxter colorectal cancer xenografts model:
1. by 1 × 107The individual colorectal cancer cell system stably expressing EGFP, is resuspended in DMEM culture fluid, and adjusting volume is 50
μ L, cell density is 1 × 107/ 50 μ L, then mix with 50 μ L Matrigel equal-volumes, obtain cell suspension, as the most standby
With,
2. use insulin syringe, 100 μ L pallium cell injections are entered A group nude mice oxter, after 2 weeks, i.e. obtain nude mice oxter knot
Rectal cancer Transplanted tumor model；
5th step, the foundation of inflammatory model:
1. B group nude mice fasting 24 hours,
2. refetch with TNBS according to B group nude mouse, add water and mend to 50 μ L, add 50 μ L dehydrated alcohol, be i.e. made into 100 μ L TNBS
Working solution, wherein every 1kg nude mice takes the TNBS of 100 mg,
3. B group nude mice is anesthetized with ether, and carries tail decorporation just, and gastric perfusion needle is inserted colon through mice anus by ventricumbent position gently,
To the distance anus 4cm of needle tubing top, make mice stand upside down, inject 100 μ L TNBS working solutions, make mice keep handstand attitude
30s, extracts gastric perfusion needle,
1.～3. 4. after one week, come again the 5th step, continue to raise one week, obtain inflammatory model；
6th step, has the foundation of the colorectal cancer animal model of inflammatory basis:
1. extract the tumor of the A group nude mice oxter of the 4th step, shred to diameter about 2mm tumor mass with Sterile ophthalmic,
2. take a tumor mass, be directly seamed on the B group nude mice Colon and rectum intestinal wall of the 5th step,
1.～3. 3. repeat the 5th step, continue to raise one week,
1.～3. the most again repeating the 5th step, continue to raise one week, i.e. in nude mouse, foundation has the Colon and rectum of inflammatory basis
Carcinoma animal model.
Comparative example 1
This comparative example directly inoculated tumour born of the same parents on the Colon and rectum intestinal wall of nude mice, (the most induced synthesis is not scorching not to carry out TNBS process
Disease).
By the nude mice the carrying out TNBS process named inflammation group in embodiment 1, comparative example 1 carries out not the carrying out tested
The named matched group of nude mice that TNBS processes, the nude mice that inflammation group and matched group carry out testing is respectively 25.
Use the detection inflammation impact on tumor growth of small animal living body imaging system.The testing result obtained such as Fig. 1 institute
Show.As shown in Figure 1, inflammation group gross tumor volume is significantly greater than matched group.Illustrate that inflammation promotes colorectal cancer growth.(* * P < 0.01,
Significant difference is had with matched group.)
After putting to death nude mice, take out tumor and weigh.Weighing results is as in figure 2 it is shown, inflammation group tumor is significantly greater than matched group.Explanation
Inflammation promotes colorectal cancer growth.(* * P < 0.01, has significant difference with matched group.)
Laparotomy exploration finds, tumor Coeliac lymphadenectomy all occurs in inflammation group laboratory animal, and matched group is showed no tumor and turns
Move.Illustrate that inflammation promotes colorectal cancer transfer.
Claims (8)
1. the method for building up of a colorectal cancer animal model with inflammatory basis, it is characterised in that step is as follows:
The first step, the structure of pBabe-CMV-EGFP-puro plasmid:
1) preparing pBabe-puro digestion products by following reaction system and condition, wherein DNA is pBabe-puro
Plasmid,
Reaction system:
DNA, 1 μ g
10 × NEbuffer3+BSA, 3 μ L
Restriction endonuclease BamHI, 1 unit
Restriction endonuclease EcoRI, 1 unit
Add sterilizing ultra-pure water to volume 30 μ L
Reaction condition: 37 DEG C of water-bath 2h
2) pBabe-puro digestion products is carried out agarose gel electrophoresis, cut glue and reclaim the DNA sheet containing 5100 bp sizes
Section, and use AxyPrep DNA gel recovery test kit to be purified, obtain pBabe-puro digestion products after purification,
3) preparing CMV-EGFP DNA fragmentation by following PCR reaction system and condition, wherein DNA profiling is pEGFP-
C1 plasmid,
PCR reaction system is as follows:
5 × PrimeSTAR Buffer(Mg2+ plus), 10 μ L
Each 2.5 mM of dNTP Mixture(), 4 μ L
Forward primer 1:5'CCGGATCCTAATAGTAATCAATTACGGG3'(10 μM), 1 μ L
Reverse primer 2:5'AAGAATTCTTACTTGTACAGCTCGTCCAT3'(10 μM), 1 μ L
DNA profiling, 100 ng
PrimeSTAR HS DNA Polymerase(2.5 U/ μ l), 0.5 μ L
Add sterilizing ultra-pure water to volume 50 μ L
Reaction condition:
98℃ 10 sec
55℃ 15 sec
72℃ 1 min/kbp
30 Cycles
Product: 1400bp
4) CMV-EGFP DNA fragmentation employing AxyPrep PCR cleaning agents box is purified, obtains not yet enzyme action
CMV-EGFP DNA fragmentation,
5) preparing CMV-EGFP digestion products by following reaction system and condition, wherein DNA is the CMV-of not yet enzyme action
EGFP DNA fragmentation,
Reaction system:
DNA, 1 μ g
10 × NEbuffer3+BSA, 3 μ L
Restriction endonuclease BamHI, 1 unit
Restriction endonuclease EcoRI, 1 unit
Add sterilizing ultra-pure water to volume 30 μ L
Reaction condition: 37 DEG C of water-bath 2h
6) CMV-EGFP digestion products is carried out agarose gel electrophoresis, cuts glue and reclaim the DNA fragmentation containing 1400 bp sizes,
And use AxyPrep DNA gel recovery test kit to be purified, obtain CMV-EGFP digestion products after purification,
7) use T4 DNA ligase and rapid ligation kit, by CMV-EGFP digestion products after purification with after purification
PBabe-puro digestion products is attached, and obtains connecting product,
8) connection product is transformed into competence Bacillus coli cells, and carries out resistance screening, obtain the Dan Ke after resistance screening
Grand bacterium colony,
9) bacterium colony after resistance screening is carried out positive colony screening, obtain pBabe-CMV-EGFP-puro plasmid；
Second step, the foundation of the EGFP process LAN system of retrovirus-mediated method:
PBabe-CMV-EGFP-puro plasmid is used with auxiliary packaging original paper vector plasmid VSVG and pMDL g/p RRE
Lipofectamine 3000 transfection reagent transfection 293T cell, obtains the EGFP process LAN system of retrovirus-mediated method, its
The mass ratio of middle plasmid is as follows: pBabe-CMV-EGFP-puro:VSVG:pMDL g/p RRE=2:1:1；
3rd step, stably expresses the foundation of the colorectal cancer cell system of EGFP:
The EGFP process LAN system of retrovirus-mediated method is added in colorectal cancer cell culture fluid, infection cell, infected
The EGFP gene of cell can be integrated in cellular genome, form the cell stably expressing EGFP, fast with final concentration 4 μ g/mL
The cell of EGFP is stably expressed in the screening of purine mycin, sets up the colorectal cancer cell system obtaining stable expression EGFP；
4th step, the foundation of nude mice oxter colorectal cancer xenografts model:
1. by 1 × 107The individual colorectal cancer cell system stably expressing EGFP, is resuspended in DMEM culture fluid, and adjusting volume is 50 μ
L, cell density is 1 × 107/ 50 μ L, then mix with 50 μ L Matrigel equal-volumes, obtain cell suspension, as the most standby
With,
2. use insulin syringe, 100 μ L pallium cell injections are entered A group nude mice oxter, after 2 weeks, i.e. obtain nude mice oxter knot
Rectal cancer Transplanted tumor model；
5th step, the foundation of inflammatory model:
1. B group nude mice fasting 24 hours,
2. refetch with TNBS according to B group nude mouse, add water and mend to 50 μ L, add 50 μ L dehydrated alcohol, be i.e. made into 100 μ L TNBS
Working solution, wherein every 1kg nude mice takes the TNBS of 100 mg,
3. B group nude mice is anesthetized with ether, and carries tail decorporation just, and gastric perfusion needle is inserted colon through mice anus by ventricumbent position gently,
To the distance anus 4cm of needle tubing top, make mice stand upside down, inject 100 μ L TNBS working solutions, make mice keep handstand attitude
30s, extracts gastric perfusion needle,
1.～3. 4. after one week, come again the 5th step, continue to raise one week, obtain inflammatory model；
6th step, has the foundation of the colorectal cancer animal model of inflammatory basis:
1. extract the tumor of the A group nude mice oxter of the 4th step, shred to diameter about 2mm tumor mass with Sterile ophthalmic,
2. take a tumor mass, be directly seamed on the B group nude mice Colon and rectum intestinal wall of the 5th step,
1.～3. 3. repeat the 5th step, continue to raise one week,
1.～3. the most again repeating the 5th step, continue to raise one week, i.e. in nude mouse, foundation has the Colon and rectum of inflammatory basis
Carcinoma animal model.
The method for building up of a kind of colorectal cancer animal model with inflammatory basis the most according to claim 1, its feature
Be the 2 of the described first step) in use AxyPrep DNA gel to reclaim comprising the concrete steps that of being purified of test kit:
1. under uviol lamp, cut the agarose gel containing target DNA, exhaust gel surface liquid with napkin and shred, meter
Calculating gel weight, this weight is as a gel volume；
2. gel is put in Eppendorf pipe, then add the Buffer DE-A of 3 gel volumes toward Eppendorf pipe, mixed
In 75 DEG C of heating after closing uniformly, it is interrupted mixing until gel piece is completely melt；
3. add the Buffer DE-B of 0.5 gel volume, mix homogeneously, obtain mixed liquor；
4. aspiration step 3. in mixed liquor, transfer to DNA and prepare in pipe, be placed in preparing pipe in 2 mL centrifuge tubes, 12,
Under 000 × g centrifugal force, centrifugal 1 min, abandons filtrate；
5. put back into 2 mL centrifuge tubes by preparing pipe, add 500 μ L Buffer W1,12, under 000 × g centrifugal force, be centrifuged 30
S, abandons filtrate；
Put back into 2 mL centrifuge tubes by preparing pipe the most again, add 700 μ L Buffer W2,12, centrifugal under 000 × g centrifugal force
30 s, abandon filtrate, add 700 μ L Buffer W2, and 12, under 000 × g centrifugal force, centrifugal 1 min, abandons filtrate；
7. put back in 2mL centrifuge tube by preparing pipe, 12, centrifugal 1 min under 000 × g centrifugal force；
8. it is placed in preparing pipe in 1.5 mL centrifuge tubes of cleaning, adds 25-30 μ L Eluent preparing film central authorities or go
Ionized water, room temperature stands 1 min, and 12, under 000 × g centrifugal force, centrifugal 1min eluted dna, i.e. completes purification.
The method for building up of a kind of colorectal cancer animal model with inflammatory basis the most according to claim 1, its feature
Be the 4 of the described first step) in use comprising the concrete steps that of being purified of AxyPrep PCR cleaning agents box:
1., in PCR, enzyme action, enzyme mark or sequencing reaction liquid, Buffer PCR-A, the Buffer PCR-A of 3 volumes is added, no
Add to 100 μ L during foot 100 μ L, then mix, transfer to prepare in pipe, be placed in preparing pipe in 2 mL centrifuge tubes, 12,000
× g is centrifuged 1 min, abandons filtrate；
2. putting back into 2 mL centrifuge tubes by preparing pipe, add 700 μ LBuffer W2,12,000 × g is centrifuged 1 min, abandons filtrate；
3. it is placed in preparing pipe in centrifuge tube, puts back into 2 mL centrifuge tubes by preparing pipe, add 400 μ L Buffer W2,12,
000 × g is centrifuged 1 min；
4. by prepare pipe be placed in cleaning 1.5 mL centrifuge tubes in, prepare periosteum central authorities add 25-30 μ LEluent or go from
Sub-water, room temperature stands 1 min, and 12,000 × g is centrifuged 1 min eluted dna.
The method for building up of a kind of colorectal cancer animal model with inflammatory basis the most according to claim 1, its feature
Be the 7 of the described first step) in CMV-EGFP digestion products after purification is entered with pBabe-puro digestion products after purification
What row connected comprises the concrete steps that:
1. by 50 ng pBabe-puro digestion products after purification and the CMV-EGFP digestion products after purification of 3 times of moles
Mixing, adding distilled water to cumulative volume is 10 l；
2. 10 l 2 × Quick Ligation Buffer are added, mixing；
3. 1 l Quick T4 DNA ligase is added, mixing；
The most at room temperature effect i.e. obtains connection product after 5 minutes, link product is stored in the environment of-20 DEG C.
The method for building up of a kind of colorectal cancer animal model with inflammatory basis the most according to claim 1, its feature
Be the 8 of the described first step) in resistance delete comprising the concrete steps that of choosing:
1. take a pipe competence Bacillus coli cells, add 3 μ L after dissolving on ice and connect product, mixing, ice bath 30min；
2. pipe is statically placed in 42 DEG C after water-bath 90s, is transferred quickly in ice, ice bath 1-2min；
3. in pipe, add 900 μ L LB, water-bath 10min in 37 DEG C；
4. pipe is placed in shaking table, 37 DEG C, shake bacterium 45min under rotating speed 210 rpm；
5. the cell that 100 μ L have converted is coated on ampicillin plate；
6. it is inverted flat board, cultivates 16-20h at 37 DEG C, i.e. complete resistance screening.
The method for building up of a kind of colorectal cancer animal model with inflammatory basis the most according to claim 1, its feature
It is the 9 of the described first step) the comprising the concrete steps that of positives colony screening:
(1) bacterium colony PCR screening:
Picking monoclonal bacterium colony, is placed in the 300 μ L LB containing ampicillin, 37 DEG C, shake bacterium 6-8h under rotating speed 280 rpm,
Obtaining bacterium solution, bacterium solution uses PrimerSTAR HS DNA polymerase to carry out PCR screening, obtains bacterium colony PCR result positive
Clone；
(2) enzyme action is identified:
Take the clone that bacterium colony PCR result is positive, use plasmid miniprep dna to extract test kit and extract plasmid, and use BamHI and
EcoRI restricted enzyme carries out enzyme action, and the enzyme action that is that can cut out 1400bp fragment identifies positive clone；
(3) order-checking is identified:
Taking enzyme action and identify positive clone, check order, sequencing result uses Clustalx software to compare with EGFP sequence,
Sequences match i.e. successfully constructs, and i.e. obtains pBabe-CMV-EGFP-puro plasmid.
The method for building up of a kind of colorectal cancer animal model with inflammatory basis the most according to claim 5, its feature
Comprising the concrete steps that of (2) middle employing plasmid miniprep dna extraction test kit extraction plasmid described in being:
1. take the bacterium solution of the positive clone of the bacterium colony PCR result of 1-4 mL overnight incubation in LB culture medium, 12,000 × g from
Under mental and physical efforts, centrifugal 1 min, abandons most supernatant；
2. add 250 μ L Buffer S1 suspended bacterial to precipitate；
3. add 250 μ L Buffer S2, spin upside down mixing 4-6 time, make thalline fully crack, until being formed bright molten
Liquid；
4. add 350 μ L Buffer S3, spin upside down mixing 6-8 time, 12, centrifugal 10 min under 000 × g centrifugal force；
5. aspiration step 4. in centrifuged supernatant and transfer to prepare in pipe, be placed in preparing pipe in 2 mL centrifuge tubes, 12,
Under 000 × g centrifugal force, centrifugal 1 min, abandons filtrate；
6. putting back into centrifuge tube by preparing pipe, add 500 μ L Buffer W1,12, under 000 × g centrifugal force, centrifugal 1 min, abandons
Filtrate；
7. putting back into centrifuge tube by preparing pipe, add 700 μ L Buffer W2,12, under 000 × g centrifugal force, centrifugal 1 min, abandons
Filtrate, adds 700 μ L Buffer W2, and 12, under 000 × g centrifugal force, centrifugal 1 min, abandons filtrate；
8. put back in 2 mL centrifuge tubes by preparing pipe, 12, centrifugal 1 min under 000 × g centrifugal force；
9. move in 1.5 mL centrifuge tubes by preparing pipe, add 60-80 μ L Eluent or deionized water, room preparing periosteum central authorities
Gentle and quiet putting 1 min, 12, under 000 × g centrifugal force, centrifugal 1 min, i.e. completes the extraction of plasmid.
The method for building up of a kind of colorectal cancer animal model with inflammatory basis the most according to claim 1, its feature
Be retrovirus-mediated method in described second step EGFP process LAN system set up comprise the concrete steps that:
1. transfection when inoculation 293T cell to degree of converging is 70-90%；
2. serum-free medium dilution Lipofectamine 3000 reagent is used；
3. serum-free medium dilution pBabe-CMV-EGFP-puro plasmid, VSVG plasmid and pMDL g/p RRE plasmid are used,
Then add P3000 reagent, mixing, obtain premixed liquid；
4. in Lipofectamine 3000 reagent diluted, add the premixed liquid of equivalent, incubated at room 5min, obtain
DNA-liposome complex；
5. DNA-liposome complex is added in cell culture fluid；
6. transfection after cultivate 24,48,72h, collect the supernatant rich in retroviral particle respectively, supernatant passes through ultracentrifugation
Concentrating virus, i.e. obtains the EGFP process LAN system of retrovirus-mediated method.
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| CN107090474A (en) * | 2017-05-22 | 2017-08-25 | 山东大学 | A kind of preparation method of abdominal aneurvsm disease animal model |
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Application publication date: 20161123 |