CN109929808A - Using LINE-1 ORF1 albumen as the screening anticancer medicine model and screening technique of target spot - Google Patents
Using LINE-1 ORF1 albumen as the screening anticancer medicine model and screening technique of target spot Download PDFInfo
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Abstract
The present invention provides a kind of using LINE-1 ORF1 albumen as the screening anticancer medicine model and screening technique of target spot.The medicaments sifting model is the breast cancer cell T47D of high expression LINE-1 ORF1.The present invention is detected using expression quantity of the In-Cell Western method to LINE-1 ORF1 albumen in cell after addition compound, a kind of detection method of easy, sensitive, stable quantitative analysis LINE-1 ORF1 expressing quantity is established, and then demonstrates a possibility that it is as LINE-1 ORF1 protein inhibitor and exciting agent high flux screening system.The inhibitor that multiple targets neoplastic cells LINE-1 ORF1 albumen have been successfully obtained using screening model of the invention, provides new tool for the research and development of anti-tumor drug.
Description
Technical field
The present invention relates to biomedicine fields, specifically, being related to a kind of using LINE-1 ORF1 albumen as the anticancer of target spot
Medicaments sifting model and screening technique.
Background technique
In the 1940s, American scientist Barbara McClintock has found one when studying the calico corn origin cause of formation
It being capable of free-moving genetic constitution, this something lost in dyeing or between different chromosomes kind independently of cell mitogen process
Pass ingredient be named as later transposons (transposon), also known as transposable element (transposable elements,
TEs)。
According to the difference of swivel base mode, transposons can be divided into DNA transposons and RNA transposons two major classes.DNA transposons is
There is the DNA fragmentation of transposase by itself coding, the DNA fragmentation of itself can be shear off from original genomic locations slotting
Enter other sites to genome, this copy mode is visually described by people as " shearing-stickup " mode.DNA transposons
The 3% of human genome is only accounted for, and the overwhelming majority is all in inactivated state.RNA transposons, also known as retrotransposon
It (Retrotransposons), is to carry out swivel base by intermediate of RNA, copy mode is usually described as " duplication-is viscous
Patch " mode that is, first by transcription synthesis RNA intermediate, then is integrated into genome by template reverse transcription synthetic DNA of the RNA
Other positions.
Retrotransposon can be divided into independence retrotransposon and nonautonomy retrotransposon.Independence
Retrotransposon can encode enzyme and albumen necessary to swivel base, being capable of complete independently transposition event.Nonautonomy reverse transcription turns
The copy mode of stand (such as Alu) is similar to the copy mode of independence retrotransposon, but itself cannot be encoded
Enzyme and albumen necessary to swivel base, and need that transposition event could be completed by means of the enzyme or albumen that encode from principal mode transposons.
Retrotransposon LINE-1 (long interspersed element -1, Long interspersed elements-1) belongs to independently
Type retrotransposon is the unique transposons with transposition activity found in human genome at present.It is in mankind's base
Because being copied in group containing about 500,000, accounts for about the 17% of genome total amount, be the highest transposons of accounting in human genome.The mankind
LINE-1 mrna length is about 6000nt, by 3 ' ends and the non-translational region (Untranslatedregion, UTR) at 5 ' ends and two
Open single open reading frame ORF1 and ORF2 is constituted.Its encoded albumen ORF1p has chaperone activity, protects in RNPs
LINE-1mRNA exempts to be degraded.ORF2p has restriction endonuclease and reverse transcriptase activity, is responsible for using LINE-1RNA as template reverse transcription
Generate position new in LINE-1cDNA insertion genome.
LINE-1 can lose itself many segments in continuous transposition event, thus can generate many 5 ' ends missing,
Sequence is inverted and the LINE-1 of the forfeiture transposition activities such as the site mutation of coding albumen, thus most in human body
LINE-1 is inactive.Nonetheless, the active LINE-1 of remaining very small part can also produce the health of people
Raw great influence.The insertion of LINE-1 can influence expression and the tune of gene by diversified forms such as mutation, missing, genetic recombination
Control, causes the unstability of genome, so as to cause the generation of genopathy or tumour.
It is found in research in recent years, can detect LINE-1RNA and its albumen in a large amount of different types of tumor tissues
The expression of (ORF1 albumen and ORF2 albumen), and this phenomenon becomes apparent in the higher cancerous issue of malignization degree,
Generation, the development of prompt LINE-1 and tumour have close relationship.Nemanja Rodic uses the immune dye of micro-array tissue
Color, discovery include breast cancer (97%LINE-1 is positive), advanced ovarian cancer (91.5%LINE-1 sun in the tumour of the LINE-1 positive
Property), ductal adenocarcinoma of pancreas (89%LINE-1 is positive), the cancer of the esophagus (64%LINE-1 is positive), lung cancer (51%LINE-1 is positive)
With colon cancer (50%LINE-1 is positive), prostate cancer (41%LINE-1 is positive) and liver cancer (19%LINE-1 is positive).
Summary of the invention
The object of the present invention is to provide a kind of using LINE-1 ORF1 albumen as the screening anticancer medicine model of target spot and screening
Method.
In order to achieve the object of the present invention, in a first aspect, the present invention provides a kind of resisting using LINE-1 ORF1 albumen as target spot
Cancer drug screening model, the medicaments sifting model are the breast cancer cell T47D of high expression LINE-1 ORF1.
Second aspect, the present invention provide the medicaments sifting model in screening using LINE-1 ORF1 albumen as the anti-of target spot
Application in cancer drug.
The action principle of the medicaments sifting model is as follows: selection endogenous height first expresses LINE-1 ORF1 breast cancer
Cell line T47D cell after 96 orifice plate inoculating cells for 24 hours adds compound, 48h is further cultured for, using In-Cell Western method
It is determined in cell according to destination protein ORF1 albumen (LINE-1 ORF1 albumen) and the fluorescent value ratio of internal reference albumen actin
The amount of ORF1 albumen, if compound, which is added, can inhibit the expression of ORF1 albumen, ORF1 albumen and internal reference albumen actin's is glimmering
Light value ratio reduces；If compound, which is added, can promote the expression of ORF1 albumen, ORF1 albumen and internal reference albumen actin's is glimmering
Light value ratio increases.
The third aspect, the present invention, which is provided, to be screened using said medicine screening model using LINE-1 ORF1 albumen as target spot
Breast cancer cell T47D is inoculated in cell culture fluid by the method for anticancer drug, after cultivating a period of time, is added into culture solution
Enter drug candidate to be measured, after being incubated for a period of time, measures the expression quantity or opposite of LINE-1 ORF1 gene or albumen in cell
Expression quantity filters out the drug with anticancer activity according to the expression quantity or relative expression quantity of gene or albumen.
Preferably, using In-Cell Western method according to the fluorescence of LINE-1 ORF1 albumen and internal reference albumen actin
Value ratio determines the relative expression quantity of LINE-1 ORF1 albumen in cell.
Further, drug candidate to be measured is first configured to drug solution, breast cancer cell T47D is in cell culture fluid
After middle culture a period of time, drug solution is added into cell culture fluid as experimental group, while to be only added and the drug
The isometric blank reagent for compounding pharmaceutical solution of solution as a control group, after being incubated for a period of time, utilizes In-Cell
Western method measures the fluorescent value of LINE-1 ORF1 albumen and internal reference albumen actin in group of cells respectively, is calculated as follows
The relative expression quantity of LINE-1 ORF1 albumen out:
The relative expression quantity of LINE-1 ORF1 albumen=[(LINE-1 ORF1 protein fluorescence valueExperimental group- secondary antibody background
ValueExperimental group)/(actin protein fluorescence valueExperimental group- secondary antibody background valueExperimental group)]/[(LINE-1 ORF1 protein fluorescence valueControl group- secondary antibody
Background valueControl group)/(actin protein fluorescence valueControl group- secondary antibody background valueControl group)] formula (I)
In the specific embodiment of the present invention, the screening technique the following steps are included:
A. cell culture
Breast cancer cell T47D is inoculated in the culture dish equipped with cell culture fluid (Ф 10cm), covers with culture to cell
It after ware, inhales and abandons culture solution, 0.25% pancreatin of 2ml is added and is digested, incubator 1min is put into, inhales and abandons digestive juice, it is thin that 6ml is added
Born of the same parents' culture solution is blown and beaten, and after cell is dispersed into single cell suspension, is counted with calculating instrument, and appropriate cell suspension inoculation is taken
In clarinet wall (microwell plate of white cannot be used herein, the autofluorescence of white tube wall surface can generate significant noise signal)
In the 96 hole microwell plates at transparent pipe bottom, 1.6 × 106A cells/well, final concentration of 10 μM of drug solution is added in rear every hole for 24 hours,
Continue after cultivating 48h, carries out In-Cell Western detection；
B.In-Cell Western detection
B1. the liquid abandoned in each hole is inhaled, every hole adds 150 μ L PBS buffer solution to wash 2 times；
B2. the fixation buffer of formaldehyde final concentration of 3.7% is prepared with 1 × PBS and 37% formaldehyde；
B3. it is rapidly added the fixed cell of fixation buffer of 150 μ L, at room temperature stationary incubation 20min；It is fixed
When buffer, carefully it is added with pipettor along tube wall, avoids separating cell；
B4. the elution buffer of Triton X-100 final concentration of 0.2% is prepared with 1 × PBS and 10%Triton X-100
Liquid；
B5. fixed buffer is removed, 150 μ L elution buffers are added, is incubated at room temperature 10-20min to guarantee to permeate
Cell；
B6. elution buffer is removed, is slowly added to 50 μ l of confining liquid along tube wall into each hole, shaking table 40rpm is at room temperature
It is incubated for 1h；Wherein, the confining liquid is 2%BSA；
B7. confining liquid is removed, every hole is added 40 μ L and contains the mixed of rabbit source LINE-1 ORF1 primary antibody and source of mouse actin primary antibody
Unification is anti-, is incubated at room temperature 1h；Wherein, the potency of the rabbit source LINE-1 ORF1 primary antibody is 1:500, the source of mouse actin primary antibody
Potency be 1:200；
B8. primary antibody is recycled, every hole adds 150 1 × PBS of μ L to be washed, and 3min/ times, washes 3 times；
B9. 40 μ L secondary antibodies are added in every hole, are incubated at room temperature 1h；Wherein, the secondary antibody is Goat anti-rabbit IRDyeTM680 (1:500 dilutions；LI-COR Cat#926-32221)；Goat anti-mouse IRDyeTM800CW (1:800 dilution；
LI-COR Cat#926-32210) two kinds of antibody mixing；
B10 recycles secondary antibody, and every hole adds 150 1 × PBS of μ L to be washed, 3min/ times, washes 3 times；
B11. it is scanned with Odyssey instrument；
C. according to testing result, the relative expression quantity of LINE-1 ORF1 albumen is calculated.
The cell culture fluid that the present invention uses is 1640 culture medium of RPMI Medium containing 10% fetal calf serum (FBS).
Wherein, 1640 culture medium of RPMI Medium is purchased from U.S. Gibco company,
Preferably, cell culture condition and incubator condition are as follows: 37 DEG C, 5%CO2。
Fourth aspect, the present invention provide the targets neoplastic cells LINE-1 ORF1 albumen arrived using above-mentioned model discrimination
The effective component of inhibitor or inhibitor medicaments composition, the inhibitor or inhibitor medicaments composition is selected from formula (1)~(3)
Shown in any compound:
5th aspect, the present invention provide the inhibitor or inhibitor medicaments composition and target LINE-1 ORF1 in preparation
Application in the anti-tumor drug of albumen.
By above-mentioned technical proposal, the present invention at least have following advantages and the utility model has the advantages that
Expression of the present invention using In-Cell Western method to LINE-1 ORF1 albumen in cell after addition compound
Amount is detected, and a kind of detection method of easy, sensitive, stable quantitative analysis LINE-1 ORF1 expressing quantity is established,
And then demonstrate a possibility that it is as LINE-1 ORF1 protein inhibitor and exciting agent high flux screening system.Utilize this hair
Bright screening model has successfully obtained the inhibitor of multiple targets neoplastic cells LINE-1 ORF1 albumen, is anti-tumor drug
Research and development provide new tool.
Detailed description of the invention
Fig. 1 is using screening system in the embodiment of the present invention 1 to the selection result of 816 compounds, and wherein inhibiting rate reaches
40% include 15 compounds.
Fig. 2 is 3 inhibitor of the LINE-1 ORF1 albumen filtered out in the embodiment of the present invention 1 using screening system
Chemical structural formula.
Fig. 3 is the LINE-1 ORF1 protein inhibitor filtered out in the embodiment of the present invention 1 using screening system.Wherein, left
Figure is that 3 compounds obtained to secondary screening carry out Western Blot verifying.Right figure is by Image J software to Western
Blot result is quantified.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
Used in the conventional means that are well known to those skilled in the art of technological means, raw materials used is commercial goods.
Embodiment 1 is using LINE-1 ORF1 albumen as the screening anticancer medicine model and screening technique of target spot
1, cell culture
Breast cancer cell T47D is inoculated in the culture dish equipped with cell culture fluid (Ф 10cm), covers with culture to cell
It after ware, inhales and abandons culture medium, 0.25% pancreatin of 2ml is added and is digested, incubator 1min is put into, inhales and abandons culture medium, 6ml training is added
Feeding base is blown and beaten, and after cell is dispersed into single cell suspension, is counted with calculating instrument, takes appropriate cell suspension inoculation in black
Tube wall (microwell plate of white pipe cannot be used, because the autofluorescence of white tube wall surface can generate significant noise signal) is thoroughly
In 96 hole microwell plates of bright tube bottom, final concentration of 10 μM of compound (preparing with DMSO) is added in rear every hole for 24 hours, continues to cultivate
After 48h, In-Cell Western experiment is carried out.
2, In-Cell Western is detected
Culture medium is abandoned 1. inhaling, every hole adds 150 μ L PBS buffer solution to wash 2 times.
2. preparing fresh fixation buffer with 1 × PBS and 37% formaldehyde: formaldehyde final concentration of 3.7%.
3. being rapidly added the fixed cell of fresh fixed buffer of 150 μ L, exempts from shake and be incubated for 20min at room temperature.
When being fixed buffer, carefully it is added with pipettor along tube wall, avoids separating cell.
4. preparing Triton X-100 elution buffer with 1 × PBS and 10%Triton X-100, (Triton X-100 is whole
0.2%) concentration is.
5. removing fixed buffer, 150 μ L Triton X-100 elution buffers are added, be incubated at room temperature 10-20min with
Guarantee can be with permeation cell.
6. removing Triton X-100 elution buffer, 2%BSA (confining liquid) 50 carefully is added along tube wall in each hole
μ l, shaking table 40rpm slowly shakes is incubated for 1h at room temperature.
7. removing confining liquid, every hole is added 40 μ L rabbit source ORF1 (1:500) and source of mouse actin (1:200) and mixes primary antibody, room
Temperature is incubated for 1h.
8. recycling primary antibody, 150 1 × PBS of μ L of every hole addition are placed on shaking table to be shaken (80rpm) slowly, 3min/ times, is washed 3 times.
9. every hole is added 40 μ L and secondary antibody is added, it is incubated at room temperature 1h.Wherein, secondary antibody is Goat anti-rabbit
IRDyeTM680 (1:500 dilutions；LI-COR Cat#926-32221)；Goat anti-mouse IRDyeTM800CW(1:800
Dilution；LI-COR Cat#926-32210) two kinds of antibody mixing.
10. recycling secondary antibody, 150 1 × PBS of μ L of every hole addition are placed on shaking table to be shaken (80rpm) slowly, 3min/ times, is washed 3 times.
11. being scanned with Odyssey instrument.
3, Analysis of test results
Control group: add 1 μ L DMSO in 200 μ L cell systems；
Experimental group: add 1 μ L compound in 200 μ L cell systems.Final concentration of 10 μM of compound.
According to testing result, the relative expression quantity of LINE-1 ORF1 albumen is calculated as follows out:
The relative expression quantity of LINE-1 ORF1 albumen=[(LINE-1 ORF1 protein fluorescence valueExperimental group- secondary antibody background
ValueExperimental group)/(actin protein fluorescence valueExperimental group- secondary antibody background valueExperimental group)]/[(LINE-1 ORF1 protein fluorescence valueControl group- secondary antibody
Background valueControl group)/(actin protein fluorescence valueControl group- secondary antibody background valueControl group)]
Fig. 1 is seen using the selection result of the screening system to 816 compounds, and wherein inhibiting rate is up to 40% including 15
A compound.
The chemistry of 3 inhibitor of the higher LINE-1 ORF1 albumen of activity filtered out using the screening system is tied
Structure formula is shown in Fig. 2.
3 kinds of LINE-1 ORF1 protein inhibitors act on the Western Blot detection knot of ORF1 albumen after T47D cell
Fruit sees Fig. 3.Wherein, left figure is that 3 compounds obtained to secondary screening carry out Western Blot verifying.Right figure is to pass through Image
J software quantifies Western Blot result.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be modified or is improved, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. using LINE-1 ORF1 albumen as the screening anticancer medicine model of target spot, which is characterized in that the medicaments sifting model is
The breast cancer cell T47D of height expression LINE-1 ORF1.
2. medicaments sifting model described in claim 1 is screening answering in the anticancer drug using LINE-1 ORF1 albumen as target spot
With.
3. using the screening of medicaments sifting model described in claim 1 using LINE-1 ORF1 albumen as the side of the anticancer drug of target spot
Method, which is characterized in that breast cancer cell T47D is inoculated in cell culture fluid, after cultivating a period of time, is added into culture solution
Enter drug candidate to be measured, after being incubated for a period of time, measures the expression quantity or opposite of LINE-1 ORF1 gene or albumen in cell
Expression quantity filters out the drug with anticancer activity according to the expression quantity or relative expression quantity of gene or albumen.
4. according to the method described in claim 3, it is characterized in that, using In-Cell Western method according to LINE-1 ORF1
The fluorescent value ratio of albumen and internal reference albumen actin determine the relative expression quantity of LINE-1 ORF1 albumen in cell.
5. according to the method described in claim 4, it is characterized in that, drug candidate to be measured is first configured to drug solution, cream
After adenocarcinoma cell T47D cultivates a period of time in cell culture fluid, drug solution is added into cell culture fluid as experiment
Group, while the blank reagent for compounding pharmaceutical solution isometric with the drug solution is only added as a control group, it incubates
After educating a period of time, LINE-1 ORF1 albumen and internal reference albumen in group of cells are measured respectively using In-Cell Western method
The relative expression quantity of LINE-1 ORF1 albumen is calculated as follows out in the fluorescent value of actin:
The relative expression quantity of LINE-1 ORF1 albumen=[(LINE-1 ORF1 protein fluorescence valueExperimental group- secondary antibody background valueExperimental group)/
(actin protein fluorescence valueExperimental group- secondary antibody background valueExperimental group)]/[(LINE-1 ORF1 protein fluorescence valueControl group- secondary antibody background
ValueControl group)/(actin protein fluorescence valueControl group- secondary antibody background valueControl group)]。
6. according to the method described in claim 5, characterized by comprising the following steps:
A. cell culture
Breast cancer cell T47D is inoculated in the culture dish equipped with cell culture fluid, the specification Ф 10cm of culture dish, to cell
It after covering with culture dish, inhales and abandons culture solution, 2ml0.25% pancreatin is added and is digested, incubator 1min is put into, inhales and abandons digestive juice, add
Enter 6ml cell culture fluid to be blown and beaten, after cell is dispersed into single cell suspension, be counted with calculating instrument, takes appropriate cell
Suspension is inoculated in the 96 hole microwell plates at clarinet wall transparent pipe bottom, and 1.6 × 106A cells/well, final concentration is added in rear every hole for 24 hours
For 10 μM of drug solution, continues after cultivating 48h, carry out In-Cell Western detection；
B.In-Cell Western detection
B1. the liquid abandoned in each hole is inhaled, every hole adds 150 μ L PBS buffer solution to wash 2 times；
B2. the fixation buffer of formaldehyde final concentration of 3.7% is prepared with 1 × PBS and 37% formaldehyde；
B3. it is rapidly added the fixed cell of fixation buffer of 150 μ L, at room temperature stationary incubation 20min；
B4. the elution buffer of Triton X-100 final concentration of 0.2% is prepared with 1 × PBS and 10%Triton X-100；
B5. fixed buffer is removed, 150 μ L elution buffers are added, is incubated at room temperature 10-20min；
B6. elution buffer is removed, is slowly added to 50 μ l of confining liquid along tube wall into each hole, shaking table 40rpm is incubated at room temperature
1h；Wherein, the confining liquid is 2%BSA；
B7. confining liquid is removed, the mixing one that 40 μ L contain rabbit source LINE-1 ORF1 primary antibody and source of mouse actin primary antibody is added in every hole
It is anti-, it is incubated at room temperature 1h；Wherein, the potency of the rabbit source LINE-1 ORF1 primary antibody is 1:500, the effect of the source of mouse actin primary antibody
Valence is 1:200；
B8. primary antibody is recycled, every hole adds 150 μ L1 × PBS to be washed, and 3min/ times, washes 3 times；
B9. 40 μ L secondary antibodies are added in every hole, are incubated at room temperature 1h；
B10 recycles secondary antibody, and every hole adds 150 μ L1 × PBS to be washed, 3min/ times, washes 3 times；
B11. it is scanned with Odyssey instrument；
C. according to testing result, the relative expression quantity of LINE-1 ORF1 albumen is calculated.
7. according to the method described in claim 6, it is characterized in that, cell culture fluid described in step a is containing 10% tire ox
1640 culture medium of RPMI Medium of serum.
8. method according to claim 6 or 7, which is characterized in that cell culture condition and incubator condition in step a are as follows:
37 DEG C, 5%CO2。
9. the inhibitor or inhibitor medicaments composition of targets neoplastic cells LINE-1 ORF1 albumen, which is characterized in that the suppression
The effective component of preparation or inhibitor medicaments composition is selected from any compound shown in formula (1)~(3):
10. inhibitor described in claim 9 or inhibitor medicaments composition are in the antitumor of preparation targeting LINE-1 ORF1 albumen
Application in drug.
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| CN101068935A (en) * | 2004-08-10 | 2007-11-07 | 肿瘤疗法科学股份有限公司 | Genes and polypeptides relating to breast cancers |
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