CN109136263A - A kind of screening promotees method, recombinant vector and the cell strain of stem cell homing drug - Google Patents
A kind of screening promotees method, recombinant vector and the cell strain of stem cell homing drug Download PDFInfo
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Abstract
The present invention relates to field of biotechnology, specially a kind of recombinant vector and the stable cell strain containing this recombinant vector can be used for screening rush stem cell homing drug.This recombinant vector contains CXCR4 promoter sequence and luc2 luciferase reporter gene；And luc2 luciferase reporter gene is located at CXCR4 promoter sequence downstream.The expression activity that stable cell strain containing above-mentioned recombinant vector and internal reference fluorescence zymophore can be used for detecting CXCR4 gene is horizontal, and then promotes the drug of stem cell homing for high flux screening.Therefore, above-mentioned stable cell strain, drug candidate, which is added, to be stimulated, pass through luc2 relative intensity of fluorescence, the activity that the drug candidate promotes the expression of CXCR4 promoter can be evaluated, the drug of myocardial infarction is treated or assisted in the treatment of to screen drug or the screening of stem cell homing, is had broad application prospects.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to can stablize the Dual-Luciferase of expression CXCR4 promoter gene
The construction method of reporter cell strain and its application, more specifically, the present invention provide and a kind of can be used to screen rush stem cell homing
The construction method of the stable cell strain of drug, and possible drug monomer is screened with it, promote stem cell homing for high flux screening
Drug provides biomaterial.
Background technique
Heart failure is carried out caused by myocardial infarction (myocardial infarction, MI) is still modern heart
The huge challenge of disease.It can be enhanced by autologous transplanting or allosome mescenchymal stem cell (mesenchymal stem cell, MSCs)
Cardiac function reduces infarct size, delays heart failure process, have broad application prospects in the treatment of MI.However, after transplanting
The lower heart retention ratio of MSCs seriously limits its therapeutic effect.Only 2% stem cell returns after studies have shown that intravenous injection
Nest is to heart, and coronary artery injection is 15%, and is injected directly into the retention ratio for also only having 11% in cardiac muscle, and most cells are dispersed in
Lung, liver and spleen.Therefore it is the pass of enhanced MSC s treatment MI effect that more stem cell Cardiomyocytes infarcted regions, which are gone back to the nest, after promotion transplanting
Key.
Gro-beta-T receptor 4 [chemokine (C-X-C motif) receptor 4, CXCR4] and its ligand matrices
- 1 α of cell derived factor (stromal cell-derived factor-1 α, SDF-1 α) plays main work in MSCs goes back to the nest
With.CXCR4 is the g protein coupled receptor of 7 cross-films, by mediating MSCs migration, proliferation in conjunction with SDF-1 alpha specific and dividing
Change.The study found that improving the ability that MSCs expression CXCR4 level can enhance cell migration and go back to the nest to the region MI, and use
CXCR4 blocking agent AMD3100 can obviously inhibit stem cell homing to myocardial infarction region, and homing cells quantity substantially reduces.Cause
This screens the candidate monomer that MSCs can be promoted to go back to the nest, the treatment for enhanced MSC s using CXCR4 expression as investigation object
Effect has a very important significance.Accordingly, the present invention constructs the medicine of the Dual-luciferase reportor systerm of the promoter containing CXCR4
Object high flux screening model, and demonstrate the reliability of the cell model.
Summary of the invention
The present invention is intended to provide a kind of recombinant vector and the stable cell strain containing this recombinant vector, can be used for screening
Promote stem cell homing drug.
This recombinant vector and stable cell strain also are used to screen drug or the screening for promoting stem cell homing by the present invention
The drug for the treatment of or adjuvant treatment myocardial infarction.
Promote the drug of stem cell homing the present invention also provides a kind of screening or screening is treated or adjuvant treatment cardiac muscle stalk
The method of dead drug.
The technical scheme is that a kind of recombinant vector, contains CXCR4 promoter sequence and luc2 luciferase reporting
Gene；The luc2 luciferase reporter gene is located at CXCR4 promoter sequence downstream.Preferably, the recombinant vector is
PGL4.17 carrier containing CXCR4 promoter sequence and luc2 luciferase reporter gene.
The construction method of this recombinant vector is to introduce different enzymes respectively at 5 ' ends of CXCR4 promoter DNA and 3 ' ends
Enzyme site, and CXCR4 promoter and luciferase report carrier are connected after digestion.Specifically, CXCR4 promoter gene is inserted
Enter luciferase reporter vector pGL4.17, constructs CXCR4 promoter recombinant vector pGL4.17-CXCR4.Preferably, described heavy
The construction method of group carrier is to introduce restriction enzyme site XhoI and HindIII respectively at 5 ' ends of CXCR4 promoter DNA and 3 ' ends；
CXCR4 promoter DNA sequence and luc2 luciferase reporter vector pGL4.17 are used into XhoI and HindIII double digestion respectively, even
It is converted after connecing with competent cell, obtains pGL4.17-CXCR4 recombinant vector by digestion and after dual identification is sequenced.Connected with T4
It connects enzyme to be attached, competent cell is bacillus coli DH 5 alpha.
A kind of stable cell strain contains above-mentioned recombinant vector and internal reference fluorescence zymophore.Preferably, host cell
For human embryonic kidney epithelial cells 293.
Preferably, internal reference fluorescence zymophore contained in the stable cell strain is the carrier for expressing sea pansy luciferase element.
The construction method of above-mentioned cell strain are as follows: with the carrier of pGL4.17-CXCR4 recombinant vector and expression internal reference luciferase
Transfection host cell, picking monoclonal cell culture, the cell strain for selecting fluorescence signal intensity high.
In a preferred method of the present invention, by pGL4.17-CXCR4 and internal reference sea pansy fluorescein carrier pRL-TK corotation
Human embryonic kidney epithelial cells 293 are contaminated, picking monoclonal expands culture, screens to obtain CXCR4 promoter pair according to luciferase intensity
Luciferase surely turns cell model 293-CXCR4P.The monomer to be measured for promoting stem cell homing is stimulated into 293-CXCR4P, with
Luc2 relative intensity of fluorescence is index, obtains the rush stem cell homing effect assessment for promoting CXCR4 expression.
Above-mentioned recombinant expression carrier and stable cell strain can be used for screening promote stem cell homing drug or screening treatment or
Assist in the treatment of myocardial infarction drug.
A method of screening promotees stem cell homing drug, using having transfected above-mentioned recombinant expression carrier and internal reference fluorescence
The stable cell strain of zymophore, drug candidate, which is added, to be stimulated, to be added without any drug as negative control group；With feminine gender
Control group is compared, and using luc2 relative intensity of fluorescence as index, reflects that the drug candidate promotes the activity of CXCR4 promoter expression.
Preferably, after drug candidate effect being added 12~48 hours, then the activity value of luc2 and internal reference luciferase are detected.
Preferably, with the activity value of double fluorescent reporter gene detection systems detection luc2 and internal reference luciferase, pass through
Luc2 relative intensity of fluorescence reflects that the drug candidate promotes the effect of CXCR4 promoter expression.
Preferably, the drug candidate that can be improved luc2 and internal reference luciferase relative activity further uses Western Blot
Method detects the expression quantity of CXCR4, verifies the activity of drug candidate.Preferably, the method for drug effect verifying are as follows: filled between rat primary
The protein level detection carried out on matter stem cell (MSCs).
The present invention by building promoter containing CXCR4 and luc2 luciferase recombinant vector, and by recombinant vector with it is interior
The obtained CXCR4 promoter Dual-Luciferase of ginseng renilla luciferase cotransfection surely turns cell model, applied to promoting drug of going back to the nest
Screening.
A preferred embodiment of the invention, by pGL4.17-CXCR4 recombinant vector and internal reference sea pansy fluorescein carrier pRL-
TK cotransfection human embryonic kidney epithelial cells 293, by the expression of luc2 and sea pansy Dual-Luciferase examining report CXCR4 promoter,
Reflect CXCR4 promoter expression by luc2 uciferase activity relative value.It is of the invention surely to turn cell and can be used for examining
The expression activity for surveying CXCR4 gene is horizontal, and then for the high-throughput drug for promoting stem cell homing with quickly screening.
If candidate drug can activate CXCR4 promoter reporter gene activity, and CXCR4 in enhanced MSC s cell
Thus expression can speculate it with certain rush stem cell homing effect, can be used for preparing rush stem cell homing drug or
The drug of preparation treatment or adjuvant treatment myocardial infarction.
Therefore, the present invention provides new approaches to enhance the medicament research and development of stem-cell therapy myocardial infarction, in clinical application
On illustrate wide application prospect.
Detailed description of the invention
Essence in order to better understand the present invention illustrates this recombinant vector below in conjunction with attached drawing, originally surely turns cell
The construction method of strain and its screening promote the application of stem cell homing drug.
Fig. 1 is the building schematic diagram of recombinant vector pGL4.17-CXCR4, and A is the building of recombinant vector pGL4.17-CXCR4
Process, B are the digestion identification electrophoretogram of connection front and back recombinant vector
Fig. 2 is that TGF β 1 (transforming growth factor-beta 1) stimulates pGL4.17-CXCR4 rotaring redyeing 293 cell CXCR4 promoter living
Property evaluation
Fig. 3 is the selection result of 293-CXCR4P monoclonal cell strain in embodiment 2
Fig. 4 is based on the candidate monomer Selection for surely turning cell 293-CXCR4P
Fig. 5 is in embodiment 4, and western Blot shows that various concentration candidate monomer 6 promotes rat primary MSCs expression
The gel imaging picture of CXCR4.
Fig. 6 is in embodiment 4, and western Blot shows that various concentration candidate monomer 6 promotes rat primary MSCs expression
The relative expression quantity of CXCR4.
Specific embodiment
The present invention provides it is a kind of by stable cell strain 293-CXCR4P screen promote stem cell homing drug new method,
Can it is high-throughput, quickly, be achieved at low cost the screening for promoting stem cell homing drug, the present invention is done into one below by way of example
The detailed description of step.It should be appreciated that specific example described herein is used only for explaining the present invention, it is not used to limit this hair
It is bright.
The building of embodiment 1pGL4.17-CXCR4 recombinant vector and double digestion identification
It is as follows to consult CXCR4 promoter sequence, artificial synthesized acquisition CXCR4 promoter sequence:
CXCR4-F:5’-CTCGAGTACCGACCACCCGCAAACAGCAGGGTCCCCTGGGCTTCCCAAGCCGCGCAC
CTCTCCGCCCCGCCCCTGCGCCCTCCTTCCTCGCGTCTGCCCCTCTCCCCCACCCCGCCTTCTCCCTCCCCGCCCC
AGCGGCGCATGCGCCGCGCTCGGAGCGTGTTTTTATAAAAGTCCGGCCGCGGCCAGAAACTTCAGTTTGTTGGCTG
CGGCAGCAGGTAGCAAAGTGACGCCGAGGGCCTGAGTGCTCCAGTAGCCACCGCATCTGGAGAACCAGCGGTTACC
AAGCTT-3’
CXCR4-R:5’-AAGCTTGGTAACCGCTGGTTCTCCAGATGCGGTGGCTACTGGAGCACTCAGGCCCTC
GGCGTCACTTTGCTACCTGCTGCCGCAGCCAACAAACTGAAGTTTCTGGCCGCGGCCGGACTTTTATAAAAACACG
CTCCGAGCGCGGCGCATGCGCCGCTGGGGCGGGGAGGGAGAAGGCGGGGTGGGGGAGAGGGGCAGACGCGAGGAAG
GAGGGCGCAGGGGCGGGGCGGAGAGGTGCGCGGCTTGGGAAGCCCAGGGGACCCTGCTGTTTGCGGGTGGTCGGTA
CTCGAG-3’
Restriction enzyme site XhoI and HindIII are introduced respectively at 5 ' ends of CXCR4 promoter sequence and 3 ' ends carries out full genome
Synthesis, length 291bp；Commercial carrier pGL4.17 carries out the double digestion of XhoI and HindIII respectively, pGL4.17 after digestion
Fragment length 5567bp.CXCR4P promoter gene and carrier are connected using T4 ligase, 16 DEG C of connections are overnight.
Connection product transformed competence colibacillus cell DH5 α, picking positive colony shake bacterium and extract plasmid, digestion and the dual mirror of sequencing
Fixed, being inserted into correct clone designation is pGL4.17-CXCR4.The building of pGL4.17-CXCR4 recombinant vector is as shown in Figure 1.Weight
Building flow chart such as Figure 1A of group carrier；Figure 1B is that electrophoretogram is identified in recombinant vector digestion, M Marker, 1 for by XhoI and
For the double digestion of HindIII to recombinant vector electrophoretic band, 2 be the recombinant vector electrophoretic band after connection.Luc2 gene is located at
The downstream of CXCR4P promoter gene.
Embodiment 2, which is established, surely turns monoclonal cell 293-CXCR4
293 kinds of human embryonic kidney epithelial cells are planted in 6 orifice plates, and when reaching 90% convergence degree, cotransfection embodiment 1 is obtained
PGL4.17-CXCR4 and sea pansy fluorescein plasmid pRL-TK as internal reference changes the G418 that final concentration 1mg/ml is added in liquid afterwards for 24 hours
Continue to cultivate, add 10d after G418, pancreatin digests attached cell, single cell suspension collected and blow and beat into, then with 1/hole density
It plants in 96 orifice plates.Picking monoclonal cell with equal densities be inoculated in 96 orifice plates expand culture, for 24 hours after, be added 60 μ of final concentration
The D-luciferin of g/mL, whole body optical imaging system shooting, fluorescence intensity results are as shown in Figure 3；Strongest gram of selection signal
Grand (the 3rd) expansion culture, and it is named as 293-CXCR4P monoclonal cell.
It uses and is known to promote the transforming growth factor TGF β 1 of CXCR4 promoter expression as positive control, induction
Stimulation has transfected the 293-CXCR4 cell of pGL4.17-CXCR4 recombinant vector and pRL-TK plasmid, detects luc2 and internal reference sea pansy
The luciferase activity (luc2 fluorescence activity/sea pansy fluorescence activity) of luciferase, as a result such as Fig. 2.Compared with negative control,
1 group of luciferase activity of TGF β obviously increases, and illustrates that gained 293-CXCR4 monoclonal cell strain can be used to detect drug monomer
Regulate and control the effect of CXCR4 promoter activity.
The rush stem cell homing of the candidate monomer of embodiment 3 acts on screening
293-CXCR4P monoclonal cell is laid on 96 orifice plates with the density in 1000/hole, to cell confluency degree about 90%
Free serum culture afterwards is added candidate traditional Chinese medicine monomer (8 μ g/ml), using ginsenoside Rb1 as positive control, any drug is not added
As negative control.
After effect for 24 hours, uciferase activity value is detected with luciferase reporter gene detection system, passes through luc2 fluorescence
Plain enzymatic activity relative value reflects CXCR4 promoter expression.
Steps are as follows: after lytic cell, firefly luciferase detection reagent II (LARII) detection luc2 fluorescence letter is added
Number, Stop&GloR reagent is then added, detects sea pansy fluorescence intensity.Positive controls and drug candidate group luc2 relative fluorescence
Activity=this sample (luc2 fluorescence activity/sea pansy fluorescence activity)/negative control (luc2 fluorescence activity/sea pansy fluorescence activity).
As a result such as Fig. 4, show that candidate monomer 2,5,6,10 can enhance CXCR4 promoter activity, wherein most with the effect of monomer 6
By force, luc2 relative intensity of fluorescence is improved to 2.8 times under 8 μ g/ml concentration.
Embodiment 4Western Blot shows that candidate monomer 6 promotes rat primary MSCs to express CXCR4
The protein level detection carried out on rat primary mescenchymal stem cell (MSCs), to carry out drug effect verifying.
Verification method is as follows: separation rat marrow in primary MSCs in vitro culture 3-5 generation, be inoculated in 6 orifice plates, for 24 hours after
Candidate monomer is added, MSCs is collected in processing for 24 hours, and Western Blot method detects the expression of CXCR4 albumen.Application density ladder
Spend MSCs in centrifugal process separation SD rat marrow.SD rat is put to death using vertebra dislocation method, with 75% ethyl alcohol soaking disinfection 8min,
Separate bilateral femur and shin bone.After rejecting bone tissue surrounding tissue, exposure ossis extracts PBS with 10ml syringe and rinses bone
PBS with bone marrow cell is collected in 10ml centrifuge tube by pulp cavity, and 1000rpm is centrifuged 10min.Supernatant is abandoned, with containing 10%
The DMEM culture solution resuspension cell of FBS, 1% penicillin/streptomycin, with 5 × 105The cell density of a/ml is uniformly inoculated in
25cm2In culture bottle, in 37 DEG C, 5%CO2It is cultivated in cell incubator.Continue to cultivate after replacing culture solution after 3rd day.Work as cell
Passage when converging 70%-80%.
It takes 3-5 to be inoculated in 6 orifice plates for MSCs, candidate monomer 6 (0,5,10,20 μm of ol/L) is added afterwards for 24 hours, after processing for 24 hours
MSCs is collected, PBS is cleaned 2 times, addition RIPA lysate, and after 4 DEG C of cracking 30min, 4 DEG C, 12000rpm centrifugation 15min are collected
Supernatant measures protein content by Bradford method.100 DEG C of denaturation 5min, by sample loading 10%SDS-PAGE glue, constant pressure
After 150mV electrophoresis 50min, constant pressure 100mV transferring film 1h to nitrocellulose filter.4 DEG C of primary antibody overnight incubations, after TBST is rinsed 3 times,
HRP marks secondary antibody to be incubated for 1h, and chemical illuminating reagent develops the color after washing film, shoots picture with gel imaging system.
Using GAPDH as internal reference, as a result as shown in Figure 5 and Figure 6.It is shown by Fig. 5 and Fig. 6, candidate monomer 6 can raise
The expression quantity of CXCR4, and be in dose dependent, illustrate that candidate monomer 6 has the expression for promoting CXCR4 and promotees the work of stem cell homing
With.
Claims (10)
1. a kind of recombinant vector, which is characterized in that contain CXCR4 promoter sequence and luc2 luciferase reporter gene；It is described
Luc2 luciferase reporter gene be located at CXCR4 promoter sequence downstream.
2. recombinant vector as claimed in claim 2, which is characterized in that contain CXCR4 promoter sequence and luc2 luciferase
The pGL4.17 carrier of reporter gene.
3. a kind of stable cell strain, which is characterized in that glimmering containing recombinant vector of any of claims 1 or 2 and expression internal reference
The carrier of light enzyme.
4. stable cell strain as claimed in claim 3, which is characterized in that the internal reference luciferase is renilla luciferase.
5. stable cell strain as claimed in claim 3, which is characterized in that its host cell is human embryonic kidney epithelial cells 293.
6. a kind of recombinant vector of any of claims 1 or 2 starts sublist in the activity of detection CXCR4 promoter expression, CXCR4
Answering in terms of the drug of the drug of the activity, screening rush stem cell homing that reach or screening treatment or adjuvant treatment myocardial infarction
With.
7. drug or screening treatment that the described in any item stable cell strains of claim 3~5 promote stem cell homing in screening
Or the application in terms of the drug of adjuvant treatment myocardial infarction.
8. a kind of screening promotees the drug of stem cell homing or screening is treated or the method for the drug of adjuvant treatment myocardial infarction,
It is characterized in that, with the described in any item stable cell strains of claim 3~5, drug candidate, which is added, to be stimulated, with negative control
Group is compared, relatively glimmering by luc2 with the activity value of double fluorescent reporter gene detection systems detection luc2 and internal reference luciferase
Luminous intensity, evaluates the activity that the drug candidate promotes the expression of CXCR4 promoter, and screening can activate CXCR4 promoter transcription living
The drug of property.
9. method according to any one of claims 8, which is characterized in that after drug candidate is added, act on 12~48 hours, then detect luc2
With the activity value of internal reference luciferase.
10. method according to any one of claims 8, which is characterized in that can be improved the candidate of luc2 and internal reference luciferase relative activity
Drug further uses the expression quantity of Western Blot method detection CXCR4, verifies the activity of drug candidate.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113265423A (en) * | 2021-05-25 | 2021-08-17 | 上海家化联合股份有限公司 | Construction and application of cell strain containing luciferase reporter gene of human GSTP1ARE |
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| 王强利等: "基于CXCR4 启动子的中药有效成分高通量筛选细胞模型构建和应用", 《中国中医药信息杂志》 * |
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| 范忠伟等: "调控SDF-1/CXCR4轴在骨髓间充质干细胞向心肌梗死部位归巢中的研究现状", 《泸州医学院学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113265423A (en) * | 2021-05-25 | 2021-08-17 | 上海家化联合股份有限公司 | Construction and application of cell strain containing luciferase reporter gene of human GSTP1ARE |
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