CN105695486A - Cell model for screening drugs with anti-chronic hypoxic pathological change activity, and application and use method thereof - Google Patents
Cell model for screening drugs with anti-chronic hypoxic pathological change activity, and application and use method thereof Download PDFInfo
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Abstract
The invention provides a cell model for screening drugs with anti-chronic hypoxic pathological change activity, and application and a use method thereof. By using an HIF-1 alpha-promoter as a target spot, the cell model established by the invention can be used for systematically, efficiently and extensively screening drugs with anti-chronic hypoxic pathological change activity.
Description
Technical field
The present invention relates to a kind of cell model for screening the medicine with anti-chronic hypoxia pathological changes activity, belong to technical field of pharmaceutical biotechnology。
Background technology
Chronic hypoxia pathological changes, such as chronic obstructive pulmonary disease, is a kind of disease with flow limitation feature, with pulmonary the abnormal inflammatory of the harmful gass such as smoke from cigarette or deleterious particle is reacted and chronic hypoxia is relevant。Along with the progress of the course of disease, long-term chronic inflammation and anoxia can cause pulmonary vasoconstriction, lung endothelium functional disorder and pulmonary artery remodeling etc. to ultimately result in the generation of pulmonary hypertension, pulmonary artery remodeling is closely related with course advancement and Complicated with Pulmonary arterial hypertension thereof。Pathological change process relates generally to body cell and coordinates oxygen change at gene transcription level, and relevant functional gene is then hypoxia inducible factor (Hypoxiainduciblefactor-1, HIF-1)。
Fast development along with life sciences, drug screening method based on examining report gene achieves bigger development in recent years and is widely applied, it relates to intact cell, the drugs impact on life entity function under living cells natural conditions, closer to internal biochemical process, it is possible to relatively accurately understand the biological characteristics of medicine。
Quick, the category filter model that build anti-chronic hypoxia pathological changes activity not only facilitate the active drug finding from Chinese medicine or existing chemical drugs or this new chemical entities and finding that treatment nerve fiber sexually transmitted disease (STD) becomes, new lead compound is provided for new drug development, it is alternatively the early stage basis that the study on mechanism offer of various Drug therapy nerve fiber sexually transmitted disease (STD) change is certain, is conducive to the development of TCM Modernization。
Lu Qiong reports the medicaments sifting model built based on HIF-1 promoter, but the base sequence of not open genetic fragment used, those skilled in the art have no way of finding out about it the concrete structure of this screening model and construction method (Lu Qiong, the structure of brain ischemia medicament screening cell model and applied research, Southwest Jiaotong University's academic dissertation, 2014)。
Summary of the invention
In order to build the screening model of a kind of medicine with anti-chronic hypoxia pathological changes activity, the invention provides a kind of gene constructs。
Gene constructs provided by the invention contains from 5 ' ends successively to 3 ' ends: HIF-1 promoter gene-583bp～+23bp sequence and reporter sequences。
For rat, HIF-1 promoter gene-583bp～+23bp sequence refers to SEQNO.1:
Further, above-mentioned reporter gene is selected from: fluorescence protein gene and/or luciferase gene。
Preferably, above-mentioned fluorescence protein gene is green fluorescent protein (GFP) gene or red fluorescent protein (RFP) gene；Above-mentioned luciferase gene is firefly luciferase gene。
In red fluorescent protein, E2-Crimson is comparatively conventional。
Invention further provides a kind of recombinant vector, this carrier contains above-mentioned gene constructs。
Carrier in the present invention refers to be transferred to by DNA fragmentation (genes of interest) DNA molecular of a kind of energy self replication of recipient cell in genetic engineering recombinant DNA technology, and conventional has plasmid, phage and animals and plants virus。
The present invention constructs a kind of stable cell further, and this cell contains in above-mentioned recombinant vector or its genome integrates above-mentioned gene constructs。
Further, any one in HEK293 cell, Hela cell, Chinese hamster ovary celI or stem cell of this cell。
Invention further provides the method building above-mentioned recombinant vector, it comprises the steps:
(1) synthesis HIF-1 promoter gene-583bp～+23bp sequence；
(2) sequence step (1) synthesized carries out enzyme action respectively with the report carrier with reporter gene, connects report carrier and promoter fragment, converts escherichia coli, resistance screening, selects positive transformant, extracts plasmid, obtains recombinant vector。
Further, in step (2), described report carrier is the pSC Basic plasmid with reporter gene。
Reporter gene described herein is with above-mentioned, namely selected from fluorescence protein gene and/or luciferase gene。
Invention further provides the method building above-mentioned stable cell, be about to the recombinant vector transfection stated
Any one in HEK293 cell, Hela cell, Chinese hamster ovary celI or stem cell。
Invention further provides the purposes of this stable cell, what this was stable can be used for screens and can improve HIF-1 expression or have the medicine of anti-chronic hypoxia pathological changes activity, such as all kinds of Chinese medicines, existing chemical drugs, new chemical entities etc.。
Invention further provides a kind of method that screening has the medicine of anti-chronic hypoxia pathological changes activity, the method comprises the steps:
(1) drug candidate is given the stable cell that the present invention builds；
(2) expression of reporter gene in described cell is detected；
(3) judged result, if described drug candidate can improve the expression of reporter gene, then shows that this drug candidate can improve HIF-1 expression or have anti-chronic hypoxia pathological changes activity。
Further,
Step (1) includes adding drug candidate in the stable cell culture system of above-mentioned structure and co-cultures；
Step (2) includes the expression of the reporter gene of test experience group, and compares with blank group, and blank group is the stable cell that the present invention with the cultivation of experimental group the same terms without described drug candidate builds；
If the expression of reporter gene is higher than matched group in experimental group, it was shown that drug candidate can improve HIF-1 expression or have anti-chronic hypoxia pathological changes activity。
It is further preferred that expression >=150% of the reporter gene of the expression/matched group of the reporter gene of experimental group, then show that drug candidate has anti-chronic hypoxia pathological changes activity。
The cell screening model that it is target spot with HIF-1 promoter that the present invention constructs, can system, effectively, in many aspects screening have anti-chronic hypoxia pathological changes activity medicine。
Accompanying drawing explanation
Fig. 1 plasmid construction schematic diagram
The structure (fluorescence field and light field, 200 ×) of Fig. 2 monoclonal cell strain
Fig. 3 builds the stable 293-pHp-E2 cell strain (fluorescence field, 100 ×) obtained
Fig. 4 red fluorescent protein (E2) cell model Function Identification (fluorescence field, 100 ×),
A:293-pHp-E2 (0 μM of CoCl2)；B:293-pHp-E2 (300 μMs of CoCl2)
Fig. 5 luciferase cell model (293-pHp-Luc) Function Identification
The selection result of Figure 65 Chinese medicine extract。
Detailed description of the invention
Material
1.1 bacterial strains and cell strain coli strain DH5 α (Sichuan University's National Biomedical material engineering investigative technique center 505 Laboratories Accession), human embryonic kidney epithelial cells HEK293 (Sichuan University's National Biomedical material engineering investigative technique center 505 Laboratories Accession)。
1.2 plasmid pSC-E2, namely the pSC plasmid (505 laboratorys build) of reporter gene E2-Crimson (E2) (red fluorescent protein) it is connected to, pSC-Luc, namely (construction method is referring to SLiu to be connected to the pSC plasmid (505 laboratorys build) of reporter gene Luciferase (Luc) (luciferase), LMa, RTan, etal.Safeandefficientlocalgenedeliveryintoskeletalmuscle viaacombinationofPluronicL64andmodifiedelectrotransfer.G eneTherapy (2014) 21, 558 565)。
1.3 main agents LATaq polymerases (TaKaRa), PfuDNA polymerase (Fermentas),Taq polymerase (Invitrogen)；Restricted enzyme (Fermentas): Mlu I, Afl II, Mun I, BamH I, Not I, Hind III, Xba I；T4DNA ligase (TaKaRa)；E.Z.N.A.TMPlasmid Mini Kit (E.Z.N.A.TMPlasmidMiniKit I, Omega), glue reclaim test kit (BioSpinGelExtractionKit, Bioflux)；Mammalian cell transfection reagent (LipofectamineTM2000, Invitrogen), Luciferase Assay Reagent box (LuciferaseAssaySystemwithReporterLysisBuffer, Promega), determination of protein concentration test kit (BCAProteinAssayKit, Thermo), G418 (Amresco), dimethyl sulfoxide (DMSO, Sigma), cobalt dichloride (CoCl2, Sigma), Tissue Culture Dish (Thermo), all size culture plate (Corning)；Other chemical reagent, except indicating, is domestic analytical pure。
1.4 instrument PCR instrument (S1000TMThermalCycler, BIO-RAD), electrophresis apparatus (DYY-III type, Liuyi Instruments Plant, Beijing), Labworks image acquisition and analysis software (ChemiDocXRS+ type, BIO-RAD), ultramicrospectrophotometer (Nanodrop2000, ThermoScientific), microscope (DMIL, Leica), inverted phase contrast fluorescence microscope (DMI4000B, Leica), CO2 incubator (MACO-15AC, SANYO), superclean bench (SW-CJ-2N type, Harbin Dong Lian company), microplate reader (Flash,ThermoScientific)。
The structure of embodiment 1 recombiant plasmid
1.1 test methods
With rat genomic dna for template, design primer introduces Mlu I and Afl II restriction enzyme site pcr amplification obtains promoter fragment respectively in the upstream and downstream of rHIF-1 promoter sequence: rHIF-1 pro (is abbreviated as Hp) (-583bp～+23bp) (+1 is gene transcription start site), the sequence of forward primer refers to SEQNO.2, and the sequence of reverse primer refers to SEQNO.3。1% agarose gel electrophoresis is identified, ethanol precipitation recovery obtains each promoter fragment。Double digestion carrier pSC-E2 and each promoter fragment (Hp) respectively, 1% agarose gel electrophoresis, glue reclaims purpose fragment, connects carrier and promoter fragment, convert escherichia coli, ampicillin plate resistance screening, selects positive transformant, carries out rapid cleavage qualification, plasmid is extracted after positive amplification culture, carry out enzyme action and PCR identifies, to identifying that successful plasmid checks order, finally obtain recombiant plasmid pHp-E2。
In like manner prepare recombiant plasmid pHp-Luc。
1.2 experimental results
As shown in the schematic of Figure 1, two kinds of reporter gene systems of this research and establishment, wherein reporter gene E2 is red fluorescent protein, observes for fast qualitative and judges；Luc is luciferase, for quantitative assay。The display construction of recombinant plasmid success of double digestion, PCR qualification result。Sending Sangon Biotech (Shanghai) Co., Ltd. to check order, through comparison, sequencing result shows that each promoter sequence inserted is included with NCBI consistent, it was shown that success construction recombination plasmid pHp-E2, pHp-Luc。
The foundation of embodiment 2 stable cell line
2.1 experimental techniques
By recombiant plasmid pHp-E2, pHp-Luc transfected HEK 293 respectively, Lipofectamine is shown in concrete operationsTM2000 reagent description。After the cell trypsinization of 36～48 hours after transfection, according to transfection efficiency, appropriate by its dispersion culture in 10cm culture dish, namely add 800ng/ μ LG418 after cell attachment and proceed by screening, after 2-3 week, cell forms spacing independent group farther out in culture dish；Under inverted microscope, select suitable population of cells, bottom culture dish, mark the position of this population of cells with marking pen；In the operation of rigorous aseptic, with knife blade, 1mL suction nozzle is whittled into suitable inclined-plane, with the rifle head with inclined-plane carefully by the whole targeted elimination of cell around labelling point；By culture medium by the cell scraped rinsing as far as possible, remove, retain the cell of labelling, add fresh culture and cultivate；When the population of cells chosen expands gradually, detect heteroproteose cell about in real time and remove in time；When this population of cells grows up to appropriately sized, with pancreatin fixed point digestion gradient dilution in 96 orifice plates, after cell attachment, picking out the hole containing individual cells under fluorescence microscope, labelling lay equal stress on a detection, cultivate；When cell grows up to appropriately sized clone in 96 orifice plates, proceeded to amplification culture in 24 orifice plates, then proceeded in 6 orifice plates amplification culture again and frozen in time。
2.2 experimental results
As in figure 2 it is shown, fluorescence field and light field observed result show the building process of the monoclonal cell strain of stable transfection, it can be seen that monoclonal is grown up gradually。
Fig. 3 again shows that, the present invention builds the HEK-293 cell (being abbreviated as 293-pHp-E2 cell) obtaining stable transfection pHp-E2。
The Function Identification of embodiment 3 cell model
3.1 experimental techniques
Positive stimulus thing CoCl2The expression of HIF-1 in cell can be raised, therefore can be used for verifying whether screening model is successfully established。If being successfully established screening model, after giving the stimulation of positive stimulus thing, in cell, the expression of HIF-1 can be raised。
For verifying the effectiveness of model, investigate the response for known positive stimulus thing of two kinds of report gene cell model 293-pHp-E2 cells and 293-pHp-Luc cell respectively。
The cultivation of 293-pHp-E2 cell and 293-pHp-Luc cell and HEK-293 cell all adopts HEK-293 cell culture condition to carry out, i.e. DMEM height sugar culture fluid (Hyclone company)+10%FBS (Sciencell company)+1 × 105The penicillin and streptomycin (Hyclone company) of U/L, 37 DEG C, 5%CO2 incubator is cultivated。Every day observation of cell quantity and form, when Growth of Cells converges to 80%, the trypsin Hyclone company with 0.25%) carry out had digestive transfer culture or need to be inoculated in 24 holes or 96 orifice plates according to experiment。Take the cell being in exponential phase to test。
Experiment sets blank group and experimental group, the multiple hole of each concentration 3。CoCl2The solution of respective concentration it is diluted to by culture medium。Experimental group is following respectively adds positive stimulus thing, and blank group adds the culture medium of respective volume。CoCl in experimental group2Final working concentration be 0,50 μMs, 150 μMs, 300 μMs, the process time is 24h。
Fluorescence microscope is utilized directly to observe the reaction to positive stimulus thing of the 293-pHp-E2 cell；Utilize the detection 293-pHp-Luc cell reaction to positive stimulus thing of Luciferase Assay Reagent box。
Luciferase assays step is as follows:
Being Lysis1 × Buffer by ReporterLysis5 × Buffer dilution, collect the cell in orifice plate with Lysis1 × Buffer, be placed in the EP pipe of 0.5mL ,-80 DEG C overnight, standby。The frozen 293-pHp-Luc cell in-80 DEG C after experimental group and matched group being processed takes out, and is placed in 4 DEG C of thawing, after melting completely on vortex instrument vortex 15s, 4 DEG C, 12000 × g is centrifuged 3min and obtains supernatant。Accurate 20 μ L of supernatant of drawing are in the microwell plate of white, and 3, each sample is parallel。Every hole 50 μ LLuciferaseAssaySubstrate, auto injection are set。Microplate reader measures the activity of luciferase。
Utilize determination of protein concentration test kit (BCAProteinAssayKit, Thermo) carry out determining the protein quantity。First the preparation of standard curve solution is carried out according to test kit description。Then the accurate titer drawing 25 μ L or sample supernatant are in 96 orifice plates, and 3, each sample is parallel, and every hole adds the working solution (workingregent) of 200 μ L, jog, hatch 30min in 37 DEG C, after being cooled to room temperature, measure the absorbance at 562nm place by microplate reader。With BSA content for abscissa, with light absorption value for vertical coordinate, drawing standard curve。The protein content of sample is gone out according to the bent Equation for Calculating of mark。
Final uciferase activity is: relative fluorescence element enzyme unit=uciferase activity/protein content alive。
3.2 experimental results
As shown in Figure 4, after processing 24h, 300 μMs of CoCl2Process group, increase in time, redness brightens gradually, namely red fluorescent protein is expressed and is gradually increased, and matched group brightness almost can be ignored, CoCl2Process group brightness is apparently higher than blank group。This shows that the present invention successfully constructs 293-pHp-E2 cell, and positive stimulus thing can be responded by this cell model。
As it is shown in figure 5, the expression of luciferase increases with the rising of positive stimulus substrate concentration, under high dose compared with untreated blank group, its difference has statistical significance (P < 0.05)。This shows that the present invention successfully constructs 293-pHp-Luc cell, and positive stimulus thing can be responded by this cell model。
Embodiment 4 cell model of the present invention is for the screening of medicine
4.1 experimental techniques
From Chinese medicine, extract isolated 17 monomer components and compile respectively as No. 1-17。With the 293-pHp-Luc cell model built, these 5 monomer components being carried out screening active ingredients, screening concentration is 1 μM, and the process time is 24h, and specific experiment step is with reference to embodiment 3。
Measure agent-feeding treatment according to above-mentioned uciferase activity assay method and respectively organize cell fluorescence element enzymatic activity, compare with negative solvent control group, calculate the impact on HIF-1 Gene Transcription in vitro of each monomer component, more than adjust and reform variability >=150% as screening activity judgment standard。
Raise rate of change=(experimental group relative luciferase activity/matched group relative luciferase activity) × 100%。
4.2 experimental results
As shown in Figure 6, in these the 17 kinds of monomer components extracted from Chinese medicine, having the rise rate of change of monomer component process group in 7 more than 150%, in the screening positive, namely to show, these 7 kinds of monomer components are likely to be of good anti-chronic hypoxia pathological changes activity。
These 7 compositions respectively 1 (astragaloside, C41H68O14), 3 (peoniflorin, C23H28O11), 4 (eugenol, C10H12O2), 7 (Hydroxy Carthamus yellow, C27H32O16), 9 (genkwanin, C16H12O5), 11 (rhodioside, C14H20O7), 12 (chimonin, C19H18O11)。
To sum up, the cell screening model that it is target spot with HIF-1 promoter that the present invention constructs, can system, effectively, in many aspects screening have anti-chronic hypoxia pathological changes activity medicine。
Claims (10)
1. a gene constructs, it is characterised in that: described gene constructs contains from 5 ' ends successively to 3 ' ends: HIF-1 promoter gene-583bp～+23bp sequence and reporter sequences。
2. gene constructs as claimed in claim 1, it is characterised in that: described reporter gene is selected from: fluorescence protein gene and/or luciferase gene；Further, described fluorescence protein gene is green fluorescence protein gene or red fluorescent protein gene, and described luciferase gene is firefly luciferase gene。
3. a recombinant vector, it is characterised in that: described recombinant vector contains the gene constructs described in any one of claim 1 or 2。
4. cell one kind stable, it is characterised in that: described cell contains the recombinant vector described in claim 3 or is integrated with the gene constructs described in any one of claim 1 or 2 in its genome。
5. cell stable as claimed in claim 4, it is characterised in that: any one in HEK293 cell, Hela cell, Chinese hamster ovary celI or stem cell of described cell。
6. build the method for recombinant vector described in claim 3, it is characterised in that: it comprises the steps:
(1) synthesis HIF-1 promoter gene-583bp～+23bp sequence；
(2) sequence step (1) synthesized carries out enzyme action respectively with the report carrier with reporter gene, connects report carrier and promoter fragment, converts escherichia coli, resistance screening, selects positive transformant, extracts plasmid, obtains recombinant vector；
Further, in step (2), described report carrier is the pSC Basic plasmid with reporter gene。
7. the method for the structure stable cell described in claim 4, it is characterised in that: by any one in the recombinant vector transfected HEK 293 described in claim 3, Hela cell, Chinese hamster ovary celI or stem cell。
8. the purposes of the stable cell described in claim 4, it is characterised in that: described cell can improve HIF-1 expression for screening or have the medicine of anti-chronic hypoxia pathological changes activity。
9. a screening can improve HIF-1 expression or the method with the active medicine of anti-chronic hypoxia pathological changes, it is characterised in that described method comprises the steps:
(1) drug candidate is given the stable cell described in claim 4 or 5；
(2) expression of reporter gene in described cell is detected；
(3) judged result, if described drug candidate can improve the expression of reporter gene, then shows that this drug candidate can improve HIF-1 expression or have anti-chronic hypoxia pathological changes activity；
Further, step (1) includes being added by drug candidate in the stable cell culture system described in claim 4 or 5 and co-cultures；
Step (2) includes the expression of the reporter gene of test experience group, and compares with blank group, described blank group be without described drug candidate with experimental group the same terms cultivate claim 4 or 5 described in cell；
If the expression of reporter gene is higher than matched group in experimental group, it was shown that drug candidate can improve HIF-1 expression or anti-chronic hypoxia pathological changes activity。
10. method according to claim 9, it is characterised in that: expression >=150% of the reporter gene of the expression/matched group of the reporter gene of experimental group, it was shown that drug candidate can improve HIF-1 expression or have anti-chronic hypoxia pathological changes activity。
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| CN114940975A (en) * | 2022-06-20 | 2022-08-26 | 中国人民解放军总医院 | TED-deleted cell line and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1580262A (en) * | 2003-08-12 | 2005-02-16 | 上海第二医科大学 | Carrier system containing artificial transcription factor regulated by oxygen deficit, and its configuration and use |
-
2016
- 2016-01-29 CN CN201610064454.4A patent/CN105695486A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1580262A (en) * | 2003-08-12 | 2005-02-16 | 上海第二医科大学 | Carrier system containing artificial transcription factor regulated by oxygen deficit, and its configuration and use |
Non-Patent Citations (7)
| Title |
|---|
| ELISABETH L.PAGÉ ET AL.: "Induction of hypoxia-inducible factor-1ɑ by transcriptional and translational mechanisms.", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
| MUZNY D.MARIE ET AL.: "EBI：AC134165", 《EBI》 * |
| WENGER,R.H. ET AL.: "GenBank:Y13656.1", 《GENBANK》 * |
| 卢琼: "脑缺血药物筛选细胞模型的构建及应用研究", 《中国优秀硕士学位论文全文数据库医药卫生科技辑》 * |
| 吉然: "如何查找基因启动子，外显子，内含子序列", 《HTTP://BLOG.SINA.COM.CN/S/BLOG_DD0944EF0101BZXP.HTML》 * |
| 吴晓兰等: "缺氧诱导因子1研究进展", 《国外医学分子生物学分册》 * |
| 新天: "使用NCBI或USCS查找基因启动子序列遇到的问题", 《HTTP://WWW.DXY.CN/BBS/TOPIC/27389142》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114940975A (en) * | 2022-06-20 | 2022-08-26 | 中国人民解放军总医院 | TED-deleted cell line and application thereof |
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