CN113403280B - KGF promoter-targeted mesenchymal stem cell screening model for treating acute lung injury - Google Patents

KGF promoter-targeted mesenchymal stem cell screening model for treating acute lung injury Download PDF

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CN113403280B
CN113403280B CN202010187546.8A CN202010187546A CN113403280B CN 113403280 B CN113403280 B CN 113403280B CN 202010187546 A CN202010187546 A CN 202010187546A CN 113403280 B CN113403280 B CN 113403280B
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kgf
promoter
mesenchymal stem
luciferase reporter
stem cells
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瞿介明
冯耘
杨晓彤
陈虹
丁永杰
陈巍
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Ruinjin Hospital Affiliated to Shanghai Jiaotong University School of Medicine Co Ltd
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Abstract

The invention discloses a KGF promoter-targeted mesenchymal stem cell screening model for treating acute lung injury. The preparation method comprises the following steps: 1) Constructing a luciferase reporter gene vector containing a KGF gene promoter; 2) Preparing a lentivirus solution containing the KGF gene promoter by using the luciferase reporter gene vector containing the KGF gene promoter in the step 1), further infecting mesenchymal stem cells, and screening stable transformants. The invention uses the mesenchymal stem cells with the function of treating diseases as a model, uses the KGF gene promoter for coding soluble factors secreted by the mesenchymal stem cells as a target spot, screens potential medicines for promoting the mesenchymal stem cells to secrete the soluble factors KGF, and lays a foundation for treating acute lung injury by using the medicines and the mesenchymal stem cells in a combined manner, enhancing the curative effect.

Description

KGF promoter-targeted mesenchymal stem cell screening model for treating acute lung injury
Technical Field
The invention relates to a cell model for screening potential drugs and a screening method of the potential drugs, in particular to a KGF promoter-targeted mesenchymal stem cell screening model for treating acute lung injury.
Background
Acute Lung Injury (ALI) and Acute Respiratory Distress Syndrome (ARDS) are life-threatening diseases and have a very high mortality rate in patients. ALI and ADRS are characterized primarily by damage to the alveolar-capillary membrane barrier, leukocyte accumulation, pulmonary edema, inflammatory cell exudation, and alveolar hemorrhage, leading to a complex relationship between the immune system and the alveolar capillaries that leads to an acute pro-inflammatory response.
Mesenchymal Stem Cells (MSCs) are cells that have the ability to self-renew, differentiate. It has been discovered that MSCs secrete soluble factors such as growth factors, anti-inflammatory factors, and antimicrobial peptides that stabilize the pulmonary microvascular barrier, enhance alveolar fluid clearance, and reduce infection. And the source of the MSCs is wide, and the MSCs can be separated from various tissues, such as bone marrow, fat, placenta and the like. According to preclinical studies, MSCs from different sources have different immune and biological functions. Human placental chorionic derived MSCs (hCMSCs) belong to placental derived MSCs and are considered as alternative sources of MSCs. Since hCMSCs can differentiate into three germ layers and are readily available, they have a better immunomodulatory function than MSCs from other sources.
Keratinocyte Growth Factor (KGF) is one of the members of the fibroblast Growth Factor family. KGF is considered to be one of the most important paracrine soluble factors of MSCs, and can stimulate the proliferation of type II lung epithelial cells in vivo and in vitro, promote the repair of the type II epithelial cells and further enhance the body resistance to lung injury. KGF can improve edema clearance by promoting the synthesis of surface active substances. In addition, the KGF can reduce the accumulation of hydroxyproline in the lung during lung injury and inhibit the expression of collagen mRNA, thereby preventing pulmonary fibrosis.
The high-flux drug screening technology has become an important subject of research in the field of drug screening at present, and the drug research process aiming at the target is the key for finding the lead compound and plays an important role in the drug research and development process. Chinese patent CN110452880A discloses a preparation method and application of an acute lung injury cell model, and specifically discloses 1) placing A549 cells in an induction culture medium for induction culture, then removing the culture medium, digesting and harvesting the cells; 2) Suspending the harvested A549 cells by using a culture medium, placing the suspension in a culture dish, adding inflammatory substances to stimulate and stimulate inflammatory reaction, and continuously culturing for 12-48 hours to obtain the medicament for screening acute lung injury. However, no cell model for screening acute lung injury drugs as described in the present application is known at present.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a mesenchymal stem cell of a targeting KGF promoter for screening potential drugs for treating acute lung injury, a preparation method and application thereof, and a method for screening potential drugs for treating acute lung injury.
In a first aspect, the mesenchymal stem cells targeting KGF promoter for screening potential drugs for treating acute lung injury are prepared by the following steps:
1) Constructing a luciferase reporter gene vector containing a KGF gene promoter;
2) Preparing a lentivirus solution containing the KGF gene promoter by using the luciferase reporter gene vector containing the KGF gene promoter in the step 1), further infecting mesenchymal stem cells, and screening stable transformants.
Preferably, the sequence of the KGF gene promoter inserted into the luciferase reporter gene vector containing the KGF gene promoter is shown as SEQ ID NO. 3.
Preferably, the luciferase reporter gene vector containing the KGF gene promoter is constructed by the following method: the KGF gene promoter sequence was inserted between the PacI and BamHI sites of the vector CV123-Luc containing the luciferase reporter gene.
Preferably, the mesenchymal stem cell is a human placental chorion-derived mesenchymal stem cell.
In a second aspect, a method for preparing a KGF promoter-targeted mesenchymal stem cell for screening potential drugs for treating acute lung injury is provided, which comprises the following steps:
1) Constructing a luciferase reporter gene vector containing a KGF gene promoter;
2) Preparing a lentivirus solution containing the KGF gene promoter by using the luciferase reporter gene vector containing the KGF gene promoter in the step 1), further infecting mesenchymal stem cells, and screening stable transformants.
Preferably, the sequence of the KGF gene promoter inserted into the luciferase reporter gene vector containing the KGF gene promoter is shown as SEQ ID NO. 3.
Preferably, the luciferase reporter gene vector containing the KGF gene promoter is constructed by the following method: the KGF gene promoter sequence was inserted between the PacI and BamHI sites of the vector CV123-Luc containing the luciferase reporter gene.
Preferably, the mesenchymal stem cell is a human placental chorion-derived mesenchymal stem cell.
In a third aspect, the provided application of the mesenchymal stem cells targeting the KGF promoter for screening potential drugs for treating acute lung injury is used for screening potential drugs for treating acute lung injury.
In a fourth aspect, the provided method for screening potential drugs for treating acute lung injury is to stimulate mesenchymal stem cells of the KGF-targeted promoter for screening potential drugs for treating acute lung injury with a substance to be tested, and after a period of action, reflect the activity of the KGF-targeted promoter by detecting fluorescence intensity according to a luciferase reporter gene, so as to obtain a compound that the KGF-targeted promoter promotes efficient expression of the luciferase reporter gene, and thus obtain the potential drugs for treating acute lung injury.
Compared with the prior art, the invention has the beneficial effects that:
1. the method for establishing the drug screening model of the invention is different from the prior art, and the target gene is generally introduced into cells such as 293T or directly takes representative cells of diseases as the model. The invention uses the mesenchymal stem cells with the function of treating diseases as a model for the first time, uses the KGF gene promoter for coding soluble factors secreted by the mesenchymal stem cells as a target spot, screens potential medicines for promoting the mesenchymal stem cells to secrete the soluble factors KGF, and lays a foundation for treating acute lung injury by using the medicines and the mesenchymal stem cells in a combined manner, enhancing the curative effect. The model of the invention can screen out the compound with the target KGF promoter from a mass compound library in a high-throughput manner, and has good application prospect for screening the drugs for treating the acute lung injury diseases.
2. The liraglutide is an analogue of GLP-1, and the liraglutide is found in previous researches to have the effects of inhibiting inflammation and promoting the expression of mesenchymal stem cells KGF-2, ang-1 and SPC so as to relieve acute lung injury. Dexamethasone is a glucocorticoid drug which is commonly used in clinic and is used for treating ALI early inflammation amplification reaction, and a certain effect is achieved, and in the previous research, dexamethasone is found to be capable of promoting KGF expression. The invention uses liraglutide and dexamethasone as medicines to verify a screening platform, and the result shows that the medicine screening platform can screen out medicines which cause KGF expression increase. This shows that the mesenchymal stem cell screening model prepared by the invention provides a good environment for the effect of the drug and the KGF promoter, and the KGF gene promoter fragment selected by the invention and the constructed plasmid vector are suitable and can be effectively combined with the drug causing KGF expression increase, thereby avoiding screen leakage.
Drawings
FIG. 1: and (3) constructing CV-123-KGF-promoter-Luc recombinant plasmid.
FIG. 2: and (3) a graph of the effect of the liraglutide targeting KGF promoter on the expression intensity of a luciferase reporter gene.
FIG. 3: and (3) a graph of the effect of the dexamethasone-targeted KGF promoter on the expression intensity of the luciferase reporter gene.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally according to conventional conditions or according to conditions recommended by the manufacturers. All percentages, ratios, proportions, or parts are by weight unless otherwise specified. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention.
Example 1
1 construction of luciferase reporter Gene vector containing KGF Gene promoter
1.1 primer design
The KGF promoter region sequence in NCBI Gene is about 3000bp, and Primer 5.0 Primer design software is applied to design the primers KGF-F and KGF-R of the KGF promoter.
KGF-F：AGATCCAGTTTGGTTAATTAAATCCTATAGATTTATTTTCGTAGTATA
AAATGAC（SEQ ID NO:1）
KGF-R：CGACCGGTACCCGGGGATCCTAGCCAGTGAGCTGTTAGTTGAATAC（SEQ ID NO:2）
1.2 Obtaining of KGF Gene promoter sequence
Carrying out PCR by using the primers KGF-F and KGF-R of the KGF promoter and an artificially synthesized promoter region sequence KGF (synthesized by Shanghai Jiehui bioengineering Co., ltd.) as a template, amplifying to obtain a promoter sequence of a target fragment KGF, and identifying a PCR product by using agarose gel electrophoresis to obtain a target product with about 3000bp containing PacI and BamHI enzyme cutting sites at two ends.
1.3 construction of recombinant plasmid carrying luciferase reporter gene of KGF promoter
After PacI and BamHI double enzyme digestion treatment is carried out on the promoter sequence KGF obtained in 1.2, the purified and recovered promoter sequence KGF is connected with a luciferase reporter gene-containing vector CV123-Luc (the CV123 vector containing the double luciferase reporter gene (purchased from Ji Kai gene) is subjected to PacI and BamHI double enzyme digestion treatment, the purified and recovered CV123-Luc is obtained), a connection reaction solution is transformed into Escherichia coli DH5 alpha competence, inverted culture is carried out for 16-20h at 37 ℃ on an LB solid culture medium, ampicillin resistance positive monoclones are picked, plasmid DNA is extracted, the positive clones are preliminarily identified by using the techniques of colony PCR, enzyme digestion and the like, and a recombinant plasmid named CV-123-KGF-promoter-Luc is obtained. The construction of the recombinant plasmid is shown in FIG. 1. The KGF promoter sequence inserted into the plasmid is shown in SEQ ID NO 3.
2 screening of hPMSCs stably expressing KGF Gene-containing promoters
2.1 preparation of lentivirus solutions containing KGF Gene promoters
24h before transfection, 293T cells in the logarithmic growth phase were trypsinized and cell density was adjusted to about 5X 10 in medium containing 10% serum 6 Cells/15 ml, reseeded in 10 cm cell culture dish at 37 deg.C with 5% CO 2 Culturing in an incubator. 24 And h, the cells can be used for transfection when the cell density reaches 70% -80%. The medium was changed to serum-free medium 2h before transfection. Adding 20. Mu.g of the prepared GV-123-KGF-promoter-Luc vector plasmid, 15. Mu.g of pHelper1.0 vector plasmid (Biovector NTCC Inc. USA, biovector 510383) and 10. Mu.g of pHelper2.0 vector plasmid (Biovector NTCC Inc. USA) to a sterilized centrifuge tube, adding a virus infection enhancing solution, adjusting the total volume to 1 ml, and incubating at room temperature for 15 min; the mixed solution is slowly dripped into the 293T cell culture solution, mixed evenly and treated at 37 ℃ with 5% CO 2 Culturing in a cell culture box; culturing 6h, discarding the culture medium containing the transfection mixture, adding 10 ml PBS solution for washing once, gently shaking the culture dish to wash the residual transfection mixture, and then pouring and discarding; slowly adding 10% serum-containing cell culture medium 20ml, and adding 5% CO at 37 deg.C 2 The incubator is used for continuously culturing 48-72 h.
2.2 concentration and purification of a lentivirus solution containing the KGF Gene promoter
Collecting 293T cell supernatant of 48 h (which can be counted as 0 h) after transfection according to cell states; centrifuging at 4000g for 10min at 4 deg.C to remove cell debris; filtering the supernatant with a 0.45 μm filter in a 40ml ultracentrifuge tube; respectively balancing samples, putting the ultracentrifuge tubes with virus supernatant into a Beckman ultracentrifuge one by one, setting the centrifugation parameters to be 25000 rpm, the centrifugation time to be 2h, and controlling the centrifugation temperature to be 4 ℃; after the centrifugation is finished, removing the supernatant, removing the liquid remained on the tube wall as much as possible, adding a virus preservation solution, and lightly and repeatedly blowing and resuspending; after full dissolution, the mixture is centrifuged at 10000 rpm at high speed for 5 min, and then the supernatant is taken and packaged.
2.3 Titer test of lentivirus solutions containing the KGF Gene promoter
The day before assay, 293T adherent cell plating, 96-well plates, 4X 10 per well, were used 4 Cells, volume 100 μ l; according to the expected titer of the virus, 8 sterile EP tubes were prepared and 90 μ l of serum-free medium was added to each tube; adding 10 μ l of virus stock solution to be measured into the first tube, mixing uniformly, adding 10 μ l into the second tube, and continuing the same operation until the last tube; selecting required cell holes, discarding 90 mul of culture medium, adding 90 mul of diluted virus solution, and culturing in an incubator; 24 After h, 100. Mu.l of complete medium was added. Resistant puromycin was added 72h post infection, maintaining drug concentration 5 μ g/ml. The culture was continued for 1 day, and the growth of the cells was observed. Viral titers were calculated by the number of viable cells after infection. And (3) the virus titer = the number of live cells/amount of virus stock, namely the measured virus titer is 2E +8TU/ml.
2.4 screening of hCMSCs stably expressing KGF promoter
Preparation of a density of 3-5X 10 with complete Medium 4 500 mul of cell suspension of human placenta chorion originated mesenchyme stem cell is inoculated to 24-well plate and cultured at 37 deg.c for 16-24 hr to reach cell confluency of 20-30%. The infectious agent is mixed into the medium and the lentivirus is added subsequently according to the virus titer. Culturing at 37 deg.C for 12-16h, replacing with complete culture medium, and continuing culturing. After 48-72h of infection, cells were cultured in puromycin-containing medium at 2. Mu.g/ml, and puromycin-containing medium was changed every 2-3 days until uninfected control cells were completely killed by puromycin, and no cells of the infected virus group died. And reducing the puromycin concentration to 1/2-1/4 of the maintenance concentration, and continuously screening and amplifying infected cells to obtain the hCMSCs stable transformant stably expressing the KGF gene promoter.
3 application of drug screening
2 the hCMSCs stable transfectants which are screened out and express KGF promoters are subjected to 2 multiplied by 10 per well 4 Mu.l of each cell was plated on a 96-well plate, and the positive control drugs liraglutide (100 nM, purchased from Maclin Co.) and dexamethasone (10 nM, purchased from Sigma Co.) were added, and a culture medium containing DMSO was added as a blank control, and 5 replicate wells were provided for each group, and after 72 hours of action, 100. Mu.l of Dual-Lumi firefly luciferase assay reagent (Dual-Lumi Dual luciferase reporter assay kit, purchased from Byunsya Co.) was added, mixed well, left to stand at room temperature for 10min, and the fluorescence intensity was measured using a multifunctional microplate reader. Then, 100. Mu.l of Dual-Lumi double-luciferase reporter gene detection working solution (Dual-Lumi Dual-luciferase reporter gene detection kit, available from Biyunnan Co., ltd.) was added thereto, mixed, incubated at room temperature for 10min, and the fluorescence intensity was measured using a multifunctional microplate reader.
According to the formula: degree of gene activation of interest = degree of activation of luciferase expression promoted by RLU calculation for RLU/renilla luciferase reporter measure measured for firefly luciferase reporter (see figure 2) and dexamethasone (see figure 3) targeting KGF.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, which is defined in the appended claims, and any other technical entity or method implemented by another person is deemed to be covered by the claims if it is exactly the same as or equivalent to the claims.
Sequence listing
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tttagagaat acatgatagg atgttttcag ctttctagct ttcaacagat gcaatgatca 600
aagagtctgt ccccctctgc tttaataaat gtggaacatt caacatttgg catatgaaga 660
gtaactgggt acatctgaac tattctgtgc tcttcattct gttccctaaa gggacagata 720
caaagagaaa tcacataaac tataatccag ttgagtagat ttaatttcct tttcctattt 780
ggtctacatg caccacctgg tggtaaagta gtgcatttca catagagaga gagagagaga 840
gagagagagt gtgtgtgtgt gtgtgtgcgc gcgcgcgcgc gtgcacgcgt gtgtgttttg 900
aggagtatat aacatgaaaa agataatttt aatatacttt cttaatactg atttagatag 960
acaacacaat tattctacac caagaatcta atacaacact tgagcactac ttcatgtata 1020
acagaacagg aagtaaagga gagaactcat gctggaaagt aaaatagctt tcacttattt 1080
accttagcaa tacatattat aattccatct ctaaattaac atttattgtt ccaatattct 1140
tattgaggca aaaatcaaac actgataatc aatgtgaaac catttcctcc tttttaaaga 1200
ataacagctt ttaaaagtag gtgccctgtt ccttatatta gtagtgagta taagtcacag 1260
atcacagaat tgtaaagcac catagaggca ttagaccaat acaccaattt tacaaatgta 1320
gcagctgaga ctctggaaca gcaggtaact tgcccgaagt catactggtc attagtggtc 1380
caacacaaat aagaatctag atcttctcac tttcagccta ggaatataat acataaaaat 1440
tgtacttata ttcatattca atgaattgtc tctgtatgat gtgataatat tcaacttatc 1500
tacacagtgc tttaaggcac tcatatgtta gttaacacat accagttttg ttctaacatt 1560
ctgagcacaa agaagtaatt gaagaaatgc atgtatcata aagctatgga aaagattttc 1620
taagtattaa aaaaccctat atatatgttc aaatgaataa ttttccaagt ataatacaac 1680
tgctatattg caaagaaggt ttaaaaaatt ccctttcccc ttctaactgc ttcagaacca 1740
gagatctgtt tctacaaagt aggtacccac ttgtagattt aagcataaaa catgttcata 1800
gctccctctc atgaccattg aatactatac aagcctcctt ttattctcgc tgttgtattc 1860
aactaacagc tcactggcta agttttaatt gcttccaatg aggtcagcaa aggtatttat 1920
cgaaaagccc tgaataaaag gctcacacac acacacaagc acacacgcgc tcacacacag 1980
agagaaaatc cttctgcctg ttgatttatg gaaacaatta tgattctgct ggagaacttt 2040
tcagctgaga aatagtttgt agctacagta gaaaggctca agttgcacca ggcagacaac 2100
agacatggaa ttcttatata tccagctgtt agcaacaaaa caagtaagtt actgttattt 2160
gtcttttaaa acaatgctga atgttttcta aaagtttatc tctttttctg attttagtac 2220
tgagcaggaa agtaaagtta caagattttt gagccatgta actatagtaa ttatgctgta 2280
gaacataaag ataaggttga agttctccag agcaatcctg acatttaact taaaaaacct 2340
gggcagttca ggagaaaata attgattaac cacaaatgta aaagcatata ataaagaagt 2400
ctagggagaa agcagttaca ctttgttagc agtaattgta aaaacttaat ttaaatcaat 2460
agttctgagt gcagtatggt tctcacaaaa tagcaattgg aatgaatggt acagtcatat 2520
tctgttttgt ctctactcat gcataaattt tataaatcta cacataaagt ggtaaagatt 2580
ttgacatcct ctgtgaaggc tgttttaaat gtatcttcaa agaataacct atactgtatt 2640
ctaatgctac tacttaccca ctaaaattta cacatacaat tttttatctt cctctctcct 2700
cccctcccta ttcttaatct ctcattgcaa acagaagtca aatagcaaac agcgtcacag 2760
caactgaact tactacgaac tgtttttatg aggatttatc aacagagtta tttaaggagg 2820
aatcctgtgt tgttatcagg aactaaaagg ataaggctaa caatttggaa agagcaacta 2880
ctctttctta aatcaatcta caattcacag ataggaagag gtcaatgacc taggagtaac 2940
aatcaactca agattcattt tcattatgtt attcatgaac acccggagca ctacactata 3000
atgcacaaat ggatactgac atggatcctg ccaactttgc tctacagatc atgctttcac 3060
attatctgtc tagtgggtac tatatcttta gcttgcaatg acatgactcc agagcaaatg 3120
gctacaaatg tgaactgttc cagccctgag cgacacacaa 3160

Claims (1)

1. A method for screening potential drugs for treating acute lung injury is characterized in that,
stimulating mesenchymal stem cells of a targeted KGF promoter for screening potential drugs for treating acute lung injury by using a substance to be tested, and reflecting the activity of the KGF gene promoter by using and detecting fluorescence intensity according to a luciferase reporter gene after acting for a period of time, so as to obtain a compound for promoting the efficient expression of the luciferase reporter gene by using the targeted KGF gene promoter, namely obtaining the potential drugs for treating acute lung injury; the drug is liraglutide or dexamethasone;
the preparation method of the mesenchymal stem cells of the targeting KGF promoter for screening potential drugs for treating acute lung injury comprises the following steps:
1) Constructing a luciferase reporter gene vector containing a KGF gene promoter;
the sequence of the KGF gene promoter inserted into the luciferase reporter gene vector containing the KGF gene promoter is shown as SEQ ID NO. 3;
the construction method of the luciferase reporter gene vector containing the KGF gene promoter comprises the following steps: inserting a KGF gene promoter sequence between PacI and BamHI sites of a vector CV123-Luc containing a luciferase reporter gene;
2) Preparing a lentivirus solution containing the KGF gene promoter by using the luciferase reporter gene vector containing the KGF gene promoter in the step 1), further infecting mesenchymal stem cells, and screening stable transformants;
the mesenchymal stem cells are human placental chorion derived mesenchymal stem cells.
CN202010187546.8A 2020-03-17 2020-03-17 KGF promoter-targeted mesenchymal stem cell screening model for treating acute lung injury Active CN113403280B (en)

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