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

文档序号:3282 发布日期:2021-09-17 浏览:57次 中文

1. A mesenchymal stem cell of a targeting KGF promoter for screening potential drugs for treating acute lung injury is characterized in that 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.

2. The KGF promoter-targeted mesenchymal stem cells for screening a potential drug for treating acute lung injury according to claim 1, wherein the KGF gene promoter inserted into the luciferase reporter gene vector containing the KGF gene promoter has a sequence shown in SEQ ID NO 3.

3. The KGF promoter-targeted mesenchymal stem cells for screening a potential drug for treating acute lung injury according to claim 1, wherein the KGF gene promoter-containing luciferase reporter gene vector is constructed by the following steps: the KGF gene promoter sequence was inserted between the PacI and BamHI sites of the vector CV123-Luc containing the luciferase reporter gene.

4. The mesenchymal stem cell of claim 1, wherein the mesenchymal stem cell is human placental chorion-derived mesenchymal stem cell used for screening KGF-targeted promoters for potential drugs for the treatment of acute lung injury.

5. A preparation method of mesenchymal stem cells of a targeting KGF promoter for screening potential drugs for treating acute lung injury is characterized by comprising 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.

6. The process according to claim 5, wherein the KGF gene promoter inserted into the luciferase reporter vector containing the KGF gene promoter has the sequence shown in SEQ ID NO 3.

7. The method according to claim 5, wherein the luciferase reporter vector containing the KGF gene promoter is constructed by: the KGF gene promoter sequence was inserted between the PacI and BamHI sites of the vector CV123-Luc containing the luciferase reporter gene.

8. The method of claim 5, wherein said mesenchymal stem cell is a human placental chorion-derived mesenchymal stem cell.

9. Use of the mesenchymal stem cells targeting the KGF promoter for screening a potential drug for the treatment of acute lung injury according to any one of claims 1 to 4 for screening a potential drug for the treatment of acute lung injury.

10. A method for screening potential drugs for treating acute lung injury is characterized in that a substance to be tested is used for stimulating mesenchymal stem cells of a KGF (KGF promoter) -targeted drug for screening potential drugs for treating acute lung injury as claimed in any one of claims 1-4, after a period of action, the activity of the KGF gene promoter is reflected by using and detecting fluorescence intensity according to a luciferase reporter gene, so that a compound for promoting the luciferase reporter gene to be efficiently expressed by the KGF-targeted gene promoter is obtained, and the potential drugs for treating acute lung injury are obtained.

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 extravasation, 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 found that MSCs secrete soluble factors such as growth factors, anti-inflammatory factors and antimicrobial peptides, which 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 chorion-derived MSCs (human chorion-derived mesenchymal stem cells, 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 surfactant synthesis. 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.

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 present invention is different from the conventional method, and conventionally, a target gene is usually introduced into 293T cells or the like as a basis, or representative cells of diseases are directly used as a 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 high flux, and has good application prospect for screening the medicine for treating the acute lung injury disease.

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 clinically and used for treating ALI early inflammation amplification reaction, and a certain effect is achieved, and the dexamethasone is found to promote the expression of KGF in previous researches. The invention uses liraglutide and dexamethasone as medicaments to verify a screening platform, and the result shows that the medicament screening platform can screen out medicaments causing 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. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out 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. 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 the promoter sequence of the KGF Gene

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, identifying a PCR product by using agarose gel electrophoresis, and obtaining a target product with two ends containing PacI and BamHI enzyme cutting sites of about 3000 bp.

1.3 construction of recombinant plasmid carrying luciferase reporter Gene of KGF promoter

After PacI and BamHI double enzyme digestion treatment, purifying and recovering the promoter sequence KGF obtained in the step 1.2, connecting the promoter sequence KGF with a luciferase reporter gene-containing vector CV123-Luc (a dual-luciferase reporter gene-containing CV123 vector (purchased from a Kjeldahl gene) through PacI and BamHI double enzyme digestion treatment, purifying and recovering the CV 123-Luc), transforming a connecting reaction solution into an Escherichia coli DH5 alpha competence, carrying out inverted culture on an LB solid culture medium at 37 ℃ for 16-20h, selecting an ampicillin resistance positive monoclonal, extracting plasmid DNA, preliminarily identifying the positive clone by using technologies such as colony PCR, enzyme digestion and the like, and obtaining a recombinant plasmid named CV-123-KGF-promoter-Luc. 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 6Cells/15 ml, reseeded in 10 cm cell culture dishes at 37 ℃ with 5% CO2Culturing in an incubator. The cell density can reach 70% -80% after 24h, and the cell can be used for transfection. 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 the pHelper1.0 vector plasmid (Biovector NTCC Inc. USA, Biovector 510383) and 10. mu.g of the pHelper2.0 vector plasmid (Biovector NTCC Inc. USA) into 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% CO2Culturing in a cell culture box; culturing for 6h, discarding the culture medium containing the transfection mixture, adding 10 ml of PBS (phosphate buffer 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.C2Continuously culturing in the incubator for 48-72h。

2.2 concentration and purification of a lentivirus solution containing the KGF Gene promoter

Collecting 293T cell supernatant 48 h after transfection (counted as 0h after transfection) according to cell state; 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 ultracentrifuge tubes with virus supernatant into a Beckman ultracentrifuge one by one, setting the centrifugation parameters to be 25000 rpm, setting 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 used4Cells, 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; after 24h, 100. mu.l of complete medium was added. Resistant puromycin was added 72h after infection to maintain the drug concentration at 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. Viral titer = number of viable cells/amount of viral stock, i.e. the measured viral titer was 2E +8 TU/ml.

2.4 screening of hCMSCs stably expressing KGF promoter

Preparation of a density of 3-5X 10 with complete Medium4500. mu.l of each/ml of cell suspension of human placental chorion-derived mesenchymal stem cells was inoculated into a 24-well plate and cultured at 37 ℃ for 16-24 hours until the degree of cell confluence was 20-30%. The infectious agent is mixed into the medium and the lentivirus is added 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 with a maintained 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 well4Mu.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 foregoing is merely a preferred embodiment of the invention and is not intended to limit the scope of the invention, which is defined by the claims appended hereto, and any other technical entity or method that is encompassed by the claims as broadly defined herein, or equivalent variations thereof, is contemplated as being encompassed by the claims.

Sequence listing

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