CN108823170B - Construction method of cell model for screening antioxidant drugs based on protein misfolding - Google Patents
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Abstract
The invention belongs to the field of cell biology, and provides a method for constructing a cell model for screening antioxidant drugs based on protein misfolding, aiming at the defect that protein misfolding cannot be quantitatively evaluated in a living cell in real time at present. Construction of fluorescent protein-tagged wild and mutant reporter genesCOMPThe gene lentiviral vector is packaged in a human embryonic kidney 293T cell, a HeLa cell is transduced, and puromycin is screened to obtain a cell model for stably expressing COMP protein. The method has the advantages that an international and independently innovative drug screening cell model is established, so that key parameters can be provided for the industrial research of protein misfolding drug screening in the future, and related scientific bases can be provided for the sustainable research of solving major diseases. The cell model has the characteristics of high chromosome integration degree, high fluorescence intensity, stable genetic character and sensitivity to antioxidant drugs, and is an ideal model for researching protein misfolding drug screening.
Description
Technical Field
The invention belongs to the field of cell biology, and particularly relates to a method for constructing a cell model for screening antioxidant drugs based on protein misfolding.
Background
The protein is the executor of all functions in the organism. Any function in the human body, from catalyzing chemical reactions to combating foreign invasion, is the result of the action of proteins. Proteins must fold precisely into the correct three-dimensional structure to perform functions in human cells. It has been found in recent years that misfolding of proteins can lead to several diseases. The relationship of protein misfolding to disease has been the leading edge of new research in molecular biology. The construction of a simple, safe and efficient drug screening cell model is a bottleneck and difficulty in the prevention and treatment of the current internationally prevalent neurodegenerative diseases such as protein misfolding diseases such as Alzheimer's disease, Parkinson's disease, hereditary bone diseases, phenylketonuria and the like.
In the process of drug screening, model construction of a drug screening model is an initial and most important link, and plays a crucial role in efficiency and effect of new drug discovery. High-throughput screening (HTS) is a high-efficiency large-scale drug screening technology that appears under the impetus of combinatorial chemistry, genomics, pharmacology, and other disciplines. HTS is largely divided into screening based on simple targets (target proteins are isolated and purified, and are performed in extracellular environments) and cell model-based screening. The screening based on the pure target (extracellular) can be carried out only under the condition that the action target is exactly known and the action target protein can be separated and purified, and the drug screening carried out on the model has definite action mechanism and single target. The cell-based screening is carried out on a cell model, is particularly suitable for complex targets with difficult functional protein separation and incomplete biochemical analysis, and is a main screening model for high-throughput screening in the future.
Multiple Epiphyseal Dysplasia (MED) is a rare inherited chondrodysplasia disease, one of the diseases caused by misfolding of proteins, and is a systemic skeletal dysplasia caused by defects in chondrocyte extracellular matrix proteins (e.g., cartilage oligomeric matrix protein; COMP) and cell membrane transporters. The COMP protein in normal humans is secreted extracellularly and functions normally, whereas in MED patients,COMPthe gene is mutated, and the mutated COMP protein is deposited on the rough endoplasmic reticulum of the chondrocyte, so that the misfolded protein is accumulated on the endoplasmic reticulum in a large quantity and cannot be normally secreted and transported to trigger endoplasmic reticulum stress reaction, and most preferablyEventually leading to apoptosis of chondrocytes and a decrease in the proliferation rate of cells, thereby inducing the onset of disease.
The present research finds thatCOMP D469del Antioxidants and anti-inflammatory drugs are effective in reducing the accumulation of COMP protein on chondrocytes, especially acetylsalicylic acid (aspirin) and resveratrol drugs, in mutant mouse models that lead to severe Pseudoachondroplasia (PSACH), but for research studies, rescue skeletal dysplasia in mutant miceCOMPThere are also limitations to MED disease caused by mutations.
Disclosure of Invention
The invention provides a construction method of a cell model for screening antioxidant drugs based on protein misfolding, aiming at the defect that the protein misfolding cannot be quantitatively evaluated in a living cell in real time at present.
The invention is realized by the following technical scheme: a method for constructing a cell model for screening antioxidant drugs based on protein misfolding adopts molecular biological gene recombination technology to construct fluorescent protein-labeled wild and mutant reporter genesCOMPThe gene lentiviral vector is packaged in a human embryonic kidney 293T cell, a HeLa cell is transduced, and puromycin is screened to obtain a cell model for stably expressing COMP protein.
The method comprises the following specific steps:
(1) transfecting a human embryonic kidney 293T cell by using a lentiviral vector carrying an antibiotic resistance gene, a reporter gene and a target gene to obtain lentiviral particles of the resistance gene and the reporter gene;
(2) infecting HeLa cells of human cervical carcinoma with the lentivirus particles obtained in the step (1), and screening antibiotics to obtain a cell model;
wherein: the lentiviral vector is an HIV-1 vector system and comprises three plasmids: a Plvx-IRES-Puro plasmid expressing a reporter gene, which contains a puromycin resistance gene and an ampicillin resistance gene, a packaging plasmid psPAX2, and an envelope protein plasmid pmd2.g, and a packaging plasmid psPAX2 containing a gag gene of HIV virus and an envelope protein genePlasmid pMD2.G contains VSV-G gene derived from herpes simplex; the antibiotic is puromycin; the report gene iseGFP-COMPA fusion gene; the gene of interest is wild typeCOMPGene and mutantCOMPGene p.D401N.
The specific screening evaluation method comprises the following steps: using an antioxidant drug acting cell model, detecting the fluorescence intensity of eGFP under the conditions of excitation wavelength of 485 nm and emission wavelength of 530 nm, determining the GFP content, using the ratio of the absorbance of cell lysate to the absorbance of cell culture medium to express the retention condition of COMP protein in cells, and evaluating the stable expression of wild type and mutant type through detectionCOMPThe content of COMP protein in a cell model of the gene is used for evaluating the effect of the antioxidant drugs.
The antioxidant drugs are acetylsalicylic acid ASA and resveratrol Res, wherein the concentration of the acetylsalicylic acid is 75 mM, the concentration of the resveratrol is 100 mu M, and the time of the drugs acting on a cell model is 24 h.
The invention establishes a setCOMPThe single base mutation cell model utilizes the cell model acted by the antioxidants acetylsalicylic acid and resveratrol, and can effectively evaluate the action effect of the antioxidants by measuring the fluorescence intensity of eGFP.
Based on the fact that the COMP protein causing hereditary bone diseases due to misfolding is taken as a research target in the process of drug screening at present, aiming at the defect that protein misfolding cannot be quantitatively evaluated in vivo cells in real time at present, the invention adopts a molecular biological gene recombination technology to construct wild and mutant reporter genes containing fluorescent protein markers for real-time quantitative tracking detection as screening markers, and then the recombinant vectors are respectively transferred into a cell line cultured in vitro to be stably expressed to obtain a drug screening cell model. A fluorescence spectrophotometer technology is used as a quantitative detection means of a molecular level, small molecules capable of changing protein folding and secretion levels are screened at a cell level, a functional reaction is used as a screening index, a drug screening cell model with international level and independent innovation is established, and the research of drug screening of protein misfolding diseases is carried out. The invention not only can provide key parameters for the industrial research of protein misfolding drug screening in the future, but also provides related scientific basis for the sustainable research of solving the serious diseases in China. The cell model has the characteristics of high chromosome integration degree, high fluorescence intensity, stable genetic character and sensitivity to antioxidant drugs, and is an ideal model for researching protein misfolding drug screening.
Drawings
FIG. 1 is a schematic diagram of the viral expression vector Plvx-IRES-Puro; FIG. 2 is a schematic representation of the lentiviral packaging plasmid psPAX 2; FIG. 3 is a schematic representation of the lentiviral envelope protein plasmid pMD2. G; FIG. 4 is a graph of the positivity of flow cytometry analysis cell models; FIG. 5 shows content comparison of wild type and mutant COMP; FIG. 6 shows the optimal concentration and optimal time for acetylsalicylic acid to act on a cell model; FIG. 7 shows the optimal concentration and optimal time for the resveratrol to act on the cell model.
Detailed Description
Through long-term research and screening, the inventor constructs a cell model which is sensitive to antioxidant drugs and can stably express COMP gene carried by lentiviral vector. The cell model is an ideal model for researching the screening of antioxidant drugs.
The invention provides a construction method of a cell model for screening antioxidant drugs based on protein misfolding. A new human cervical cancer cell model capable of expressing antibiotic resistance genes and reporter genes carried by a lentiviral vector can be used for screening antioxidant drugs.
The invention is realized by constructionCOMPThe method comprises the following steps of (1) packaging a virus in a 293T cell by using a gene lentiviral vector, transducing a HeLa cell, screening puromycin to a cell model for stably expressing COMP protein, and then evaluating the content of COMP in a mutant cell by utilizing an antioxidant drug acetylsalicylic acid and resveratrol acting cell model to obtain the optimal action concentration and time of the antioxidant drug.
Example 1: packaging lentiviral particles
The lentivirus system comprises three plasmids: the Plvx-IRES-Puro plasmid (fig. 1) expressing the reporter gene, the packaging plasmid psPAX2 (fig. 2), the envelope protein plasmid pmd2.g (fig. 3). Wherein the Plvx-IRES-Puro plasmid contains puromycin resistance gene and ampicillin resistance gene, the packaging plasmid psPAX2 contains gag gene of HIV virus, encodes major structural proteins of virus, and the envelope protein plasmid pMD2.G contains VSV-G gene of herpes simplex origin, providing envelope proteins required for virus packaging.
Constructing a reporter gene: the method comprises the steps of using a plasmid with known eGFP-COMP fusion gene as a template to carry out polymerase chain reaction amplification on a target fragment (TAKARA), recovering the target fragment (Shanghai bio-engineering Co., Ltd.), connecting the enzyme digestion target fragment and a vector by using T4 DNA Ligase, standing overnight at normal temperature, using a connection product for a transformation experiment, carrying out bacterial liquid PCR and enzyme digestion identification on the constructed plasmid, and carrying out sequencing identification on the constructed plasmid by the Shanghai bio-engineering Co., Ltd.
Packaging of lentivirus: paving 293T cells in a 25T culture bottle 24h before transfection, and replacing the cells with a culture solution (10% fetal calf serum) of antibiotic-free DMEM when the cells grow to about 80% adherent thereto; B. adding Opti-MEM 233.5 μ L and FuGENE 6 Transfection Reagent 16.5 μ L into a sterile 1.5 mL EP tube, mixing gently, incubating at room temperature for 5 min, adding Opti-MEM 250 μ L, pMD2.G plasmid 0.5 μ g, psPAX2 plasmid 2.5 μ g, recombinant plasmid COMP-Plvx-IRES-Puro 2.5 μ g and incubated Transfection Reagent into a sterile 1.5 mL EP tube, mixing gently, incubating at room temperature for 20 min, adding into a 25T culture flask, shaking, and culturing in a 37 deg.C (5% CO 2) culture flask; c.48 h later, collecting cell culture supernatant, adding fresh culture solution, and continuing to culture; collecting cell culture supernatant after 72 h, centrifuging the supernatant at 3000 g for 10 min, precipitating cell debris, collecting supernatant, filtering with 0.45 μ M filter for sterilization, packaging, and storing at-80 deg.C;
example 2: viral transduction of HeLa cells
Collecting the lentivirus containing the recombinant COMP in the supernatant, further transducing HeLa cells by using the packaged virus, and screening stably transfected cell strains by the following specific operations:
24h before transduction, the HeLa cells are paved in a 6-well plate, when the cells grow to about 80% by adherence, old culture medium is sucked, fresh MEM culture medium (10% fetal bovine serum, 1% Penicillin-Streptomyces) is added for 1.8 mL, recombinant COMP lentivirus 200 mu L and Hexadimethrine bromide (transfection enhancer) 1.6 mu L are added, and after gentle and uniform mixing, the cells are cultured in a 37 ℃ culture box with 5% CO 2;
after 24h, absorbing the culture medium containing the virus, replacing 2 mL of fresh culture medium, and continuing culturing;
after 48 h, the medium was changed and 2 mL of fresh medium was added along with Puromycin (2. mu.g/mL);
the medium (containing puro) was then changed every 3-4 days until stable monoclonal cells were screened.
Through flow cytometry analysis, the positive rate of the transduced HeLa cells reaches more than 99 percent (figure 4), and the cell model can be used for later-stage drug screening research.
Example 3: evaluation of intracellular COMP content in cell models by fluorescence spectrophotometer
In the stably transfected HeLa cell line, the COMP protein and eGFP are expressed in a fusion mode, so that the content of the COMP protein can be indirectly reflected by detecting the content of GFP.
We examined the GFP content using a Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies), and initially discovered that GFP best reflects the relative Fluorescence intensity of GFP when excited at 485 nm and emitted at 530 nm.
We collected wild type and mutant cells and cell culture fluid, diluted with Tris-EDTA solution, and measured GFP Fluorescence intensity using Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies), and analyzed the ratio of intracellular GFP to GFP in cell culture fluid to represent changes in intracellular COMP.
The results show that: the GFP content ratio of the mutant COMP is obviously higher than that of the wild-type COMP, which indicates that the GFP content (namely the COMP content) of the mutant COMP in cells is high, and the mutant COMP protein is retained in the cells (figure 5).
Example 4: evaluating COMP content in mutant cell by using cell model of antioxidant drug action
Uniformly spreading cells in a 24-well plate (20 × 104 cells/well) 24h before drug treatment, replacing fresh serum-free culture solution when the cells grow to about 80% by adherence, treating the cells with drugs with different time gradients or different concentrations, collecting the cell culture solution and the total protein of the cells after the cells are treated, then diluting 100 μ L of the collected cell culture solution by 10 times with Tris-EDTA (PH 8.0), and diluting 50 μ L of cell lysate by 20 times with Tris-EDTA (PH 8.0); the intracellular retention of COMP protein was measured by using a Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies), and the ratio of the absorbance of cell lysate to the absorbance of cell culture medium was used to indicate whether the COMP protein retention was affected after drug treatment, three independent replicates of each set of experiments, and the significant difference was analyzed by SPSS 17.
Literature has demonstrated that the antioxidants acetylsalicylic acid (ASA) and resveratrol (Res) are able to reduce intracellular retention of mutant COMP, reduce chondrocyte death and restore chondrocyte proliferation in a COMP mutant mouse model [ Posey K L, et al.
1. After a cell model is treated by acetylsalicylic acid (ASA), and a COMP cell model is treated by acetylsalicylic acid drugs with the concentrations of 1 mM, 5 mM, 10 mM, 25 mM, 50 mM, 75 mM and 100mM in sequence for 24h, the content of GFP is detected by a Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies), and the result is shown in FIG. 6A, different concentrations of ASA drugs hardly affect wild-type COMP cells, while for mutant COMP cells, the content of GFP in the cells increases at low concentration, the content of GFP in the cells starts to decrease after 10 mM along with the increase of the concentration, and the content of GFP in the cells tends to be stable at 75 mM and 100mM, which shows that the drug concentration has a good effect on the mutant COMP cells at 75-100 mM.
To further explore the optimal time for ASA to act on cells, we selected a 75 mM ASA cell model and set different time gradients, and as shown in fig. 6B, as the time is prolonged, ASA drug is significantly reduced in wild cells at 12 h, and then the change is not significant, probably because the drug has a certain promotion effect on protein secretion at 12 h, while the mutant cells are significantly reduced, i.e. the retention in the cells is reduced, and then the change is increased at 12 h and 24h, the results show that: when 75 mM ASA drug acts for 24 hours, the drug has good effect on mutant cells.
2. We treated a cell model with the antioxidant resveratrol by first exploring the optimal concentration of drug-affected cells. We set different concentrations of Res drug treatment COMP cell model 48 h before examining the GFP content using the Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies). As shown in FIG. 7A, different concentrations of Res drug hardly affected wild-type COMP cells, while low concentrations of the Res drug increased the intracellular GFP content of mutant COMP cells, but with increasing concentration, the intracellular GFP content began to decrease after 25 μ M, and after 100 μ M, the intracellular GFP content leveled off, indicating that the drug concentration at 100 μ M had a good effect on mutant COMP cells.
To further explore the optimal time for Res acting cells, we selected a 100 μ M Res acting cell model and set different time gradients, and the results are shown in FIG. 7B, as the time is prolonged, Res drugs do not act on wild cells significantly, while mutated cells act on drugs for 24h, the drug retention in cells is reduced, and the subsequent change is not significant, and the results show that: when 100 mu M Res medicine acts for 24 hours, the medicine has good effect on mutant cells.
Various aspects of the invention are described above. However, it should be understood that equivalent changes and modifications can be made thereto by those skilled in the art without departing from the spirit of the present invention, and the changes and modifications also fall within the scope of the appended claims of the present application.
Claims (2)
1. The application of a cell model for screening antioxidant drugs based on protein misfolding in screening antioxidant drugs is characterized in that: the cell model construction method comprises the following steps: construction of fluorescent protein-labeled wild and mutant reporter genes by molecular biological gene recombination technologyCOMPThe gene lentiviral vector is packaged with virus in a 293T cell of a human embryonic kidney, a HeLa cell is transduced, and puromycin is screened to obtain a cell model for stably expressing COMP protein;
the method comprises the following specific steps:
(1) transfecting a human embryonic kidney 293T cell by using a lentiviral vector carrying an antibiotic resistance gene, a reporter gene and a target gene to obtain lentiviral particles of the resistance gene and the reporter gene;
(2) infecting HeLa cells of human cervical carcinoma with the lentivirus particles obtained in the step (1), and screening antibiotics to obtain a cell model;
wherein: the lentiviral vector is an HIV-1 vector system and comprises three plasmids: a Plvx-IRES-Puro plasmid expressing a reporter gene, a packaging plasmid psPAX2, an envelope protein plasmid pmd2.G, wherein the Plvx-IRES-Puro plasmid contains a puromycin resistance gene and an ampicillin resistance gene, the packaging plasmid psPAX2 contains a gag gene of HIV virus, and the envelope protein plasmid pmd2.G contains a VSV-G gene derived from herpes simplex; the antibiotic is puromycin; the report gene iseGFP-COMPA fusion gene; the gene of interest is wild typeCOMPGene and mutantCOMPGene p.D401N;
the method for specifically screening and evaluating the antioxidant drugs by the cell model comprises the following steps: using an antioxidant drug acting cell model, detecting the fluorescence intensity of eGFP under the conditions of excitation wavelength of 485 nm and emission wavelength of 530 nm, determining the GFP content, using the ratio of the absorbance of cell lysate to the absorbance of cell culture medium to express the retention condition of COMP protein in cells, and evaluating the stable expression of wild type and mutant type through detectionCOMPThe content of COMP protein in a cell model of the gene is used for evaluating the effect of the antioxidant drugs;
the antioxidant drugs are acetylsalicylic acid ASA and resveratrol Res.
2. The use of the protein misfolding based cell model for screening of antioxidant drugs as claimed in claim 1 for screening of antioxidant drugs, wherein: the concentration of the acetylsalicylic acid is 75 mM, the concentration of the resveratrol is 100 mu M, and the time of the drug acting on the cell model is 24 h.
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| Antioxidant and anti-inflammatory agents mitigate pathology in a mouse model of pseudoachondroplasia.;Karen L. Posey等;《 Hum Mol Genet.》;20151231;第24卷(第14期);摘要，第3919页表1和第3926页材料和方法部分 * |
| COMP mutation screening as an aid for the clinical diagnosis and counselling of patients with a suspected diagnosis of pseudoachondroplasia or multiple epiphyseal dysplasia;第5期;《Eur J Hum Genet.》;20050630;第13卷(第5期);摘要和表2 * |
| 慢病毒介导的稳定过表达人谷氨酰半胱氨酸连接酶催化亚基基因的WRL68细胞株构建;范誉等;《环境与健康杂志》;20151020(第10期);61-65，99页 * |
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