CN114350617A - Application of Raw264.7 cell line for stably expressing different human ApoE genotypes and preparation method thereof - Google Patents
Application of Raw264.7 cell line for stably expressing different human ApoE genotypes and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an application of a Raw264.7 cell line for stably expressing different human ApoE genotypes in the research of the influence of the ApoE genotypes on inflammatory immunity, and a preparation method of the stable cell line, wherein the preparation method comprises the following steps: human different ApoE genotype plasmids ApoE2, ApoE3, ApoE4 and control plasmid N1 carrying EGFP tags were transfected into raw264.7 cells using transfection reagents; after adding a positive screening agent G418 for selective culture, carrying out multiple sorting in a single cell sorting mode of a flow cytometer to obtain a stable cell strain, and carrying out ApoE genotype and expression identification; the stable cell is applied to research the influence of the ApoE genotype on the activation of NLRP3 inflammatory corpuscle, the synthesis of endogenous melatonin, the energy metabolism of mitochondria, the elimination of pathogens and the like. The cell strain provides a good tool for further researching the biological characteristics of different ApoE genotypes in the field of inflammatory immunity and drug research and development.
Description
Technical Field
The invention relates to the technical field of biology, in particular to application of a Raw264.7 cell line for stably expressing different human ApoE genotypes and a preparation method thereof.
Background
Human ApoE, also called apolipoprotein E, is an arginine-rich polymorphic basic protein consisting of 299 amino acids and having a molecular weight of 34KDa, is mainly synthesized by the liver, and recently, the synthesis of brain, kidney, skeleton, adrenal gland and macrophage is discovered. The ApoE gene is polymorphic and contains 3 common alleles: ε 2, ε 3 and ε 4, resulting in six phenotypes E2/2, E3/3, E4/4, E2/3, E2/4, E3/4. There are three common subtypes of ApoE protein in humans, namely ApoE2, ApoE3 and ApoE 4. They differ only at position 112/158, with ApoE3 having cysteine at position 112 and arginine at position 158, and ApoE2 having cysteine at both positions and ApoE4 having arginine at both positions, the difference in amino acid positions resulting in differences in the structure and function of the three subtypes. ApoE plays an important role primarily in the homeostatic regulation of lipids, such as cholesterol transport and lipoprotein metabolism. Recent studies have found that ApoE functions are not limited to cholesterol homeostasis, but are also involved in immune responses, such as host susceptibility to infection, lipid antigen presentation, and modulation of inflammation. Current studies have shown that ApoE is associated with the pathogenesis of asthma. The expression level of ApoE is obviously increased in lung tissues of an asthma animal model, and different ApoE genotypes are obviously related to the severity of asthma. Different genotypes of human ApoE can regulate and control House Dust Mite (HDM) induced airway inflammation to different degrees, and ApoE gene polymorphism may affect the severity of asthma disease to different degrees.
However, no good in vitro immune cell model exists so far, and a good cytological research platform can be provided for researching the influence of different ApoE gene subtypes on inflammatory immunity.
Disclosure of Invention
The invention aims to solve the technical problem of providing a Raw264.7 cell line for stably expressing different human ApoE genotypes and application thereof, and the Raw264.7 cell line for stably expressing different human ApoE genotypes is prepared by utilizing a cell transfection and flow sorting method, is a good in-vitro immune cell model for stably expressing different human ApoE and provides a good cell platform for researching the influence of different ApoE gene subtypes on inflammatory immunity.
In order to solve the technical problems, the invention adopts a technical scheme that: provides an application of Raw264.7 cell lines for stably expressing different human ApoE gene types in research on the influence of different ApoE gene subtypes on inflammatory immunity.
In a preferred embodiment of the invention, the activation of NLRP3 inflammasome is ApoE genotype dependent in raw264.7 cell lines stably expressing different ApoE genotypes of human origin, with or without LPS or staphylococcus aureus stimulation.
In a preferred embodiment of the invention, the synthesis of endogenous melatonin is ApoE genotype dependent in raw264.7 cell lines stably expressing different ApoE genotypes of human origin, with or without stimulation by LPS or staphylococcus aureus.
In a preferred embodiment of the invention, the energy metabolism of mitochondria in Raw264.7 cells stably expressing different ApoE genotypes of human origin is ApoE genotype dependent in Raw264.7 cells with or without stimulation by LPS or Staphylococcus aureus
In a preferred embodiment of the invention, the Raw264.7 cell line, which stably expresses different ApoE genotypes of human origin, is ApoE genotype-dependent on phagocytosis of the pathogen Staphylococcus aureus.
In order to solve the technical problem, the invention adopts another technical scheme that: provides a preparation method of the Raw264.7 cell line for stably expressing different human ApoE genotypes, which comprises the following steps:
(1) culturing in vitro Raw264.7 macrophage cell line;
(2) transfecting different human ApoE genotype plasmids ApoE2-EGFP, ApoE3-EGFP, ApoE4-EGFP and a control group plasmid N-EGFP into Raw264.7 cells by using a transfection reagent;
(3) adding a positive screening reagent G418 after 24 hours, carrying out selective culture for 1-2 weeks, and repeatedly sorting for many times in a single cell sorting mode of a flow cytometer to obtain stable transfected cells with the purity of more than 98%;
(4) the transfected cells were genotyped and expressed for ApoE using confocal laser microscopy, Sanger sequencing and Western Blot.
In a preferred embodiment of the present invention, in step (2), the transfection reagent is Lipofectamine 3000.
In a preferred embodiment of the present invention, in step (3), the medium for selective culture is G418 complete medium comprising DMEM, 10% fetal bovine serum, 2mM glutamine, 100 units/ml penicillin, 0.1mg/L streptomycin.
In a preferred embodiment of the present invention, in step (3), the concentration of the positive selection reagent G418 is 800. mu.g/ml.
In a preferred embodiment of the present invention, in step (3), the method for screening cells is a single cell sorting mode using a sorting flow cytometer, and the cell screening is required to achieve a cell density of 10 for each group6One per ml.
In the step (4), the method for identifying the screened positive cells comprises the following steps of detecting the ApoE genotype and expression in each group of cells by using a laser confocal microscope, Sanger sequencing and Western Blot:
(1) identifying ApoE expression of the screened cells by using a laser confocal microscope: respectively inoculating the sorted cells into a glass bottom culture dish special for a laser confocal microscope, and observing the transfected positive cells with different ApoE genotypes with green fluorescence by using the laser confocal microscope after 24 hours;
(2) extracting DNA of the stably transformed cells, performing PCR amplification by taking a DNA sample as a template, determining the DNA sequence of the amplified PCR fragment by using a Life technologies 3730xl sequencer by adopting a Sanger sequencing technology, and judging the ApoE genotype according to two SNP loci of rs429358 and rs 7412;
(3) expression of ApoE in each cell group was detected using Western Blot: when the number of cells reaches 5×106And each group of cell proteins are separated by 12% polyacrylamide gel electrophoresis after the cells are digested by cell lysate, the proteins are extracted, and after electrophoresis, membrane transfer and sealing are carried out, a coat anti-ApoE antibody and an HRP-anti-coat secondary antibody are incubated, the expression of the ApoE proteins in Raw264.7 cells stably expressing different human ApoE plasmids (ApoE2-EGFP, ApoE3-EGFP and ApoE4-EGFP) is detected.
The invention has the beneficial effects that:
(1) the invention establishes an immune cell Raw264.7 cell strain capable of stably expressing different human ApoE gene types, and the cell strain provides a good tool for further deep research on the biological characteristics and drug development of different ApoE gene subtypes in the field of inflammatory immunity;
(2) the invention takes plasmid as a carrier, prepares an immune cell Raw264.7 cell strain capable of stably expressing different human ApoE genotypes by using a cell transfection and flow cell sorting method, and the ApoE protein is fused and expresses an EGFP label, so that living cells can be directly observed under a laser confocal microscope.
Drawings
Fig. 1 is a schematic illustration of ApoE genotype affecting endogenous melatonin synthesis, differentially modulating NLRP3 inflammatory body activation, mitochondrial energy metabolism, and pathogen clearance;
FIG. 2 is a drawing showing that the ApoE expression diagram is detected by a confocal microscope (a) and a western blotting (b) after a Raw264.7 cell line transfected with different human ApoE genotypes is screened, and the ApoE genotypes of different subtype cell strains are identified by Sanger sequencing (c-e);
FIG. 3 is a graph of the effect of ApoE genotype on the activation of NLRP3 inflammasome in Raw264.7 cell lines stably expressing different ApoE genotypes of human origin, with or without stimulation by LPS or Staphylococcus aureus;
FIG. 4 is a graph of the effect of ApoE genotype on the synthesis of endogenous melatonin in Raw264.7 cell lines stably expressing different human ApoE genotypes, with or without stimulation by LPS or Staphylococcus aureus;
FIG. 5 is a graph of the effect of ApoE genotype on mitochondrial respiratory function (OCR) with or without stimulation by LPS or Staphylococcus aureus in a Raw264.7 cell line stably expressing different ApoE genotypes of human origin;
FIG. 6 is a diagram showing the result of phagocytosis of pathogen Staphylococcus aureus by RAW264.7 cell line stably expressed by different ApoE genotypes of human origin.
Detailed Description
The following detailed description of the preferred embodiments of the present invention, taken in conjunction with the accompanying drawings, will make the advantages and features of the invention easier to understand by those skilled in the art, and thus will clearly and clearly define the scope of the invention.
Example 1: establishment of Raw264.7 cell line stably expressed by different human ApoE genotypes
1. Materials and methods
1.1 cell culture
The Raw264.7 cell line was purchased from the cell bank of Chinese academy of sciences (Shanghai). At 5% CO2Raw264.7 cells were cultured in DMEM medium containing 10% FBS and 1% double antibody in a 37 ℃ incubator.
1.2 reagents
Lipofectamine 3000 transfection kit was purchased from Saimer Feishel (Invitrogen), DMEM, G418 and mammalian DNA kits were purchased from Sigma, opti-MEM and FBS from Gibico, Elisa kit was purchased from Wuhan cloud cloning, fluorescent secondary antibody was purchased from Jackson ImmunoResearch Laboratories, USA, ApoE primary antibody was purchased from Calbiochem, USA.
1.3 establishment of different human ApoE genotype stable transgenic Raw264.7 cell lines
The Raw264.7 cell line was seeded in a 60mm dish and the transfection procedure was performed when the degree of cell confluence was observed to be about 80%. The method comprises the following specific steps: after 24 hours of culture, ApoE2-EGFP, ApoE3-EGFP, ApoE4-EGFP and control N1-EGFP were transfected into the mouse macrophage line Raw264.7, respectively, using the transfection reagent lipofectamine 3000; adding 800ug/ml positive screening reagent G418 after 24 hours, after 1-2 weeks of selective culture, sorting out green fluorescence labeled Raw264.7 cells by using a sorting flow cytometer single cell sorting mode, and culturing in a complete culture medium containing G418 (including Dubucco's Modified Eagle's Medium (DMEM), 10% Fetal Bovine Serum (FBS), 2mM glutamine (L-glutamine), 100 unit/ml penicillin (penicill), 0.1mg/L streptomycin (streptomycin)); when the confluency of the cells is 95 percent, sorting Raw264.7 cells which are successfully transfected and have green fluorescence by using a sorting flow cytometer, and culturing in a complete culture medium containing 800ug/ml G418; the stable transfected cells were then screened 3 times repeatedly by G418 and sorting flow to achieve more than 98%. Raw264.7 cell lines stably expressing ApoE2, ApoE3 and ApoE4 were selected and observed by confocal laser microscopy.
1.4 Sanger sequencing to identify different ApoE genotypes of stably expressing cell lines
Stable cell DNA was extracted according to GenEluteTM mammalian genomic DNA Miniprep kit instructions and amplified by PCR. 1U SAP and 6U Exo I were added to 8ul PCR product and incubated at 37 ℃ for 60min and 70 ℃ for 10min for purification, and the purified PCR product included 2ul BigDye3.1 mix and 2ul 1uM ApoE primers. Then amplifying at 96 ℃ for 1min, at 96 ℃ for 10s, at 50 ℃ for 5s and at 60 ℃ for 4min for 28 cycles. And finally, performing sequence analysis by using a DNA analyzer.
TABLE 1 amplification primers for rs429385 and rs7412
1.5 Western Blotting detection of protein expression level of ApoE in stably transfected cells
The cells seeded in the culture plate were lysed with RIPA containing protease inhibitor and EDTA, and the obtained samples were subjected to protein concentration determination using the BCA kit. Equal loading (25 ug/well) of the gel on a 12% SDS-PAGE, after completion of the electrophoresis, the protein was transferred to a PVDF membrane, which was then blocked with 5% skim milk for 2 hours after the completion of the membrane transfer, and then incubated with a primary anti-coat anti-ApoE antibody at room temperature for 3 hours and then overnight at 4 ℃. After washing three times with TBST the secondary antibody HRP-anti-goat was incubated the next day, after 1 hour washing three times with TBST, after completion of which the expression of ApoE protein in Raw264.7 cells stably expressing different human-derived ApoE plasmids (ApoE2-EGFP, ApoE3-EGFP, ApoE4-EGFP) was examined by imaging with ECL.
Example 2: application of Raw264.7 cell line stably expressed by different human ApoE genotypes
2.1 Effect of different ApoE genotypes of human origin on the activation of the inflammatory bodies of Raw264.7 cell line NLRP3
Raw264.7 cells stably expressed by different human ApoE genotypes are subjected to ELISA (enzyme-linked immunosorbent assay) to detect the levels of IL-1 beta and IL-18 related cytokines of NLRP3 in culture supernatants of stably transfected cells after being stimulated or not to be stimulated by LPS (1 mu g/ml) or staphylococcus aureus (the infection multiple is 1: 10) for 8 hours. Pre-coating IL-1 beta and IL-18 monoclonal antibodies on a microplate, adding 50ul of samples into each well, removing unbound conjugate by washing for three times to stop incubation, adding horseradish peroxidase conjugated secondary antibody for incubation for 30 minutes, adding a substrate solution, and carrying out color development after terminating the reaction; western Blotting examined the changes in the expression of NLRP3, Caspase1(p20) and IL-1 beta (p17) proteins in cell lysates.
2.2 Effect of different ApoE genotypes of human origin on the Synthesis of endogenous melatonin in the Raw264.7 cell line
The level of melatonin in the culture supernatant of stably transfected cells was determined by ELISA 8 hours after stable expression of Raw264.7 cells of different ApoE genotypes of human origin with or without stimulation by LPS (1. mu.g/ml) or Staphylococcus aureus (fold of infection 1: 10). Adding 100ul of sample into each hole of an ELISA kit, washing for three times to remove unbound conjugate to stop incubation, adding horseradish peroxidase conjugated secondary antibody for incubation for 30 minutes, adding a substrate solution, and performing color development after reaction is stopped; western Blotting detects the change of expression of key enzymes ASMT and AANAT for synthesizing melatonin in cell lysate.
2.3 Effect of different ApoE genotypes of human origin on mitochondrial energy metabolism in Raw264.7 cell line
Raw264.7 cells stably expressing different human ApoE genotypes at 2.5X 104Cells/well were plated on an Agilent Seahorse XF96 well plate, and after 8 hours of stimulation with or without LPS (1 μ g/ml) or staphylococcus aureus (fold 1: 10 infection), mitochondrial respiratory function of cells was measured according to the protocol of the Mito Stress, and finally data analysis was performed using an XF96 cell energy metabolism analyzer.
2.4 Effect of different human ApoE genotypes on the phagocytosis of Staphylococcus aureus in Raw264.7 cell line
Inoculating the stable transfected cells on a 12-well plate glass, adhering the glass to the wall for 12 hours, then infecting the cells for 8 hours in a culture medium without antibiotics by using CellTracker blue (the infection multiple is 1: 10), then washing off the staphylococcus aureus which is not combined with the cells by vigorous shaking, fixing the cells by using 4% paraformaldehyde, and finally shooting and detecting by using a confocal microscope.
As shown in figure 1, ApoE genotype variability induces activation of NLRP3 inflammasome, modulation of mitochondrial energy metabolism, and clearance responses to pathogens when stimulated by inflammation; meanwhile, the ApoE genotype difference induces the expression of melatonin synthase ASMT, thereby affecting the endogenous melatonin level.
2. Results of the experiment
1) ApoE protein expression and genotype identification
The expression of EGFP in the stably transfected cells was detected by confocal microscopy (a in FIG. 2), the expression level of ApoE in the stably transfected cells was detected by western blotting technique (b in FIG. 2), and the gene sequence of the stably transfected cells was determined by sanger sequencing (c-e in FIG. 2). The above results indicate that the flow-sorted stably transfected cells, ApoE2-EGFP, ApoE3-EGFP and ApoE4-EGFP, have similar ApoE protein expression levels, but are significantly improved compared to N1-EGFP.
2) Effect of different ApoE genotypes of human origin on activation of inflammatory bodies of Raw264.7 cell line NLRP3
As tested by western blotting and Elisa in FIG. 3, in the absence of LPS or Staphylococcus aureus, the expression levels of NLRP3, caspase-1(p20), mature IL-1 β (p17) (a-d; g-j) and secreted IL-18(f, k) in the culture supernatant were higher in ApoE 2-stable cells and ApoE 4-stable cells in ApoE 3-stable cells, while the expression levels of secreted IL-1 β (e, j) in the culture supernatant were similar in the three stable cells. 8 hours after LPS or Staphylococcus aureus stimulated the stably transfected cells, NLRP3, caspase-1(p20), and mature IL-1 β (p17) (a-d; g-j), as well as the NLRP 3-associated cytokines IL-1 β and IL-18 expression levels in the culture supernatant were significantly elevated (e, f; j, k) on average compared to unstimulated cells, and the level of activation of NLRP3 inflammasome in ApoE3 stably transfected cells was significantly higher than that of ApoE2 stably transfected cells and ApoE4 stably transfected cells. These results indicate that activation of NLRP3 inflammasome is associated with ApoE genotype with or without LPS or staphylococcus aureus stimulation.
3) Effect of different ApoE genotypes of human origin on the Synthesis of melatonin endogenous to the Raw264.7 cell line
In unstimulated raw264.7 cells, ApoE3 stabilized melatonin synthesis critical enzymes ASMT and AANAT in cells (shown as a-c, e-g in fig. 4), and endogenous melatonin levels (shown as d, h in fig. 4) were significantly higher than those expressing ApoE2 and ApoE4 stabilized cells. Furthermore, protein expression of ASMT and mean melatonin levels increased in all stable cell lines (shown as a-d, g-h in fig. 4) and ApoE3 stabilized cells with the highest levels of endogenous melatonin synthase and melatonin, under stimulation with LPS or staphylococcus aureus, although AANAT protein expression was not affected. These data indicate that melatonin biosynthesis is also dependent on ApoE genotype.
4) Effect of different human ApoE genotypes on mitochondrial energy metabolism in Raw264.7 cell lines
The cellular mitochondrial respiratory function, as measured by Mito Stress (FIG. 5), was significantly lower for ApoE4 than for ApoE2 and ApoE3 than for cells with stable basal mitochondrial respiration rates (a, b; d, e) and maximal respiration (a, c; d, f) regardless of stimulation by LPS or Staphylococcus aureus. Indicating that mitochondrial energy metabolism in RAW264.7 cells is ApoE gene dependent.
5) Effect of RAW264.7 cell lines stably expressed with different ApoE genotypes on phagocytosis of the pathogen Staphylococcus aureus
The laser confocal results show that the phagocytosis of staphylococcus aureus by ApoE3 stably transfected cells is obviously higher than that by ApoE2 and ApoE4 stably transfected cells, as shown in a and b in fig. 6. In addition, as shown in c-e in FIG. 6, the expression level of the scavenger receptors SR-A and CD36, which are critical to the phagocytosis of Staphylococcus aureus, in ApoE3 stably transfected cells was significantly higher than that of ApoE2 and ApoE4, as detected by western blotting results.
The invention has many applications, and the above description is only a preferred embodiment of the invention. It should be noted that the above examples are only for illustrating the present invention, and are not intended to limit the scope of the present invention. It will be apparent to those skilled in the art that various modifications can be made without departing from the principles of the invention and these modifications are to be considered within the scope of the invention.
Claims (10)
1. An application of Raw264.7 cell line for stably expressing different human ApoE gene types in the research of the influence of different ApoE gene subtypes on inflammatory immunity.
2. The use according to claim 1, characterized in that the activation of NLRP3 inflammasome is ApoE genotype dependent on the raw264.7 cell line stably expressing different ApoE genotypes of human origin, with or without stimulation by LPS or staphylococcus aureus.
3. The use according to claim 1, characterized in that the synthesis of endogenous melatonin is ApoE genotype dependent in raw264.7 cell lines stably expressing different ApoE genotypes of human origin, with or without stimulation by LPS or staphylococcus aureus.
4. The use according to claim 1, wherein the mitochondrial energy metabolism in raw264.7 cells stably expressing different ApoE genotypes of human origin is ApoE genotype dependent, with or without LPS or staphylococcus aureus stimulation.
5. The use according to claim 1, characterized in that the raw264.7 cell line, stably expressing different ApoE genotypes of human origin, is ApoE genotype dependent on the phagocytosis of the pathogen staphylococcus aureus.
6. A method for preparing a raw264.7 cell line stably expressing different ApoE genotypes of human origin according to claim 1, comprising the steps of:
(1) culturing in vitro Raw264.7 macrophage cell line;
(2) transfecting different human ApoE genotype plasmids ApoE2-EGFP, ApoE3-EGFP, ApoE4-EGFP and a control group plasmid N-EGFP into Raw264.7 cells by using a transfection reagent;
(3) adding a positive screening reagent G418 after 24 hours, carrying out selective culture for 1-2 weeks, and repeatedly sorting for many times in a single cell sorting mode of a flow cytometer to obtain stable transfected cells with the purity of more than 98%;
(4) the transfected cells were genotyped and expressed for ApoE using confocal laser microscopy, Sanger sequencing and Western Blot.
7. The method for preparing Raw264.7 cell line stably expressing different ApoE genotypes of human origin according to claim 6, wherein in step (2), the transfection reagent is Lipofectamine 3000.
8. The method for preparing Raw264.7 cell line stably expressing different ApoE genotypes of human origin according to claim 6, wherein the culture medium for selective culture in step (3) is G418 complete medium comprising DMEM, 10% fetal bovine serum, 2mM glutamine, 100 units/ml penicillin, 0.1mg/L streptomycin.
9. The method for preparing Raw264.7 cell line stably expressing different ApoE genotypes of human according to claim 6, wherein the concentration of the positive selection reagent G418 in step (3) is 800 μ G/ml.
10. The method of claim 6, wherein in step (3), the cells are selected by single cell sorting using a sorting flow cytometer, and the cell selection is performed in such a way that the cell density of each group reaches 106One per ml.
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| CN107488635A (en) * | 2017-09-22 | 2017-12-19 | 中国人民解放军第三军医大学第三附属医院 | Monoclonal cell and its production and use |
| CN113151356A (en) * | 2021-04-29 | 2021-07-23 | 安徽医科大学第一附属医院 | CHO cell line for stably expressing different human ApoE and preparation method thereof |
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| CN107488635A (en) * | 2017-09-22 | 2017-12-19 | 中国人民解放军第三军医大学第三附属医院 | Monoclonal cell and its production and use |
| CN113151356A (en) * | 2021-04-29 | 2021-07-23 | 安徽医科大学第一附属医院 | CHO cell line for stably expressing different human ApoE and preparation method thereof |
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| EMILY IGEL等: "Distinct pro-inflammatory properties of myeloid cell–derived apolipoprotein E2 and E4 in atherosclerosis promotion", 《J BIOL CHEM》, vol. 297, no. 3, pages 3 * |
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