CN116855545A - Application of NFIL3 gene targeted K562 cells in erythroid hematopoietic differentiation - Google Patents
Application of NFIL3 gene targeted K562 cells in erythroid hematopoietic differentiation Download PDFInfo
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Abstract
The application discloses an application of NFIL3 gene targeted K562 cells in erythroid hematopoietic differentiation in the technical field of biological medicine, wherein the application is to apply the NFIL3 gene targeted K562 cells in erythroid hematopoietic differentiation. The application provides that the NFIL3 gene has a remarkable relation with the amount of hemoglobin produced by K562 cells for the first time. Up-regulating the expression of NFIL3 gene was found to have the effect of reducing the amount of hemoglobin in the corresponding K562 cells. Meanwhile, the designed NFIL3siRNA carrier expression carrier can increase the hemoglobin content of K562 cells and improve the erythroid hematopoietic differentiation capacity of the K562 cells.
Description
Technical Field
The application belongs to the technical field of biological medicines, and particularly relates to application of a target K562 cell of an NFIL3 gene in erythroid hematopoietic differentiation.
Background
Erythroid differentiation refers to the process of developing from hematopoietic stem cells to mature erythrocytes. This process is regulated by intracellular genes and cytokines, and chronic myelogenous leukemia cell K562 was established in 1975 from pleural effusions of 1 chronic myelogenous leukemia patient in primitive cell crisis, with philadelphia chromosome. K562 cells can differentiate towards erythroid, granulocytic and megakaryocytic cell lines under certain conditions, showing specific maturation markers for the respective cell types.
The nuclear factor interleukin-3 (nfil 3) is a key transcriptional inhibitor, critical for the development of NK cells and innate lymphocytes. NFIL3 was found to inhibit hypoxia-induced apoptotic cell death by targeting insulin-like growth factor 2 receptor, but no report was made at this stage on targeting K562 cells of the NFIL3 gene to affect erythroid hematopoietic differentiation.
Disclosure of Invention
In view of the above, the present application provides an application of the NFIL3 gene-targeted K562 cells in erythroid hematopoietic differentiation to overcome the defects of the prior art, and in order to solve the above-mentioned problems, the present application provides an application of the NFIL3 gene-targeted K562 cells in erythroid hematopoietic differentiation.
In order to achieve the above purpose, the technical scheme adopted by the application is as follows: the application provides an application of a target K562 cell of an NFIL3 gene in erythroid hematopoietic differentiation, wherein the application is to apply the target K562 cell of the NFIL3 gene in erythroid hematopoietic differentiation.
Further, the method for application comprises the following steps:
(1) K562 cells were cultured and then plated using six well plates;
(2) Transfecting NFIL3 expression vector in K562 cells using a kit;
(3) After 24h transfection of NFIL3 expression vector, K562 cells were induced to erythroid differentiation using Hemin at a final concentration of 50 μm;
(4) Collecting K562 cells induced for different times;
(5) Detecting the induction condition and the expression condition of the red line.
Preferably, the NFIL3 expression vector is NFIL3siRNA.
Preferably, the concentration of the NFIL3 expression vector is 5 μg/mL.
Further, the expression level of the NFIL3 gene is inversely related to the erythroid differentiation ability of K562 cells.
In another aspect, the application provides the use of the NFIL3 gene for the preparation of a medicament for increasing erythroid differentiation of K562 cells.
On the other hand, the application also provides a kit for the hematopoietic differentiation of the target K562 cells of the NFIL3 gene in erythroid, which comprises the following primer sequences:
NFIL3 gene forward amplification primer: TCCTCAGTAGAACACACGCAG;
NFIL3 gene reverse amplification primer: TCATCTCTTGGCTCCCTTGT.
The beneficial effects obtained by the application are as follows:
according to the application, research and analysis show that the NFIL3 gene has a remarkable relation with the amount of hemoglobin produced by K562 cells for the first time. Up-regulating the expression of NFIL3 gene was found to have the effect of reducing the amount of hemoglobin in the corresponding K562 cells. Meanwhile, the designed NFIL3siRNA expression vector can increase the hemoglobin content of K562 cells and improve the erythroid hematopoietic differentiation capacity of the K562 cells.
In summary, NFIL3 is a potential new target for chronic granulocytic leukemia treatment. The application has great significance for researching leukemia occurrence, diagnosis and treatment.
Drawings
FIG. 1 is a graph showing the results of the gene expression level of NFIL 3;
FIG. 2 is a graph of hemoglobin measurement results;
FIG. 3 is a graph showing the results of benzidine staining after 24h transfection of silencing vector NFIL3siRNA into K562 cells;
FIG. 4 is a heat map of significantly differentially expressed genes associated with erythroid differentiation after NFIL3 silencing.
The accompanying drawings are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate the application and together with the embodiments of the application, serve to explain the application.
Detailed Description
The following description of the embodiments of the present application will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the application; all other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
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 present application. The preferred methods and materials described herein are illustrative only and should not be construed as limiting the application.
The experimental methods in the following examples are all conventional methods unless otherwise specified; the test materials and test strains used in the examples described below, unless otherwise specified, were commercially available. NFIL3siRNA was purchased from the company, sharp biotechnology limited, guangzhou. NCBI number of NFIL3 gene is: gene ID 4783.
The application provides an application of a target K562 cell of an NFIL3 gene in erythroid hematopoietic differentiation, wherein the application is to apply the target K562 cell of the NFIL3 gene in erythroid hematopoietic differentiation.
Further, the method for application comprises the following steps:
(1) K562 cells were cultured and then plated using six well plates;
(2) Transfecting NFIL3 expression vector in K562 cells using a kit;
(3) After 24h transfection of NFIL3 expression vector, K562 cells were induced to erythroid differentiation using Hemin at a final concentration of 50 μm;
(4) Collecting K562 cells induced for different times;
(5) Detecting the induction condition and the expression condition of the red line.
Preferably, the NFIL3 expression vector is NFIL3siRNA.
Preferably, the concentration of the NFIL3 expression vector is 5 μg/mL.
Further, the expression level of the NFIL3 gene is inversely related to the erythroid differentiation ability of K562 cells.
In another aspect, the application provides the use of the NFIL3 gene for the preparation of a medicament for increasing erythroid differentiation of K562 cells.
On the other hand, the application also provides a kit for the hematopoietic differentiation of the target K562 cells of the NFIL3 gene in erythroid, which comprises the following primer sequences:
NFIL3 gene forward amplification primer: TCCTCAGTAGAACACACGCAG;
NFIL3 gene reverse amplification primer: TCATCTCTTGGCTCCCTTGT.
Example 1
(1) Resuscitating K562 cells
Taking half an hour of bench in advance, taking out K562 cells in a liquid nitrogen tank, quickly thawing in a water bath at 37 ℃, transferring to a biosafety cabinet for operation, transferring the cells to a 15mL centrifuge tube, centrifuging at 1000rpm for 4min, and discarding the supernatant. The cells were transferred into a cell culture dish, the dish was gently shaken from top to bottom and from the side to uniformly distribute the cells, and finally placed into an incubator for culturing.
(2) Passage cell
Taking out cells from the incubator, observing the form and density (about 80%), transferring the cells into a centrifuge tube by blowing at 1000rpm for 4min, discarding the supernatant, adding 2mL of complete culture medium, uniformly mixing by blowing, discarding 1mL, transferring the rest cell suspension into a cell culture dish, slightly shaking uniformly, and culturing in the incubator.
(3) Cell cryopreservation
Transferring the cells into a centrifuge tube by using a pipetting gun at 1000rpm for 4min, discarding the supernatant, adding 400 mu L of complete culture medium, 500 mu LFBS and 100 mu LDMSO respectively, lightly blowing, transferring into a freezing storage tube, immediately placing into the freezing storage box, transferring into a refrigerator at-80 ℃ for one night, taking out in a liquid nitrogen container every morning, and writing corresponding positions on a record book for marking.
(4) Construction of NFIL3 Gene vector
The NFIL3 recombinant vector was constructed using the ClonExpress-II One Step Cloning Kit kit (Vazyme, C112). Wherein the vector is pCMV-Tag2B plasmid, which is transferred into slow virus.
(5) K562 cells (over-expressing NFIL 3) were infected with lentivirus.
Virus transfection:
human chronic myelogenous leukemia cell line K562 culture medium IMDM containing 10% Fetal Bovine Serum (FBS), 100U penicillin, 100 μg streptomycin, was added to 100ml of culture medium. 37 ℃,5% CO 2 Culturing under saturated humidity. Cells in logarithmic growth phase were taken and tested. The experimental method is as follows:
1. collecting K562 cells in good condition into a 15mL centrifuge tube, and rotating at 600rpm/min for 5 minutes;
2. the supernatant was discarded, resuspended in 2mL of 1 XPBS, washed 2 times, and spun at 600rpm/min for 5 minutes;
3. the supernatant was discarded, 1mL of a serum-free 1640 basal medium without diabody was added, and the cells were gently resuspended;
4. after cell counting, plates were performed according to the experimental design, and 500 μl of basal medium was added per well. Cells per well
About 1X 10 5 And each.
5. And adding a proper amount of empty virus control and a proper amount of polybrene into the control group, and adding a proper amount of slow virus which over-expresses NFIL3 and a proper amount of polybrene into the experimental group. And a blank (medium and K562 cells only) was set.
Virus loading (μl) = (cell number x MOI value/virus stock low). MOI values of K562 cells: 20.
6. placing the 24-well plate at 37deg.C with 5% CO 2 Is cultured in a constant temperature incubator.
7. After 24 hours of virus infection, liquid exchange is carried out, cells in each hole are collected into a 15mL centrifuge tube which is marked, the cells are washed for 2 times by 1 XPBS, the centrifuged cells are respectively added into a complete culture medium containing double antibodies, and the cells are placed into a 24-hole plate again for culture.
8. Changing the liquid according to the cell state.
9. After 72h of virus infection, the fluorescence intensity of the cells is observed under a fluorescence microscope, and the 1D efficiency of virus infection is judged. Cells transfected for 48h were collected and NFIL3 cell overexpression was verified by RT-qPCR.
(6) Cell transfection (silencing NFIL 3)
The cultured K562 cells were counted for observation, followed by plating (density 60%) and siRNA transfected according to lipo3000 kit instructions. Cells transfected for 48h were collected and NFIL3 gene expression was verified by RT-qPCR. And performing cell number analysis and RNA-seq gene expression analysis.
The specific method of RT-qPCR is as follows:
total RNA extraction
(1) The cell suspension was transferred from the flask into a centrifuge tube, centrifuged at 1000rpm for 4min, the cells were collected in a 1.5mL centrifuge tube, the supernatant was discarded, and washed twice with PBS.
(2) 1mL Trizol was added and the mixture was vortexed on a vortexing apparatus for 5min to allow complete lysis of the cells and left at room temperature for 5min.
(3) 200. Mu.L of chloroform was added thereto, and the mixture was left for 2min,12000g and centrifuged at 4℃for 15min.
(4) 350. Mu.L of the aqueous phase was transferred to a fresh EP tube, 300. Mu.L of isopropanol was added, and after mixing, the mixture was allowed to stand at room temperature for 10min, followed by centrifugation at 12000g at 4℃for 10min.
(5) The supernatant was discarded, 1mL of 75% ethanol was added, 12000g was centrifuged at 4℃for 5min. The supernatant was discarded and finally 10-20. Mu.L DEPC water was added to dissolve the RNA thoroughly.
Reverse transcription
The experimental procedure using the norfirazan reverse transcription kit is as follows:
(1) The purity and concentration of RNA were measured using an ultra-micro nucleic acid analyzer.
(2) Genomic DNA was removed as shown in Table 1. Blowing and mixing well at 42 ℃ for 2min.
TABLE 1 preparation of reverse transcription reaction System
(3) A reverse transcription system is configured, and the reverse transcription system,
TABLE 2 cDNA Synthesis reaction System
The procedure is as in Table 2. The reaction was carried out on a PCR apparatus at 37℃for 15min and 85℃for 5sec by using a centrifuge for instantaneous separation.
qPCR reaction
The procedure was as in tables 3-4 using the Norfirazan qPCR kit.
TABLE 3 two-step qPCR reaction System
| Reagent(s) | Volume (mu L) |
| 2×SYBR qPCR Master MIX | 5μL |
| Forward Primer(10μM) | 0.2μL |
| Reverse Primer(10μM) | 0.2μL |
| ddH 2 O | 3.6μL |
| cDNA | 1μL |
TABLE 4 two-step qPCR reaction procedure
As shown in FIG. 1, the gene expression level of NFIL3 is increased after the overexpression of the experimental group, and the silenced NFIL3 gene is down-regulated as shown in FIG. 3, and the number of erythroid differentiated cells is significantly higher after the silencing of the NFIL3 gene in the experimental group as compared with that of the blank vector, as shown in FIG. 4, and the gene expression of a large number of related gene groups is significantly affected.
Example 2
(1) With reference to the prior art schemes and specifications, the specific experimental method is as follows:
1. preparing a detection working solution: preparing a standard product working solution: hb (1 mg/mL) was taken out, room temperature was restored, 10. Mu.L was taken out, and ddH was added 2 O, namely standard working solution (13.2 mug/mL), and preparing color development working solution: and taking Hb Assay buffer to restore to room temperature. The chromogenic substrate was dissolved in Hb Assay.
2. Preparing a sample: cells of the study group and the control group collected as described above were 1X 10 6 And each. The cells were lysed on ice for 30min by washing twice and taking 100. Mu.L of RIP lysate, the supernatant.
3. Hb loading was performed using the hemoglobin detection kit to detect cellular hemoglobin concentration.
Blank control group: 8 mu L ddH 2 O+40. Mu.L of color development working solution+acid assay buffer 160. Mu.L;
standard well: 8 mu L of standard working solution +40 mu L of chromogenic working solution + Acid Assay buffer160 mu L;
sample to be measured: after 8. Mu.L of sample to be tested +40. Mu.L of chromogenic working solution + Acid Assay buffer 160. Mu.L and gentle mixing of the above samples, incubation was carried out at 37℃for 10min. Acid Assay buffer160 μl was added to each well and mixed well.
4. The microplate reader detects the absorbance at 530.
Hemoglobin concentration calculation formula (mg/L) = (OD sample-OD blank)/(OD standard-OD blank) ×13.2X100 (mg/L). The 530mm absorbance between the different groupings was calculated.
As shown in fig. 2, over-expression of NFIL3 reduced the production of hemoglobin in K562 cells, and silencing NFIL3 reduced the production of hemoglobin in K562 cells.
Although embodiments of the present application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the application, the scope of which is defined in the appended claims and their equivalents.
The application and its embodiments have been described above with no limitation, but only one of the embodiments of the application is shown in the drawings, and the practical solution and application are not limited thereto. In summary, those skilled in the art, having benefit of this disclosure, will appreciate that the application can be practiced without the specific details disclosed herein.
Claims (7)
- Use of a targeted K562 cell of nfil3 gene in erythroid differentiation, characterized in that: the application is to apply the NFIL3 gene targeted K562 cells to erythroid hematopoietic differentiation.
- 2. The use of the NFIL3 gene-targeted K562 cells according to claim 1 in erythroid hematopoietic differentiation, characterized in that: the method for application comprises the following steps:(1) K562 cells were cultured and then plated using six well plates;(2) Transfecting NFIL3 expression vector in K562 cells using a kit;(3) After 24h transfection of NFIL3 expression vector, K562 cells were induced to erythroid differentiation using Hemin at a final concentration of 50 μm;(4) Collecting K562 cells induced for different times;(5) Detecting the induction condition and the expression condition of the red line.
- 3. The use of the NFIL3 gene-targeted K562 cells according to claim 2 in erythroid hematopoietic differentiation, characterized in that: the NFIL3 expression vector is NFIL3siRNA.
- 4. Use of the NFIL3 gene-targeted K562 cells according to claim 3 in erythroid hematopoietic differentiation, characterized in that: the concentration of the NFIL3 expression vector is 5 mug/mL.
- 5. The use of the NFIL3 gene-targeted K562 cells according to claim 4 in erythroid hematopoietic differentiation, characterized in that: the expression level of the NFIL3 gene is inversely related to the erythroid differentiation ability of K562 cells.
- 6. The use of NFIL3 gene-targeted K562 cells in erythroid differentiation according to claim 5, characterized in that: the application is that the NFIL3 gene is used for preparing medicines for improving erythroid hematopoietic differentiation of K562 cells.
- 7. The use of NFIL3 gene-targeted K562 cells in erythroid differentiation according to claim 6, characterized in that: kit for preparing erythroid hematopoietic differentiation studies using NFIL3 gene targeting K562 cells, comprising the primer sequences:NFIL3 gene forward amplification primer: TCCTCAGTAGAACACACGCAG;NFIL3 gene reverse amplification primer: TCATCTCTTGGCTCCCTTGT.
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