Bifluorescence expression human chronic myelogenous leukemia cell K562 stably transfected cell strain and preparation method and application thereof

Abstract

The invention discloses a bifluorescence expression human chronic myelogenous leukemia cell K562 stably transfected cell strain as well as a preparation method and application thereof, and particularly relates to the field of biological medicines. The method comprises the following steps: transfecting luciferase Luciferase and red fluorescent protein mCherry gene sequences into human chronic myeloid leukemia cells K562 by utilizing a lentiviral vector, screening to obtain a cell strain capable of stably expressing Luciferase and mCherry, and constructing a mouse in-vivo tumor model by utilizing the cell strain. When the stably transfected cell strain constructed by the method is inoculated into a mouse body, non-invasive monitoring can be performed on growth of tumors in the body, so that occurrence, progress, infiltration, metastasis and anti-tumor effects of related treatment of chronic myeloid leukemia cells can be known in real time, meanwhile, the cell line has two fluorescent tracing systems with different luminescence principles, and the cell line has a good application prospect. The method can adapt to more detection methods, more accurate and stable disease signals can be obtained, and a reliable research means is provided for development of tumor generation, development and metastasis mechanisms and related drugs and treatment methods.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to a dual-fluorescence expressing human chronic myeloid leukemia cell K562 stable cell line, screening and tumor model construction. Background Art

[0002] Transgenic stable cell lines refer to cell lines that stably overexpress or inhibit specific genes by integrating exogenous DNA fragments into host chromosomes through genetic engineering technology. The construction of stable cell lines generally involves loading the target gene sequence or the shRNA sequence that inhibits the target gene onto a vector with characteristic resistance, and then integrating the target sequence into the chromosome of the host cell through a lentiviral system. The resistance markers contained in the vector, such as puromycin, are used for screening to obtain cell lines that stably express or silence specific genes, thereby achieving continuous and stable regulation of the target gene.

[0003] Cancer research requires the establishment of tumor-specific in vivo animal models, and cancer cell inoculation is often used to evaluate new treatments in vivo. The construction of tumor cell lines that stably express detectable fluorescent proteins provides a new direction for monitoring longitudinal tumor development. Detection of fluorescent proteins can be used to visualize various types of cancer processes, including primary tumor growth, tumor cell movement and invasion, metastasis, and tumor-host interactions. Stably transfected fluorescent cell lines can be used to evaluate the efficacy of anticancer treatments through whole-body imaging. They are valuable tools for in vivo evaluation of new treatments and are of great significance for evaluating new cancer therapies. Summary of the invention

[0004] The present invention provides a dual-fluorescence expressing human chronic myeloid leukemia cell K562 stably transfected cell line and a preparation method and application thereof. The cell line can stably and highly express red fluorescent protein mCherry and luciferase Luciferase. The specific technical scheme is as follows:

[0005] The first aspect of the present invention provides a dual-fluorescence expressing human chronic myeloid leukemia cell K562 stable cell line, characterized in that a lentiviral vector simultaneously expressing red fluorescent protein mCherry and luciferase Luciferase is transfected into human chronic myeloid leukemia cells K562 to obtain the cell line.

[0006] The second aspect of the present invention provides a method for preparing a dual-fluorescence expressing human chronic myeloid leukemia cell K562 stably transfected cell line, comprising the following steps:

[0007] Constructing a lentiviral vector for expressing luciferase and red fluorescent protein mCherry;

[0008] The lentiviral vector and packaging plasmid are co-transfected into viral host cells, and the lentivirus is amplified and collected;

[0009] The collected lentivirus was used to transfect human chronic myeloid leukemia cells K562 to obtain the dual-fluorescence expressing human chronic myeloid leukemia cell K562 stable cell line.

[0010] Furthermore, the construction method of the lentiviral vector is:

[0011] The lentiviral vector LV-EF1a-WPRE was digested to obtain a linearized vector;

[0012] Primers were designed respectively to amplify target gene fragments encoding red fluorescent protein mCherry and luciferase Luciferase;

[0013] The recovered linearized vector and the target gene fragment are homologously recombined to obtain a lentiviral vector LV-EF1a-Luciferase-mCherry-WPRE carrying the target gene fragment.

[0014] Preferably, the viral host cell is a 293T cell.

[0015] Furthermore, the method of transfecting human chronic myeloid leukemia cells K562 with the lentivirus is:

[0016] The collected lentivirus is prepared into a virus infection solution with a complete culture medium at an MOI value of 3-200, and the virus infection solution is used to transfect human chronic myeloid leukemia cells K562;

[0017] After infection, the culture medium containing puromycin was replaced and culture was continued. The transfection of polyclonal cells was verified by detecting the proportion of red fluorescent cells and luciferase activity.

[0018] Monoclonal cells were isolated from polyclonal cells that were successfully transfected and verified, and dual-fluorescence expressing human chronic myeloid leukemia K562 stable cell lines with high expression of red fluorescent protein and luciferase activity were screened.

[0019] Preferably, the MOI value is 150.

[0020] Preferably, the concentration of puromycin is 2 μg / mL.

[0021] The third aspect of the present invention provides a method for constructing a tumor model, which is obtained by inoculating the dual-fluorescence expressing human chronic myeloid leukemia cell K562 stable cell line into an animal.

[0022] Preferably, the animal is a mouse.

[0023] Furthermore, the inoculation method of the above mouse tumor model is injection into the mouse tail vein.

[0024] It should be noted that the above-mentioned mouse tumor model is obtained by inoculating the screened monoclonal dual-fluorescence expressing human chronic myeloid leukemia cell K562 stable cell line into the tail vein of the mouse. After inoculation, the tumor growth is detected using a live imaging device once a week. If the results show that the two types of photon signals at the inoculation site of the mouse gradually increase over time, the tumor model is successfully constructed.

[0025] The fourth aspect of the present invention provides the use of the above-mentioned dual-fluorescence expressing human chronic myeloid leukemia cell K562 stable cell line for preparing a drug for treating chronic myeloid leukemia.

[0026] The process of preparing the drug for treating chronic myeloid leukemia in the present invention also includes evaluating and screening the therapeutic effects of related drugs or treatment plans.

[0027] The beneficial effects of the present invention are:

[0028] 1. The present invention stably transfers luciferase and red fluorescent protein into K562 human chronic myeloid leukemia cells through a lentiviral expression vector, thereby obtaining a K562-Luciferase-mCherry stable cell line. The stable cell line established by this method can be transplanted into mice to monitor and evaluate the occurrence, development, infiltration, metastasis of chronic myeloid leukemia in real time, as well as the therapeutic effects of corresponding treatment measures.

[0029] 2. The mouse tumor model prepared by using the dual-fluorescence-expressing human chronic myeloid leukemia cell K562 stable cell line of the present invention has excellent application prospects in the study of the occurrence and development mechanism of chronic myeloid leukemia, the screening of therapeutic drugs and the screening of different treatment schemes. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the map of the lentiviral vector LV-EF1a-Luciferase-mCherry-WPRE;

[0031] Figure 2 This is the red fluorescence ratio detection of K562-Luciferase-mCherry monoclonal cells and polyclonal cells. In the figure, PC represents polyclonal cells, and MC represents monoclonal cells with different numbers (1, 2, 6, etc.);

[0032] Figure 3 The red fluorescence intensity of K562-Luciferase-mCherry monoclonal cells and polyclonal cells is detected. In the figure, WT represents wild-type cells, PC represents polyclonal cells, and MC represents monoclonal cells with different numbers (1, 2, 6, etc.);

[0033] Figure 4 Luciferase activity detection for K562-Luciferase-mCherry monoclonal cells and polyclonal cells;

[0034] Figure 5 These are the results of in vivo imaging tests on days 33 and 39 after mice were inoculated with K562-Luciferase-mCherry-22 monoclonal cells through the tail vein. DETAILED DESCRIPTION

[0035] The present invention is further described below in conjunction with specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments, and do not constitute any limitation on the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention can be modified and replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

[0036] The reagents and instruments used in the following specific embodiments, unless otherwise specified, can all be purchased through conventional channels. The reagents or reaction raw materials used, unless otherwise specified, all use the initial concentration of each raw material in the purchased goods.

[0037] Example 1: Construction of a lentiviral vector containing the genes Luciferase and mCherry

[0038] (1) The lentiviral vector LV-EF1a-WPRE was digested with BamHI restriction endonuclease and SalI restriction endonuclease to obtain a linearized vector.

[0039] (2) Design primers to amplify the red fluorescent protein mCherry and luciferase to obtain the target gene fragments.

[0040] (3) Recover and purify the linearized vector and target gene fragment using a gel recovery kit.

[0041] (4) Take 2 μL of the target gene fragment and 3 μL of the linearized vector, and use the full-strand gold homologous recombination reagent to connect and recombine the target fragment and the linearized vector. The recombination condition is incubated at 50°C for 60 min to obtain the lentiviral vector LV-EF1a-Luciferase-mCherry-WPRE carrying the target gene fragment. The vector map is as follows: Figure 1 shown.

[0042] Example 2: Infection of 293T cells

[0043] (1) One day before transfection, seed 293T cells in a 10 cm culture dish so that the cell confluence reaches 90% on the day of transfection.

[0044] (2) On the day of transfection, replace the culture medium from 293T cells with growth medium.

[0045] (3) For each transfection sample, prepare Lipofectamine complex and use lentiviral vector LV-EF1a-Luciferase-mCherry-WPRE and packaging plasmids [psPAX2] and [pMD2.G] for transfection.

[0046] (4) Add the DNA-Lipofectamine complex to the cells, shake gently to mix, and then place the cells in an incubator for culture.

[0047] (5) On the next day, replace the culture medium containing DNA-Lipofectamine complex with 2 mL of complete culture medium and continue to culture in the incubator.

[0048] (6) 48-72 h after transfection, collect the supernatant containing the virus.

[0049] (7) Centrifuge the viral supernatant at 3000 rpm for 15 min at 4 °C to precipitate debris, and then filter the viral supernatant through a Millex HV 0.45 μm filter.

[0050] (8) The filtered viral supernatant can be titered and used in subsequent infection experiments, or stored in 200 μL aliquots at -80 °C for later use.

[0051] Example 3: Preliminary experiment of lentivirus infection of cells (MOI exploration)

[0052] (1) Plating: Take K562 cells in the logarithmic growth phase and plate them at 1×10 4 Each well was inoculated into a 96-well plate and cultured overnight in a 37°C, 5% CO2 incubator to ensure that the cell confluence was between 30% and 50% on the next day.

[0053] (2) Group design: The MOI was designed to be 0, 25, 50, 100, and 150, respectively. Five gradient groups were set, with 2 replicates in each group.

[0054] (3) Infection (1 / 2 small volume infection method): Remove the lentivirus and place it on ice to slowly thaw. Prepare the virus infection solution with complete culture medium according to the MOI value. Add 50 μL of the virus infection solution to each well and place it in a 37 ℃, 5% CO2 incubator. After culturing for 4 h, remove the culture plate.

[0055] Continue to add 50 μL of complete culture medium to each well. After 24 hours of infection, remove the culture medium in each group of experiments and replace it with fresh complete cell culture medium for continued culture. After 72 hours of infection, observe the cells under a microscope, take pictures and collect the cells.

[0056] The PE-CF594 fluorescence channel was selected by flow cytometry to detect the fluorescent cell ratio and fluorescence value. The infection conditions and MOI values ​​corresponding to the group with a fluorescent cell ratio ≥ 80% and good growth status were selected for subsequent experiments.

[0057] In the present invention, the MOI value corresponding to the group with good growth status is 150.

[0058] Example 4: Puromycin minimum killing concentration screening

[0059] (1) Suspension cell plating: Take K562 cells in the logarithmic growth phase that have not been infected with the virus and plate them at 5×10 5 Each well was inoculated into a 12-well plate.

[0060] (2) Treatment: Complete culture medium containing different concentrations of puromycin was added, and the final concentration gradient of puromycin was set to 0, 0.5, 1, 2, 4, 6, 8, and 10 μg / mL. The cells were cultured in a 37°C, 5% CO2 incubator. The puromycin concentration intervention treatment was maintained for 3-4 days. The medium was changed every other day. The cells were observed and photographed every day to screen out the lowest concentration of puromycin that could kill more than 90% of K562 cells for subsequent experiments.

[0061] In the present invention, the lowest concentration of puromycin that can kill more than 90% of K562 cells is 2 μg / mL.

[0062] Example 5: Polyclonal cell construction

[0063] (1) Plating: Take K562 cells in the logarithmic growth phase and plate them at 1×10 per well. 5 The cells were inoculated into 24-well plates and cultured in a 37°C, 5% CO2 incubator. Subsequent experiments were performed when the cell confluence grew to 40%-60%.

[0064] (2) Infection (suspended cells): Remove the virus and place it on ice to slowly thaw. Prepare the virus infection solution with complete culture medium according to the MOI value. Harvest K562 cells in the logarithmic growth phase, count them, and resuspend the cell pellet with 250 μL of the prepared virus infection solution. Plate them into a 24-well plate, with 2×10 cells per well. 5 cells.

[0065] Seal the plate, place it in a flat-angle centrifuge, centrifuge at 1200 g for 1 h, and then place it in an incubator for normal culture. Remove the culture plate after 4 h, continue to add 250 μL of complete culture medium to each well, and change the medium after 24 h of infection. Set up a control group (MOI=0) and culture it normally without treatment.

[0066] (3) 72 hours after infection, replace the culture medium with fresh complete medium containing 2 μg / mL puromycin and continue culturing. When the cell confluence is ≥90%, subculture and expand.

[0067] (4) Collect wild-type cells and polyclonal cells.

[0068] The PE-CF594 fluorescence channel was selected by flow cytometry to detect the ratio of fluorescent cells. The results are as follows: Figure 2 As shown, the red fluorescence ratio of polyclonal cells was >95%.

[0069] Example 6: Detection of luciferase activity in polyclonal cells

[0070] (1) Reagent preparation: First, prepare 200× D-luciferin sodium salt stock solution (30 mg / mL) with sterile water. After thoroughly mixing and dissolving, dilute it 1:100 with preheated complete culture medium to prepare 2× working solution (300 μg / mL) for later use.

[0071] (2) Plating: Collect polyclonal cells and wild-type K562 cells, count and resuspend the cells to 1×10 5 100 μL of cell suspension was added to a black 96-well cell culture plate, with 3 replicates for each group.

[0072] (3) Detection: Add 100 μL of 300 μg / mL D-luciferin sodium salt to each well and mix thoroughly by pipetting. After 2 min, place the well plate in the instrument for imaging and analysis.

[0073] Example 7: Monoclonal cell screening

[0074] Take the successfully verified polyclonal cells and inoculate them into 96-well plates at 1 cell / 100 μL / well for culture (the culture medium contains 2 μg / mL puromycin), and change the medium once a week. When the cell confluence grows to 70%~80%, the cells are passaged and expanded. Example 8: Detection of red fluorescence ratio, intensity and luciferase activity in monoclonal cells

[0075] The red fluorescent cell ratio and intensity were detected by flow cytometry. Figure 2 , Figure 3 As shown, the red fluorescence ratio of monoclonal cells is greater than 99%, and the red fluorescence intensity is relatively strong, with a value greater than 5889.

[0076] The luciferase activity in monoclonal cells was detected by microplate reader. Figure 4 As shown, the luciferase fluorescence intensity of the monoclonal cells was strong, with a value of >19913.

[0077] Monoclonal cells with red fluorescent cell ratio>99% and stable high expression of luciferase were screened.

[0078] Example 9: Construction of a chronic myeloid leukemia cell K562 tumor model

[0079] In this example, a chronic myeloid leukemia cell K562 tumor model was constructed using monoclonal cell K562-Luciferase-mCherry-22 with a red fluorescent cell ratio of >99% and stable high expression of luciferase.

[0080] (1) Resuspend K562-Luciferase-mCherry-22 cells in serum-free medium and adjust the cell concentration to 5×10 7 / mL, and keep on ice for later use.

[0081] (2) Wipe the tail of the M-NSG mouse with 75% alcohol, draw 200 μL of cell fluid with a syringe, and insert the needle at 1 / 3 to 1 / 2 of the distal end of the tail. Press the injected mouse tail tightly on the table with your left hand. When inserting the needle with your right hand, the needle tip should be parallel to the table and slightly downward. Once inserted, lift the needle slightly upward and insert it along the blood vessel. The needle can be observed to move forward with the naked eye. If the needle moves forward in the blood vessel, you can clearly feel that the needle moves smoothly without any resistance. Do not insert the needle too deep. Immediately after the needle enters the skin, move the needle slightly upward and insert it parallel to the skin. If there is a sense of emptiness when the needle is inserted and there is no resistance when pushing the liquid, it means that the injection is successful.

[0082] (3) After inoculation, move the M-NSG mice into a feeding box for routine feeding.

[0083] The tumor growth was monitored and weighed once a week using an in vivo imaging device. If the results showed that both types of photon signals at the inoculation site of the mouse gradually increased over time, the tumor model was successfully constructed.

[0084] like Figure 5 As shown in the figure, after the K562-Luciferase-mCherry-22 monoclonal cells were inoculated into the tail vein of mice, the in vivo imaging detection results on the 33rd and 39th days showed that both the red fluorescence and luciferase photon signals increased over time; and on the 39th day, both types of photon signals were strong, with the luciferase intensity value >6×10 10 , red fluorescence intensity value>8×10 9 .

[0085] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A dual-fluorescence expressing human chronic myeloid leukemia cell K562 stable cell line, characterized in that: The lentiviral vector simultaneously expressing red fluorescent protein mCherry and luciferase Luciferase was transfected into human chronic myeloid leukemia cells K562.

2. A method for preparing a dual-fluorescence expressing human chronic myeloid leukemia cell K562 stable cell line, characterized in that: The following steps are involved: Constructing a lentiviral vector for expressing luciferase and red fluorescent protein mCherry; The lentiviral vector and packaging plasmid are co-transfected into viral host cells, and the lentivirus is amplified and collected; The collected lentivirus was used to transfect human chronic myeloid leukemia cells K562 to obtain the dual-fluorescence expressing human chronic myeloid leukemia cell K562 stable cell line.

3. The preparation method according to claim 2, characterized in that: The construction method of the lentiviral vector is: The lentiviral vector LV-EF1a-WPRE was digested to obtain a linearized vector; Primers were designed respectively to amplify target gene fragments encoding red fluorescent protein mCherry and luciferase Luciferase; The recovered linearized vector and the target gene fragment are homologously recombined to obtain a lentiviral vector LV-EF1a-Luciferase-mCherry-WPRE carrying the target gene fragment.

4. The preparation method according to claim 2, characterized in that: The method for transfecting human chronic myeloid leukemia cells K562 with lentivirus is as follows: The collected lentivirus is prepared into a virus infection solution with a complete culture medium at an MOI value of 3-200, and the virus infection solution is used to transfect human chronic myeloid leukemia cells K562; After infection, the culture medium containing puromycin was replaced and culture was continued. The transfection of polyclonal cells was verified by detecting the proportion of red fluorescent cells and luciferase activity. Monoclonal cells were isolated from polyclonal cells that were successfully transfected and verified, and dual-fluorescence expressing human chronic myeloid leukemia K562 stable cell lines with high expression of red fluorescent protein and luciferase activity were screened.

5. The preparation method according to claim 2, characterized in that: The virus host cell is 293T cell.

6. A method for constructing a tumor model, characterized in that: The dual-fluorescence expressing human chronic myeloid leukemia cell K562 stable cell line according to claim 1 is inoculated into an animal to obtain the cell line.

7. The use of a dual-fluorescence expressing human chronic myeloid leukemia cell K562 stable cell line according to claim 1, characterized in that: Used for preparing medicines for treating chronic myeloid leukemia.

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