CN103468643A - Human colonic cancer SW480 cell line capable of stably expressing luciferase and construction method of human colonic cancer SW480 cell line - Google Patents

Abstract

The invention relates to a human colorectal cancer SW480-luc cell line stably expressing luciferase and a construction method thereof. This cell line uses cationic liposomes to transfect the PGL4.51[luc2/CMV/neo] plasmid carrying luciferase reporter gene and neomycin resistance gene into human colorectal cancer SW480 cell line; G418, obtained through more than 3 monocloning operations. The cell line has been successfully constructed and can be stably passaged. The luminescence can be observed in the in vivo imaging system, and the luminescence intensity is proportional to the number of cells, and the in vivo (nude mouse xenograft tumor) model constructed with cells can also be observed in the imaging system It is feasible to dynamically observe biological activities such as tumor growth and metastasis in vivo.

Description

Human colorectal cancer SW480 cell line stably expressing luciferase and its construction method

technical field

The invention belongs to the technical field of cell engineering, and relates to the construction of a human colorectal cancer SW480 cell line, in particular to a method for constructing a human colorectal cancer SW480 cell line stably expressing luciferase.

Background technique

In vivo bioluminescent imaging is an emerging imaging technology based on the use of light-emitting proteins such as luciferase to catalyze a chemical reaction that releases photons. The most commonly used protein is firefly luciferase, and its mechanism is an enzymatic reaction, namely luciferase In the presence of ATP and oxygen, it catalyzes the oxidation reaction of the luciferase substrate and emits light. Therefore, this luminescent phenomenon must be carried out in living cells. The light from this reaction is distinct, with an emission spectrum between 400nm and 620nm, easily recorded by a CCD camera. Researchers can conduct scientific studies with luciferase-labeled cells, viruses, DNA, gene expression, and animal models. Using this imaging system can directly and rapidly observe the biological activities of tumors in vivo.

BALB/c nude mice belong to animals with deficient T cell immune function, and the autofluorescence interference produced by fur is less when used for in vivo imaging, so it is an ideal experimental animal for establishing xenograft tumor models in vivo.

Colorectal cancer, including colon cancer and rectal cancer, is one of the common malignant tumors. The incidence rate in my country is high. Surgery is the main treatment method at present, and postoperative metastasis is an important factor affecting the prognosis of patients. Therefore, the study of colorectal cancer cells Biological activity is important in finding effective treatments for colorectal cancer. The establishment of human tumor animal models using immunodeficient animals is an important tool for studying biological characteristics such as tumor growth and metastasis in vivo. Therefore, for colorectal cancer cells labeled with luciferase, it is of great significance for the study of colorectal cancer to observe tumor luminescence in an in vivo imaging system and dynamically observe biological activities such as tumor growth and metastasis in vivo.

Contents of the invention

The present invention aims to provide a human colorectal cancer SW480 cell line stably expressing luciferase and its construction method, so as to realize the observation of tumor luminescence in an in vivo imaging system, dynamic observation of biological activities such as tumor growth and metastasis in a living body.

The present invention is achieved through the following schemes:

The above-mentioned human colorectal cancer SW480-luc cell line stably expressing luciferase is obtained by the following method: using cationic liposomes to transfect the plasmid carrying the luciferase reporter gene into the human colorectal cancer SW480 cell line; The final cells were added with G418, and after more than 3 monocloning operations, a human colorectal cancer SW480-luc cell line stably expressing luciferase was obtained.

The human colorectal cancer SW480-luc cell line stably expressing luciferase, wherein the PGL4.51 plasmid carrying a luciferase reporter gene and a neomycin resistance gene is transfected into human colorectal cancer by using cationic liposomes SW480 cell line.

In the human colorectal cancer SW480-luc cell line stably expressing luciferase, the optimum working concentration of G418 for the SW480 cell line is 700ug/ml.

The method for constructing the above-mentioned human colorectal cancer SW480-luc cell line stably expressing luciferase specifically comprises the following steps:

First, the determination of the optimal screening concentration of G418

SW480 cells were routinely cultured in 1640 medium containing 10% calf serum; cells in the logarithmic growth phase were taken, and the cell concentration was adjusted to 1×104/ml, 100 μl per well were inoculated in a 96-well plate, and placed in a cell incubator for routine culture ; 24 hours after cell inoculation, double-dilute G418, establish 0-1mg/mL10 gradients, add to 96-well plate, set 6 replicate wells for each concentration; observe cell growth every day, and make cells within 10-14 days The lowest G418 concentration for all death is the working concentration for screening transfected SW480 cells;

Second, cell transfection

Take SW480 cells in the logarithmic growth phase, inoculate the cells in a 6-well plate one day before transfection, and transfect after the cell confluence reaches 70%-90% within 24 hours; transfection is performed according to the operation guide of LipofectamineTM2000 reagent; After transfection, the cells were placed in the incubator for routine culture;

Third, G418 selection of resistant cells and their monoclonalization

48 hours after plasmid transfection, the cells were subcultured into 24-well plates at a ratio of 1:10, and untransfected cells were subcultured at the same density as a control; after the cells adhered to the wall, G418 was added, and the cells were replaced in time according to the cell death and the nutritional status of the medium. solution; after all the cells in the control group died, the primary resistant clones were obtained. The resistant clone was tested for luciferase activity, and the clone expressing luciferase was prepared into a single cell suspension, inoculated in a 96-well plate, and transferred to a 24-well plate for expansion after proliferation. This monoclonalization process was repeated three times to obtain SW480-luc cells stably expressing luciferase.

The method for constructing the human colorectal cancer SW480-luc cell line stably expressing luciferase also includes the identification of luciferase activity. The specific identification method is: the concentration of SW480-luc cells is 5.0×105/ml, 2.5×105 /ml, 1.25×105/ml, 0.625×105/ml, 0.313×105/ml and 0.156×105/ml, 100μl per well was added to a 96-well white plate, and 3 replicate wells were set up for each group; 0.5μl of 30mg/ml was added to each well ml of fluorescein substrate, incubated at 37°C for 10 min, and measured the fluorescence value with a fluorescent illuminometer; compared the fluorescence intensity of each clone, and analyzed the correlation between the fluorescence intensity and the number of cells. The optimum working concentration of G418 for SW480 cell line is 700ug/ml.

Beneficial effect:

In this study, SW480-luc stably expressing luciferase was subcutaneously inoculated into nude mice to successfully establish an in vivo imaging animal model, which provided convenient conditions for the next step of dynamic observation of tumor growth. The SW480-lun cell line has been successfully constructed and can be stably passaged. The luminescence can be observed in the in vivo imaging system, and the luminescence intensity is proportional to the number of cells. In addition, the in vivo (nude mouse xenograft tumor) model constructed with cells can also be observed in the imaging system. The luminescence of the tumor can be observed, making it feasible to dynamically observe biological activities such as the growth and metastasis of the tumor in the living body.

Description of drawings

Fig. 1 is the in vivo imaging step of the embodiment of the present invention;

Fig. 2 is the luminescence value of 6 positive clones of the embodiment of the present invention;

Fig. 3 shows the luminescence of SW480-luc cells in nude mice according to the embodiment of the present invention.

Detailed ways

The human colorectal cancer SW480 cell line (SW480-luc) stably expressing luciferase of the present invention is obtained by the following method: using cationic liposomes to transfect the plasmid carrying the luciferase reporter gene and neomycin resistance gene Into the human colorectal cancer SW480 cell line; the transfected cells were added with G418, and after more than 3 monoclonal operations, the human colorectal cancer SW480-luc cell line stably expressing luciferase was obtained.

The construction method and identification of the human colorectal cancer SW480 cell line (SW480-luc) stably expressing luciferase specifically includes the following steps:

First, the determination of the optimal screening concentration of G418

SW480 cells were routinely cultured in 1640 medium containing 10% calf serum; cells in the logarithmic growth phase were taken, and the cell concentration was adjusted to 1×104/ml, 100 μl per well were inoculated in a 96-well plate, and placed in a cell incubator for routine culture ; 24 hours after cell inoculation, double-dilute G418, establish 0-1mg/mL10 gradients, add to 96-well plate, set 6 replicate wells for each concentration; observe cell growth every day, and make cells within 10-14 days The lowest G418 concentration for all death is the working concentration for screening transfected SW480 cells.

Second, cell transfection

Take SW480 cells in the logarithmic growth phase, inoculate the cells in a 6-well plate one day before transfection, and transfect after the cell confluence reaches 70%-90% within 24 hours; transfection is performed according to the operation guide of LipofectamineTM2000 reagent, That is: add 500ug PGL4.51 plasmid to 500μl OPTI-MEM, mix thoroughly, and make solution I; add 7μl PLUS to solution I, incubate at room temperature for 5min; add 21μlLTX, incubate at room temperature for 30min, then add to the wells of a 6-well plate; cells after transfection Placed in an incubator for routine cultivation.

Third, G418 selection of resistant cells and their monoclonalization

48 hours after plasmid transfection, the cells were subcultured into 24-well plates at a ratio of 1:10, and untransfected cells were subcultured at the same density as a control; after the cells adhered to the wall, G418 was added, and the cells were replaced in time according to the cell death and the nutritional status of the medium. solution; after all the cells in the control group died, the primary resistant clones were obtained. The resistant clone was tested for luciferase activity, and the clone expressing luciferase was prepared into a single cell suspension, inoculated in a 96-well plate, and transferred to a 24-well plate for expansion after proliferation. This monoclonalization process was repeated three times to obtain SW480-luc cells stably expressing luciferase.

Fourth, luciferase activity identification

The concentration of SW480-luc cells is 5.0×105/ml, 2.5×105/ml, 1.25×105/ml, 0.625×105/ml, 0.313×105/ml and 0.156×105/ml, add 100μl per well into 96-well white plate In each group, 3 replicate wells were set; each well was added with 0.5 μl of 30 mg/ml fluorescein substrate, incubated at 37°C for 10 minutes, and the fluorescence value was measured by a fluorescent light meter; the fluorescence intensity of each clone was compared, and the relationship between the fluorescence intensity and the number of cells was analyzed. correlation between.

In this study, we used cationic liposomes to transfect the PGL4.51[luc2/CMV/neo] plasmid carrying the luciferase reporter gene and neomycin resistance gene into the human colorectal cancer SW480 cell line, as shown in Figure 1 In vivo imaging steps: plasmid PGL4.51 map and in vivo imaging system for observing luminescence. Because different cell lines have different tolerance to G418, this experiment screened through 10 concentration gradient experiments of 0-1mg/ml, and determined that the optimal working concentration of G418 for SW480 cell line was 700ug/ml. It should be noted that the transfected cells must undergo more than 3 monoclonal operations to obtain genetically stable cell clones. In this experiment, 7 resistant clones were preliminarily screened out. After measuring the fluorescence value, 1 was found to be a false positive clone, and 6 were positive clones; refer to the luminescence values of the 6 positive clones in Figure 2, the right side of the figure is the luminous intensity judgment value, The luminous intensity is stronger from bottom to top. The luciferase-catalyzed substrate reaction is a high-energy-consuming process, and the luciferase activity should not be too high. Therefore, this experiment chose SW480-luc clone 1 with a slightly lower fluorescence value for animal experiments. BABL/c nude mice belong to animals with deficient T cell immune function, and the autofluorescence interference produced by fur is less when used for in vivo imaging, so it is an ideal experimental animal for establishing xenograft tumor models in vivo. In this study, SW480-luc stably expressing luciferase was inoculated subcutaneously into nude mice to successfully establish an in vivo imaging animal model, which provided convenient conditions for the next step of dynamic observation of tumor growth. The luminescence of SW480-luc cells in nude mice is shown in sequence with reference to Figure 3 The conditions observed on the 3rd day, 10th day, 17th day and 24th day after inoculation respectively.

The SW480-lun cell line has been successfully constructed and can be stably passaged. The luminescence can be observed in the in vivo imaging system, and the luminescence intensity is proportional to the number of cells. In addition, the in vivo (nude mouse xenograft tumor) model constructed with cells can also be observed in the imaging system. The luminescence of the tumor can be observed, making it feasible to dynamically observe biological activities such as the growth and metastasis of the tumor in the living body.

Claims (6)

1. A human colorectal cancer SW480-luc cell line stably expressing luciferase is obtained by the following method: using cationic liposomes to transfect the plasmid carrying the luciferase reporter gene into the human colorectal cancer SW480 cell line; The transfected cells were added with G418, and the human colorectal cancer SW480-luc cell line stably expressing luciferase was obtained through more than 3 monocloning operations. 2. the human colorectal cancer SW480-luc cell line stably expressing luciferase as claimed in claim 1, is characterized in that: utilize cationic liposome to carry the PGL4. 51 plasmid was transfected into human colorectal cancer SW480 cell line. 3. The human colorectal cancer SW480-luc cell line stably expressing luciferase according to claim 1, wherein the optimum working concentration of G418 for SW480 cell line is 700ug/ml. 4. The construction method of the human colorectal cancer SW480-luc cell line stably expressing luciferase as claimed in claim 1, specifically comprising the following steps: First, the determination of the optimal screening concentration of G418 SW480 cells were routinely cultured in 1640 medium containing 10% calf serum; cells in the logarithmic growth phase were taken, and the cell concentration was adjusted to 1×10 ⁴ /ml, 100 μl per well were inoculated in a 96-well plate, and placed in a cell culture incubator for routine Culture; 24 hours after cell inoculation, double-dilute G418, establish 10 gradients of 0-1mg/mL, add to 96-well plate, set 6 replicate wells for each concentration; observe cell growth every day, use within 10-14 days The lowest concentration of G418 at which all cells die is the working concentration for screening transfected SW480 cells; Second, cell transfection Take SW480 cells in the logarithmic growth phase, inoculate the cells in a 6-well plate one day before transfection, and transfect after the cell confluence reaches 70%-90% within 24 hours; transfection is performed according to the operation guide of LipofectamineTM2000 reagent; After transfection, the cells were placed in the incubator for routine culture; Third, G418 selection of resistant cells and their monoclonalization 48 hours after plasmid transfection, the cells were subcultured into 24-well plates at a ratio of 1:10, and untransfected cells were subcultured at the same density as a control; after the cells adhered to the wall, G418 was added, and the cells were replaced in time according to the cell death and the nutritional status of the medium. After all the cells in the control group died, a preliminary resistant clone was obtained; the luciferase activity of the resistant clone was detected, and the clone expressing luciferase was prepared into a single cell suspension, seeded in a 96-well plate, and waited for its proliferation. Transfer to a 24-well plate for expanded culture; repeat this monoclonalization process three times to obtain SW480-luc cells stably expressing luciferase. 5. The construction method of the human colorectal cancer SW480-luc cell line stably expressing luciferase as claimed in claim 4, further comprising luciferase activity identification, the specific identification method is: The concentration of SW480-luc cells is 5.0×105/ml, 2.5×105/ml, 1.25×105/ml, 0.625×105/ml, 0.313×105/ml and 0.156×105/ml, add 100μl per well into 96-well white plate In each group, 3 replicate wells were set; each well was added with 0.5 μl of 30 mg/ml fluorescein substrate, incubated at 37°C for 10 minutes, and the fluorescence value was measured by a fluorescent light meter; the fluorescence intensity of each clone was compared, and the relationship between the fluorescence intensity and the number of cells was analyzed. correlation between. 6. The method for constructing a human colorectal cancer SW480-luc cell line stably expressing luciferase according to claim 4 or 5, characterized in that: the optimum working concentration of G418 for SW480 cell line is 700ug/ml.

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