CN109536452B - Visual nasopharyngeal carcinoma cell and application thereof - Google Patents
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Abstract
The invention discloses a visible nasopharyngeal carcinoma cell and application thereof, wherein the visible nasopharyngeal carcinoma cell is named as S18-1C3, is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: C2018189. The cell line S18-1C3 has the capability of continuously expressing high fluorescence intensity, and the fluorescence intensity of the cell line is not lost along with the continuous passage of nasopharyngeal carcinoma cells; the integration and expression of the fluorescent gene do not affect the main biological characters of nasopharyngeal carcinoma cells; S18-1C3 also has high cell proliferation capacity, cell migration and invasion capacity and strong reporter gene expression capacity, and is an ideal cell model for fluorescence tracing nasopharyngeal carcinoma metastasis or proliferation. The S18-1C3 can be used for screening or verifying nasopharyngeal carcinoma medicaments, can be used for researching the mechanism of nasopharyngeal carcinoma metastasis or proliferation, and can also be used for researching the action mechanism of the nasopharyngeal carcinoma medicaments.
Description
Technical Field
The invention relates to the technical field of medical models, in particular to a visual nasopharyngeal carcinoma cell and application thereof.
Background
Nasopharyngeal carcinoma is a cancer that occurs in the nasopharyngeal cavity or upper pharyngeal portion. According to WHO's rough estimate, about 80% of nasopharyngeal carcinomas occur in china. In south China, the incidence rate is 25-30/100,000 people/year, while in the Caucasian population in North America, the incidence rate is less than 1/100,000 people/year. At present, the distal metastasis of nasopharyngeal carcinoma has become the main cause of nasopharyngeal carcinoma death, so the research on the nasopharyngeal carcinoma metastasis mechanism and corresponding drugs is very urgent, and the visualized nasopharyngeal carcinoma cell metastasis cell model is the basis for relevant research.
Common labels for cell visualization markers include both fluorescent proteins and luciferases. GFP is a green fluorescent protein (greenfluorescent protein GFP) cloned from the bioluminescent jellyfish Aequorea victoria, encoding 283 amino acid residues, with a molecular weight of 27kDa and capable of emitting green fluorescence without the assistance of other Aequorea proteins, without the need for substrates, and without the need for other factors. Luciferase (Luciferase, Luc) is a generic term for enzymes that produce bioluminescence in nature, the most representative of which is the Luciferase in the firefly known as Photinus pyralis. In the corresponding chemical reaction, the fluorescence is generated by oxidation of luciferin, and in some cases Adenosine Triphosphate (ATP) is also included in the reaction system.
On the basis of nasopharyngeal carcinoma cell strains, a nasopharyngeal carcinoma cell model which can be stably expressed in vitro and in vivo for a long time, has high experiment repeatability and is labeled by fluorescence is established by adopting GFP and Luc double-label stable transfection, is used for tracing the formation and evolution process of nasopharyngeal carcinoma, and can be effectively used for carrying out the research on nasopharyngeal carcinoma metastasis.
Disclosure of Invention
The invention aims to provide a visual nasopharyngeal carcinoma cell and application thereof.
The technical scheme adopted by the invention is as follows:
a human nasopharyngeal carcinoma cell S18-1C3, which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: C2018189.
the human nasopharyngeal carcinoma cell S18-1C3 is applied as a nasopharyngeal carcinoma cell model.
The human nasopharyngeal carcinoma cell S18-1C3 is used as a cell model for researching nasopharyngeal carcinoma metastasis or/and proliferation.
The human nasopharyngeal carcinoma cell S18-1C3 is applied to screening or verifying nasopharyngeal carcinoma medicines.
The application of the human nasopharyngeal carcinoma cell S18-1C3 in researching nasopharyngeal carcinoma metastasis or/and proliferation mechanism.
Further, the mechanism of nasopharyngeal carcinoma metastasis or/and proliferation includes interaction between signal pathways and proteins related to nasopharyngeal carcinoma metastasis or/and proliferation.
The human nasopharyngeal carcinoma cells S18-1C3 are applied to research on action mechanisms of nasopharyngeal carcinoma medicines.
Furthermore, the action mechanism of the nasopharyngeal darcinoma medicine comprises a target point and an influenced signal path which are acted with the nasopharyngeal darcinoma medicine.
The human nasopharyngeal carcinoma cell S18-1C3 is applied to the visualization research of nasopharyngeal carcinoma.
Further, the nasopharyngeal carcinoma visualization research is to observe the change of the nasopharyngeal carcinoma through the reporter gene in S18-1C 3.
Applicants deposited cell line S18-1C3 at the China center for type culture Collection at the university of Wuhan, where the cell lines provided by applicants were received in 2018, 9/19. The preservation number given to the culture by the preservation center is CCTCC NO: c2018189, a proposed classification named human nasopharyngeal carcinoma cells S18-1C3, the deposited cells were identified as viable by 9/28 in 2018.
The invention has the beneficial effects that:
the cell line S18-1C3 has the capability of continuously expressing high fluorescence intensity, and the fluorescence intensity of the cell line is not lost along with the continuous passage of nasopharyngeal carcinoma cells; the integration and expression of the fluorescent gene do not affect the main biological characters of nasopharyngeal carcinoma cells; the S18-1C3 also has high cell proliferation capacity and cell migration and invasion capacity and strong reporter gene expression capacity, is an ideal cell model for fluorescence tracing nasopharyngeal carcinoma metastasis or proliferation, and is an ideal cell model for visual research of nasopharyngeal carcinoma. The S18-1C3 can be used for screening or verifying nasopharyngeal carcinoma medicaments, can be used for researching the mechanism of nasopharyngeal carcinoma metastasis or proliferation, and can also be used for researching the action mechanism of the nasopharyngeal carcinoma medicaments.
Drawings
FIG. 1 schematic diagram of the structure of the lentiviral packaging plasmid pGreenfire;
FIG. 2 Structure of lentiviral packaging plasmid pLP 1;
FIG. 3 schematic representation of the structure of the lentiviral packaging plasmid pLP 2;
FIG. 4 is a schematic diagram of the structure of the lentiviral packaging plasmid pCMV-VSV-G;
FIG. 5S 18-1C3 by fluorescent screening;
FIG. 6 is a growth curve for each cell line; in the figure, S18 represents the original human nasopharyngeal carcinoma cell S18, 1C3 represents the cell line S18-1C3, and 1F7 represents the cell line S18-1F 7;
FIG. 7 shows the results of the scratch test on each cell line, wherein S18 represents original human nasopharyngeal carcinoma cell S18, 1C3 represents cell line S18-1C3, 1F7 represents cell line S18-1F7, and Distance of migration represents migration Distance of each cell line; (ii) a
FIG. 8Transwell-Migration experiment; in the figure, S18 represents the original human nasopharyngeal carcinoma cell S18, 1C3 represents the cell line S18-1C3, and 1F7 represents the cell line S18-1F 7;
FIG. 9 shows the expression of Luciferase in each cell line;
FIG. 10 is a visual representation of S18-1C3 cells in a mouse plantar lympholysis transfer model;
FIG. 11 is a visual representation of S18-1C3 cells in a mouse spleen liver metastasis model.
Detailed Description
The present invention will be further described with reference to the following examples.
The invention is further illustrated below with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental methods and reagents not specifying the specific conditions in the examples were generally carried out under the conventional conditions, for example, in Sambrook. J; huang Petang et al, molecular cloning, the third edition (Beijing: science Press 2002) described in the conditions or manufacturer suggested conditions or configuration.
EXAMPLE 1 acquisition of human nasopharyngeal carcinoma cells S18-1C3
One, packaging of pseudolentivirus particles
(1) Lentivirus packaging plasmid (lentivirus lvector): a lentiviral vector System (SBI) was used, consisting of plasmids pGreenfire (FIG. 1), pLP1 (FIG. 2), pLP2 (FIG. 3) and pCMV-VSV-G (FIG. 4). Wherein the plasmid pGreenfire (vector component) contains a GFP gene, a Luciferase gene and a Puromycin (Puromycin) resistance gene and is driven by a CMV promoter; pLP1 contains the pol gene of the HIV virus, encoding a virus-specific enzyme; the gag gene, encodes the major viral structural protein; pLP2 contains the rev gene of the HIV virus, encoding a regulatory factor that regulates the expression of the gag and pol genes. pCMV-VSV-G contains a VSV-G gene derived from herpes simplex and provides an envelope protein required for virus packaging.
(2) Amplification of plasmid DNA: the plasmids pGreenfire, pLP1, pLP2 and pCMV-VSV-G were transformed into E.coli Stbl3 by the conventional method, positive clones were selected with the corresponding antibiotics, plasmid DNA extraction was performed with a plasmid extraction kit (Magen Co.), the obtained plasmid was dissolved in sterilized TE solution, the concentration and purity thereof were measured by ultraviolet absorption to ensure the A extraction of plasmid DNA260/A280Between 1.8 and 2.0.
(3) Packaging and purifying pseudolentivirus particles: 24 hours before transfection, human embryonic kidney cells 293T cells (purchased from the Shanghai cell Bank of the Chinese academy) in the logarithmic growth phase were digested with trypsin (Invitrogen), and the cell density was adjusted to 2.5X 10 by using a DMEM complete medium (Invitrogen) containing 10% FBS (Gibco)4Cells/ml, seeded in 6-well plates at 37 ℃ with 5% CO2Culturing in an incubator. The cell density can reach 70-80% to be used for transfection. 2h before transfection, the original medium was aspirated and 2ml of fresh complete medium was added. Then, 3. mu.g of pGreenfire vector, 0.3ug of pLP1 vector, 0.3. mu.g of pLP2 vector, 0.6ug of pCMV-VSV-G and 200. mu.l of DMEM (Inv)itrogen) were mixed well. Taking 4uLTransInTMEL (all type gold) was added to the plasmid mixed solution and mixed, and incubated at room temperature for 20 minutes. Transferring the above DNA and mixed solution to the prepared 293T cell culture solution, mixing, and culturing at 37 deg.C in 5% CO2After culturing in a cell culture incubator for 48 hours, the cell supernatant was collected, centrifuged at 400g and filtered through a sterile filter of 0.45 μm to obtain pseudolentiviral particles. The pseudolentiviral vector is an HIV-1 vector system, the antibiotic is puromycin, and the reporter genes are GFP and Luciferase.
Second, screening of human nasopharyngeal carcinoma cells S18-1C3
(1) Viral infection of S18 cells: pancreatin digestion of human nasopharyngeal carcinoma S18 cells in logarithmic growth phase to obtain cell suspension (cell density of 5 × 10)4/mL) were seeded in 6-well plates and cultured until the degree of cell confluence reached about 30%. Adding appropriate amount of the pseudolentivirus particles and Polybrene (Polybrene), culturing for 24 hr, changing culture medium, infecting for 72 hr, expanding cells, adding Puromycin (Puromycin) 2 μ g/mL to the expanded cells, and screening, and observing fluorescence expression of the screened cells.
(2) Fluorescent expressed S18 cells were digested with pancreatin and single-clone screened. The digested cells were diluted to 100/10 ml, mixed well and inoculated to 100. mu.l per well in 96-well plates. And continuously culturing for about two weeks, and selecting cell clones with fluorescence expression when the cells in the plate holes grow to cell clones, wherein the percentage of the cell clones with strong fluorescence expression in the total cell clones is 2%.
(3) Amplification and expression stability identification of fluorescence expression cells: the cell clone with the fluorescent expression in the previous step is subjected to multiple monoclonal screening, and two S18 cells with strong fluorescent expression are screened from 960 cell clones of 10 96-well plates; are respectively named as S18-1C3 and S18-1F 7.
The result of continuous passage of S18-1C3 for over 200 days in vitro shows that fluorescence intensity and expression rate of nasopharyngeal carcinoma cells are not obviously changed, and a cell line S18-1C3 (figure 5) for stably expressing green fluorescence is obtained, so that the nasopharyngeal carcinoma cells S18-1C3 of the inventor have the characteristics of high chromosome integration degree, high fluorescence intensity, stable genetic character and the like, and the nasopharyngeal carcinoma cells S18-1C3 of the inventor have the capability of continuously expressing high fluorescence intensity, and the fluorescence intensity of the nasopharyngeal carcinoma cells cannot be lost along with the continuous passage of the nasopharyngeal carcinoma cells. Similarly, the invention also obtains a cell line S18-1F7 stably expressing green fluorescence.
Example 2 Performance testing of human nasopharyngeal carcinoma cells S18-1C3
First, biological function detection
(1) General biological trait detection
General biological property observation is carried out on the cell lines S18-1C3 and S18-1F7 obtained in the above way, and the two cell lines are general biological properties of nasopharyngeal carcinoma cells, are epithelial-like tumor cells and grow in an adherent manner. The integration and expression of the exogenous reporter gene are proved not to affect the general biological properties of the human nasopharyngeal carcinoma cells S18-1C3 and S18-1F7, S18-1C3 and S18-1F 7.
(2) Detection of cell proliferation Capacity
The method comprises the following steps: s18, S18-1C3 and S18-1F7 in logarithmic growth phase at 3X 104Adding 100 mul/well of 96-well plate, setting 4 multiple wells for each cell, taking out and replacing fresh basal medium at 6h, 24h, 48h, 72h, 96h and 120h, respectively, adding CCK-8 at the amount of 10 mul/well, incubating for 30min, detecting light absorption at 450nm, and setting blank control wells.
As a result: the growth profile of each cell line is shown in FIG. 6, where FIG. 6-A is the profile and FIG. 6-B is the corresponding bar graph, from which it can be seen that the proliferation potency of the S18-1C3 cell line is significantly higher than that of the S18-1F7 cell line and its parent cell line, S18.
(3) Measurement of cell migration Capacity
1. Scratch test
The method comprises the following steps: adding 5X 10 of the solution into each hole of a 6-hole plate5Each cell was cultured in 2 duplicate wells for about 24 hours. Making cell streak with 200 μ l tip attached to ruler, washing cells with sterile 1 × PBS for 3 times, removing streaked cells, adding culture medium, and adding 5% CO at 37 deg.C2An incubator. And sampling for 0, 8 and 24h to take pictures.
As a result: the migration ability test results of the cell lines are shown in fig. 7, wherein fig. 7-a is the migration situation of the cell lines under the microscope, fig. 7-B is the histogram of the migration distances of the cell lines at the corresponding time points, and it can be seen from the results that after 24 hours, the migration distance of the cell line S18-1C3 of the present invention can reach 18 μm, while the migration distance of the cell line S18-1F7 and the original mother cell line S18 can only reach 13 μm, and the migration ability of the cell line S18-1C3 of the present invention is significantly enhanced compared with that of the mother cell S18 and S18-1F7 (fig. 7), and the cell line is more suitable for corresponding researches as a nasopharyngeal carcinoma model, such as researches on metastasis or/and proliferation of nasopharyngeal carcinoma, researches on screening or verifying nasopharyngeal carcinoma drugs, and researches on the mechanism of action of the nasopharyngeal carcinoma drugs.
2. Cell Migration invasion assay (Transwell-Migration assay)
The method comprises the following steps: after digesting the cells, adjusting the density value to 1 × 105 cells/ml, placing the cells into a 24-well plate special for Transwell, taking 200 μ l of cell suspension, uniformly and slowly dripping the cell suspension into the Transwell chamber in all directions, adding 3 multiple wells in each group, immediately and slowly adding 800 μ l of a culture medium containing 1% FBS into the lower chamber of the 24-well plate, culturing the cells for 24h, taking out the Transwell chamber, discarding the culture solution in the wells, slightly wiping the cells in the upper chamber (only by Z-shaped or circular wiping for 1-2 times) by using a cotton swab dipped with 1 × PBS, and placing the cells into a clean 24-well plate. Washed 1 time with 1ml DPBS, 800-. 0.1% crystal violet at 500 ul/well was added to the lower chamber and stained for 20min, the upper non-migrated cells were gently wiped off with a cotton swab and washed 1 time with 1ml PBS. Cell counts were observed at random in five fields under a 100-fold microscope.
As a result: the results of the measurement of the migration invasion ability of each cell line are shown in FIG. 8, wherein FIG. 8-A is the migration invasion ability of each cell line under the microscope, FIG. 8-B is the bar chart of the number of cells migrated and invaded in the same-sized field of each cell line, and it can be seen that the average number of S18-1C3 cells per field is more than 400, the average number of S18 mother strain per field is less than 100, and the average number of S18-1F7 cells per field is less than 150. The results further demonstrate that the migration invasion ability of the cell line S18-1C3 is significantly enhanced compared with that of the parent cells S18 and S18-1F7 (FIG. 8), and the cell line is an ideal model for nasopharyngeal carcinoma research.
Secondly, detecting the expression condition of Luciferase (Luciferase) in vitro
The method comprises the following steps: the expression conditions of the reporter genes Luciferase in cell lines S18, S18-1C3 and S18-1F7 are respectively detected in vitro by a Luciferase kit (ABM), and the cell number of each cell line is respectively set to be 0, 1250, 2500, 5000 and 1 × 104、2*104、3*104、4*104、5*104。
As a result: as shown in FIG. 9, there is no expression of the reporter gene Luciferase in the original mother cell line S18, while the expression of Luciferase in the cell lines S18-1C3 and S18-1F7 is obvious, and the expression amount is proportional to the number of cells. The cell line S18-1C3 of the invention is proved to be capable of well expressing the target reporter gene Luciferase.
Third, the visual application of human nasopharyngeal carcinoma cells S18-1C3 in mice
(1) Living body imaging observation of lymph node metastasis of human nasopharyngeal carcinoma cells S18-1C3 in mice
The method comprises the following steps: collecting well-grown cells S18-1C3, digesting, counting, diluting with 1 × PBS to 5 × 106pieces/mL, placed on ice. Cell inoculation: mouse foot pads were sterilized with 75% alcohol. 20 μ L of cells were suspended, i.e., 1X 105Individual cells were injected into the left posterior sole of nude mice. After 49 days of tumor cell inoculation, mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital, followed by intraperitoneal injection of 15mg/ml D-fluorescein (potassium salt) at 5 ul/g. Luciferase signals were detected after injection into the body for 10-20min (until the light signal reached the strongest plateau).
As a result: the detection result is shown in figure 10, S18-1C3 cell lines expressing luciferase can bring good visual imaging in 16 mouse plantar lymph node metastasis models, S18-1C3 cells can proliferate and metastasize quickly in the mouse plantar lymph node metastasis models, and meanwhile, high-level expression genes can be stably expressed, so that the living body imaging effect is good, compared with the method using mother plant cells S18, the method can be used for visually observing the occurrence and development of mouse tumors, and can be used for judging whether the tumors are planted successfully or not and observing the occurrence and development process of the tumors under the condition that the mice are not killed.
(2) Living body imaging observation of spleen and liver metastasis condition of human nasopharyngeal carcinoma cells S18-1C3 in mice
The method comprises the following steps: wiping the skin of the operative field of the mouse with 75 percent alcohol for disinfection; the epidermis and peritoneum were cut at the lower part of the left rib with a surgical scissors, a wound of about 1.0cm was opened, the spleen was exposed to the abdomen, the spleen was gently pulled out of the abdominal cavity with a tissue forceps, the cancer cells were injected into the spleen of mice slowly by about 1.5cm along the spleen with a 1ml syringe, and 0.1ml (5X 10) of the cell suspension was injected into each mouse6One), injection time was about 3 minutes, spleen capsule was seen to swell, whiten, spleen was gently returned to place after injection and wound was closed. Mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital 29 days after tumor cell inoculation, followed by intraperitoneal injection of 15mg/ml D-fluorescein (potassium salt) 5 ul/g. Luciferase signals were detected after injection into the body for 10-20min (until the light signal reached the strongest plateau).
As a result: as shown in FIG. 11, the detection result shows that S18-1C3 cells expressing luciferase also have good visual imaging in a mouse spleen liver metastasis model (FIG. 11), S18-1C3 cells can be proliferated and transferred quickly in the mouse spleen liver metastasis model, and simultaneously can stably express marker genes at high levels, so that the living imaging effect is good, and the spleen liver metastasis condition of nasopharyngeal carcinoma cells can be observed visually through living imaging.
In conclusion, the nasopharyngeal carcinoma cells S18-1C3 of the inventor have the characteristics of high chromosome integration, high fluorescence intensity, stable genetic character and the like, and the cell line S18-1C3 of the invention has the capability of continuously expressing high fluorescence intensity, so that the fluorescence intensity of the cell line is not lost along with continuous passage of the nasopharyngeal carcinoma cells; the integration and expression of the fluorescent gene do not affect the main biological characters of nasopharyngeal carcinoma cells, and the fluorescent tracer nasopharyngeal carcinoma cell model is an ideal fluorescent tracer nasopharyngeal carcinoma cell model. The screening of the invention refers to screening of a plurality of parameters of the expression intensity, cell morphology, growth, differentiation, migration, apoptosis and the like of the reporter gene on the premise of the expression of the marker gene; particularly, the method is used for screening cell migration parameters to finally obtain an ideal cell model S18-1C3 for nasopharyngeal carcinoma research, namely S18-1C3 has good cell proliferation capacity, cell migration invasion capacity and strong reporter gene expression capacity.
The S18-1C3 also has high cell proliferation capacity and cell migration and invasion capacity and strong reporter gene expression capacity, is an ideal cell model for fluorescence tracing nasopharyngeal carcinoma metastasis or proliferation, and is an ideal cell model for visual research of nasopharyngeal carcinoma. The S18-1C3 can be used for screening or verifying nasopharyngeal carcinoma medicaments, can be used for researching the mechanism of nasopharyngeal carcinoma metastasis or proliferation, and can also be used for researching the action mechanism of the nasopharyngeal carcinoma medicaments.
Applicants deposited cell line S18-1C3 at the China center for type culture Collection at the university of Wuhan, where the cell lines provided by applicants were received in 2018, 9/19. The preservation number given to the culture by the preservation center is CCTCC NO: c2018189, a proposed classification named human nasopharyngeal carcinoma cells S18-1C3, the deposited cells were identified as viable by 9/28 in 2018.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A human nasopharyngeal carcinoma cell S18-1C3, which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: C2018189.
2. use of the human nasopharyngeal carcinoma cell of claim 1, S18-1C3 as a nasopharyngeal carcinoma cell model.
3. Use of the human nasopharyngeal carcinoma cells S18-1C3 of claim 1 as a cell model for studying metastasis or/and proliferation of nasopharyngeal carcinoma.
4. The use of the human nasopharyngeal carcinoma cell S18-1C3 of claim 1 in screening or verifying drugs for nasopharyngeal carcinoma.
5. Use of the human nasopharyngeal carcinoma cell S18-1C3 of claim 1 in studying the metastasis or/and proliferation mechanism of nasopharyngeal carcinoma.
6. The use of claim 5, wherein the mechanism of nasopharyngeal carcinoma metastasis or/and proliferation comprises signal pathway, protein interaction associated with nasopharyngeal carcinoma metastasis or/and proliferation.
7. Use of the human nasopharyngeal carcinoma cells S18-1C3 of claim 1 in studying the mechanism of action of nasopharyngeal carcinoma drugs.
8. The use of claim 7, wherein the mechanism of action of the nasopharyngeal carcinoma drug comprises interaction of nasopharyngeal carcinoma cells S18-1C3 with a nasopharyngeal carcinoma drug, and signaling pathway affected by the nasopharyngeal carcinoma drug.
9. The use of the human nasopharyngeal carcinoma cell S18-1C3 of claim 1 in the visualization study of nasopharyngeal carcinoma.
10. The use of claim 9, wherein the nasopharyngeal carcinoma visualization is performed by observing the nasopharyngeal carcinoma changes with the reporter gene of S18-1C 3.
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