CN107828729B - Human lung cancer A549 derived cell strain and preparation method and application thereof - Google Patents
Human lung cancer A549 derived cell strain and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a human lung cancer A549 derived cell strain, which is named as a human non-small cell lung cancer cell line A549-SE06, and the preservation number of the cell strain is CGMCC No. 14315. In addition, the invention also discloses a preparation method and application of the cell strain. The invention can be used for analyzing the morphological and biological phenotype change after the lung cancer cell mutation; researching the molecular mechanism and molecular expression change affecting the tumor cell proliferation invasion process; studying the association between the relevant molecular proteins and the clinical manifestations of the patient and their impact on the prognosis of the patient; the application of the protein molecule in researching lung cancer tumor cell proliferation molecular mechanism and the influence of specific protein molecules on clinical prognosis; the application in preparing lung cancer tumor animal models; the application in preparing lung cancer diagnostic reagent; and the application in developing lung cancer tumor drug targets. The experiment proves that the lung cancer A549 derivative cell strain A549-SE06 of the inventor has higher proliferation rate and stronger proliferation capacity than the A549 cell strain, the growth speed is accelerated, and the tumor forming time of a nude mouse is shortened.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a lung cancer cell strain, and particularly relates to a human lung cancer A549 derived cell strain.
Background
Lung cancer is a common malignancy, and in the annual new-born cancer population, lung cancer accounts for approximately 12.7%, men and women account for 2.1:1, and approximately 75% of patients are diagnosed at age 60 or older. Lung cancer patients generally have a poor prognosis, with a five-year survival rate of between 6-18%. To further study the mechanism of development and progression of lung cancer, the applicant purchased the a549 cell line from the cell bank of the chinese academy of sciences and performed animal experiments in nude mice. The A549 cell line is established by D.J.Griad through lung cancer tissue transplantation culture, and a 58-year-old white man male is mainly used for lung cancer related research, such as research on the killing effect of a medicament on lung cancer cells or tumor formation of a nude mouse. The initial tumor formation rate of the experiment is stable, after multiple passages, the tumor formation capacity is gradually weakened until no tumor formation is realized, 5 nude mice are inoculated under the axilla in 2016 and 5 months for tumor formation experiment, no tumor formation is realized in 2 months, the mice are not treated, four tumors are found in the 5 nude mice when the mice are prepared and treated in the fourth month, the tumor growth is rapidly observed and observed continuously, the mice are killed, the tumors are ground and cultured, when the cell growth state is good, the tumor cell strains are selected by single cloning, the separated cells and the original A549 cells are subjected to contrast experiment and cell identification, the cells are found to be A549 cell mutation lines, western blotting detection shows that part of proteins of the mutation cell lines are abnormally expressed, and animal experiments on the mutation cell lines and the original A549 cells show that the tumor growth of the mutation cell lines is rapid, the tumor formation rate of the original A549 cells is extremely low, and the growth is nonuniform. The A549 mutant can be used for replacing the original A549 cell for preclinical research of lung cancer.
Disclosure of Invention
The invention aims to solve the technical problem of providing a human lung cancer A549 derived cell strain.
The second technical problem to be solved by the invention is to provide a preparation method of a human lung cancer A549 derived cell strain.
The invention solves the technical problem of providing the application of the human lung cancer A549 derived cell strain.
In order to solve the technical problems, the invention adopts the following technical scheme:
in one aspect of the invention, a human lung cancer A549-derived cell strain is provided, which is named as a human non-small cell lung cancer cell line A549-SE06 with the collection number of CGMCC number 14315. The human lung cancer A549 derived cell strain has been preserved in China general microbiological culture Collection center (preservation number is CGMCC No. 14315. preservation place: China, Beijing) in 2017, 8 months and 18 days.
The human non-small cell lung cancer cell line A549-SE0The biological characteristics of 6 are: the cells are epithelioid, polygonal and adherent, round, full and rapid, and the percentage of cells in each phase of the cell cycle is G0-G1Stage 35.45%, S stage 47.25%, G2-M phase 17.30%, showing tumor cell proliferation properties.
In a second aspect of the present invention, there is provided a method for preparing a human lung cancer a 549-derived cell strain, comprising the steps of: step one, a human lung cancer A549 cell line is inoculated under the skin of a nude mouse and becomes tumor after several months; step two, taking the formed tumor, shearing and digesting the tumor, and adding the tumor into a 1640 culture medium for culture; and step three, obtaining a single cell, namely the human lung cancer A549 derived cell strain, after multiple times of adherent purification and monoclonal selection.
As a preferred technical scheme of the invention, the preparation method of the human lung cancer A549 derived cell strain specifically comprises the following steps:
(1) the human lung cancer A549 cell line is inoculated under the skin of a nude mouse and becomes tumor after several months;
(2) taking subcutaneous tumor masses of A549 nude mice, shearing and digesting, centrifuging to remove trypsin, transferring to a cell culture bottle, and adding a 1640 culture medium;
(3) culturing for a period of time, removing the tissue in the bottle after a large number of cells adhere to the wall, and adding 1640 culture solution;
(4) when the cells grow to about 70-80%, digesting the cells by using trypsin digestion solution, centrifuging and then resuspending by using a serum-free culture medium; adding the cell suspension into a culture bottle, standing for 15-20min, observing under an inverted microscope, introducing the cell suspension into another culture bottle when part of cells adhere to the wall and do not float after slight shaking;
(5) repeating the process in the step (4) for multiple times, gradually separating fibroblasts from other cells, and selecting single clones from the cells obtained by adherence of the last batch of suspension to obtain the separated and purified human lung cancer A549 derived cell strain.
In a preferred embodiment of the present invention, the culture conditions in steps (2), (3), (4) and (5) are: 37 ℃ and 5% CO2And culturing under 95% humidity.
In a third aspect of the invention, the invention provides the use of the human lung cancer A549 derived cell strain, including the application in researching lung cancer tumor cell proliferation molecular mechanism and the influence of specific protein molecules on clinical prognosis; the application in preparing lung cancer tumor animal models; the application in preparing lung cancer diagnostic reagent; and the application in developing lung cancer tumor drug targets. The invention can be used for analyzing the morphological and biological phenotype change after the lung cancer cell mutation; researching the molecular mechanism and molecular expression change affecting the tumor cell proliferation invasion process; the correlation between the relevant molecular proteins and the clinical manifestations of the patients and their impact on the prognosis of the patients were studied.
Morphological observation and biological characteristic identification are carried out on the A549 derived cell strain established by the method, and the result shows that the cell morphology of the A549 derived cell strain has slight change, the growth speed is accelerated, and the tumor formation time of a nude mouse is shortened. Experiments prove that the lung cancer A549 derivative cell strain A549-SE06 has faster proliferation rate and stronger proliferation capacity than the A549 cell strain.
Drawings
FIG. 1 is an observation view (X100) of A549 cells under an inverted microscope in example 2 of the present invention;
FIG. 2 is an observation (. times.100) of A549-SE06 cells under an inverted microscope in example 2 of the present invention;
FIG. 3 is a graph showing the proliferation profiles of A549 cells and A549-SE06 cells in example 2 of the present invention;
FIG. 4 is a graph showing cycle results of A549 cells in example 2 of the present invention;
FIG. 5 is a graph showing cycle results of A549-SE06 cells in example 2 of the present invention;
FIG. 6 is a graph showing the results of animal nodulation of A549 cells and A549-SE06 cells in example 2 of the present invention.
The human non-small cell lung cancer cell line with the code number of A549-SE06 has been preserved in the China general microbiological culture Collection center (CGMCC No. 14315; preservation site: No. 3 of the national institute of sciences, Ministry of China, West Lu No.1 of the Chaoyang district, Beijing, China) in 8.8.18.2017.
Detailed Description
The following examples are presented to enable those skilled in the art to more fully understand the present invention and are not intended to limit the invention in any way.
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified.
Example 1
The invention adopts a purification method of multiple adherence and single clone selection to establish a human lung cancer A549 derived cell strain. The method comprises the following specific steps:
(1) human lung carcinoma A549 cell line (purchased from Shanghai cell bank of Chinese academy of sciences, cryopreserved by the laboratory), recovered and treated with DMEM culture solution (containing 10% fetal calf serum, penicillin 100U/ml and streptomycin 100 μ g/ml) at 37 deg.C and 5% CO2Culturing under 95% humidity;
(2) digesting A549 cells in logarithmic growth phase with trypsin digestion solution, counting, and adjusting density to 5 × 10 per nude mouse6Mu.l, mixed in physiological saline, and inoculated subcutaneously into nude mice. The nude mice do not grow tumor mass within the set time of the experiment, and after the nude mice are continuously raised for several months, the nude mice suddenly grow tumor. Shearing and digesting tumor, adding 1640 culture medium (containing 10% fetal calf serum, penicillin 100U/ml and streptomycin 100 μ g/ml) at 37 deg.C and 5% CO2Culturing under 95% humidity;
(3) culturing for a period of time, removing the tissue in the bottle after observing that a large number of cells adhere to the wall, and adding a new 1640 culture solution;
(4) when the cells grew to around 80%, the cells were digested with trypsin digest, centrifuged and resuspended in 1640 medium without serum (Gibco, 61870036). Adding the cell suspension into a culture bottle, standing for 20min, observing under an inverted microscope, introducing the cell suspension into another culture bottle when partial cells adhere to the wall and do not float even if slightly shaking.
(5) Repeating the process in the step (4) for multiple times, gradually separating fibroblasts from other cells, and selecting single clones from the cells obtained by adherence of the last batch of suspension to obtain the separated and purified human lung cancer A549 derived cell strain.
When the cells grow stably, freezing and storing partial cells in liquid nitrogen, wherein the freezing and storing liquid is prepared according to FBS: 1640 medium: the DMSO ratio is 5:4:1, the freezing and storing process is gradually cooled, and the freezing and storing process is carried out by adopting the sequence of 4 ℃ for 30 minutes → 20 ℃ for 1 hour → -80 ℃ overnight → liquid nitrogen. The recovery of the cryopreserved cells was performed according to the following procedure: adding at least 10ml of culture solution containing 10% fetal calf serum into a 10cm cell culture dish; taking out the cryopreservation tube filled with the cells from the liquid nitrogen, immediately placing the tube into a water bath at 37 ℃, gently shaking the tube to thaw the frozen stock within 1 minute, wiping the outer wall of the cryopreservation tube with an alcohol cotton ball, and taking the tube into a super clean bench; the thawed cell suspension was centrifuged, the frozen stock solution was removed, and the cell suspension was resuspended in 1640 culture medium. Transferring the cell suspension into culture dish containing culture solution, shaking slightly, mixing, placing in carbon dioxide incubator at 37 deg.C and 5% CO2Culturing under 95% humidity condition, and continuously culturing after about 24 hours of cell adherence by changing 5-10ml of fresh culture solution.
Example 2 morphological observation and biological characterization of the human lung cancer a549 derived cell strain established by the method of the present invention:
1. microscopic observation of cell morphology
A549 cells and A549 derived cells in logarithmic growth phase are taken in bottles respectively, and after liquid change, the living cell morphology is observed under an inverted phase contrast microscope and photographed. A549 cells are shown in FIG. 1, and A549-SE 06-derived cells are shown in FIG. 2. As shown in FIG. 2, the cells of the non-small cell lung cancer cell line A549-SE06 of the present invention are epithelioid, polygonal and adherently growing, round and plump and grow rapidly.
2. CCK8 colorimetric method for measuring cell proliferation rate
Respectively taking A549 cells and A549 derived cells in logarithmic growth phase, digesting, counting and adjusting the cell density to be 3 multiplied by 104one/mL, inoculated in 96-well culture plates, 100. mu.l/well. Standing at 37 deg.C and CO2Culturing in a constant temperature incubator with the volume fraction of 5 percent. 0h, 24h, 48h and 72h into the hole respectivelyAdd 10. mu.l of CCK8 detection reagent, incubate for 3 hours in incubator, determine absorbance values (A450 and A620) with full-wavelength microplate reader, repeat the experiment 3 times, take the average. The cell proliferation curves of the two cells are obtained by plotting the time as the abscissa and the absorbance value (A450-A620) as the ordinate, and the result shows that the A549-SE06 derived cell strain has a faster proliferation rate than the A549 cell strain after 24h of culture, and the CCK8 proliferation curve is shown in figure 3.
3. Flow cytometer for detecting cell cycle
A549 cells and A549-SE06 cells in logarithmic growth phase were plated on a 6cm dish, and after 24 hours, the cells were digested with trypsin and centrifuged to the bottom of a 1.5ml EP tube. The cells were fixed by adding 1ml of 70% ethanol pre-cooled at-20 ℃ (PBS formulation), the fixed cells were left overnight at-20 ℃, the cells were removed the next day, centrifuged at 1000rpm x 5min to remove ethanol and rinsed once with PBS to remove residual ethanol. A little PBS remained in the tube, the cells were mixed evenly and then added into 300ul PI solution containing 10ug/ml RNase to resuspend the cells, and the flow detection was performed after the cells were protected from light for 30 min. Cell cycle as shown in fig. 4 and 5, the cell cycle distribution is shown in table 1: the results show that S-phase cells of the A549-SE-06 cell strain are obviously increased to 47.25 percent, while A549 cells are 45.85 percent, and G0-G1-phase cells of the A549-SE-06 cells are obviously reduced, which indicates that the A549-SE-06 cells have stronger proliferation capacity.
TABLE 1 cell cycle distribution tables of A549 and A549-SE-06
4. Nude mice are inoculated subcutaneously to observe cell tumorigenesis capacity
A549 cells and A549-SE06 cells in logarithmic growth phase are taken, counted and adjusted to the density of 5 multiplied by 10 for each nude mouse 6200 mul, mixed in normal saline, and inoculated subcutaneously on the left and right sides of the same nude mouse. Comparing the tumorigenicity capacity of the two cells, as shown in fig. 6, the tumorigenicity of the A549-SE06 cell is larger than that of the A549 cell, and the result shows that the A549-SE06 cell of the invention accelerates the growth speed of the tumor and shortens the tumorigenicity time of a nude mouse.
Claims (5)
1. A human lung cancer A549 derived cell strain is named as a human non-small cell lung cancer cell line A549-SE06, and the preservation number is CGMCC number 14315.
2. The use of the human lung cancer A549-derived cell line of claim 1 in studying lung cancer tumor cell proliferation molecular mechanism and the influence of specific protein molecules on clinical prognosis.
3. The use of the human lung cancer A549-derived cell line of claim 1 in preparing a lung cancer tumor animal model.
4. The use of the human lung cancer A549-derived cell line of claim 1 in preparing a lung cancer diagnostic reagent.
5. The use of the human lung cancer A549-derived cell line of claim 1 in developing a lung cancer tumor drug target.
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| CN104694476A (en) * | 2013-12-05 | 2015-06-10 | 上海睿智化学研究有限公司 | Human non-small cell lung cancer cell line, and establishment method and application thereof |
| CN106065397A (en) * | 2010-06-07 | 2016-11-02 | 广州呼吸疾病研究所 | Human lung adenocarcinoma derived cell strain A1015 |
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| CN106065397A (en) * | 2010-06-07 | 2016-11-02 | 广州呼吸疾病研究所 | Human lung adenocarcinoma derived cell strain A1015 |
| WO2012040587A2 (en) * | 2010-09-23 | 2012-03-29 | President And Fellows Of Harvard College | Compositions and methods for altering matrix rigidity to regulate cancer cell growth and phenotype |
| CN104694476A (en) * | 2013-12-05 | 2015-06-10 | 上海睿智化学研究有限公司 | Human non-small cell lung cancer cell line, and establishment method and application thereof |
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