CN113969263A - Human tumor-associated fibroblast cell line and uses thereof - Google Patents
Human tumor-associated fibroblast cell line and uses thereof Download PDFInfo
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Abstract
The invention belongs to the field of tumor biology, and relates to human tumor-associated fibroblasts and application thereof. The human tumor-associated fibroblast cells of the present invention have the following biological properties: the cells are in a long fusiform or starburst shape, the cytoplasm is rich and acidophilic, the cell nucleus is positioned in the center of a cell body, the cell nucleus is circular or oblong, the cells are arranged in a radial or bundle shape, part of the cells are disorderly arranged, the polarity disappears, and the obvious cross overlapping phenomenon exists; stable passage within ten generations; the cell expression protein alpha-SMA, FAP, E-cadherin and Vimentin are positive. The invention has the advantages that: can be cultured in vitro for a long time and keep the cell characteristics unchanged, is a powerful tool for the relevant research of the tumor-related fibroblasts, and is a powerful tool for researching the tumor-related fibroblasts mediated tumorigenesis development and the relevant molecular action mechanism, developing a tumor treatment drug target, developing a new tumor biological treatment technology and detecting the research of relevant biological engineering products.
Description
Technical Field
The invention belongs to the field of tumor biology, and relates to a human tumor-associated fibroblast and application thereof.
Background
The tumor microenvironment provides a powerful support for tumors in the process of occurrence and development of the tumors, and the tumor-related fibroblasts are one of the most important components in the tumor microenvironment, are fusiform cells, can be differentiated from various types of cells under the action of external factors and the inside of the cells, and can be used as sources of human tumor-related fibroblasts, such as normal fibroblasts, adipocytes, pericytes and the like. The human tumor-related fibroblast can construct and remodel the structure of extracellular matrix, has a certain secretion function, and can secrete cytokines and inflammatory factors such as interleukin 6(IL-6), chemokine (CXCL12) and the like, so that the tumor tissue morphology is maintained, and the multidrug resistance, invasion and proliferation of tumor cells can be mediated. Because of its important role in mediating tumors, the attention on the treatment of tumor-associated fibroblasts is now increasing.
The establishment of the human tumor-related fibroblast cell line provides abundant experimental materials for solving the biological characteristics of the tumor-related fibroblast cells, researching the drug resistance, invasion and development matrixes of the tumor-related fibroblast cells, and the like, thereby providing help for establishing a standard treatment mode. Moreover, current studies indicate that the appearance of tumor-associated fibroblasts may precede the development of tumors. Since tumor-associated fibroblasts have some heterogeneity and have different functions even when the cell morphology is the same, it is difficult to directly study the cells at a tissue level. Therefore, enrichment of the human tumor-associated fibroblast cell bank plays an important role in order to conduct detailed research on a cellular level. Understanding how tumor-associated fibroblasts mediate tumor invasion and progression allows for the targeted inhibition of this process.
In view of the above, the present inventors propose to provide a human tumor-associated fibroblast cell line that can be expanded in large quantities and subcultured in vitro for a long period of time, and applications thereof.
Disclosure of Invention
The present invention aims to provide a human tumor-associated fibroblast cell line that can be expanded in large quantities and subcultured in vitro for a long period of time, and its use, based on the current state of the art. Can be used for preparing human tumor-related fibroblast models, biological therapeutic drugs and diagnostic reagents.
The technical scheme adopted by the invention for solving the technical problems is as follows:
separating and culturing human tumor-related fibroblasts from tumor tissues of hospitalized colon cancer patients, digesting the tumor tissues by collagenase according to a conventional method to obtain adherent tumor single cells, and adding a DMEM complete culture medium into the tumor single cells for subculture to obtain the human tumor-related fibroblasts; under an optical microscope, the tumor-associated fibroblasts are in a long fusiform or starburst shape, abundant cytoplasm and acidophilic property, nuclei are located in the center of a cell body, the cytoplasm is circular or oblong, the cells are arranged in a radial or bundle shape, part of the cells are disorderly arranged, the polarity disappears, and an obvious cross overlapping phenomenon exists.
More specifically, the invention provides a human tumor-associated fibroblast, which has at least one of the following biological properties:
the cells are in a long fusiform or starburst shape, the cytoplasm is rich and acidophilic, the cell nucleus is positioned in the center of a cell body, the cell nucleus is circular or oblong, the cells are arranged in a radial or bundle shape, part of the cells are disorderly arranged, the polarity disappears, and the obvious cross overlapping phenomenon exists;
stable passage within ten generations;
the cell expression protein alpha-SMA, FAP, E-cadherin and Vimentin are positive.
The human tumor-related fibroblast cell line is named as human tumor-related fibroblast CAF95 and is preserved in China center for type culture Collection of the university of Wuhan at 7-month and 14-month 2020 with the preservation number of CCTCCNO: C2020130.
In embodiments of the invention, the surface proteins α -SMA, FAP, E-cadherin, Vimentin are positively expressed in a human tumor-associated fibroblast cell line as described above.
The invention also provides the use of the human tumor-associated fibroblast, which includes but is not limited to the following uses:
the human tumor-associated fibroblast cell strain is used for preparing a human tumor-associated fibroblast cell model.
The human tumor-related fibroblast cell strain is used for preparing a tumor animal model by co-culturing a tumor-related fibroblast cell conditioned medium and tumor cells.
The human tumor-related fibroblast cell strain is used for screening and/or evaluating/preparing tumor treatment medicines by taking a tumor-related fibroblast cell conditioned medium and co-culturing with tumor cells.
The human tumor-related fibroblast cell strain is used for developing tumor-related fibroblast drug targets.
The application of the human tumor-associated fibroblast cell line in preparing a tumor diagnostic reagent.
The application of the human tumor-related fibroblast strain and tumor cell co-culture in screening tumor biotherapeutic drugs/reagents.
The application of the human tumor-associated fibroblast cell line in developing a new tumor treatment technology.
The human tumor-associated fibroblast strain is used for developing and detecting tumor-associated fibroblast bioengineering products.
The cell line of the invention has multiple uses: can be used for researching the relevant molecular action mechanism of the tumor-related fibroblast-mediated tumorigenesis development, researching, screening and evaluating the drugs for the tumor-related fibroblast targeted therapy, establishing a 3D culture model with the tumor cells, and providing necessary cell experimental tools closer to the growth environment of tumor tissues, new biological therapy technology, detection of relevant biological engineering products and the like.
The invention is further described with reference to the following drawings and detailed description, but the invention is not limited thereto, and equivalents in the art may be substituted in accordance with the present disclosure without departing from the scope of the invention.
Drawings
FIG. 1 shows the morphology of primary and secondary cells cultured in the form of elongated spindle or starburst cells, abundant cytoplasm and eosinophilia, with nuclei in the center of the cell body, round or oblong, and cells in radial or beam arrangement.
FIG. 2 shows that the expression of fibroblast-associated proteins is detected by Western blotting, and the expressions of alpha-SMA, FAP and Vimentin are all positive.
FIG. 3 is a scratch test that shows that tumor-associated fibroblasts promote the invasion function of tumor cells.
FIG. 4 is a transwell experiment demonstrating that tumor-associated fibroblasts promote tumor cell invasion function.
FIG. 5 is a tube forming experiment demonstrating that tumor-associated fibroblasts promote the tube forming function of HUVEC cells.
The CCK experiment in FIG. 6 shows that tumor-associated fibroblasts promote multidrug resistance in tumor cells.
Detailed description of the preferred embodiments
Example 1: protein expression condition of human tumor-associated fibroblasts detected by western blotting method
1) Taking cells in logarithmic phase, removing culture medium by suction, washing the cells with PBS for three times, adding a proper amount of protein lysate, and cracking the cells for 15 minutes on ice;
2) cells were scraped with a clean-swabbed cell scraper and then transferred to a 1.5ml ep tube;
3) placing the EP tube in a 4-degree centrifuge at 12000rpm, centrifuging for 10 minutes, and taking the supernatant and placing the supernatant in a 1.5ml EP tube;
4) and (3) carrying out protein quantification by using a BCA method, adding a5 xSDS-PAGE protein loading buffer solution according to the volume of a sample, fully and uniformly mixing the buffer solution, and boiling the mixture for 10-15min in a metal bath kettle at 95 ℃. Centrifuging at 1000rpm for 5min until the sample is cooled, and optionally storing in refrigerator at-20 deg.C;
5) preparing glue: the glass plate is vertically placed and aligned, placed on a sponge cushion of a frame to be clamped tightly, cut to prevent liquid leakage, separation glue is slowly added into the inner side by a suction pipe, and after the liquid level reaches a certain position, liquid sealing treatment is carried out on the separation glue by using isopropanol or double distilled water. Standing for a period of time, pouring out water or isopropanol when a separation line appears between isopropanol or water and glue, pouring out isopropanol, performing pouring-out cleaning for 2-3 times by using double distilled water, and completely absorbing water with paper. Adding concentrated glue, wherein the concentrated glue is solidified after about 20 minutes, wiping the periphery of the glass plate clean, carefully installing on an electrophoresis rack, filling the inner tank with electrophoresis liquid, checking leakage firstly, and adding the electrophoresis liquid into the outer tank if no leakage exists;
6) uniformly mixing the prepared samples, softly adding 10-20 mu l of samples into sample holes by using a gun head, and adding 4-5 mu l of marker;
7) opening an electrophoresis apparatus switch, setting the electrophoresis condition to be 80V, electrophoresing the sample to the position of the separation gel for about half an hour, enabling bromophenol blue to become a straight line, adjusting the electrophoresis condition to be 120V at the moment, and generally setting the electrophoresis time to be about 1 hour;
8) after the electrophoresis is finished, the membrane is rotated, the cut rubber block is placed, the PVDF membrane is placed on the PVDF membrane, the bubbles are driven out by a glass rod gently, and the membrane rotating condition is set to be 300mA for 80 min;
9) after the membrane is transferred, TBST is used for preparing 5% of skimmed milk, sealing is carried out, and the skimmed milk is shaken and sealed on a shaking table at room temperature for 1-2 hours generally;
10) overnight incubation with a solution containing the primary antibody, alpha-SMA (1:1000 dilution formulation) (product of sigma, cat. No. A5228); FAP (1:1000 dilution preparation) (product of Abcam, Cat. AB 53066); vimentin (1:1000 diluted preparation) (product of CST company, cat # 5741S), washed three times with TBST for ten minutes each time, and incubated with the corresponding secondary antibody for one hour;
11) and uniformly mixing the solution A and the solution B in the HRP fluorescence detection kit in a dark room according to the ratio of 1: 1. Placing the film on a clean flat plate, completely sucking the film and surrounding liquid by paper, dripping a proper amount of luminous liquid on the film, uniformly dripping, placing the film and a bottom vessel in an imager, carrying out exposure and picture shooting, and carrying out relevant adjustment on exposure time according to requirements to obtain an optimal picture;
12) the experimental result shows that the alpha-SMA, FAP and Vimentin expressions of the human tumor-related fibroblasts are all positive. Thus proving the identity of the tumor-associated fibroblast.
Example 2: tumor-associated fibroblasts promote tumor metastasis
Method and device
II, I, scratch experiment:
1) the cells were digested with pancreatin containing 0.25% EDTA, and the cell suspension was adjusted to 5X 10^5 cells/ml after resuspension of the cells in basal medium.
2) The counted cell suspension was spread evenly in 12-well plates in an amount of 1ml per well.
3) Cells were scraped using a sterile wound replicator after 24h attachment. After scraping, PBS was added to the wells and the plates were gently shaken to remove the scraped cells, and repeated 3 times.
4) The experimental group was added with 95CAF-CM, placed under a microscope for taking a photograph and sampling, and the sampling position was marked. And continuing to culture after the photographing is finished.
5) After 48h of incubator culture, the same sampling point was photographed again.
6) The scratch healing rate was calculated as follows: the healing rate (%) (scratch distance at 0 h-scratch distance at end time)/scratch distance at 0h x 100%.
II transwell invasion test
1) The cells were digested with pancreatin containing 0.25% EDTA, and the cell suspension was adjusted to a range of 1-4X 105 cells/ml after resuspension of the cells in the basal medium.
2) The counted cell suspension was spread evenly in 12-well plates in an amount of 1ml per well.
3) The matrigel was diluted to 1mg/ml, 100. mu.l of each supernatant chamber was added, and the mixture was placed in an incubator at 37 ℃ for 1 hour to coagulate it.
4) 500. mu.l of 95CAF-CM containing 10% fetal bovine serum was added to the lower chamber of the Transwell plate and 100. mu.l of the cell suspension was added to the upper chamber. The number of cells in the upper chamber was finally adjusted to 1X 104 cells/well.
5) After normal culture for 48h, the cells that did not pass through the membrane in the upper chamber were gently wiped off with a cotton swab, and wiped clean as much as possible.
6) The chamber was placed in the lower chamber with 500. mu.l of 0.1% crystal violet solution and placed in an incubator for 30 minutes.
7) After the membrane-covered cells were completely stained with crystal violet, the cells were rinsed slowly 3 times with PBS and then the chamber was air-dried.
8) The chamber was placed under a high power microscope, 5 high power fields were randomly selected for photographing, the number of the plated cells was counted, the number of migration of each group of cells was represented by the obtained average, and each experiment was repeated three times.
Three, result in
The conditioned medium of 95-CAF obviously promotes the invasion capacity of tumor cells.
Example 3: HUVEC tubulation promoted by tumor-associated fibroblasts
Method and device
1. Taking 95-CAF in logarithmic phase, adding 1% double antibody into DMEM medium, culturing for 48h, filtering the obtained medium, and adding 10% serum;
2. HUVEC cells were stimulated with 95-CAF conditioned medium for half an hour;
3. spreading matrigel with 96-well plate, spreading matrigel at low temperature, rapidly, uniformly, fully on ground, uniformly thin, precooling the gun head, 50 μ l per hole, spreading, and polymerizing in 37-degree incubator for 30 min;
4. after the stimulation is finished, removing CM and pbs, digesting, counting, controlling the number of cells in each hole to be the same, and planting the cells in the matrigel of the laid 96-hole plate;
5. the plated cells were cultured in a 37 ℃ incubator and observed under a microscope for 4h and 6 h.
Second, result in
HUVEC in 95-CAF conditioned medium in 96-well plates started to form tubes after 4h and peaked after 6h, compared to the control group. The tube forming capability is obviously improved.
Experimental example 4: tumor-associated fibroblasts promote tumor cell drug resistance
Method and device
1. Inoculating 3-6 th generation colon CAFs at a concentration of 1.5 x10 ^ 5/ml to 75cm2In the culture bottle, the cells need to be normally changed, after the cell density reaches 70%, Phosphate Buffered Saline (PBS) is used for washing twice and serum-free culture medium is changed for culturing for 48 hours, supernatant is collected after 48 hours, cell debris is removed by centrifugation at 1000r/5min, and CAFsCM is obtained and is subpackaged at 4 ℃ for storage;
2. grouping experiments: divided into a control group and an experimental group. Control group, human colon cancer cells HCT116, HT29 cultured in conventional complete medium. Experimental groups, which are renamed HCT116-CAFs-CM and HT29-CAFs-CM after CAFsCM stimulates HCT116 and HT2948 hours;
3. cell counting and plating: irradiating the ultraclean bench for 30 minutes by conventional ultraviolet rays, wiping experiment consumables required by the experiment by alcohol before the experiment, sucking old culture solution in a culture bottle by a disposable pipette, washing for 2 times by PBS, adding 0.25% pancreatin (containing 0.02% EDTA) to cover the bottom of the bottle, placing the bottle in a constant-temperature incubator for digestion until cells fall off, stopping digestion immediately, sucking a proper amount of culture solution by the disposable pipette to wash the cells stained at the bottom of the culture bottle, and preparing cell suspension. Using a disposable cell counting plate to suck 10ul of cell suspension to be detected; the cell counting plate was inserted into an electronic cell counter and read automatically. Inoculating colon cancer cells of a control group and an experimental group on a 96-hole cell culture plate at the concentration of 1.5 multiplied by 10^ 5/ml, and adding medicine after the cells adhere to the wall;
4. removing the previously prepared OXA from the temperature of 4 ℃ and the concentration of the mother liquor is 10 mM;
the drug gradient for each group of cells was:
HCT116 and HCT116-CAFs-CM groups 0, 25,50,100,200 (unit: uM);
HT29 and HT29-CAFs- CM groups 0, 40,80,160,320 (unit: uM);
CCK detection: after 48h of drug action, the supernatant was aspirated off, and the CCK8 solution was diluted in serum-free medium at a ratio of 1: and 9, incubating in a dark constant temperature incubator, reading an absorbance OD value under a multifunctional microplate reader when the CCK solution in the blank control hole is orange, and the wavelength is 450 nm.
Second, result in
The tumor cells HCT116 and HT29 are stimulated by CAF-CM, and the drug resistance is remarkably improved.
Claims (8)
1. The human tumor-associated fibroblast is characterized in that the cell strain is named 95-CAF and has been preserved in China center for type culture Collection of Wuhan university with the preservation number of CCTCC NO: C2020130; the human tumor-associated fibroblast has at least one of the following biological properties:
(1) the cells are in a long fusiform or starburst shape, the cytoplasm is rich and acidophilic, the cell nucleus is positioned in the center of a cell body, the cell nucleus is circular or oblong, the cells are arranged in a radial or bundle shape, part of the cells are disorderly arranged, the polarity disappears, and the obvious cross overlapping phenomenon exists;
(2) stable passage within ten generations;
(3) the cell expression protein alpha-SMA, FAP, E-cadherin and Vimentin are positive.
2. Use of the human tumor-associated fibroblast cell line of claim 1 in the preparation of a human tumor-associated fibroblast cell model.
3. Use according to claim 2, characterized in that it comprises the following steps:
taking a tumor-related fibroblast conditioned medium;
co-culturing with tumor cells;
and (5) preparing a tumor animal model.
4. Use according to claim 2, characterized in that said human tumor-associated fibroblast model is used for screening and/or evaluating/preparing a medicament for the treatment of tumors.
5. The use according to claim 2, wherein said human tumor-associated fibroblast model is used for the development of a tumor-associated fibroblast drug target.
6. Use of the human tumor-associated fibroblast cell line of claim 1 in the preparation of a tumor diagnostic reagent.
7. Use of the human tumor-associated fibroblast cell line of claim 1 in the preparation of a medicament for the biotherapeutic treatment of tumors.
8. Use of the human tumor-associated fibroblast cell line of claim 1 in the preparation of a biological engineering product for detecting tumor-associated.
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