CN106676074B - Method for in-vitro amplification of liver cancer stem cells - Google Patents
Method for in-vitro amplification of liver cancer stem cells Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
- C12N5/0695—Stem cells; Progenitor cells; Precursor cells
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Abstract
The invention discloses a method for in vitro amplification of liver cancer stem cells, which comprises the following steps: adding the obtained human liver cancer cells in a single cell state into a culture medium to prepare a cell suspension; dropwise adding the cell suspension onto a porous cellulose support, placing the porous cellulose support into a cell culture box for culture and attachment after the cell suspension permeates into the porous cellulose support, then adding a culture medium, and placing the porous cellulose support into the cell culture box for culture for several days to obtain three-dimensional cultured cells; the transformed liver cancer stem cells are detected by detecting the change of the expression conditions of liver cancer stem cell marker molecules EpCAM, OCT4, CD44 and CD133 in three-dimensional cultured cells by using Western Blot, quantitative PCR, flow cytometry and IHC and/or microscopic observation. The invention also discloses a liver cancer stem cell and application thereof. The liver cancer stem cells obtained by the invention have drug resistance.
Description
Technical Field
The invention belongs to the research field of oncology and stem cell, and particularly relates to a method for amplifying liver cancer stem cells in vitro.
Background
Liver cancer is a terminal liver disease, the disease condition is dangerous, the death rate is as high as 80%, and the liver cancer is one of malignant tumors seriously harming human health. China is a country with high incidence of liver diseases, more than 1 hundred million patients with various liver diseases exist at present, and 55 of 100 new liver cancers are Chinese people in every year around the world. The presence of tumor stem cells has been considered to be a significant cause of malignant tumor development. The liver cancer stem cells can resist cancer treatment drugs and chemotherapy radiation, and increase the tumor recurrence and metastasis probability after operation. In summary, the main problem to be solved by the present invention is how to develop a suitable cell culture and screening method to effectively separate and screen liver cancer stem cells, and thus, the present invention is used for research on liver cancer pathogenesis, drugs, immunotherapy, etc.
The previous research shows that EpCAM, OCT4, CD44 and CD133 are the most basic molecular markers of liver cancer stem cells and are closely related to the activities of liver cancer cell invasion, metastasis and the like. Therefore, how to develop an anti-tumor drug targeting liver cancer stem cells is an important opportunity for treating liver cancer. However, because the distribution ratio of liver cancer stem cells in liver cancer tissues is low, how to culture liver cancer stem cells in vitro becomes a problem to be solved urgently in the technical development of liver cancer stem cells.
In recent years, three-dimensional cell culture techniques have matured. The three-dimensional culture technology is that a carrier with a three-dimensional structure and cells are cultured together in vitro, so that the cells can migrate and grow in a three-dimensional space of a carrier scaffold to form a cell-carrier compound with the three-dimensional structure characteristic. At present, the three-dimensional cell culture technology is widely applied in the fields of tumor biology, cartilage and bone tissues, circulation systems, hearts, nervous systems and the like, and the breadth and depth of biomedical research are expanded.
In the traditional two-dimensional cell culture system, cells gradually lose original properties under in-vitro culture conditions and are often inconsistent with the in-vivo conditions of organisms, and animal experiments are influenced by various in-vivo and in-vitro complex factors together, so that the single-factor regulation and control mechanism is difficult to study. The three-dimensional cell culture technology is a novel technology between two-dimensional cell culture and animal experiments, can simulate the in-vivo environment to the maximum extent, and can show the intuitiveness and condition controllability of cell culture.
Disclosure of Invention
The purpose of the invention is as follows: the first purpose of the invention is to provide a method for expanding liver cancer stem cells in vitro.
The invention also aims to provide the application of the liver cancer stem cells in-vitro screening of anti-tumor drugs or in-vitro drug sensitivity experiments of tumor cells.
The technical scheme is as follows: in order to solve the above problems, the present invention provides a method for expanding liver cancer stem cells in vitro, comprising the steps of:
1) adding the obtained human liver cancer cells in a single cell state into a culture medium to prepare a cell suspension;
2) dropwise adding the cell suspension onto a porous cellulose support, placing the porous cellulose support into a cell culture box for culture and attachment after the cell suspension permeates into the porous cellulose support, adding a culture medium, and placing the porous cellulose support into the cell culture box for culture for several days to obtain three-dimensional cultured cells;
3) changes in expression of the liver tumor stem cell marker molecules EpCAM, OCT4, CD44 and CD133 in three-dimensional cultured cells were measured using Western Blot, RT-PCR, flow cytometry and IHC, and the transformed liver cancer stem cells were detected using microscopic observation methods.
The porous cellulose scaffold takes cellulose as a main material, or takes cellulose as a main material and other biological materials with good biocompatibility as auxiliary materials.
Wherein the cellulose material can be methylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose and cellulose ether ester material.
The porous cellulose scaffold can be prepared by a solution casting/particle leaching method, and can also be prepared by a fused deposition method, a 3D printing technology, a stereolithography method and the like.
Wherein the porous cellulose scaffold is a sponge carrier with mutually communicated pores, and the pore diameter is 50-500 mu m.
Among them, the pore diameter of the porous cellulose scaffold is preferably 50 to 200. mu.m. The pore size distribution is uniform, cells are easy to enter the pores, and space is provided for growing cell microspheres.
Wherein the rigidity of the porous cellulose scaffold is 5-100 kPa.
Wherein the rigidity of the porous cellulose scaffold is preferably 5 to 30 kPa.
Wherein the liver cancer cell is HepG2, HUH7, PLC/PRF/5 or other types of liver cancer cells.
Wherein the culture medium is a corresponding culture medium used in the culture of the liver cancer cell line or a tumor stem cell culture medium without serum.
Wherein the culture conditions comprise 35-39 deg.C, 3% -21% oxygen content, and 5% carbon dioxide.
The invention also comprises the liver cancer stem cells obtained by transformation by the method.
The invention also comprises the application of the liver cancer stem cell in-vitro screening of anti-tumor drugs or in-vitro drug sensitivity experiments of tumor cells.
Has the advantages that: compared with the prior art, the invention has the following advantages:
1. the invention adopts the porous cellulose scaffold to carry out three-dimensional culture on the human liver cancer cells, and directly obtains the liver cancer stem cell microspheres, and the obtained microsphere cells contain a large amount of EpCAM, OCT4, CD44 and CD133 positive cells and obviously have the characteristics of the liver cancer stem cells.
2. The amplification method is simple and convenient to operate, cells do not need to be subjected to complex pretreatment, additional physical and chemical pressure cannot be caused to the cells, and the cells are in a normal physiological state.
3. The amplification method can amplify the liver cancer stem cells to the maximum extent, and provides a new idea for diagnosis and treatment of liver cancer. The method has high yield and quick time effect, and can reduce the price cost without using commercial stem cell culture products.
4. The porous cellulose scaffold is used in the amplification method, so that a three-dimensional growth environment can be provided for cells, and the in-vivo microenvironment can be simulated.
5. The liver cancer stem cell obtained by the invention has good drug resistance to 5-FU and cisplatin, and can be used for drug sensitivity test of antitumor drugs.
Drawings
FIG. 1 Western Blot technique for detecting the protein expression levels of EpCAM, OCT4, CD44 and CD133 in two-dimensional and three-dimensional cultured HepG2, HUH7, PLC/PRF/5 cells;
FIG. 2 Real time-PCR technique to detect EpCAM and OCT4mRNA expression levels in HepG2, HUH7 cells cultured in two and three dimensions;
FIG. 3 the IHC technique measures EpCAM and CD44 expression levels in two-and three-dimensional cultured HepG2 and HUH7 cells;
FIG. 4 flow cytometry detection of CD44 and CD133 expression levels in two and three dimensional cultured HepG2 cells;
FIG. 5 flow cytometry detection of CD44 and CD133 expression levels in two-and three-dimensional cultured PLC/PRF/5 cells;
FIG. 6 microscopic observation of the two-and three-dimensional cultured HepG2 and HUH7 cells of example 1 under bright field conditions.
FIG. 7 is a drug resistance experiment of two-dimensional and three-dimensional cultured liver cancer cell PLC/PRF/5 cells;
FIG. 8 shows the drug resistance test of HepG2 cells cultured in two-and three-dimensions.
Detailed Description
The present invention will be further described below.
Example 1: method for in-vitro amplification of liver cancer stem cells
1) Digesting liver cancer cells (HepG 2, HUH7 and PLC/PRF/5) which are in good growth state and are in logarithmic growth phase with 0.25% of pancreatin at 37 ℃ for 5min to obtain human liver cancer cells in a single cell state, removing digestive juice containing the pancreatin, and adding the human liver cancer cells into a DMEM complete culture medium containing 10% of fetal calf serum to prepare cell suspension;
2) spreading appropriate amount of cell suspension in cell culture dish, placing at 37 deg.C and 5% CO 220% oxygen content, cell incubator for 7 days, as two-dimensional cultured cell control. Replacing DMEM complete culture medium every other day; wherein the DMEM complete culture medium is a high-glucose DMEM culture medium added with 10% fetal calf serum, 100U/ml penicillin and 100mg/ml streptomycin.
3) 60 μ L of cell suspension (cell density 10)5One/ml) was added dropwise onto a porous cellulose support, which was left at 37 ℃ with 5% CO 220% oxygen, adding DMEM complete culture medium after being attached for 4 hours in a cell culture box, placing at 37 ℃ and 5% CO2And culturing in an incubator with 20% oxygen for 7 days to obtain the three-dimensional cultured cells. The medium was changed every other day. The cellulose of the porous cellulose scaffold is mainly methyl cellulose, the aperture is 50 mu m, and the rigidity is 15 kPa;
4) the cells obtained by two-dimensional culture and three-dimensional culture are respectively collected, the expression changes of liver cancer cell marker molecules EpCAM, OCT4, CD44 and CD133 under two-dimensional or three-dimensional culture conditions are measured by using methods such as Western Blot, RT-PCR, flow cytometry and IHC, and the characterization changes of the three-dimensional cultured cells are observed by a microscope.
Example 2A method for in vitro expansion of liver cancer Stem cells
1) Digesting liver cancer cells (HepG 2, HUH7 and PLC/PRF/5) which are in good growth state and are in logarithmic growth phase with 0.25% of pancreatin at 37 ℃ for 5min to obtain human liver cancer cells in a single cell state, removing digestive juice containing the pancreatin, and adding the human liver cancer cells into a DMEM complete culture medium containing 10% of fetal calf serum to prepare cell suspension;
2) spreading appropriate amount of cell suspension in cell culture dish, and standing at 35 deg.C and 5% CO 25% oxygen, cultured in a cell culture box for 7 days, and used as a two-dimensional cell culture pairAnd (6) irradiating. Replacing the DMEM complete culture medium every other day; wherein the DMEM complete culture medium is a high-glucose DMEM culture medium added with 10% fetal calf serum, 100U/ml penicillin and 100mg/ml streptomycin.
3) 60 μ L of cell suspension (cell density 10)5One/ml) was added dropwise to a porous cellulose support (at 37 ℃ C., 5% CO)2Adding DMEM complete culture medium after attaching in 21% oxygen cell incubator for 4 hours, placing at 35 ℃ and 5% CO2And culturing for 7 days in a 5% oxygen incubator to obtain the three-dimensional cultured cells. DMEM complete medium was changed every other day. The cellulose of the porous cellulose scaffold is mainly hydroxypropyl cellulose, the aperture is 100 mu m, and the rigidity is 30 kPa;
4) the cells obtained by two-dimensional culture and three-dimensional culture are respectively collected, the expression changes of the liver cancer stem cell marker molecules EpCAM, OCT4, CD44 and CD133 under two-dimensional or three-dimensional culture conditions are measured by using methods such as Western Blot, RT-PCR, flow cytometry and IHC, and the characterization changes of the three-dimensional cultured cells are observed by a microscope.
Example 3A method for in vitro expansion of liver cancer Stem cells
1) Digesting liver cancer cells (HepG 2, HUH7 and PLC/PRF/5) which are in good growth state and are in logarithmic growth phase with 0.25% of pancreatin at 37 ℃ for 5min to obtain human liver cancer cells in a single cell state, removing digestive juice containing the pancreatin, and adding the human liver cancer cells into a DMEM complete culture medium containing 10% of fetal calf serum to prepare cell suspension;
2) spreading appropriate amount of cell suspension in cell culture dish, and standing at 35 deg.C and 5% CO 210% oxygen, cell incubator for 7 days, as two-dimensional cell culture control. Replacing serum-free tumor stem cell culture medium every other day;
3) 60 μ L of cell suspension (cell density 10)5One/ml) was added dropwise to a porous cellulose support (at 37 ℃ C., 5% CO)2Adding serum-free tumor stem cell culture medium after attaching in 21% oxygen cell culture box for 4 hours, placing at 35 deg.C and 5% CO2And culturing for 7 days in a 10% oxygen incubator to obtain the three-dimensional cultured cells. And replacing the serum-free tumor stem cell culture medium every other day. The porous celluloseThe cellulose of the stent is mainly hydroxypropyl methylcellulose, the aperture is 500 mu m, and the rigidity is 100 kPa;
4) the cells obtained by two-dimensional culture and three-dimensional culture are respectively collected, the expression changes of the liver cancer stem cell marker molecules EpCAM, OCT4, CD44 and CD133 under two-dimensional or three-dimensional culture conditions are measured by using methods such as Western Blot, RT-PCR, flow cytometry and IHC, and the characterization changes of the three-dimensional cultured cells are observed by a microscope.
Example 4
1) Digesting liver cancer cells (HepG 2, HUH7 and PLC/PRF/5) which are in good growth state and are in logarithmic growth phase with 0.25% of pancreatin at 37 ℃ for 5min to obtain human liver cancer cells in a single cell state, removing digestive juice containing the pancreatin, and adding the human liver cancer cells into a DMEM complete culture medium containing 10% of fetal calf serum to prepare cell suspension;
2) spreading appropriate amount of cell suspension in cell culture dish, standing at 39 deg.C and 5% CO 210% oxygen, cell incubator for 7 days, as two-dimensional cell culture control. Replacing serum-free tumor stem cell culture medium every other day;
3) 60 μ L of cell suspension (cell density 10)5One/ml) was added dropwise to a porous cellulose support (placed at 39 ℃, 5% CO)2Adding serum-free tumor stem cell culture medium after attaching in 21% oxygen cell culture box for 4 hours, placing at 39 deg.C and 5% CO2And culturing for 7 days in a 10% oxygen incubator to obtain the three-dimensional cultured cells. And replacing the serum-free tumor stem cell culture medium every other day. The cellulose of the porous cellulose scaffold is mainly cellulose ether ester, the aperture is 500 mu m, and the rigidity is 100 kPa;
4) the cells obtained by two-dimensional culture and three-dimensional culture are collected respectively, and the change of the expression conditions of the liver cancer stem cell marker molecules EpCAM, OCT4, CD44 and CD133 under two-dimensional or three-dimensional culture conditions is measured by using methods such as Western Blot, RT-PCR, flow cytometry, IHC and the like. And observing the characterization change of the three-dimensional cultured cells by a microscope.
Experimental example 1 detection by Western Blot technique
1.1 extraction of Total protein
After digesting the two-dimensional and three-dimensional cells obtained in example 1 with trypsin, the cells were washed 2 times in cold PBS, 200. mu.l of ice-cold lysis buffer was added each, mixed well, ice-cooled for 30min, shaken for 10s every 5min, and fully lysed. Centrifuge for 25 min. And (5) packaging the supernatant and storing at-20 ℃ for later use.
1.2 determination of protein concentration by BCA method
1.2.1 according to the quantity of a sample, adding 50 volumes of BCA reagent A into 1 volume of BCA reagent B, preparing a proper amount of BCA working solution, and then fully mixing the solution by using a gun head.
1.2.2 completely dissolve the protein standard, take 10. mu.L of the protein standard to dilute to 100. mu.L, make the final concentration to be 0.5 mg/ml.
1.2.3 Add the standard 0, 1, 2, 4, 8, 12, 16, 20. mu.l in sequence to the standard wells of a 96-well plate, make up to 20. mu.L per well with the standard diluent.
1.2.4 Add 10. mu.L of the sample to be tested to the sample wells of a 96-well plate and dilute to 20. mu.L with the standard.
1.2.5 Add 200. mu.L of BCA working solution to each well and incubate at 37 ℃ for 30 min.
1.2.6 optical density values at 520nm wavelength were determined for the samples and standards. And calculating the protein concentration of each group of samples according to the concentration standard curve of the protein standard.
1.3 SDS-PAGE electrophoresis
1.3.1 mu.L of the protein lysate adjusted to the same concentration was mixed with 5. mu.L of SDS loading buffer, and the clean Bio-Rad 1.5cm glass plate was mounted on the gel frame according to the instructions.
6.6mL double distilled water
30% acrylamide 8.0mL
1M Tris-Cl(PH8.8） 5.0mL
10%SDS 0.2mL
0.2mL of 10% ammonium persulfate
TEMED 24μL
1.3.4 the separation gel is carefully injected into the interspace between the glass plates with a tip, stopping at approximately two thirds of the height of the glass plates, after which a few ml of double distilled water are added, in order to prevent the inhibition of the polymerization of the gel by the air.
1.3.5 after the polymerization of the separation gel was completed, the double distilled water covering the gel was poured off, and the remaining liquid was sucked up with filter paper as far as possible, taking care not to touch the separation gel.
6.8mL of double distilled water
30% acrylamide 1.66mL
1 M Tris-Cl（PH8.8） 1.26mL
10%SDS 0.1mL
0.1mL of 10% ammonium persulfate
TEMED 16μL
1.3.2 after polymerization of the concentrated gels, the sample comb was carefully removed and 1xTris-Gly electrophoresis buffer was added to check for leakage.
1.3.8 absorbing a proper amount of sample supernatant and adding the sample supernatant into a sample hole, adding a pre-stained protein Marker into a hole beside the sample, and adding 1xSDS (sodium dodecyl sulfate) loading buffer solution into a hole without the sample supernatant to keep the gel surface balance.
1.3.9 the power is turned on and the voltage is initially set to 80V, which can be increased to 120V after the protein sample enters the separation gel. Referring to the location of the prestained Marker, the electrophoresis was stopped when the band of interest entered the optimal separation zone of the gel (about 2/3 of the gel).
1.4 transfer film
1.4.1 precooling the membrane transferring liquid by 4 degrees in advance.
1.4.2 smash open the transfer box on the tray, lay the porous fiber pad that has been soaked with the transfer membrane buffer solution on the inner face near the cathode side, put three layers of filter paper soaked with the transfer membrane buffer solution on it, pay attention to the clean air bubbles.
1.4.3 carefully pry open the glass plate, place the glue in a tray containing the membrane transfer solution, cut the separation glue containing the target strip, soak the separation glue in the membrane transfer solution, and place the soaked separation glue on filter paper.
1.4.4 PVDF membrane soaked by methanol and membrane transferring liquid is laid on the gel, and no air bubbles can be generated between the gel and the membrane.
1.4.5 two more layers of Whatman filter paper soaked with the spin-coating solution were placed on the PVDF membrane, and the air bubbles were removed.
And 1.4.6 placing a second sponge cushion, closing the transfer printing clamp, placing the second sponge cushion into a transfer groove, and filling the transfer printing groove with a transfer printing liquid.
1.4.7 turning on the power supply, 100V, 60 min;
1.4.8 after the transfer of the membrane, the PVDF membrane was removed and labeled, and the membrane was washed with TBST for 10min 3 times.
1.5 blocking, antigen-antibody reaction
1.5.1 blocking: putting the PVDF membrane into a plate, adding a confining liquid containing 5% skimmed milk powder, and shaking for 1.5-2h by a shaking table.
1.5.2 after the end of blocking, the membrane was washed with TBST for 10min 3 times.
1.5.3 the membrane was placed in a dish containing primary antibody and incubated overnight with shaking at 4 degrees.
1.5.4 the next day, remove primary antibody by pipetting, and wash with TBST for 10min 3 times.
1.5.5 diluting the secondary antibody with 5% skimmed milk powder confining liquid, and shaking the secondary antibody for reaction for 1-2h in a shaking table at room temperature.
1.5.6 after the secondary antibody reaction, the secondary antibody was recovered. The membrane was then washed with TBST for 5-10min x 3.
1.6 color development
1.6.1 two liquids A, B from ECL chemiluminescence kit were mixed at 1: 1 equal volume mixing, preparing working solution for standby.
1.6.2 taking out the PVDF film from the TBST, throwing away redundant liquid, putting the film containing protein with the right side facing upwards on a preservative film, dripping a proper amount of working solution, covering the film with the preservative film, putting the film in a developing clamp, and closing the developing clamp.
1.6.3, the film enters a darkroom for development, the photosensitive film is placed in a development clamp, the exposure time is adjusted according to the strength of the protein strip, and then the film is sequentially placed in a developing solution and a fixing solution to develop and fix the film.
Protein expression levels of EpCAM, OCT4, CD44 and CD133 were examined in the two-and three-dimensional cultured HepG2, HUH7, PLC cells of example 1 by using the conventional Western Blot technique described above. As can be seen from FIG. 1, the protein expression levels of EpCAM, OCT4, CD44 and CD133 were increased in each cell under the three-dimensional culture conditions. Specifically, the Western Blot band gray value of each molecule is higher than that of the two-dimensional culture condition in the three-dimensional culture.
Experimental example 2 detection by RT-PCR technique
1. RNA extraction
1.1 adding 1ml of precooled TRIzol to the cell discarded culture supernatant obtained under the two-dimensional and three-dimensional culture conditions obtained in the example 1, and repeatedly blowing and beating the cell discarded culture supernatant by using a 1ml gun head until a particle-free transparent solution is obtained, namely a cracked cell sap;
1.2 transferring the fully-lysed cell sap into a 1.5ml centrifuge tube, and standing at room temperature for 5min to completely separate nucleic acid protein complexes;
1.3 adding chloroform (0.2 ml of chloroform is added to 1ml of TRIzol), covering a tube cover of a centrifuge tube tightly, shaking up and down by hands for 15s to ensure that the solution is milky without layering, and standing for 3min at room temperature;
1.412,000 g, centrifuging at 4 ℃ for 15 min;
1.5 taking out the centrifuge tube, wherein the sample is divided into three layers: a colorless supernatant phase, a middle white layer and a pink lower organic phase;
1.6 carefully pipette colorless supernatant and phase into another centrifuge tube, the volume of water phase is about 60% of that of Trizol reagent;
1.7 adding isopropanol with the same volume into the obtained supernatant, gently mixing uniformly, and standing for 10min at room temperature; centrifuging at 4 deg.C for 10min at 12,000 g;
1.8 carefully remove the supernatant, slowly add 1ml 70% ethanol (made with DEPC treated water) along the tube wall, mix gently;
1.912,000 g, centrifuging at 4 ℃ for 10 min;
1.10 carefully sucking up the supernatant, drying the precipitate at room temperature for about 5min, properly (the RNA is hard to dissolve if the RNA is not aired too dry), adding 30-50 mu L of RNase-free water to dissolve the RNA precipitate, and storing at-70 ℃ after the RNA precipitate is completely dissolved.
2. Determination of RNA concentration and purity
2.1 taking 5 μ L of RNA sample to 595 μ L of 1 × TE Buffer, and determining the absorption values of the sample at 260nm and 280 nm;
2.2 the concentration of RNA (OD 260X dilution factor X0.04. mu.g/. mu.L) (cuvette optical diameter 1 cm), OD260/280 in the range of 1.8 to 2.1 was considered to be high in the purity of extracted RNA.
3. First strand cDNA Synthesis (10. mu.L system):
3.1 reaction System
Number of reactant doses
1 reverse transcription buffer 2. mu.L
2 upstream primer 0.2. mu.L
3 downstream primer 0.2. mu.L
4 dNTP 0.1 μL
5 reverse transcriptase MMLV 0.5 uL
6 DEPC water 5. mu.L
7 RNA template 2. mu.L
8 Total volume 10. mu.L
The solution was mixed by flicking the bottom of the tube and briefly centrifuged at 6000 rpm.
3.2 the mixture was dry-bathed at 70 ℃ for 3 minutes before the addition of the reverse transcriptase MMLV, immediately after removal in an ice-water bath until the temperature inside and outside the tube was the same, and then 0.5. mu.l of reverse transcriptase was added and the mixture was bathed at 37 ℃ for 60 minutes.
3.3 taking out the cDNA, immediately carrying out dry bath at 95 ℃ for 3 minutes to obtain a reverse transcription final solution which is a cDNA solution, and storing at-80 ℃ for later use.
4 Real time-PCR experiment
4.1 making 2 multiple holes for one sample gene according to a certain sequence;
4.2 to a 0.1ml PCR tube, the following components were added in order:
2X Real time PCR Master Mix(SYBR Green) 10μL
template (10-fold cDNA dilution) 1. mu.L
Primer MIX (F/R each 10. mu.M) 2. mu.L
0.1% DEPC Water 7. mu.L
Total volume 20μL
4.3 PCR amplification procedure
And (3) testing the prepared sample on a computer, and setting different reaction conditions according to different detection genes. The conditions are all conventional PCR experimental conditions. EpCAM, and OCT4mRNA expression levels were detected in the two-and three-dimensional cultured HepG2, HUH7 cells of example 1 using realtome-PCR technology. In the three-dimensional cultured cells, the expression level of EpCAM and OCT4 is obviously increased. EPCAM mRNA expression levels were increased 24-fold and OCT4mRNA expression levels were increased 74-fold in HepG2 cells. In HUH7 cells, OCT4mRNA expression levels increased 24-fold (fig. 2).
Experimental example 3 detection by IHC technique
1. And (3) performing paraffin slicing on the cell slide fixing sample or the three-dimensional culture slice sample, then placing the cell slide fixing sample or the three-dimensional culture slice sample in a 70 ℃ oven, baking the cell slide for 2 hours, performing gradient alcohol dewaxing, and washing the cell slide fixing sample or the three-dimensional culture slice sample with PBS for three times, wherein each time lasts for 3 min.
2. Adding a certain amount of citrate buffer solution with the pH =6.0 into a microwave box, heating the citrate buffer solution to boiling by microwave, placing the dewaxed and hydrated tissue slices on a high-temperature-resistant plastic slice frame, putting the tissue slices into the boiling buffer solution, carrying out medium-grade microwave treatment for 10 minutes, taking out the flow water of the microwave box for natural cooling, taking out the slide from the buffer solution, firstly washing the slide twice by using distilled water, and then washing the slide for 2 x 3 minutes by using PBS.
3. Adding 1 drop of 3% H into each slice2O2Incubate at room temperature for 10min to block endogenous peroxidase activity, wash with PBS for 3 × 3 min.
4.1 drop of immunohistochemical nonspecific staining blocking agent was added dropwise to each section, and incubation was carried out at room temperature for 10 min.
5. PBS was removed and 1 drop of the corresponding primary antibody (dilution 1: 500) was added to each section and incubated overnight at 4 ℃.
6. PBS rinse 3X 5 min. PBS was removed and 1 drop of polymer enhancer was added to each section and incubated at room temperature for 20 min. PBS rinse 3X 3 min.
7. PBS was removed and 1 drop of enzyme-labeled anti-mouse/rabbit polymer was added to each section and incubated at room temperature for 30 min. PBS rinse 3X 5 min.
8. PBS was removed and 1 drop of freshly prepared DAB solution (diaminobenzidine) was added to each section and observed under the microscope for 10 min.
9. Hematoxylin counterstaining, 0.1% HCl differentiation, tap water washing, bluing, slice dehydration and drying by gradient alcohol, xylene transparency, neutral gum sealing, air drying and observation.
EpCAM and CD44 expression levels were detected in two-and three-dimensional cultured HepG2 and HUH7 cells of example 1 using IHC technology. In the three-dimensional cultured cells, the expression level of EpCAM and CD44 was significantly increased (fig. 3). In the cells cultured in two dimensions, the expression levels of EpCAM and CD44 are extremely low, and the expression levels are hardly observed in stained sections, and in the cells cultured in three dimensions, the positive rate of the target staining molecule is obviously increased.
Experimental example 4 flow cytometry assay
1. HepG2 and PLC/PRF/5 cells were cultured in two dimensions and three dimensions, respectively, under the same culture conditions and for the same time as in example 1.
2. Cells were harvested by trypsinization and the remaining cells were washed once with 1mL of PBS buffer and all added to 15mL tubes. For the three-dimensional cultured cells, the porous methylcellulose scaffold is mechanically separated after trypsinization, so that cell clusters are scattered into digestive juice, and scaffold fragments are filtered out to obtain the three-dimensional cultured cells.
3.1200 rpm centrifugation for 5min, remove the supernatant, add 5mL PBS buffer heavy suspension cells, again centrifugation and discard the supernatant, repeat twice, finally heavy suspension cells in 0.1mL PBS, and transfer to 1.5mL centrifuge tube.
4.1 UL primary antibody was added at 1:500 and incubated on the vertical mix for 1 hour at room temperature.
5.1200rpm, centrifuging for 5min in a mini-centrifuge, removing the supernatant, adding 1mL PBS buffer solution to resuspend the cells, centrifuging again and discarding the supernatant, repeating for 3 times, and finally resuspending the washed cells in 0.1mL PBS.
6. The fluorescent secondary antibody was added to the cell suspension at a ratio of 1:800 and incubated at room temperature for 1 hour.
7.1200rpm centrifugation for 5min, remove the supernatant, add 1mL PBS buffer heavy suspension cells, again centrifugation and abandon the supernatant, repeat 3 times, finally heavy suspension cells in 0.2mL PBS.
8. The cells were packed in a tin foil to the exclusion of light, added to a flow tube and analyzed on a flow cytometer.
The expression levels of CD44 and CD133 were detected in the two-and three-dimensional cultured HepG2 and PLC/PRF/5 cells prepared in example 1 using flow cytometry. The number of collected cells was 1 ten thousand. Negative controls of single-staining secondary antibodies and groups of single-staining CD44 and single-staining CD133 are arranged on cell samples cultured in two dimensions and three dimensions, and after false positive results are eliminated on the premise of unifying experimental conditions and detection parameters, the expression quantity of CD44 and CD133 in each group of cells cultured in three dimensions is obviously increased in positive percentage of flow detection (fig. 4-fig. 5).
Experimental example 5
The two-and three-dimensional cultured HepG2 and HUH7 cells of example 1 were observed under bright field conditions using a microscope. As can be seen from FIG. 6, the cells cultured in two dimensions attached to the bottom of the dish and expanded in shape, while the cells cultured in three dimensions suspended in the culture medium grow in clusters, which appear as a plurality of cells aggregated together into a micro-cell sphere.
Experimental example 6
Liver cancer stem cells of PLC/PRF/5 cells and HepG2 cells cultured in a three-dimensional way and liver cancer stem cells of liver cancer cells PLC/PRF/5 and HepG2 cells cultured in a 1.96 pore plate are added with 5-FU or cisplatin and cultured for 72 hours on the 4 th day of culture; the control (control) is the three-dimensional cultured liver cancer stem cells and the two-dimensional cultured liver cancer cells without adding corresponding anti-tumor drugs;
2. on day 7 of the two-dimensional and three-dimensional culture, 20. mu.L of MTT solution (5 mg/ml in PBS < pH =7.4 >) was added to each well;
2. after further incubation for 4 hours, the culture was terminated and the culture supernatant from the wells was carefully aspirated. Adding 150 mu L DMSO into each hole, and oscillating for 10 minutes to fully melt the crystal;
3. color comparison: selecting 490nm wavelength, measuring the light absorption value of each pore on an enzyme linked immunosorbent assay, and recording the result;
4. and calculating the cell viability (cell viability) under different treatment conditions according to the absorbance values obtained by detection.
The experiments on drug resistance of the liver cancer cells cultured in two dimensions and the liver cancer stem cells cultured in three dimensions were carried out, and it can be seen from fig. 7 and 8 that the drug resistance of the liver cancer stem cells cultured in three dimensions was much higher than that of the liver cancer stem cells cultured in two dimensions.
The results of the above experiments 1 to 6 show that the liver cancer cells can be transformed into liver cancer stem cells by using the three-dimensional culture technique.
The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.
Claims (1)
1. A method for expanding liver cancer stem cells in vitro, comprising the steps of:
1) digesting liver cancer cells which are in good growth state and in logarithmic growth phase with 0.25% of pancreatin for 5min at 37 ℃ to obtain human liver cancer cells in single cell state, removing digestive juice containing pancreatin, and adding into a DMEM complete culture medium containing 10% of fetal calf serum to prepare cell suspension; the liver cancer cells are HepG2, HUH7 and PLC/PRF/5;
2) spreading appropriate amount of cell suspension in cell culture dish, placing at 37 deg.C and 5% CO220% oxygen content, culturing for 7 days in a cell culture box, serving as a two-dimensional cell culture control, and replacing a DMEM complete culture medium every other day; wherein the DMEM complete culture medium is a high-glucose DMEM culture medium added with 10% fetal calf serum, 100U/ml penicillin and 100mg/ml streptomycin;
3) 60 μ l of cell suspension with a cell density of 105Dropping the mixture into a porous cellulose bracket, placing the porous cellulose bracket at 37 ℃ and 5% CO220% oxygen, adding DMEM complete culture medium after being attached for 4 hours in a cell culture box, placing at 37 ℃ and 5% CO2，2Culturing in an incubator with 0% oxygen for 7 days to obtain three-dimensional cultured cells, and replacing culture medium every other day, wherein cellulose of the porous cellulose scaffold is mainly methylcellulose, the aperture is 50 μm, and the rigidity is 15 kPa;
4) the cells obtained by two-dimensional culture and three-dimensional culture are respectively collected, the expression changes of the liver cancer stem cell marker molecules EpCAM, OCT4, CD44 and CD133 under two-dimensional or three-dimensional culture conditions are measured by using methods such as Western Blot, RT-PCR, flow cytometry and IHC, and the characterization changes of the three-dimensional cultured cells are observed by a microscope.
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