CN113774025B - Construction method of colorectal cancer cisplatin resistant strain - Google Patents
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Abstract
The invention relates to a construction method of colorectal cancer cisplatin resistant strain, belonging to the technical field of bioengineering. The invention provides a novel construction method of drug-resistant strains, which combines a magnetic bead sorting method with a conventional drug-resistant strain screening method, selects P-glycoprotein (P-gp) as a drug-resistant target, marks cells with an antibody of a coding gene ABCB1, and separates a cell group with drug resistance from a parent strain through magnetic bead sorting. The method has the advantages of short construction period, high success rate and stable drug resistance of the constructed strain.
Description
Technical Field
The invention relates to a construction method of colorectal cancer cisplatin resistant strain, belonging to the technical field of bioengineering.
Background
At present, two main methods exist for screening drug-resistant strains: concentration gradient increment method and high concentration impact method.
Concentration gradient increasing method: the IC50 of parent cells is measured by MTT, 1/10 of the IC50 concentration is selected as the initial concentration, the cells are impacted, the cells are recovered to be cultivated after 24 hours of treatment, the cells are impacted for a plurality of times with the same concentration until the cells can stably grow in the concentration, the drug concentration is gradually increased, the cells are treated by repeating the steps until the drug resistance index of the cells reaches a target value, the impact is stopped, and the cultivated cells normally grow in the maintenance concentration and the IC50 value is stable.
High concentration impact method: the cell is impacted by the high-concentration drug in a short period of gradient increasing (for example, 1h, 2h, 3h, 4h and 5 h), the cell is recovered to be in a normal state after 1 time of impact, and then the cell is impacted, and when the cell can normally grow under a certain concentration of the drug and reaches a target drug resistance index, the drug treatment is stopped, and the medium containing the drug with the maintained concentration is replaced for normal culture.
However, the existing screening method for drug-resistant strains has the following disadvantages:
(1) The concentration gradient increasing method induces cells to generate drug resistance for a long period of time, usually 6-8 months;
(2) The main purpose of constructing drug-resistant strain cells is to explore the drug-resistant process of tumor cells to clinical chemotherapeutic drugs so as to achieve the aim of optimizing treatment, but the action process of a gradient increasing method is different from the basic principle of large-dose short treatment course of clinical chemotherapeutic drugs;
(3) The high-concentration impact method has the defects that the adjustment of the cell state is difficult and the drug resistance of the cells is unstable due to the higher drug concentration of the impact cells.
Chemotherapy is one of methods for treating malignant tumors, mainly kills tumor cells by chemical drugs, but in the chemotherapy process, some tumor cells generate drug resistance, so that the treatment effect of the tumor is greatly influenced, and therefore, the exploration of a drug resistance mechanism of the tumor has very important significance for improving the treatment effect and selecting the correct chemotherapy method. Cisplatin belongs to a broad-spectrum chemotherapeutic drug and is commonly used in the treatment of colorectal cancer. Screening colorectal cancer cell cisplatin resistant strains can be used for analyzing and researching molecular mechanisms and pathological mechanisms of colon cancer cell drug resistance, can also be used for screening other anti-colon cancer drugs or target screening, and has high research application value and clinical significance.
Under the action of the medicine, the physical and chemical properties of the cells can be changed, and P-glycoprotein (P-gp) appears on the surface of cell membranes, so that the cells gradually generate medicine resistance. The P-gp protein is called as multi-drug resistant protein 1 (MDRP 1), is encoded by ABCB1, plays a role of an efflux pump, and actively pumps out the drugs entering cells under the energy of ATP (adenosine triphosphate) so as to reduce the damage of the drugs to the cells.
The invention aims to treat colorectal cancer cells with low concentration of cisplatin, marks the colorectal cancer cells with ABCB1 antibodies, and separates out ABCB1 marked cell groups by a magnetic bead separation method, thus obtaining colorectal cancer cells with a certain drug resistance index initially. And then, the screening or maintenance culture of the drug-resistant strain is continued on the basis, so that the screening efficiency and stability of the drug-resistant cells are improved.
Disclosure of Invention
In order to solve the technical problems, the invention provides a construction method of a colorectal cancer cisplatin resistant strain. The invention provides a novel drug-resistant strain construction method, which combines a magnetic bead sorting method with a conventional drug-resistant strain screening method; p-glycoprotein (P-gp) is selected as a drug-resistant target, cells are marked by an antibody of the coding gene ABCB1, and a cell group with drug resistance is separated from a parent strain through magnetic bead separation. The method has the advantages of high construction period, high success rate and stable drug resistance of the constructed strain.
The technical scheme of the invention is as follows:
the invention provides a construction method of a cisplatin resistant strain, which comprises the following steps:
(1) Determining the IC50 value of the tumor cells after the cisplatin action, and selecting cisplatin pretreatment tumor cells with the concentration of 0.8-1.2 times of the IC50 value;
(2) And then labeling tumor cells pretreated by cisplatin by using an ABCB1 antibody, and separating out an ABCB1 labeled cell group to obtain the cisplatin resistant strain.
Further, in the step (1), the tumor cells are human colorectal cancer cells.
Further, the determination of the IC50 value of tumor cells after cisplatin action is performed using the MTT method, comprising the steps of:
1) Digesting the log phase cells with 0.25% pancreatin, collecting the cells into a centrifuge tube, and counting;
2) Cell plating: taking 1 96-well plate, arranging 5 parallel holes in each group of samples according to experimental grouping, and paving 5000 cells in each hole into the 96-well plate;
3) Placing 5% CO 2 Culturing in a 37 ℃ incubator for 24 hours, and adding medicine;
4) Cisplatin with different concentration gradients is prepared and added into a 96-well plate;
5) After 72h, taking out the 96-well plate, adding 10 mu L of MTT solution into each well in a dark place, and placing the mixture into a cell incubator for incubation for 4h;
6) Taking out the 96-well plate, carefully discarding the culture solution in the well, and adding 150 mu L of DMSO into each well to dissolve the purple crystals;
7) MTT value was measured at 490nm wavelength;
8) Data were processed with GraphPad and IC50 calculated as the concentration of cisplatin used in step (2).
Further, the pretreatment tumor cells are specifically: expanding and culturing tumor cells in a 10-dish, adding cisplatin for 24 hours, removing cisplatin, replacing a normal culture medium, and culturing for 24 hours, wherein the following steps are 1:1 passage; and collecting the cells after the cells grow to the logarithmic phase.
Further, in the step (2), the ABCB 1-labeled cell population is isolated using magnetic bead sorting or flow sorting.
Still further, the separating of the ABCB 1-labeled cell population using magnetic bead sorting comprises the steps of:
1) Taking cells in logarithmic growth phase, digesting the cells with pancreatin, collecting the cells in a centrifuge tube, and counting the cells;
2) Taking 1×10 8 Centrifuging the cells at 300g for 10min, and removing the supernatant;
3) Re-suspending with 1mL buffer, adding 100 μL PE-CD243Antibody, and mixing;
4) Incubating for 10min at 4 ℃ in dark;
5) Centrifuging 300g for 10min, removing supernatant, and adding 1mL buffer to wash off unlabeled antibody;
6) Centrifuging at 300g for 10min, and repeatedly cleaning for 1 time;
7) The supernatant was discarded and 800. Mu.L buffer was added to resuspend the cells;
8) Adding 200 mu L of Anti-PE magnetic beads, uniformly mixing, and incubating for 15min at 4 ℃;
9) Washing the cells twice with 1mL buffer, centrifuging for 10min at 300g, and discarding the supernatant;
10 Add 500 μl buffer to resuspend cells;
11 Placing the MACS separator on a magnetic frame, placing an MS separation column in the separator, dripping the cell suspension into the column, adsorbing the cells marked by the CD243 on the separation column, flushing 3 times by using a buffer to remove redundant cells, and adding 500 mu L each time;
12 Taking the MS separation column out of the separator, putting the MS separation column into a collecting pipe, adding 1mL buffer, eluting cells marked with CD243 by using a propeller, and obtaining positive cells of the CD243, namely ABCB1 marked cells by the collecting pipe;
13 Flow identification of the sorted cells.
The invention also provides a strain constructed by the construction method.
Further, the strain is HCT116/DDP.
Further, the strain is HCT15/DDP.
Further, the strain is HCT8/DDP.
The invention has the beneficial effects that:
1. the method for constructing the drug-resistant strain greatly shortens the period of the drug-resistant strain, and the conventional construction period needs 6-8 months, and the method for constructing the drug-resistant strain can succeed in construction for 2-3 months;
2. the method has high success rate of constructing the drug-resistant strain, and does not conflict with the clinical chemotherapy principle;
3. the drug-resistant strain constructed by the method has stable drug resistance, does not recover the drug resistance after being maintained for a period of time, and has stable cell state.
Drawings
Fig. 1: IC50 profile of cisplatin effect on HCT116 cells for MTT detection.
Fig. 2: IC50 profile of cisplatin effect on HCT116/DDP cells for MTT assay.
Fig. 3: IC50 profile of cisplatin effect on HCT15 cells for MTT detection.
Fig. 4: IC50 profile of cisplatin effect on HCT15/DDP cells for MTT assay.
Fig. 5: IC50 profile of cisplatin effect on HCT8 cells for MTT detection.
Fig. 6: IC50 profile of cisplatin effect on HCT8/DDP cells for MTT assay.
Detailed Description
(1) In the invention, besides the MTT method can detect the IC50, reagents such as MTS, CCK8 and the like can be used, the reagents are generally selected according to the characteristics of cells and medicines, the MTT method has low cost, and the MTT method is generally selected without special requirements.
(2) In the invention, the magnetic bead sorting is fully applied to Meitian and gentle, other brands of magnetic bead sorting reagents or instruments can be selected, and the flow sorting can be used for replacing the magnetic bead sorting, and the principle is the immunological combination of antigen and antibody.
(3) Determination of IC50 by MTT method
1) Log phase cells were digested with 0.25% pancreatin, collected into centrifuge tubes and counted.
2) Cell plating: 1 96-well plate was taken, 5 parallel wells were set per group of samples according to experimental grouping, and 5000 cells per well were plated into 96-well plates.
3) Placing 5% CO 2 Culturing in a 37 ℃ incubator for 24 hours, and adding the medicine.
4) Cisplatin was formulated in different concentration gradients and added to 96-well plates.
5) After 72h, the 96-well plate was removed, 10. Mu.L of MTT solution was added to each well in the dark, and incubated in a cell incubator for 4h.
6) The 96-well plates were removed and the culture medium was carefully discarded and 150 μl DMSO was added to dissolve the purple crystals per well.
7) MTT value was measured at 490nm wavelength.
8) Data were processed with GraphPad and IC50 calculated.
(4) MACS magnetic bead sorting CD243 (ABCB 1) positive cell population
1) Cells in the logarithmic growth phase were digested with pancreatin, collected in centrifuge tubes, and counted.
2) Taking 1×10 8 The individual cells were centrifuged at 300g for 10min and the supernatant was removed.
3) Resuspended in 1mL buffer, 100. Mu.L PE-CD243 (ABCB 1) Antibody was added and mixed.
4) Incubate at 4℃for 10min in the dark.
5) 300g was centrifuged for 10min, the supernatant removed, and 1mL buffer was added to wash away unlabeled antibody.
6) Centrifugation at 300g for 10min and repeated washing 1 time.
7) The supernatant was discarded and 800. Mu.L buffer was added to resuspend the cells.
8) 200. Mu.L of Anti-PE magnetic beads were added, mixed well and incubated at 4℃for 15min.
9) The cells were washed twice with 1mL buffer, centrifuged at 300g for 10min, and the supernatant discarded.
10 500. Mu.L buffer was added to resuspend cells.
11 Placing MACS separator on a magnetic rack, placing MS separation column into the separator, dropping cell suspension into the column, adsorbing CD243 labeled cells on the separation column, washing 3 times with buffer to remove redundant cells, and adding 500 μl each time.
12 The MS separation column was removed from the separator, placed in a collection tube, 1mL buffer was added, and CD 243-labeled cells were eluted using a propeller. The collection tube yielded positive cells for CD 243.
13 Flow identification of the sorted cells.
Example 1: construction of HCT116 cisplatin-resistant Strain (HCT 116/DDP)
1. Resuscitation of HCT116 cells
1) The frozen cells were removed from the liquid nitrogen tank, placed in a 37℃water bath, thawed by rapid shaking, and mixed well with 5mL of DMEM complete medium (90% DMEM+10% FBS+1% P/S).
2) Centrifuging at 1000rpm for 4min, removing supernatant, adding 1mL of DMEM complete medium, adding cell suspension into 10cm culture dish, adding 7mL of medium, and adding 5% CO 2 Culturing in an incubator at 37 ℃.
2. MTT assay HCT116 parental cell cisplatin resistant drugs have an IC50 value of 10.55. Mu.g/mL, so 10. Mu.g/mL cisplatin was selected to pre-treat the cells.
3. Cell expansion culture is carried out on a 10-dish, cisplatin with the concentration of 10 mug/mL is added for 24 hours, medicines are removed, normal culture medium is replaced, and after the cell expansion culture is carried out for 24 hours, the cell expansion culture is carried out according to the following steps of 1: passage 1.
4. And after the cells grow to the logarithmic growth phase, collecting the cells for MACS magnetic bead sorting.
5. The sorted CD243 positive cells were re-inoculated into a petri dish for resumption of culture.
6. Determination of HCT116/CD243 after normal cell State + The IC50 of the cells was 94.46. Mu.g/mL, the drug resistance index was 8.95, and the cells were maintained in culture by selecting a concentration of about 10. Mu.g/mL at 1/10 of the IC50, which had been characteristic of the drug-resistant strain.
7. Waiting for HCT116/CD243 + Cells were able to grow normally in maintenance culture concentration and were re-assayed after 5 passages.
After 5 passages, the cell resistance index is still stable, and the construction of HCT116 cisplatin resistant strain (HCT 116/DDP) is successful.
Table 1: HCT116 cisplatin resistant Strain (HCT/DDP) MTT results
Fig. 1 and 2 are graphs of drug resistance obtained from the cell viability calculated from the data of table 1, and IC50 (half-lethal dose) of HCT116 and HCT116 resistant strain were calculated according to the formula of the drug resistance curve, respectively, and the ratio of the two was the drug resistance index of 8.95.
Example 2: construction of HCT15 cisplatin-resistant Strain (HCT 15/DDP)
1. Resuscitation of HCT15 cells
1) The frozen cells were removed from the liquid nitrogen tank, placed in a 37℃water bath, thawed by rapid shaking, and mixed well with 5mL of DMEM complete medium (90% 1640+10% FBS+1% P/S).
2) Centrifuging at 1000rpm for 4min, removing supernatant, adding 1mL of DMEM complete medium, adding cell suspension into 10cm culture dish, adding 7mL of medium, and adding 5% CO 2 Culturing in an incubator at 37 ℃.
2. MTT assay HCT15 parental cells anti-cisplatin drug IC50 value was 10. Mu.g/mL, so 10. Mu.g/mL cisplatin was chosen to pre-treat the cells.
3. Cell expansion culture is carried out on a 10-dish, cisplatin with the concentration of 10 mug/mL is added for 24 hours, medicines are removed, normal culture medium is replaced, and after the cell expansion culture is carried out for 24 hours, the cell expansion culture is carried out according to the following steps of 1: passage 1.
4. And after the cells grow to the logarithmic growth phase, collecting the cells for MACS magnetic bead sorting.
5. The sorted CD243 positive cells were re-inoculated into a petri dish for resumption of culture.
6. Determination of HCT15/CD243 after normal cell State + The IC50 of the cells was 31. Mu.g/mL, the drug resistance index was 3.1, the characteristics of the drug resistant strain were already possessed, and the 1/10IC50 concentration was selected to be about 3. Mu.g/mL to maintain the cultured cells.
7. Waiting for HCT15/CD243 + Cells were able to grow normally in maintenance culture concentration and were re-assayed after 5 passages.
After 5 passages, the cell resistance index is still stable, and the HCT15 cisplatin resistant strain (HCT 15/DDP) is successfully constructed.
Table 2: HCT15 cisplatin resistant Strain (HCT/DDP) MTT results
Fig. 3 and 4 are graphs showing the drug resistance obtained from the cell viability calculated from the data of table 2, and IC50 of HCT15 and HCT15 resistant strain were calculated according to the formula of the drug resistance curve, respectively, and the ratio of the two was 3.1.
Example 3: construction of HCT8 cisplatin-resistant Strain (HCT 8/DDP)
1. Resuscitation of HCT8 cells
3) The frozen cells were removed from the liquid nitrogen tank, placed in a 37℃water bath, thawed by rapid shaking, and mixed well with 5mL of DMEM complete medium (90% 1640+10% FBS+1% P/S).
4) Centrifuging at 1000rpm for 4min, removing supernatant, adding 1mL of DMEM complete medium, adding cell suspension into 10cm culture dish, adding 7mL of medium, and adding 5% CO 2 Culturing in an incubator at 37 ℃.
2. MTT assay HCT8 parental cells anti-cisplatin drug IC50 value was 9.2. Mu.g/mL, so 10. Mu.g/mL cisplatin was chosen to pre-treat the cells.
3. Cell expansion culture is carried out on a 10-dish, cisplatin with the concentration of 10 mug/mL is added for 24 hours, medicines are removed, normal culture medium is replaced, and after the cell expansion culture is carried out for 24 hours, the cell expansion culture is carried out according to the following steps of 1: passage 1.
4. And after the cells grow to the logarithmic growth phase, collecting the cells for MACS magnetic bead sorting.
5. The sorted CD243 positive cells were re-inoculated into a petri dish for resumption of culture.
6. Determination of HCT15/CD243 after normal cell State + The IC50 of the cells was 43.65. Mu.g/mL, the drug resistance index was 4.75, and the cells were maintained in culture at a concentration of about 4. Mu.g/mL with 1/10 of the IC50, which had been characteristic of drug resistant strains.
7. Treat HCT 15-CD243 + Cells were able to grow normally in maintenance culture concentration and were re-assayed after 5 passages.
After 5 passages, the cell resistance index is still stable, and the HCT8 cisplatin resistant strain (HCT 8/DDP) is successfully constructed.
Table 3: HCT8 cisplatin resistant Strain (HCT/DDP) MTT results
Fig. 5 and 6 are graphs showing the cell viability calculated from the data of table 3, and IC50 of HCT8 and HCT8/DDP resistant strain were calculated according to the formula of the drug resistance curve, respectively, and the ratio of the two was 4.75.
It should be understood that the foregoing description is only of the preferred embodiments of the present invention and is not intended to limit the scope of the invention, but is intended to cover any and all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (2)
1. The construction method of the cisplatin resistant strain is characterized by comprising the following steps of:
(1) Determining the IC50 value of tumor cells after cisplatin action, and selecting cisplatin with a concentration which is 0.8-1.2 times of the IC50 value to pretreat the tumor cells, wherein the tumor cells are human colorectal cancer cells;
(2) Labeling tumor cells pretreated by cisplatin by using an ABCB1 antibody, and separating out an ABCB1 labeled cell group by using magnetic bead separation to obtain a cisplatin resistant strain;
the method for separating the ABCB1 marked cell population by using magnetic bead separation comprises the following steps:
1) Taking cells in logarithmic growth phase, digesting the cells with pancreatin, collecting the cells in a centrifuge tube, and counting the cells;
2) Taking 1×10 8 Centrifuging the cells at 300g for 10min, and removing the supernatant;
3) Re-suspending with 1ml buffer, adding 100 μLPE-CD243Antibody, and mixing;
4) Incubating for 10min at 4 ℃ in dark;
5) Centrifuging at 300g for 10min, removing supernatant, and adding 1ml buffer to wash off unlabeled antibody;
6) Centrifuging at 300g for 10min, and repeatedly cleaning for 1 time;
7) The supernatant was discarded and 800. Mu.Lbuffer was added to resuspend the cells;
8) Adding 200 mu LAnti-PE magnetic beads, uniformly mixing, and incubating for 15min at 4 ℃;
9) Washing the cells twice with 1ml buffer, centrifuging for 10min at 300g, and discarding the supernatant;
10 Add 500 μl buffer to resuspend cells;
11 Placing the MACS separator on a magnetic frame, placing an MS separation column in the separator, dripping the cell suspension into the column, adsorbing the cells marked by the CD243 on the separation column, flushing 3 times by using a buffer to remove redundant cells, and adding 500 mu L each time;
the MS separation column is taken off from the separator, put into a collecting pipe, 1ml buffer is added, cells marked with CD243 are eluted by using a propeller, and positive cells of CD243, namely ABCB1 marked cells, are obtained by the collecting pipe.
2. The method of claim 1, wherein the determination of IC50 values of tumor cells after cisplatin action is performed using MTT method, comprising the steps of:
1) Digesting the log phase cells with 0.25% pancreatin, collecting the cells into a centrifuge tube, and counting;
2) Cell plating: taking 1 96-well plate, arranging 5 parallel holes in each group of samples according to experimental grouping, and paving 5000 cells in each hole into the 96-well plate;
3) Placing 5% CO 2 Culturing in a 37 ℃ incubator for 24 hours, and adding medicine;
4) Cisplatin with different concentration gradients is prepared and added into a 96-well plate;
5) After 72 hours, taking out the 96-well plate, adding 10 mu LMTT solution into each well in a dark place, and placing the mixture into a cell incubator for 4 hours;
6) Taking out the 96-well plate, discarding the culture solution in the well, and adding 150 mu LDMSO into each well to dissolve purple crystals;
7) MTT value was measured at 490nm wavelength;
8) The IC50 is calculated.
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