阅读说明：本技术 一种结直肠癌顺铂耐药株的构建方法 (Method for constructing colorectal cancer cisplatin resistant strain ) 是由 胡清云 刘彩云 许澎 于 2021-08-26 设计创作，主要内容包括：本发明涉及一种结直肠癌顺铂耐药株的构建方法,属于生物工程技术领域。本发明提供了一种新型的耐药株构建方法,将磁珠分选法与常规的耐药株筛选法结合,选用P-糖蛋白(P-gp)作为耐药靶点,用其编码基因ABCB1的抗体标记细胞,通过磁珠分选将具备耐药性的细胞群从亲本株中分离出来。该方法构建周期短、成功率高、构建菌株耐药性稳定。(The invention relates to a method for constructing a colorectal cancer cisplatin resistant strain, belonging to the technical field of biological engineering. 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, selects P-glycoprotein (P-gp) as a drug-resistant target, marks cells by using an antibody of a coding gene ABCB1 thereof, and separates a cell population 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.)

1. A method for constructing a cisplatin-resistant strain is characterized by comprising the following steps:

(1) determining the IC50 value of the tumor cells after cisplatin action, selecting a concentration of 0.8-1.2 times the IC50 value of cisplatin-pretreated tumor cells;

(2) and labeling the tumor cells pretreated by the cisplatin by using an ABCB1 antibody, and separating the ABCB 1-labeled cell population to obtain the cisplatin-resistant strain.

2. The method of claim 1, wherein in step (1), the tumor cell is a human colorectal cancer cell.

3. The method of constructing according to claim 1 or 2, wherein the determination of the IC50 value of the tumor cell after cisplatin action is performed by MTT method, comprising the steps of:

1) digesting the logarithmic phase cells by 0.25% of pancreatin, collecting the cells into a centrifuge tube, and counting;

2) cell plating: taking 1 96-well plate, grouping according to the experiment, arranging 5 parallel wells for each group of samples, and paving 5000 cells in each well into the 96-well plate;

3) adding 5% CO₂Culturing in an incubator at 37 ℃ for 24h, and adding the medicine;

4) preparing cisplatin with different concentration gradients, and adding the cisplatin 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 putting the well in a cell culture box for incubation for 4 h;

6) taking out the 96-well plate, discarding culture solution in the hole, and adding 150 mu L DMSO into each hole to dissolve purple crystals;

7) measuring the MTT value at 490nm wavelength;

8) the IC50 is calculated.

4. The method of claim 1 or 2, wherein in step (2), the ABCB1 labeled cell population is isolated by magnetic bead sorting or flow sorting.

5. The method for constructing the antibody of claim 4, wherein the step of separating the ABCB1 labeled cell population by magnetic bead sorting comprises the following steps:

1) digesting the cells in logarithmic phase with pancreatin, collecting the cells in a centrifuge tube, and counting;

2) take 1X 10⁸Centrifuging the cells at 300g for 10min, and removing supernatant;

3) resuspending with 1mLbuffer, adding 100 μ LPE-CD243 Antibody, and mixing;

4) incubating at 4 deg.C in dark for 10 min;

5) centrifuging at 300g for 10min, removing supernatant, and adding 1mLbuffer to wash off unlabeled antibody;

6) centrifuging at 300g for 10min, and repeatedly cleaning for 1 time;

7) discarding the supernatant, and adding 800 μ lb buffer to resuspend the cells;

8) adding 200 mu of Lanti-PE magnetic beads, mixing uniformly, and incubating for 15min at 4 ℃;

9) washing the cells twice by using 1mLbuffer, centrifuging for 10min at 300g, and removing the supernatant;

10) 500 μ Lbuffer was added to resuspend the cells;

11) placing the MACS separator on a magnetic frame, placing an MS sorting column into the separator, dropwise adding the cell suspension on the column, adsorbing the cells marked by the CD243 on the sorting column, washing for 3 times by using a buffer to remove redundant cells, and adding 500 mu L of the cells each time;

12) the MS sort column was removed from the separator and placed in a collection tube, 1mLbuffer was added, and CD243 labeled cells were eluted using a propeller, and the collection tube yielded CD243 positive cells, i.e., ABCB1 labeled cells.

6. A strain constructed by the construction method according to any one of claims 1 to 5.

7. The strain of claim 6, wherein the strain is HCT 116/DDP.

8. The strain of claim 6, wherein the strain is HCT 15/DDP.

9. The strain of claim 6, wherein the strain is HCT 8/DDP.

Technical Field

The invention relates to a method for constructing a colorectal cancer cisplatin resistant strain, belonging to the technical field of biological engineering.

Background

At present, two methods are mainly used for screening drug-resistant strains: concentration gradient increasing method and high concentration impact method.

Concentration gradient increasing method: determining the IC50 of parent cells by MTT, selecting 1/10IC50 concentration as initial concentration to shock the cells, restoring culture after 24h, shocking for several times at the same concentration until the cells can stably grow in the concentration, then gradually increasing the drug concentration, repeating the steps to treat the cells until the drug resistance index of the cells reaches the target value, stopping shock, and culturing the cells to normally grow in the maintenance concentration and the IC50 value is stable.

High-concentration impact method: the method comprises the steps of impacting cells with high-concentration drugs in a short time period (such as 1h, 2h, 3h, 4h and 5h) with gradient increasing, recovering culture to a normal state every 1 time of impact, then impacting, stopping drug treatment when the cells can normally grow under the drugs with certain concentrations and reach target drug resistance indexes, and replacing a culture medium containing the drugs with maintenance concentrations for normal culture.

However, the existing drug-resistant strain screening method has the following defects:

(1) the process of inducing the cell to generate the drug resistance by the concentration gradient increasing method is long, and generally, the time is 6 to 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 purpose of optimizing treatment, but the action process of a gradient increasing method is different from the basic principle of large dose and short course of clinical chemotherapeutic drugs;

(3) in the high-concentration impact method, the drug concentration of the impact cells is high, so that the adjustment of the cell state is difficult, and the drug resistance of the cells is unstable.

Chemotherapy is one of the methods for treating malignant tumors, mainly kills tumor cells by chemical drugs, but some tumor cells can generate drug resistance in the chemotherapy process, so that the treatment effect of the tumors is greatly influenced, and therefore, the exploration of the drug resistance mechanism of the tumors has very important significance for improving the chemotherapy effect and selecting the correct chemotherapy method. Cisplatin is a broad-spectrum chemotherapeutic drug commonly used in the treatment of colorectal cancer. The screening of the colorectal cancer cell cisplatin-resistant strain 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 screening targets, and has high research application value and clinical significance.

Under the action of medicine, the physical and chemical properties of cell change, and P-glucoprotein (P-gp) appears on the surface of cell membrane, so that the cell can gradually produce drug resistance. The P-gp protein is also called multidrug resistance protein 1(MDRP1), is coded by ABCB1, plays a role of an efflux pump, and actively pumps out the medicine entering the cell to the outside of the cell under the energy supply of ATP, thereby reducing the damage of the medicine to the cell.

The invention aims to treat the colorectal cancer cells by using low-concentration cisplatin, labels the colorectal cancer cells by using an ABCB1 antibody, and separates the cell population labeled by the ABCB1 by a magnetic bead sorting method, so that the colorectal cancer cells with a certain drug resistance index can be obtained preliminarily. And then, on the basis, the screening or the maintenance culture of the drug-resistant strains is continued so as to improve the screening efficiency and the stability of the drug-resistant cells.

Disclosure of Invention

In order to solve the technical problems, the invention provides a method for constructing 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, the cell is marked by an antibody of a coding gene ABCB1, and a cell population with drug resistance is separated from a parent strain by magnetic bead sorting. The method has the advantages of short 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 method for constructing a cisplatin-resistant strain, which comprises the following steps:

(1) determining the IC50 value of the tumor cells after cisplatin action, selecting a concentration of 0.8-1.2 times the IC50 value of cisplatin-pretreated tumor cells;

(2) and labeling the tumor cells pretreated by the cisplatin by using an ABCB1 antibody, and separating the ABCB 1-labeled cell population to obtain the cisplatin-resistant strain.

Further, in the step (1), the tumor cell is a human colorectal cancer cell.

Further, the determination of the IC50 value of the tumor cells after the cisplatin action is carried out by using an MTT method, and comprises the following steps:

1) digesting the logarithmic phase cells by 0.25% of pancreatin, collecting the cells into a centrifuge tube, and counting;

2) cell plating: taking 1 96-well plate, grouping according to the experiment, arranging 5 parallel wells for each group of samples, and paving 5000 cells in each well into the 96-well plate;

3) adding 5% CO₂Culturing in an incubator at 37 ℃ for 24h, and adding the medicine;

4) preparing cisplatin with different concentration gradients, and adding the cisplatin 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 putting the well in a cell culture box for incubation for 4 h;

6) taking out a 96-well plate, carefully removing culture solution in the well, and adding 150 mu L of DMSO into each well to dissolve purple crystals;

7) measuring the MTT value at 490nm wavelength;

8) data were processed with GraphPad and IC50 was calculated as the concentration of cisplatin used in step (2).

Further, the pre-treatment of tumor cells is specifically: expanding and culturing tumor cells in 10 dishes, adding cisplatin for treating for 24h, removing the cisplatin, replacing a normal culture medium, culturing for 24h, and then, adding a culture medium with the ratio of 1: 1, passage; when the cells grow to logarithmic phase, the cells are collected.

Further, in the step (2), the ABCB1 labeled cell population is separated by magnetic bead sorting or flow sorting.

Further, the separation of the ABCB1 labeled cell population by magnetic bead sorting comprises the following steps:

1) digesting the cells in logarithmic phase with pancreatin, collecting the cells in a centrifuge tube, and counting;

2) take 1X 10⁸Centrifuging the cells at 300g for 10min, and removing supernatant;

3) resuspending with 1mL buffer, adding 100. mu.L PE-CD243 Antibody, and mixing;

4) incubating at 4 deg.C in dark for 10 min;

5) centrifuging for 10min at 300g, removing supernatant, and adding 1mL buffer to wash off unlabeled antibody;

6) centrifuging at 300g for 10min, and repeatedly cleaning for 1 time;

7) discarding the supernatant, and adding 800. mu.L of buffer to resuspend the cells;

8) adding 200 μ L Anti-PE magnetic beads, mixing, and incubating at 4 deg.C for 15 min;

9) washing the cells twice with 1mL buffer, centrifuging for 10min at 300g, and discarding the supernatant;

10) add 500. mu.L of buffer to resuspend the cells;

11) placing the MACS separator on a magnetic frame, placing an MS sorting column into the separator, dropwise adding the cell suspension on the column, adsorbing the cells marked by the CD243 on the sorting column, washing for 3 times by using a buffer to remove redundant cells, and adding 500 mu L of the cells each time;

12) taking the MS sorting column off the separator, putting the MS sorting column into a collecting pipe, adding 1mL of buffer, eluting the cells marked with the CD243 by using a propeller, and obtaining the positive cells of the CD243, namely the cells marked with ABCB1, from the collecting pipe;

13) flow-identifying the sorted cells.

The invention also provides a strain constructed by the construction method.

Further, the strain is HCT 116/DDP.

Further, the strain is HCT 15/DDP.

Further, the strain is HCT 8/DDP.

The invention has the beneficial effects that:

1. the method for constructing the drug-resistant plant greatly shortens the period of the drug-resistant plant, the conventional construction period needs 6-8 months, and the method for constructing the drug-resistant plant can be successfully constructed within 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, the drug resistance can not recover after the drug-resistant strain is maintained and cultured for a period of time, and the cell state is also stable.

Drawings

FIG. 1: MTT detects the IC50 profile of cisplatin on HCT116 cells.

FIG. 2: MTT assay IC50 graph of cisplatin effect on HCT116/DDP cells.

FIG. 3: MTT detects the IC50 profile of cisplatin on HCT15 cells.

FIG. 4: MTT assay IC50 profile of cisplatin on HCT15/DDP cells.

FIG. 5: MTT detects the IC50 profile of cisplatin on HCT8 cells.

FIG. 6: MTT assay IC50 profile of cisplatin on HCT8/DDP cells.

Detailed Description

(1) In the invention, besides the MTT method which can detect 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 cost of the MTT method is lower, and the MTT method is generally selected without special requirements.

(2) In the invention, the magnetic bead sorting is totally used in beautiful weather and gentle, other brands of magnetic bead sorting reagents or instruments can be selected, and the magnetic bead sorting can be replaced by flow sorting, and the principle is the immune combination of antigen and antibody.

(3) Determination of IC50 by MTT method

1) The log phase cells were digested with 0.25% pancreatin, collected into centrifuge tubes and counted.

2) Cell plating: 1 96-well plate is taken and divided into groups according to the experiment, 5 parallel wells are arranged in each group of samples, and 5000 cells in each well are paved into the 96-well plate.

3) Adding 5% CO₂And adding the medicine after culturing for 24 hours in an incubator at 37 ℃.

4) Cisplatin with different concentration gradients is added into a 96-well plate.

5) After 72h, the 96-well plate was removed, 10. mu.L of MTT solution was added to each well in the dark, and the plate was incubated in a cell incubator for 4 h.

6) The 96-well plate was removed, the medium in the well carefully discarded, and 150. mu.L of DMSO was added to each well to dissolve the purple crystals.

7) The MTT value was determined at 490nm wavelength.

8) Data were processed with GraphPad and IC50 was calculated.

(4) MACS magnetic bead sorting of CD243(ABCB1) Positive cell populations

1) Cells in the logarithmic growth phase were digested with pancreatin, collected in a centrifuge tube and counted.

2) Take 1X 10⁸The cells were centrifuged at 300g for 10min and the supernatant was discarded.

3) Resuspend with 1mL buffer, add 100. mu.L PE-CD243(ABCB1) Antibody, and mix well.

4) Incubate at 4 ℃ for 10min in the dark.

5) The mixture was centrifuged at 300g for 10min, the supernatant was removed, and 1mL of buffer was added to wash off the unlabeled antibody.

6) Centrifuge at 300g for 10min and repeat washing 1 time.

7) The supernatant was discarded and 800. mu.L of buffer was added to resuspend the cells.

8) Adding 200. mu.L Anti-PE magnetic beads, mixing well, and incubating at 4 ℃ for 15 min.

9) Cells were washed twice with 1mL buffer, centrifuged at 300g for 10min, and the supernatant discarded.

10) 500 μ L of buffer was added to resuspend the cells.

11) The MACS separator was placed on a magnetic frame, the MS sorting column was placed in the separator, the cell suspension was added dropwise over the column, at which time the CD243 labeled cells were adsorbed onto the sorting column, and the buffer was used to wash 3 times to remove excess cells, 500. mu.L each time.

12) The MS sort column was removed from the separator, placed in a collection tube, 1mL buffer was added, and CD243 labeled cells were eluted using a propeller. The collection tube yielded positive cells for CD 243.

13) Flow-identifying the sorted cells.

Example 1: construction of HCT116 cisplatin-resistant Strain (HCT116/DDP)

1. Resuscitation of HCT116 cells

1) The frozen cells were taken out of the liquid nitrogen tank, placed in a 37 ℃ water bath kettle to be rapidly shaken and thawed, and 5mL of DMEM complete medium (90% DMEM + 10% FBS + 1% P/S) was added and mixed well.

2) Centrifuging at 1000rpm for 4min, discarding supernatant, adding 1mL DMEM complete culture medium, adding cell suspension into 10cm culture dish, supplementing 7mL culture medium, and adding 5% CO₂And culturing in an incubator at 37 ℃.

2. The HCT116 parental cells were selected to pre-treat cells with 10. mu.g/mL cisplatin as a result of an MTT assay that had an IC50 value of 10.55. mu.g/mL for cisplatin-resistant drugs.

3. And (3) expanding cells in 10 dishes, adding 10 mu g/mL cis-platinum, treating for 24h, removing the medicament, replacing a normal culture medium, and culturing for 24h according to the proportion of 1: passage 1.

4. And collecting the cells for MACS magnetic bead sorting when the cells grow to the logarithmic growth phase.

5. The sorted CD243 positive cells are re-inoculated into culture dishes for culture recovery.

6. The cell state is normalPost assay HCT116/CD243⁺The IC50 of the cell is 94.46 mu g/mL, the drug resistance index is 8.95, the cell has the characteristics of a drug-resistant strain, and the concentration of 1/10IC50 is about 10 mu g/mL to maintain the cultured cell.

7. Waiting for HCT116/CD243⁺Cells were able to grow normally in maintenance culture concentration and were assayed after 5 passages.

After 5 passages, the cell drug resistance index is still stable, and the HCT116 cisplatin-resistant strain (HCT116/DDP) is successfully constructed.

Table 1: MTT results for HCT116 cisplatin-resistant Strain (HCT116/DDP)

Fig. 1 and 2 are drug resistance curves obtained from the cell viability calculated according to the data in table 1, and the IC50 (semi-lethal dose) of HCT116 and HCT116 drug-resistant strains is calculated according to the formula of the drug resistance curve, and the ratio of the two is the drug resistance index of 8.95.

Example 2: construction of HCT15 cisplatin-resistant Strain (HCT15/DDP)

1. Recovery of HCT15 cells

1) The frozen cells were taken out of the liquid nitrogen tank, placed in a 37 ℃ water bath kettle to be rapidly shaken and thawed, and 5mL of DMEM complete medium (90% 1640+ 10% FBS + 1% P/S) was added and mixed well.

2) Centrifuging at 1000rpm for 4min, discarding supernatant, adding 1mL DMEM complete culture medium, adding cell suspension into 10cm culture dish, supplementing 7mL culture medium, and adding 5% CO₂And culturing in an incubator at 37 ℃.

2. The IC50 value of HCT15 parent cell cisplatin-resistant drug determined by the MTT method was 10. mu.g/mL, therefore 10. mu.g/mL cisplatin was selected to pre-treat the cells.

3. And (3) expanding cells in 10 dishes, adding 10 mu g/mL cis-platinum, treating for 24h, removing the medicament, replacing a normal culture medium, and culturing for 24h according to the proportion of 1: passage 1.

4. And collecting the cells for MACS magnetic bead sorting when the cells grow to the logarithmic growth phase.

5. The sorted CD243 positive cells are re-inoculated into culture dishes for culture recovery.

6. Determination of HCT15/CD243 after Normal cellular status⁺The IC50 of the cell is 31 mug/mL, the drug resistance index is 3.1, the cell has the characteristics of a drug-resistant strain, and the 1/10IC50 concentration is about 3 mug/mL to maintain the cultured cell.

7. Waiting for HCT15/CD243⁺Cells were able to grow normally in maintenance culture concentration and were assayed after 5 passages.

After 5 passages, the cell drug resistance index is still stable, and the HCT15 cis-platinum drug-resistant strain (HCT15/DDP) is successfully constructed.

Table 2: MTT results for the cisplatin-resistant Strain of HCT15 (HCT116/DDP)

Fig. 3 and 4 are drug resistance curves obtained from the cell viability calculated according to the data in table 2, and then the IC50 of HCT15 and HCT15 drug-resistant strains are calculated according to the formula of the drug resistance curves, and the ratio of the two is the drug resistance index of 3.1.

Example 3: construction of HCT8 cisplatin-resistant Strain (HCT8/DDP)

1. Recovery of HCT8 cells

3) The frozen cells were taken out of the liquid nitrogen tank, placed in a 37 ℃ water bath kettle to be rapidly shaken and thawed, and 5mL of DMEM complete medium (90% 1640+ 10% FBS + 1% P/S) was added and mixed well.

4) Centrifuging at 1000rpm for 4min, discarding supernatant, adding 1mL DMEM complete culture medium, adding cell suspension into 10cm culture dish, supplementing 7mL culture medium, and adding 5% CO₂And culturing in an incubator at 37 ℃.

2. The IC50 value of HCT8 parent cell cisplatin-resistant drug determined by the MTT method was 9.2. mu.g/mL, therefore 10. mu.g/mL cisplatin was selected to pre-treat the cells.

3. And (3) expanding cells in 10 dishes, adding 10 mu g/mL cis-platinum, treating for 24h, removing the medicament, replacing a normal culture medium, and culturing for 24h according to the proportion of 1: passage 1.

4. And collecting the cells for MACS magnetic bead sorting when the cells grow to the logarithmic growth phase.

5. The sorted CD243 positive cells are re-inoculated into culture dishes for culture recovery.

6. Determination of HCT15/CD243 after Normal cellular status⁺The IC50 of the cell is 43.65 mug/mL, the drug resistance index is 4.75, the cell has the characteristics of a drug-resistant strain, and the concentration of 1/10IC50 is about 4 mug/mL to maintain the cultured cell.

7. Waiting for HCT15/CD243⁺Cells were able to grow normally in maintenance culture concentration and were assayed after 5 passages.

After 5 passages, the cell drug resistance index is still stable, and the HCT8 cis-platinum drug-resistant strain (HCT8/DDP) is successfully constructed.

Table 3: MTT results for the cisplatin-resistant Strain of HCT8 (HCT116/DDP)

FIGS. 5 and 6 are graphs of drug resistance curves obtained from the cell viability calculated according to the data in Table 3, and the IC50 of HCT8 and HCT8/DDP drug-resistant strains are calculated according to the formula of the drug resistance curves, and the ratio of the two is the drug resistance index of 4.75.

It should be understood that the above-mentioned embodiments are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.