CN108853123B - Application of aspirin in preventing or treating colon cancer - Google Patents
Application of aspirin in preventing or treating colon cancer Download PDFInfo
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- A61K31/612—Salicylic acid; Derivatives thereof having the hydroxy group in position 2 esterified, e.g. salicylsulfuric acid
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Abstract
The invention relates to application of aspirin in preventing or treating colon cancer, and also relates to a pharmaceutical composition containing aspirin. Aspirin can relieve the reduction of platelet activation rate, and inhibit the migration ability and invasion ability of tumor cells. The invention researches the biological regulation mechanism of aspirin to the microenvironment of colon cancer tumor and the medicinal effect of aspirin to colon cancer, and provides a new idea for the deep research and development of aspirin and the clinical treatment of colon cancer.
Description
Technical Field
The invention relates to the field of biological medicines, in particular to application of aspirin in prevention or treatment of colon cancer.
Background
Aspirin (Aspirin, acetylsalicylic acid) is a derivative of salicylic acid, and is a white crystal or crystalline powder, odorless or slightly smelly with acetic acid, slightly soluble in water, easily soluble in ethanol, soluble in ether and chloroform, and acidic in aqueous solution. As early as 1853, people synthesized acetylsalicylic acid for the first time through salicylic acid and acetic anhydride. Over the years, people gradually find that the acetylsalicylic acid has good effects of resisting inflammation, relieving fever and pain and the like. Until 1899, acetylsalicylic acid began to be widely used in the clinic and was more commonly known as Aspirin (Aspirin).
The clinical application of aspirin for over a hundred years has proved that the aspirin has better effect on relieving mild or moderate pain, such as toothache, headache, neuralgia, muscular soreness, arthralgia and dysmenorrheal, and is also used for defervescence of fever diseases such as cold, flu and the like. At present, aspirin derivatives are endlessly available, and are widely used in clinical treatment for relieving fever and pain. In recent years, aspirin has an inhibiting effect on platelet aggregation, can prevent the formation of thrombus, and is clinically used for preventing the formation of thrombus after transient ischemic attack, myocardial infarction, artificial heart valve and venous fistula or other operations.
Colon cancer is a common malignancy of the digestive tract that occurs in the colon, usually at the junction of the rectum and the sigmoid colon. The highest incidence rate of the disease in the age group of 40-50 years, the ratio of male to female is 2-3: 1. the incidence rate is 3 rd of gastrointestinal tumors. Colon cancer is mainly classified into adenocarcinoma, mucinous adenocarcinoma, and undifferentiated carcinoma. The general morphology is polypoid, ulcer type, etc. The colon cancer can circulate along the intestinal wall, spread up and down along the longitudinal diameter of the intestinal canal or infiltrate into the deep layer of the intestinal wall, and can be planted in the abdominal cavity or spread and transferred along the suture line and the incision surface besides the lymphatic vessel, blood flow transfer and local invasion. Patients with chronic colitis, colonic polyps, male obesity, etc. are susceptible people.
In recent years, aspirin has been found to have a certain clinical effect in the prevention and treatment of a part of tumors. However, aspirin has been reported in the literature for the prevention and treatment of colon cancer. The application method and the treatment effect of aspirin in the prevention or treatment of colon cancer are researched, and a new thought is provided for clinically treating colon cancer.
Disclosure of Invention
Aiming at the defects in the prior art, the invention relates to application of aspirin in treatment of colon cancer.
The present invention provides the use of aspirin in the manufacture of a medicament for the prophylaxis or treatment of colon cancer or a patient at risk of colon cancer.
In one aspect, the aspirin is present in the patient in an amount of 10 to 100 mg/kg/d; preferably 50 mg/kg/d.
In one aspect, the aspirin content in the medicine is 20-500 mu M; preferably 50 to 300. mu.M.
In one aspect, the medicament further comprises a platelet aggregation inhibitor; ticagrelor is preferred.
In one aspect, the platelet aggregation inhibitor is present in the patient in an amount of 5 to 50 mg/kg/d; preferably 10 mg/kg/d.
In one aspect, the platelet aggregation inhibitor is present in the medicament in an amount of 5 to 50 μ M; preferably 20. mu.M.
In one aspect, in the medicine, the mass ratio of the aspirin to the ticagrelor is (10-2): 1, preferably 5: 1, or the molar ratio of the two is (20-5): 1, preferably 10: 1.
in one aspect, the colon cancer tumor is inhibited after the patient receives the drug.
In one aspect, the subject has decreased body weight gain and decreased platelet activation following administration of the agent, and the subject has decreased expression of HIF-1 α, GLUT-1, and ALDH-1, and the subject has inhibited tumor cell migration and invasion.
In one aspect, the patient is selected from a human or non-human animal; preferably a non-human animal; more preferably a mouse.
The invention provides a pharmaceutical composition comprising aspirin and a pharmaceutically acceptable carrier.
In one aspect, the aspirin content is 20-500 μ M; preferably 50 to 300. mu.M.
In one aspect, the pharmaceutical composition further comprises a platelet aggregation inhibitor; ticagrelor is preferred. .
In one aspect, the platelet aggregation inhibitor is present in the pharmaceutical composition in an amount of 5 to 50 μ M; preferably 20. mu.M.
In one aspect, in the pharmaceutical composition, the mass ratio of the aspirin to the ticagrelor is (10-2): 1, preferably 5: 1, or the molar ratio of the two is (20-5): 1, preferably 10: 1.
in one aspect, the pharmaceutical composition may be formulated into a pharmaceutical formulation according to conventional methods. In the preparation process, the aspirin is preferably mixed with a pharmaceutically acceptable carrier or diluted with a carrier. When the carrier serves as a diluent, it may be solid, semi-solid or liquid. The preparation is selected from tablet, pill, powder, capsule, suspension, emulsion, solution, aerosol, injectable solution, etc.
The aspirin-based colon cancer treatment method has the advantages that aspirin can inhibit platelet activation, transplanted tumor mice treated by aspirin are lower in weight reduction degree and lower in platelet activation rate compared with a control group, expressions of HIF-1 α, GLUT-1 and ALDH-1 are reduced, and migration capacity and invasion capacity of tumor cells are inhibited.
Drawings
FIG. 1: the body weight of each group of mice changed 7-31d after tumor inoculation. Control group is blank group, i.e. no tumor inoculation + no drug treatment; the TI group was a control group, i.e. transplantable tumor + no drug treatment; the TI + Aspirin group is an Aspirin whole-course administration group, namely, the intragastric administration treatment of the transplanted tumor and Aspirin before, during and after transplantation; the TI + TiCagrelior group is a Ticagrelor whole-course administration group, namely, the stomach irrigation treatment of the Ticagrelor before and after transplantation and the transplantation of the tumor; the TI, Aspirin and TiCagrelior group is an Aspirin and Ticagrelor whole administration group, namely, the intragastric administration treatment of the transplanted tumor, the Aspirin before and after transplantation and the Ticagrelor. TI stands for tuminocculation.
FIG. 2: the body weight of each group of mice changed 7-31d after tumor inoculation. The meaning of Control group, TI + Aspirin group, TI + TiCagrellor group, TI + Aspirin + TiCagrellor group and TI is the same as that of the previous figure. Note: p<0.05vs.Control，#p<TI 0.05vs. unlabeled for p>0.05。
FIG. 3: tumor microvascular morphology (400 ×) was transplanted in each group of mice. The meanings of the TI group, the TI + Aspirin group, the TI + TiCagrelalor group, the TI + Aspirin + TiCagrelalor group and the TI are the same as those in the previous figure.
FIG. 4: tumor vessel branch number and total vessel length were transplanted for each group of mice. The meanings of the TI group, the TI + Aspirin group, the TI + TiCagrelalor group, the TI + Aspirin + TiCagrelalor group and the TI are the same as those in the previous figure.
FIG. 5: the platelet activation rate results were flow-tested for each group of mice. The meaning of Control group, TI + Aspirin group, TI + TiCagrellor group, TI + Aspirin + TiCagrellor group and TI is the same as that of the previous figure.
FIG. 6: platelet activation rates in each group of mice. Control group, TI + Aspirin group, TI + TiCaThe meanings of the grelor group, TI + Aspirin + TiCagrelior group and TI are the same as those in the previous figure. Note: p<0.05vs.Control，**p<0.01vs.Control，#p<TI 0.05vs. unlabeled for p>0.01。
FIG. 7: platelet activation rates in each group of mice. The meaning of Control group, TI + Aspirin group, TI + TiCagrellor group, TI + Aspirin + TiCagrellor group and TI is the same as that of the previous figure. Note: p<0.05vs.Control，**p<0.01vs.Control，#p<TI 0.05vs. unlabeled for p>0.01。
FIG. 8: platelet activation rates in each group of mice. The meaning of Control group, TI + Aspirin group, TI + TiCagrellor group, TI + Aspirin + TiCagrellor group and TI is the same as that of the previous figure. Note: p<0.05vs.Control，**p<0.01vs.Control，##p<TI 0.01vs. p, not labeled>0.01。
FIG. 9: platelet activation rates in each group of mice. The meaning of Control group, TI + Aspirin group, TI + TiCagrellor group, TI + Aspirin + TiCagrellor group and TI is the same as that of the previous figure. Note: p<0.05vs.Control，**p<0.01vs.Control，##p<TI 0.01vs. p, not labeled>0.01。
FIG. 10: results of immunofluorescence double-label staining of tumorigenic tissues of mice of each group with CD41 and CD62P (400 ×). Group2 is a control Group, Group3 is a Group administered with aspirin in the whole course, Group 4 is a Group administered with ticagrelor in the whole course, and Group 5 is a Group administered with aspirin and ticagrelor in the whole course.
FIG. 11: immunohistochemical staining results (400 ×) were obtained for each group of mice. The meanings of the TI group, the TI + Aspirin group, the TI + TiCagrelalor group, the TI + Aspirin + TiCagrelalor group and the TI are the same as those in the previous figure.
FIG. 12: representative picture of staining location (400 ×), GULT1 (No. 5-20, 400 ×, cell membrane staining).
FIG. 13 is a photograph showing a representative staining site (400X), HIF1- α (No. 3-2, 400X, cell nucleus stained).
FIG. 14: representative picture of stained sites (400X), ALDH-1 (No. 3-1, 400X, cytoplasmic staining).
FIG. 15: statistical map of migration capacity of each group of cells. Control group: colon cancer cells + 10% PRP group; aspirin-low group: colon cancer cells + 10% PRP + aspirin low dose group; aspirin-high group: colon cancer cells + 10% PRP + aspirin high dose group; ticagrelir group: colon cancer cells + 10% PRP + ticagrelor group; aspirin + TiCagrelir group: colon cancer cells + 10% PRP + aspirin + ticagrelor group. Note: denotes p < 0.05; denotes p < 0.01; denotes p < 0.001; denotes p < 0.0001.
FIG. 16: statistical map of the invasion capacity of each group of cells. The meaning of Control group, Aspirin-low group, Aspirin-high group, TiCagrilor group, and Aspirin + TiCagrilor group is the same as that in the previous figure. Note: denotes p < 0.05; denotes p < 0.01; denotes p < 0.001; denotes p < 0.0001.
FIG. 17: flow detection is carried out on the ALDH-1 expression condition of the tumor cells treated differently. The meaning of Control group, Aspirin-low group, Aspirin-high group, TiCagrilor group, and Aspirin + TiCagrilor group is the same as that in the previous figure. Note: denotes p < 0.05; denotes p < 0.01; denotes p < 0.001; denotes p < 0.0001.
Detailed Description
The test materials used in the following test methods are readily available from commercial companies unless otherwise specified. Many modifications may be made to the present invention by those skilled in the art in conjunction with the well-known techniques without departing from the spirit of the invention, and such modifications are intended to be within the scope of the present invention.
Example 1 experiment of influence of aspirin on microenvironment of colon cancer graft tumor
First, experimental material
Experimental animals: BALB/C nude mice, 6-8 weeks old, female, 25-30g in body weight.
Experimental cells: human colorectal cancer HCT116 cell line, in 10% fetal calf serum RPMI1640 culture medium at 37 deg.C and 5% CO2Culturing, carrying out passage 1 time for 2-3d, and taking cells in logarithmic growth phase for experiment.
The preparation of the medicine comprises the following steps: (1) aspirin dissolved in 0.9% physiological saline solution to prepare 1.0mg/ml solution. (2) Ticagrelor dissolved in 500ml of 0.9% physiological saline to prepare a 1mg/ml solution.
Second, grouping experiments
1. Blank group: tumor-free nude mice + no drug treatment
2. Control group: transplanted tumor + no-drug treatment
3. The aspirin whole course administration group comprises: tumor transplantation + treatment by aspirin gavage before, during and after inoculation.
4. Ticagrelor whole course administration group: transplanted tumor + pre-inoculation, mid-post-inoculation ticagrelor gavage treatment.
5. Aspirin + ticagrelor full administration group: performing intragastric administration on transplanted tumors, aspirin before and after inoculation and ticagrelor; the mass ratio of aspirin to ticagrelor is 5: 1.
the administration scheme is as follows: the two medicines are both administered with the medicine for intragastric administration every day at 7 days before the inoculation day, and the medicine for intragastric administration is continued on and after the inoculation day; the daily gavage dose of aspirin is 50 mg/kg/d; the daily gavage dosage of ticagrelor was 10 mg/kg/d.
Tumor inoculation: taking several BALB/C nude mice, injecting HCT116 single cell suspension subcutaneously in right groin, the number of which is 5 × 106And (4) respectively.
Example 2 weight Change in mice
First, experiment method
Feeding and drug treatment were performed according to the experimental set-up and the change in body weight of the mice was measured and recorded. Second, experimental results
FIG. 1 and FIG. 2 show the body weight changes of the mice in each group from 7 to 31 days after tumor inoculation.
The results show that there was no significant difference in body weight between groups of mice 7-17d after tumor inoculation compared to the blank group of normal mice (p > 0.05); at 21d, the control group (TI) had significantly reduced body weight (p < 0.05); at 24d, the control group (TI) and Ticagrelor whole course dosing group (TI + Ticagrelor) had significantly reduced body weight (p < 0.05); at 28d, the control group (TI group), Aspirin whole course dosing group (TI + Aspirin) and Ticagrelor whole course dosing group (TI + Ticagrelor) significantly reduced body weight (p < 0.05); at 31d, the control group (TI group) and the Aspirin whole course dosing group (TI + Aspirin) were significantly reduced in body weight (p < 0.05). There was no significant difference in body weight (p >0.05) for each drug-treated group compared to the control group (TI).
Example 3 evaluation of transplanted tumor microvascular morphology and function
First, preparation of experiment
Each experimental group was randomly selected from 6 nude mice. TRITC-dextran was injected into the tail vein at 100 mg/kg. FITC-lectin 10mg/kg was injected into the tail vein after 3 h.
Second, Experimental methods
10min after the injection of the drug, the mice are killed by cervical spondylolysis, tumor masses are taken out, the sections (6 mu m) are immediately frozen, the frozen sections are observed under a laser confocal microscope, 5 non-repetitive visual fields are randomly selected for each section under a high power lens (x 400) to acquire images, the blood vessel morphology is evaluated through FITC-lectin green fluorescence, and the blood vessel permeability is evaluated through the proportion of the area of TRITC-dextran leaked to the outside of the blood vessel to the total area.
Third, experimental results
Fig. 3 shows the morphology of the transplanted tumor microvasculature in each group of mice, and fig. 4 shows the number of blood vessel branches and the total length of blood vessels in each group of mice.
The results showed that compared to the model control group (TI) mice, the Aspirin whole course dosing group (TI + Aspirin) mice had an insignificant increase in the number of transplanted tumor vessel branches and total length of vessels (p >0.05), and the Ticagrelor whole course dosing group (TI + Ticagrelor) and the Aspirin and Ticagrelor combined whole course dosing group (TI + Aspirin + Ticagrelor) mice had an insignificant decrease in the number of transplanted tumor vessel branches and total length of vessels (p > 0.05).
The vascular permeability is evaluated by the proportion of the area of TRITC-dextran leaked to the outside of blood vessels to the total area, and pictures show that the vascular leakage of transplanted tumors of mice in a model control group (TI) is obvious, the vascular leakage of transplanted tumors of mice in an Aspirin whole course administration group (TI + Aspirin) and a Ticagrelor whole course administration group (TI + TiCagrel) is reduced, and the vascular leakage of transplanted tumors of mice in an Aspirin and Ticagrelor whole course administration group (TI + Aspirin + TiCagrel) is obvious.
Example 4 detection of platelet activation Rate
First, preparation of experiment
For each experimental group, 6 mice were randomly selected to puncture for blood sampling, and the blood sampling was placed in an anticoagulant tube containing EDTA to detect the platelet activation rate.
Second, Experimental methods
2 tubes were taken and T1 tubes were charged with 10. mu.L of FITC-CD41 antibody and 10. mu.L of PE-CD62p antibody. Adding corresponding isotype control antibody in a T1 test tube into a C1 test tube, adding 0.5mL of whole blood into 2 test tubes respectively, uniformly mixing, incubating for 20min in the dark at room temperature, adding 1.5mL of PBS buffer solution, uniformly mixing, centrifuging, discarding supernatant, adding 1mL of 1% paraformaldehyde with precooling at 4 ℃ and 3 muL of Triton-100 respectively, keeping out of the light, cleaning completely before loading, and loading on a machine for detection within 24 h.
Third, experimental results
FIG. 5 is a graph showing the results of platelet activation rates in each group of mice by flow assay, and FIGS. 6, 7, 8 and 9 are statistical graphs of platelet activation rates in each group of mice. CD41+Cell fraction + CD62+The cell ratio is the ratio of activated platelet cells.
The results show that the platelet activation rates of the model control group (TI), the Aspirin whole course dosing group (TI + Aspirin) and the Ticagrelor whole course dosing group (TI + Ticagrelor) are significantly reduced (p <0.05) compared with the blank group normal mice after tumor inoculation; the platelet activation rate of the Aspirin and Ticagrelor combined whole-course administration group (TI + Aspirin + TiCagrel) has no significant difference (p > 0.05).
Compared with a model control group (TI) mouse, the platelet activation rates of an Aspirin whole course administration group (TI + Aspirin) and a Ticagrelor whole course administration group (TI + Ticagrelor) are improved, but the difference is not significant (p is more than 0.05); the platelet activation rate of the Aspirin and Ticagrelor combined whole-course administration group (TI + Aspirin + TiCagrel) is remarkably improved (p is less than 0.01).
The results show that when aspirin and ticagrelor are independently administered, the reduction of the platelet activation rate of mice caused by tumor inoculation is relieved, but is not obvious; and the aspirin and the ticagrelor can be used for remarkably relieving the reduction of the platelet activation rate of mice caused by tumor inoculation after the aspirin and the ticagrelor are jointly administered in the whole process.
Example 5 immunofluorescence detection of platelet distribution at tumor site
First, preparation of experiment
Each experimental group randomly selected 6 mice, extracted tumor tissue, and placed on a glass slide for later use.
Second, Experimental methods
1. Serum blocking: dropping normal goat serum on the tissue part of the slide, and sealing at room temperature for 30 min.
2. The blocking solution was aspirated off the absorbent paper, washed away, and FITC-CD41 antibody was added to the sections, incubated overnight at 4 ℃ and washed 3 times with PBS for 5min each.
3. Non-immune serum was added dropwise and incubated at 37 ℃ for 10 minutes.
4. The PE-CD62p antibody was added dropwise and incubated overnight at 4 ℃. PBS was washed 3 times for 5min each.
5. DAPI counterstained nuclei.
6. And (5) sealing by using a water-soluble sealing agent.
Second, experimental results
FIG. 10 shows the results of immunofluorescence double-label staining of tumor-forming tissues of various groups of mice. CD62P expression was reduced in Group3-5 compared to Group2, with Group 5 expressing the lowest. Group3-5 showed increased expression of CD41 compared to Group2, with Group 5 showing the highest expression level.
CD62P is a marker for the level of platelet activation and CD41 is a marker for the overall level of platelets. As can be seen from the results of immunofluorescence staining of the groups of fig. 10, aspirin and ticagrelor inhibited platelet activation and promoted platelet aggregation at the tumor site.
Example 6 detection of hypoxia, carbohydrate metabolism and tumor Stem cell-associated markers in transplanted tumors (immunohistochemistry)
First, preparation of experiment
And 6 mice are randomly selected from each experimental group, a tumor part tissue sample is extracted, and the positive piece is mouse liver tissue. The samples are firstly preserved in neutral formalin after being extracted, and then the samples are subjected to conventional dehydration operation and are embedded by paraffin after the conventional dehydration operation is finished.
Second, Experimental methods
1. Tissue sections were removed and hydrated by dewaxing with xylene and gradient alcohol.
2、3％H2O2Action 10min, to block endogenous peroxidase; washing with distilled water, and soaking in PBS for 3 min.
3. Antigen retrieval: placing the processed slices into a pressure cooker containing citrate buffer solution (pH 6.0), timing for 2.5min from the beginning of steam release by adopting a high-pressure repairing method, naturally cooling, and spraying tap water to room temperature.
4. Washing with distilled water for 3min × 3 times, and washing with PBS for 3min × 3 times; sealing the normal sheep serum working solution, and incubating at room temperature for 30-40 min.
5. Adding primary antibody (1: 100) dropwise, and incubating overnight in a refrigerator at 4 ℃; PBST washing for 3min × 3 times; dripping corresponding secondary antibody at room temperature for 30 min; PBST washing 3min x 3 times.
6. DAB solution color development; hematoxylin counterstaining, washing with tap water, performing hydrochloric acid alcohol differentiation, and returning blue; and sealing the sheet after dehydration and transparency.
7. And (5) observing and photographing under a microscope.
Third, experimental results
Fig. 11 is a picture of the immunohistochemical staining results of the mice in each group, table 1 is an evaluation of the immunohistochemical staining results of the mice in each group, and fig. 12, 13, and 14 are representative pictures of the partial staining positions.
The result shows that Glut1 is mainly localized on the cell membrane of tumor cells, and the tumor cells close to the edge of the tumor envelope have stronger expression compared with the cells in the interior of the tumor, HIF1- α is mainly localized on the cell nucleus of the tumor cells, the cytoplasm of partial cells is stained, the expression level of ALDH-1 is lower, a small part of tumor cells are positive to cytoplasm, and a part of cells in blood vessels are unknown positive.
Compared with a model control group (TI) mouse, the expressions of the hypoxia marker (HIF-1 α), the glucose metabolism marker (GLUT-1) and the tumor stem cell marker (ALDH-1) of the tumor in the Aspirin whole-course administration group (TI + Aspirin), the Ticagrelor whole-course administration group (TI + TiCagrel) and the combination of Aspirin and Ticagrelor whole-course administration group (TI + Aspirin + TiCagrel) are reduced, but the three treatment groups have no significant difference.
The results show that the expression of HIF-1 α, GLUT-1 and ALDH-1 of tumors can be reduced by singly administering aspirin, singly administering ticagrelor and jointly and completely administering the aspirin and the ticagrelor, and tumors can be inhibited.
TABLE 1 evaluation summary of immunohistochemical staining results
Example 7 Effect of Aspirin on Colon cancer cell invasion and migration potential in Co-culture System
First, experiment grouping
1. Group of colon cancer cells: RPMI1640 medium + 10% FBS + 10% PRP (platelet rich plasma) group.
2. Colon cancer cells + 10% PRP + aspirin low dose group: RPMI1640 medium + 10% FBS + 10% PRP + 50. mu.M aspirin.
3. Colon cancer cells + 10% PRP + aspirin high dose group: RPMI1640 medium + 10% FBS + 10% PRP +300 μ M aspirin.
4. Colon cancer cells + 10% PRP + ticagrelor group: PRMI1640 medium + 10% FBS + 10% PRP + 20. mu.M ticagrelor
5. Colon cancer cells + 10% PRP + aspirin group + ticagrelor group: PRMI1640 culture medium, 10% FBS, 10% PRP, 200 mu M aspirin and 20 mu M ticagrelor II and experimental method
1. Transwell migration detection
(1) Transwell chamber preparation.
(2) After 48h of infection, colon cancer cells were prepared as cell suspensions. Adjusting cell density to 1X 106/mL。
(3) Inoculating cells; adding 200 mu L of cell suspension into a Transwell chamber; the lower chamber was filled with 500. mu.L of 10% serum medium at 37 ℃ CO2The incubator is used for 24 h.
(4) The Transwell chamber was removed, the culture medium was discarded, washed 2 times with PBS, fixed with formaldehyde for 30min, and the chamber was air-dried. Stained with 0.1% crystal violet for 20min and washed 3 times with PBS.
(5) And (5) photographing and observing.
2. Transwell invasion detection
(1) The Matrigel was thawed and thawed at 4 ℃.
(2) According to a serum-free culture medium: matrigel ═ 7: 1 as a glue layer, dripping 50 mu L of glue into each hole of the small chamber, and standing for 1-2 h at 37 ℃.
(3) The working solution was prepared in the upper and lower layers, 200. mu.L of serum-free medium was prepared in the upper layer (i.e., in the chamber), and 500. mu.L of 10% serum medium was prepared in the lower layer.
(4) After the Matrigel had solidified, the culture medium without solidification in the supernatant was removed and aspirated.
(6) And adding the lower layer working solution into the lower layer 24-hole plate, placing the lower layer 24-hole plate into the small chamber, and adding the upper layer working solution.
(7) After leaving for about 20 hours, the culture medium in the chamber was discarded, and washed with PBS 2 times.
(8) Removing the Matrigel and the cells on the upper chamber surface, fixing for 20min by methanol, dyeing for 15-20 min by 0.1% crystal violet, and washing for more than 3 times by clear water.
(9) And (5) photographing and observing.
3. Detection of colon cancer tumor stem cell marker ALDH-1
(1) Digesting the cells with 0.25% pancreatin, collecting after terminating digestion, centrifuging at 1500rpm for 5min, discarding the supernatant, and collecting the cells.
(2) The cells were resuspended 2 times in pre-cooled PBS, centrifuged at 1500rpm for 5min, and washed.
(3) Is prepared to have a concentration of 25X 106Taking 50 mu L of cell suspension, adding 10% PRP respectively, and obtaining the final platelet concentration of 200X 106ml, then add aspirin with corresponding concentration, and react for 15min under magnetic bar stirring.
(4) CD61-FITC labeling: after 5. mu.L of CD41a-PE was added and mixed well, the mixture was incubated for 15min at room temperature in the dark.
(5) CD31-FITC labeling: add 5. mu.L of CD31-FITC and mix well, avoid light, incubate for 15min at room temperature.
(6) The cells were resuspended 2 times in pre-cooled PBS, centrifuged at 1500rpm for 5min, and washed.
(7) Adding 500. mu.L PBS to re-suspend into single cell suspension, detecting by flow cytometry, and analyzing by software.
Third, experimental results
1. Transwell detection of colon cancer cell migration capacity
FIG. 15 is a statistical chart of cell migration ability. The results show that compared with the Control group, the cell migration inhibition effect is achieved at both low and high aspirin concentrations in 24-hour cell treatment time, the cell migration inhibition ability at the high aspirin concentration is higher than that in the low aspirin concentration group, and the ticagrelor inhibition effect is better than that in the aspirin concentration group. After 48 hours of cell treatment, the effect of inhibiting the cell migration capacity of each treatment group is more obvious. The inhibiting effect of the aspirin group and the ticagrelor group is improved by 8.7 percent compared with that of the ticagrelor group alone.
2. Transwell detection of colon cancer cell invasion capacity
FIG. 16 is a statistical chart of cell invasion capacity. The results show that compared with the Control group, the cell invasion inhibiting effect is achieved at both low and high aspirin concentrations in 24-hour cell treatment time, the cell invasion inhibiting ability at the high aspirin concentration is higher than that in the low aspirin concentration group, and the ticagrelor inhibiting effect is better than that in the aspirin low aspirin concentration group. After 48 hours of cell treatment time, the effect of inhibiting cell invasion capacity of each treatment group was consistent with 24 hours. The inhibiting effect of the aspirin group and the ticagrelor group is improved by 6.8 percent compared with that of the ticagrelor group alone.
3. Detection of colon cancer tumor stem cell marker ALDH-1
FIG. 17 shows the concentration of 25X 106The tumor cells are subjected to different treatments and then flow-detection is carried out on the statistical result of ALDH-1 positive rate. The results of the two graphs show that aspirin can inhibit the expression of the ALDH-1 protein at both the treatment time of 24h and the treatment time of 48h, the high-dose aspirin has better effect of inhibiting the expression of the ALDH-1 protein than the low-dose aspirin, and the treatment time of 48h has better effect than the treatment time of 24 h. The ticagrelor group shows that the effect of ticagrelor on inhibiting ALDH-1 is obvious in 24h and 48h treatment time, and the effect of the 48h treatment time is better than that of the 24h treatment time. The inhibiting effect of the aspirin group and the ticagrelor group is better than that of the ticagrelor group aloneThe group was increased by another 10.3%.
Claims (1)
1. Use of aspirin in the manufacture of a medicament for the prevention or treatment of colon cancer or a patient at risk of colon cancer,
the dosage of the aspirin in the patient is 50 mg/kg/d; or the content of the aspirin in the medicine is 200 mu M;
the medicament further comprises a platelet aggregation inhibitor selected from ticagrelor;
the amount of said platelet aggregation inhibitor in said patient is 10 mg/kg/d; alternatively, the amount of the platelet aggregation inhibitor in the drug is 20 μ M;
in the medicine, the mass ratio of the aspirin to the ticagrelor is 5: 1; or the molar ratio of the two is 10: 1.
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| CN101229141A (en) * | 2008-02-29 | 2008-07-30 | 常州制药厂有限公司 | Aspirin sustained release tablet and preparing method thereof |
| CN104950112A (en) * | 2015-06-11 | 2015-09-30 | 浙江大学 | Application of FasL protein serving as biomarker to qualitative Aspirin sensitivity detection of colorectal cancer stem cell |
| CN104971070A (en) * | 2014-04-04 | 2015-10-14 | 北京大学 | Oral nano composition of ticagrelor |
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| CN101229141A (en) * | 2008-02-29 | 2008-07-30 | 常州制药厂有限公司 | Aspirin sustained release tablet and preparing method thereof |
| CN104971070A (en) * | 2014-04-04 | 2015-10-14 | 北京大学 | Oral nano composition of ticagrelor |
| CN104950112A (en) * | 2015-06-11 | 2015-09-30 | 浙江大学 | Application of FasL protein serving as biomarker to qualitative Aspirin sensitivity detection of colorectal cancer stem cell |
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