Application of the aspirin in colon cancerous precaution or treatment
Technical field
The present invention relates to biomedicine fields, and in particular to application of the aspirin in colon cancerous precaution or treatment.
Background technique
Aspirin (Aspirin, acetylsalicylic acid) is salicylic derivative, material property be a kind of white crystals or
Crystalline powder, odorless or micro-strip acetic acid is smelly, is slightly soluble in water, is soluble in ethyl alcohol, dissolves in ether, chloroform, aqueous solution is in acidity.
Early in 1853, people synthesized acetylsalicylic acid by salicylic acid and acetic anhydride for the first time.Between several years, people gradually have found acetyl
Salicylic acid has the effects that good anti-inflammatory, antipyretic analgesic.Until 1899, acetylsalicylic acid started to be widely applied to face
Bed, and it is renamed as aspirin (Aspirin).
Aspirin was after clinical application in over one hundred year, it has proved that alleviating slight or moderate pain, such as toothache, head
Bitterly, the effect of neuralgia, DOMS, arthralgia and dysmenorrhea is preferable, is equally used for bringing down a fever for the fever diseases such as flu, influenza
Deng.The derivative drug of aspirin emerges one after another at present, is widely used in the clinical treatment of antipyretic analgesic.Discovered in recent years Ah Si
Woods has inhibiting effect to platelet aggregation, can prevent the formation of thrombus, clinically for preventing transient ischemic attack, the heart
The formation of thrombus after flesh infarct, heart valve prosthesis and venous fistula or other operations.
Colon cancer is the common malignant tumor of digestive tract for betiding colon site, and usually morbidity is in rectum and sigmoid colon
Intersection.With the disease incidence highest of 40~50 years old age group, the ratio between men and women is 2~3:1.Disease incidence accounts for the of gastroenteric tumor
3.Colon cancer is broadly divided into gland cancer, mucinous adenocarcinoma, undifferentiated carcinoma.General form is in polypoid, ulcer type etc..Colon cancer can edge
Intestinal wall, which is gone in ring, to be developed, and is indulged diameter or more sprawling along intestinal tube or is infiltrated to intestinal wall deep layer, except through lymphatic vessel, blood flow shifts and local Invasion
It outside, can also be to intraperitoneal plantation or along suture, cut sides diffusion transfer.Patients with chronic colitis, polyp of colon patient, Nan Xingfei
Fat persons etc. are Susceptible population.
In recent years, people start to find that aspirin has certain clinical effectiveness in preventing, treating for Partial tumors.
But aspirin is for also rarely having document report in the preventing, treating of colon cancer.Aspirin is studied in the present invention to tie
Intestinal cancer prevention or application method and therapeutic effect in treatment, provide a new thinking for clinical treatment colon cancer.
Summary of the invention
The present invention aiming at the shortcomings in the prior art, is related to application of the aspirin in treatment of colon cancer.
The present invention provides aspirin in preparation for preventing or treating with colon cancer or with colon cancer risk
Purposes in the drug of patient.
In an aspect, dosage of the aspirin in the patient is 10~100mg/kg/d;It is preferred that 50mg/kg/d.
In an aspect, content of the aspirin in the drug is 20~500 μM;It is preferred that 50~300 μM.
In an aspect, the drug further includes platelet aggregation inhibitor;It is preferred that ticagrelor.
In an aspect, dosage of the platelet aggregation inhibitor in the patient is 5~50mg/kg/d;It is excellent
Select 10mg/kg/d.
In an aspect, content of the platelet aggregation inhibitor in the drug is 5~50 μM;It is preferred that 20 μM.
In an aspect, in the drug, the quality proportioning of the aspirin and both ticagrelors is
(10~2):1, preferably 5:1, or both mol ratio is (20~5):1, preferably 10:1.
In an aspect, after the patient receives the drug, colon cancer tumours are suppressed.
In an aspect, after the patient receives the drug, the reduction degree of the patient's weight slows down, described
The degree that Platelet activation rate reduces is alleviated, and the expression of HIF-1 α, GLUT-1 and ALDH-1 are lowered in the patient, described
The transfer ability and invasive ability of the tumour cell of patient are suppressed.
In an aspect, the patient is selected from people or non-human animal;It is preferred that non-human animal;More preferable mouse.
The present invention provides a kind of pharmaceutical compositions, and it includes aspirin and pharmaceutically acceptable carrier.
In an aspect, the content of the aspirin is 20~500 μM;It is preferred that 50~300 μM.
In an aspect, described pharmaceutical composition also includes platelet aggregation inhibitor;It is preferred that ticagrelor.
In an aspect, content of the platelet aggregation inhibitor in described pharmaceutical composition is 5~50 μM;It is excellent
Select 20 μM.
In an aspect, in described pharmaceutical composition, the quality of both the aspirin and described ticagrelor
Proportion is (10~2):1, preferably 5:1, or both mol ratio is (20~5):1, preferably 10:1.
In an aspect, pharmaceutical preparation can be made in described pharmaceutical composition according to conventional methods.In production process, preferably
The aspirin is mixed with pharmaceutically acceptable carrier or is diluted with carrier.It when the carrier serves as a diluent, can be with
For solid, semisolid or liquid.Preparation is selected from tablet, pill, pulvis, capsule, suspension, emulsion, solution, aerosol, note
It penetrates with forms such as solution.
The positive effect of the present invention includes:Aspirin can inhibit platelet activation, receive the shifting of aspirin for treatment
Tumor mouse is planted, compared to the control group, the reduction degree of weight slows down, and the degree that platelet activation rate reduces is alleviated, HIF-1 α,
The expression of GLUT-1 and ALDH-1 is lowered, and the transfer ability and invasive ability of tumour cell are suppressed.Present invention research Ah Si
Woods studies influence of the aspirin to colon cancer medicinal effects, existing to the bioelectric detecting mechanism of colon cancer tumours microenvironment
Have in technology and be rarely reported, this has opened up new Research Prospects for the treatment of colon cancer, is depth research and development and the knot of aspirin
The clinical treatment of intestinal cancer provides new thinking.
Detailed description of the invention
Fig. 1:The changes of weight of each group mouse 7-31d after tumor inoculation.Control group is blank group, i.e., is not inoculated with swollen
Tumor+without drug-treated;TI group is control group, i.e. transplantable tumor+drug-treated is not added;TI+Aspirin group is that aspirin is complete
Journey administration group, i.e., before transplantable tumor+transplanting in after aspirin stomach-filling processing;TI+Ticagrelor group be ticagrelor whole process to
Medicine group, i.e., before transplantable tumor+transplanting in after ticagrelor stomach-filling processing;TI+Aspirin+Ticagrelor group is aspirin+replace
Ge Ruiluo whole process administration group, i.e., before transplantable tumor+transplanting in after the processing of aspirin+ticagrelor stomach-filling.TI represents tumor
inoculation。
Fig. 2:The changes of weight of each group mouse 7-31d after tumor inoculation.Control group, TI group, TI+Aspirin group,
TI+Ticagrelor group, TI+Aspirin+Ticagrelor group, the meaning of TI are consistent with preceding figure.Note:*p<
0.05vs.Control,#p<0.05vs.TI, not marking indicates p>0.05.
Fig. 3:Each group mice-transplanted tumor Microvascular architecture (400 ×).TI group, TI+Aspirin group, TI+Ticagrelor
Group, TI+Aspirin+Ticagrelor group, the meaning of TI are consistent with preceding figure.
Fig. 4:Each group mice-transplanted tumor vessel branch number and blood vessel total length.TI group, TI+Aspirin group, TI+
Ticagrelor group, TI+Aspirin+Ticagrelor group, the meaning of TI are consistent with preceding figure.
Fig. 5:Each group mouse flow cytometer detection platelet activation rate result.Control group, TI group, TI+Aspirin group, TI+
Ticagrelor group, TI+Aspirin+Ticagrelor group, the meaning of TI are consistent with preceding figure.
Fig. 6:Each group mouse platelets activation rate.Control group, TI group, TI+Aspirin group, TI+Ticagrelor group,
TI+Aspirin+Ticagrelor group, the meaning of TI are consistent with preceding figure.Note:*p<0.05vs.Control, * * p<
0.01vs.Control,#p<0.05vs.TI, not marking indicates p>0.01.
Fig. 7:Each group mouse platelets activation rate.Control group, TI group, TI+Aspirin group, TI+Ticagrelor group,
TI+Aspirin+Ticagrelor group, the meaning of TI are consistent with preceding figure.Note:*p<0.05vs.Control, * * p<
0.01vs.Control,#p<0.05vs.TI, not marking indicates p>0.01.
Fig. 8:Each group mouse platelets activation rate.Control group, TI group, TI+Aspirin group, TI+Ticagrelor group,
TI+Aspirin+Ticagrelor group, the meaning of TI are consistent with preceding figure.Note:*p<0.05vs.Control, * * p<
0.01vs.Control,##p<0.01vs.TI, not marking indicates p>0.01.
Fig. 9:Each group mouse platelets activation rate.Control group, TI group, TI+Aspirin group, TI+Ticagrelor group,
TI+Aspirin+Ticagrelor group, the meaning of TI are consistent with preceding figure.Note:*p<0.05vs.Control, * * p<
0.01vs.Control,##p<0.01vs.TI, not marking indicates p>0.01.
Figure 10:Each group mouse is at tumor tissue CD41 and CD62P Double immunofluorescence coloration result (400 ×).Group 2 is
Control group, Group 3 are aspirin whole process administration group, and Group 4 is ticagrelor whole process administration group, and Group 5 is Ah Si
Woods+ticagrelor whole process administration group.
Figure 11:Each group mouse immune histochemical staining result (400 ×).TI group, TI+Aspirin group, TI+Ticagrelor
Group, TI+Aspirin+Ticagrelor group, the meaning of TI are consistent with preceding figure.
Figure 12:Dyeing site representativeness picture (400 ×), GULT1 (number 5-20,400 ×, cell membrane coloring).
Figure 13:Dyeing site representativeness picture (400 ×), HIF1- α (number 3-2,400 ×, nucleus coloring).
Figure 14:Dyeing site representativeness picture (400 ×), ALDH-1 (number 3-1,400 ×, cytoplasm coloring).
Figure 15:Group of cells transfer ability statistical chart.Control group:Colon cancer cell+10%PRP group;Aspirin-
Low group:Colon cancer cell+10%PRP+ aspirin low dose group;Aspirin-high group:Colon cancer cell+10%PRP+
Aspirin high dose group;Ticagrelor group:Colon cancer cell+10%PRP+ ticagrelor group;Aspirin+
Ticagrelor group:Colon cancer cell+10%PRP+ aspirin group+ticagrelor group.Note:* p is indicated<0.05;* indicates p<
0.01;* * indicates p<0.001;* * * indicates p<0.0001.
Figure 16:Group of cells invasive ability statistical chart.Control group, Aspirin-low group, Aspirin-high group,
Ticagrelor group, the meaning of Aspirin+Ticagrelor group are consistent with preceding figure.Note:* p is indicated<0.05;* indicates p<
0.01;* * indicates p<0.001;* * * indicates p<0.0001.
Figure 17:Flow cytometer detection different disposal tumour cell ALDH-1 expression.Control group, Aspirin-low group,
Aspirin-high group, Ticagrelor group, the meaning of Aspirin+Ticagrelor group are consistent with preceding figure.Note:* p is indicated<
0.05;* indicates p<0.01;* * indicates p<0.001;* * * indicates p<0.0001.
Specific embodiment
Experimental material used in following experimental methods unless otherwise instructed, can be obtained easily from commercial company.?
In the case where spirit of that invention, those skilled in the art combine well-known technique, and many modifications can be made to the present invention,
Such modification is also fallen within protection scope of the present invention.
The experiment of embodiment 1, aspirin to colon cancer transplantable tumor microenvironment
One, experimental material
Experimental animal:BALB/C nude mouse, 6-8 week old, female, weight 25-30g.
Experimental cell:Human large intestine cancer HCT116 cell line is placed in 10% fetal calf serum RPMI1640 culture solution, 37 DEG C,
5%CO2Culture, 2-3d are passed on 1 time, and logarithmic growth phase cell is for testing.
Medicine preparation:(1) aspirin is dissolved in 0.9% physiological saline, is configured to 1.0mg/ml solution.(2) auspicious for lattice
Lip river is dissolved in 0.9% physiological saline 500ml, is configured to 1mg/ml solution.
Two, experimental group
1, blank group:The not nude mice of inoculated tumour+without drug-treated
2, control group:Transplantation tumor+drug-treated is not added
3, aspirin whole process administration group:Aspirin stomach-filling processing after in front of transplantation tumor+inoculation.
4, ticagrelor whole process administration group:Ticagrelor stomach-filling processing after in front of transplantation tumor+inoculation.
5, aspirin+ticagrelor whole process administration group:Aspirin+ticagrelor fills after in front of transplantation tumor+inoculation
Stomach processing;The quality proportioning of aspirin and ticagrelor is 5:1.
Dosage regimen:Two kinds of drugs inoculation a few days ago 7d start to give drug stomach-filling daily, in inoculation day on the day of and it
Afterwards, continue to give drug stomach-filling;The daily given low of aspirin is 50mg/kg/d;The daily given low of ticagrelor is
10mg/kg/d。
Tumor inoculation:BALB/C nude mice several are taken, HCT116 single cell suspension, quantity is subcutaneously injected in right side groin
It is 5 × 106It is a.
Embodiment 2, mouse weight variation
One, experimental method
It is set empirically and carries out raising and drug-treated, measure and record mouse weight variation.Two, experimental result
Fig. 1, Fig. 2 are the changes of weight of each group mouse 7-31d after tumor inoculation.
The results show that comparing blank group normal mouse, the 7-17d after tumor inoculation, each group mouse weight is without significant difference
(p>0.05);When 21d, control group (TI) weight is substantially reduced (p<0.05);When 24d, control group (TI) and ticagrelor are whole
Administration group (TI+Ticagrelor) weight is substantially reduced (p<0.05);When 28d, control group (TI group), the administration of aspirin whole process
Group (TI+Aspirin) and ticagrelor whole process administration group (TI+Ticagrelor) weight are substantially reduced (p<0.05);When 31d,
Control group (TI group) and aspirin whole process administration group (TI+Aspirin) weight are substantially reduced (p<0.05).Compared to the control group
(TI), each drug-treated group weight is without significant difference (p>0.05).
Embodiment 3, transplantable tumor Microvascular architecture and functional assessment
One, Preparatory work of experiment
Each experimental group randomly selects 6 nude mices respectively.Tail vein injection TRITC-dextran 100mg/kg.3h rear molding is quiet
Arteries and veins injects FITC-lectin 10mg/kg.
Two, experimental method
After drug injection terminates 10min, cervical dislocation method puts to death mouse, takes out tumor mass, immediately frozen section (6 μm), laser
Frozen section is observed under Laser Scanning Confocal Microscope, every slice randomly selects 5 unduplicated visuals field under high power lens (× 400) and adopts
Collect image, by FITC-lectin green fluorescence assessment of vessel morphology, by leaking into extravascular TRITC-dextran's
Area accounts for the ratio of the gross area, assesses vascular permeability.
Three, experimental result
Fig. 3 is each group mice-transplanted tumor Microvascular architecture, and Fig. 4 is each group mice-transplanted tumor vessel branch number and blood vessel total length.
The results show that comparing model control group (TI) mouse, aspirin whole process administration group (TI+Aspirin) mouse is moved
Plant tumor vessel branch number and the non-limiting raising (p of blood vessel total length>0.05), ticagrelor whole process administration group (TI+
Ticagrelor) and aspirin and ticagrelor combine whole administration group (TI+Aspirin+Ticagrelor) mouse transplanting
Tumor vessel branch number and the non-limiting reduction (p of blood vessel total length>0.05).
Area by leaking into extravascular TRITC-dextran accounts for the ratio of the gross area, assesses Vascular permeability situation,
Picture display model control group (TI) mice-transplanted tumor vascular leakage is obvious, aspirin whole process administration group (TI+Aspirin) and
Ticagrelor whole process administration group (TI+Ticagrelor) mice-transplanted tumor vascular leakage mitigates, aspirin and ticagrelor connection
It is obvious to close whole administration group (TI+Aspirin+Ticagrelor) mice-transplanted tumor vascular leakage.
The detection of embodiment 4, platelet activation rate
One, Preparatory work of experiment
Each experimental group randomly selects 6 mouse punctures respectively and takes blood, is placed in anticoagulant tube containing EDTA living to detect blood platelet
Rate.
Two, experimental method
2 test tubes are taken, 10 μ L FITC-CD41 antibody and 10 μ L PE-CD62p antibody are added in T1 test tube.Add in C1 test tube
Enter corresponding isotype control Ab in T1 test tube, 0.5mL whole blood is added into 2 test tubes respectively, is protected from light and incubates at room temperature after mixing
20min is educated, 1.5mL PBS buffer solution is added, is mixed, centrifugation is abandoned supernatant and is separately added into again containing 4 DEG C of 1% paraformaldehyde 1mL of pre-cooling
+ 3 μ L Triton-100, are protected from light, will clean up before upper machine, upper machine testing interior for 24 hours.
Three, experimental result
Fig. 5 is each group mouse flow cytometer detection platelet activation rate result figure, and Fig. 6, Fig. 7, Fig. 8, Fig. 9 are that each group mouse blood is small
Plate activation rate statistical chart.CD41+Cell proportion+CD62+Cell proportion is activated blood platelet cell proportion.
The results show that comparing blank group normal mouse, after tumor inoculation, model control group (TI), aspirin are whole
Administration group (TI+Aspirin) and ticagrelor whole process administration group (TI+Ticagrelor) platelet activation rate significantly reduce (p<
0.05);Aspirin and ticagrelor combine whole administration group (TI+Aspirin+Ticagrelor) platelet activation rate without aobvious
Write difference (p>0.05).
Compared to model control group (TI) mouse, aspirin whole process administration group (TI+Aspirin) and ticagrelor whole process are given
Medicine group (TI+Ticagrelor) platelet activation rate increases, but the not significant (p of difference>0.05);Aspirin and replace lattice
Rui Luo combines the extremely significant raising (p of whole administration group (TI+Aspirin+Ticagrelor) platelet activation rate<0.01).
The result shows that when aspirin and ticagrelor are respectively administered alone, to mouse platelets caused by tumor inoculation
Activation rate reduction has relaxation effect, but not significant;And aspirin and the whole administration of ticagrelor joint can significantly alleviate tumour
Mouse platelets activation rate caused by being inoculated with reduces.
Embodiment 5, the distribution of Immunofluorescence test tumor locus blood platelet
One, Preparatory work of experiment
Each experimental group randomly selects 6 mouse respectively, extracts tumor locus tissue, is placed on slide, spare.
Two, experimental method
1, serum is closed:Normal Goat Serum is added dropwise in slide tissue site, room temperature closes 30min.
2, blotting paper sops up confining liquid, does not wash, and FITC-CD41 antibody is added in slice, and after 4 DEG C are incubated overnight, PBS rinses 3
It is secondary, each 5min.
3, non-immune serum is added dropwise, 37 DEG C are incubated for 10 minutes.
4, PE-CD62p antibody is added dropwise, 4 DEG C are incubated overnight.PBS is rinsed 3 times, each 5min.
5, DAPI redyes nucleus.
6, water-soluble mountant mounting.
Two, experimental result
Figure 10 is the double mark coloration results of each group mouse tumor formation histogenic immunity fluorescence.Compared with Group2 group, Group3-5 group
CD62P express reduce, wherein 5 groups of expression of Group are minimum.Compared with 2 groups of Group, the CD41 expression of Group3-5 group has
Increased, wherein 5 groups of expression highests of Group.
CD62P is level of platelet activation mark, and CD41 is blood platelet aggregate level mark.From Figure 10 each group immunofluorescence
Coloration result it can be concluded that, aspirin and ticagrelor can inhibit platelet activation, and can promote tumor locus blood
Platelet aggregation.
The detection (immunohistochemistry) of embodiment 6, transplantable tumor tumor hypoxia, glycometabolism and tumor stem cell correlation marker
One, Preparatory work of experiment
Each experimental group randomly selects 6 mouse respectively, extracts tumor locus tissue samples, and positive piece is mouse liver group
It knits.The above sample is first stored in neutral formalin after extraction, and sample carries out conventional dehydrating operations later, uses stone after the completion
Wax embedding.
Two, experimental method
1, histotomy is taken, with dimethylbenzene and graded ethanol dewaxing aquation histotomy.
2,3%H2O210min is acted on, to block endogenous peroxydase;Distillation washing, PBS impregnate 3min.
3, antigen retrieval:The slice handled well, which is put into, fills citrate buffer (pH 6.0) pressure cooker, using high pressure
Repairing method, the timing 2.5min since the steam bleeding, natural cooling, tap water shower to room temperature.
4, distillation washing 3min × 3 time, PBS wash 3min × 3 time;The closing of normal sheep serum working solution, is incubated at room temperature 30-
40min。
5, primary antibody (1 is added dropwise:100), 4 DEG C of refrigerators are incubated overnight;PBST washes 3min × 3 time;Corresponding secondary antibody, room temperature is added dropwise
30min;PBST washes 3min × 3 time.
6, DAB solution develops the color;Haematoxylin is redyed, and is originally washed, and indigo plant is returned in hydrochloride alcohol differentiation;It is dehydrated transparent rear mounting.
7, microscopically observation is taken pictures.
Three, experimental result
Figure 11 is each group mouse immune histochemical staining result picture, and table 1 is each group mouse immune histochemical staining outcome evaluation,
Figure 12, Figure 13, Figure 14 are part dyeing site representativeness pictures.
The results show that Glut1 is primarily targeted for the cell membrane of tumour cell, and close to tumour by the tumour cell of film edge
There is stronger expression compared to the cell of inside tumor.HIF1- α is primarily targeted for the nucleus of tumour cell, part cell
Cytoplasm dyeing.ALDH-1 expression quantity is lower, and the cytoplasm of seldom Partial tumors cell is positive, the intravascular unknown cell positive in part.
Compared to model control group (TI) mouse, aspirin whole process administration group (TI+Aspirin), ticagrelor whole process are given
Medicine group (TI+Ticagrelor) and aspirin and ticagrelor combine whole administration group (TI+Aspirin+Ticagrelor)
The hypoxia markers (HIF-1 α) and glycometabolism label (GLUT-1) and tumor stem cell of plantation tumor mark (ALDH- in tumour body
1) expression decreases, but three treatment groups are between each other without significant difference.
The result shows that aspirin is administered alone, ticagrelor is administered alone, aspirin and ticagrelor joint are whole
Administration can lower the expression of tumour HIF-1 α, GLUT-1 and ALDH-1, inhibit tumour.
Table 1, immunohistochemical staining outcome evaluation summarize
Embodiment 7, aspirin invade colon cancer cell under co-culture system and the influence of migration potential is tested
One, experimental group
1, colon cancer cell group:RPMI1640 culture medium+10%FBS+10%PRP (platelet rich plasma) group.
2, colon cancer cell+10%PRP+ aspirin low dose group:RPMI1640 culture medium+10%FBS+10%PRP+
50 μM of aspirin.
3, colon cancer cell+10%PRP+ aspirin high dose group:RPMI1640 culture medium+10%FBS+10%PRP+
300 μM of aspirin.
4, colon cancer cell+10%PRP+ ticagrelor group:+ 10%FBS+10%PRP+20 μM of PRMI1640 culture medium replaces
Ge Ruiluo
5, colon cancer cell+10%PRP+ aspirin group+ticagrelor group:PRMI1640 culture medium+10%FBS+
+ 20 μM of ticagrelors of 10%PRP+200 μM of aspirin two, experimental method
1, Transwell migration detection
(1) cell Transwell prepares.
(2) after infecting 48h, colon cancer cell prepares cell suspension.Cell density is adjusted to 1 × 106/mL。
(3) inoculating cell;Take 200 μ L of cell suspension that the cell Transwell is added;The training of 500 μ L, 10% serum is added in lower room
Base is supported, in 37 DEG C of CO2Incubator culture is for 24 hours.
(4) cell Transwell is taken out, culture solution is discarded, PBS is washed 2 times, and the fixed 30min of formaldehyde air-dries cell.With
0.1% violet staining 20min, PBS are washed 3 times.
(5) it takes pictures observation.
2, Transwell invasion detection
(1) Matrigel thaws, 4 DEG C of defrostings.
(2) serum free medium is pressed:Matrigel=7:1 mixes as glue-line, is added drop-wise in cell with the 50 every holes μ L, in
37 DEG C of 1~2h of placement.
(3) upper and lower level working solution, upper layer (i.e. in cell) 200 μ L free serum culture basigamy cell liquid, 500 μ L of lower layer are configured
10% blood serum medium.
(4) it after Matrigel solidification, takes out and sucks the culture medium for not having solidification in supernatant.
(6) lower layer's working solution is added in 24 orifice plate of lower layer, is put into cell, upper layer working solution is added.
(7) small indoor culture solution is discarded after placing 20h or so, PBS is washed 2 times.
(8) remove the Matrigel and cell in upper chamber face, the fixed 20min of methanol, with 0.1% violet staining 15~
20min, clear water are washed 3 times or more.
(9) it takes pictures observation.
3, the detection of colon cancer tumours stem cell labeling object ALDH-1
(1) it with 0.25% trypsin digestion cell, is collected after terminating digestion, 1500rpm is centrifuged 5min, discards supernatant, and collects
Cell.
(2) it is resuspended cell 2 times with pre-cooling PBS, 1500rpm is centrifuged 5min, washs cell.
(3) being configured to concentration is 25 × 106Suspension cell, take 50 μ L cell suspensions, be separately added into 10%PRP, whole blood
Platelet concentration 200 × 106Then ml adds the aspirin of respective concentration, react 15min under bar magnet stirring.
(4) CD61-FITC is marked:It after the CD41a-PE mixing of 5 μ L is added, is protected from light, is incubated at room temperature 15min.
(5) CD31-FITC is marked:It after the CD31-FITC mixing of 5 μ L is added, is protected from light, is incubated at room temperature 15min.
(6) it is resuspended cell 2 times with pre-cooling PBS, 1500rpm is centrifuged 5min, washs cell.
(7) 500 μ L PBS are added to be resuspended into single cell suspension, flow cytomery, software analysis.
Three, experimental result
1, Transwell detects colon cancer cell transfer ability
Figure 15 is cell migration ability statistical chart.The results show that 24 hour cells handle the time compared with Control group,
Aspirin low concentration and high concentration, which have, inhibits cell migration effect, and high concentration inhibits cell migration ability higher than low dense
Degree group, ticagrelor inhibitory effect are better than aspirin high concentration group.After 48 hour cells handle the time, each processing group inhibits thin
Born of the same parents' transfer ability effect is more obvious.And aspirin group+ticagrelor group inhibitory effect is than individual ticagrelor group
8.7% is improved again.
2, Transwell detects colon cancer cell invasive ability
Figure 16 is cell invasion ability statistical chart.The results show that 24 hour cells handle the time compared with Control group,
Aspirin low concentration and high concentration, which have, inhibits cell invasion effect, and high concentration inhibits cell invasion ability higher than low dense
Degree group, ticagrelor inhibitory effect are better than aspirin low concentration group.After 48 hour cells handle the time, each processing group inhibits thin
Born of the same parents' invasive ability effect is consistent with 24 hours.And aspirin group+ticagrelor group inhibitory effect is than individually replacing lattice auspicious
Lip river group improves 6.8% again.
3, the detection of colon cancer tumours stem cell labeling object ALDH-1
Figure 17 is that concentration is 25 × 106Tumour cell flow cytometer detection ALDH-1 positive rate after different disposal statistics knot
Fruit.Two figures can inhibit ALDH-1 protein expression with 48h processing time aspirin for 24 hours as the result is shown, and high dose Ah
Department woods gets well than low-dosage aspirin inhibits ALDH-1 protein expression effect, and the 48h processing time is than effect for 24 hours
It is good.Ticagrelor group, which is shown, inhibits ALDH-1 significant effect with 48h processing time ticagrelor for 24 hours, and 48h handles time ratio for 24 hours
Effect will be got well.And aspirin group+ticagrelor group inhibitory effect improves 10.3% than individual ticagrelor group again.