CN108303552A - Detection method of the cucoline to pancreatic carcinoma SW1990 Proliferation and apoptosis - Google Patents
Detection method of the cucoline to pancreatic carcinoma SW1990 Proliferation and apoptosis Download PDFInfo
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- CN108303552A CN108303552A CN201810312223.XA CN201810312223A CN108303552A CN 108303552 A CN108303552 A CN 108303552A CN 201810312223 A CN201810312223 A CN 201810312223A CN 108303552 A CN108303552 A CN 108303552A
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/485—Morphinan derivatives, e.g. morphine, codeine
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
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- General Physics & Mathematics (AREA)
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention belongs to the pharmaceutical product technical field containing organic effective component, a kind of cucoline is disclosed to the detection method of pancreatic carcinoma SW1990 Proliferation and apoptosis, change of the cucoline to Cell Proliferation of Pancreatic Cancer Cell apoptosis is inquired by various experiment in vitro；8 methods of CCK detect various concentration gradient, the influence that cucoline survives to pancreatic cancer cell under the different role time；The influence that plate clone experiment detection cucoline forms cell colony；The influence of flow cytometry and immunoblot experiment detection cucoline to Apoptosis.Cell proliferation rate is reduced as cucoline dosage increases, and cell colony forming quantity gradually decreases, and apoptosis rate gradually increases；Western blot results show that PARP protein expression levels gradually increase.Cucoline is capable of the generation of effective inducing cell apoptosis in the proliferation that can effectively inhibit cancer of pancreas to a certain degree, shows that cucoline may play an important roll in the treatment of cancer of pancreas.
Description
Technical field
The invention belongs to containing organic effective component pharmaceutical product technical field more particularly to a kind of cucoline to pancreas
The detection method of cancerous cell line SW1990 Proliferation and apoptosis.
Background technology
Currently, the prior art commonly used in the trade is such：Cancer of pancreas is a kind of tumor in digestive tract of high malignancy, hair
Sick rate and the death rate are in the trend risen year by year, and overall five year survival rate is less than 5%, and Mass median life span is 6 months.Closely
The method of various treatment cancers of pancreas is in the ascendant over year, and cancer of pancreas early stage lacks manifest symptom, and most of cases have been lost when making a definite diagnosis
Go the chance of radical surgery.Operative treatment needs the degree for different stadium and tumor focus local Invasion, takes difference
Modus operandi, however perform the operation poor prognosis, easily recurrence as treatment difficult point.Radiotherapy is mainly used for the part that can not be performed the operation
The complex treatment of advanced pancreatic carcinoma, the complex treatment of postoperative Tumoral survival or recurrent cases, and advanced pancreatic cancer are appeased
Subtract disease treatment；Common Chemotherapy drug is the scheme based on gemcitabine or tegafur, gimeracil and oteracil potassium (S1), and chemicotherapy side effect is big, unpromising
Pancreas cancer patients bring significant existence to benefit.Immunization therapy is a kind of new treatment means of rising in recent years, can human activin
Self immune system, therapeutic strategy include Nonspecific immunotherapy for bronchus, tumor vaccine, adoptive immunity cell therapy and list
Clonal antibody is treated, the important antitumor means as a kind of auxiliary.Traditional Chinese medicine can enhance as a kind of auxiliary treating method
Chemotherapeutical medicine curative effect reduces chemotherapy adverse effect, effectively mitigates patient pain, prevents complication, to improve minimal invasive treatment's matter
Amount extends patient's median survival interval.From the point of view of the document delivered at present, though Chinese medicine cancer of pancreas achieves certain achievement,
But there is also problems, such as:1. because not yet establishing unified standard to cancer of pancreas diagnosis and treatment, controlling for standard there is no at present
Criterion and method are treated, and the polycentric randomized control study of large sample is lacked to the research of therapy；2. Chinese medicine preparation is without apparent prominent
Broken Journal of Sex Research, it is less for the novel effective Chinese medicine preparation of cancer of pancreas or traditional Chinese medicine monomer at present, it there is no breakthrough；In 3.
Traditional Chinese medicine still mainly plays auxiliary treatment in terms of doctor trained in Western medicine combined treatment.Although radical surgery is still current treatment of pancreatic cancer
The best approach, but its is single ineffective, and smaller scope of application.Chinese medicine is improving patient clinical symptom, slows down chemicotherapy
Adverse reaction in terms of have outstanding performance, but clinical traditional Chinese medicine is using less, and lacks its Study on Molecular Mechanism, therefore should reinforce Chinese medicine
The clinical application of medicine and Study on Molecular Mechanism actively carry out combination of Chinese tradiational and Western medicine complex treatment and prolong to improve patients ' life quality
Longevity period.Cucoline can inhibit Several Kinds of Malignancy cell, to cervical carcinoma, osteosarcoma, lung cancer, breast cancer, liver cancer, gastric cancer
Etc. kinds of tumors have apparent inhibiting effect.
In conclusion problem of the existing technology is：The therapeutic effect of the method for existing treatment cancer of pancreas is undesirable.
Solve the difficulty and meaning of above-mentioned technical problem：The present invention passes through a series of experiment in vitro Primary Studies cucoline
To the influence in terms of cancer of pancreas SW1990 cell Proliferations and apoptosis, positive spy is done to develop and finding novel anti-pancreatic cancer medicament
Rope.Cucoline can inhibit Several Kinds of Malignancy cell, a variety of to cervical carcinoma, osteosarcoma, lung cancer, breast cancer, liver cancer, gastric cancer etc.
Tumour has apparent inhibiting effect.
Invention content：
Influences of the SIN to cellular morphology is observed on the basis of original experimental technique, then by inverted microscope；
Hoechst33258 fluorescent stainings detect Apoptosis；JC-1 fluorescence probes detect mitochondrial membrane potential in anoxic；PI staining for flow
Cell art detects the cell cycle；WesternBlot detects the variation of more multicycle, apoptosis-related protein；CM-H2DCFDA fluorescence
Probe label detection reactive oxygen species (Reactiveoxygen species, ROS) level etc..It is right in vitro to observe SIN
Human pancreas cancer cell strain is proliferated and the effect of apoptosis.The biology meaning that SIN human pancreatic cancer cells play is studied by experiment in vivo
Justice, by establishing Xenografts in nude mice model, animal level observe various concentration SIN to pancreatic cancer cell angiogenesis,
The influence of the biological characteristics such as proliferation, apoptosis.
Cucoline bioavilability is relatively low, and metabolism is rapid in vivo, causes it that cannot play drug effect in time.As biology is made
Medicine engineering technology is constantly progressive, and using natural drug as lead compound, is carried out structural modification and modified form to it, is
It solves above-mentioned problem and provides new direction.It can change the original physicochemical property of drug by modification, clinically there is pole
Its important role.More natural drug such as tanshinone, matrine are currently known after carrying out structure of modification, biology
Activity is greatly improved compared with pro-drug.The structure of cucoline is modified and optimized in recent years, structural modification synthesis
Derivative is more.
Chemotherapy is one of the main means of current oncotherapy, but many antitumor drugs have larger side effect,
And it is expensive.Therefore, the small antitumour auxiliary drug of toxic side effect is found from cheap Chinese herbal medicine to reduce this
The dosage of a little antitumor drugs, reduces the hot spot that its side effect has become research.Modern experimental studies have shown that cucoline with
A variety of antitumor drugs have synergistic effect.Cucoline is combined 5 FU 5 fluorouracil and is eaten to human colon cancer cell line LoVo cells and people
Anticancer effect during pipe cancerous cell line Eca-109 cells are tested in vivo and in vitro is all higher than and is applied alone, and shares and be not improved chemotherapy
Side effect, molecular mechanism may be synergistic activation mitochondrial apoptosis access, enhancing up-regulation Bax and lower Bcl-2 albumen
Caused by expression.Cucoline can also enhance the human gastric cancer cell line MKN-28 that 5 FU 5 fluorouracil mediates in vitro and integral experiment
The growth inhibition and apoptosis of cell, mechanism of action is again by activation mitochondrial apoptosis access, and reduces thymidylic acid and close
It is transcribed at enzyme mRNA.Separately some researches show that, cucoline (2.5,5mmol/L) can with when m- dosage correlation inhibit people's uterine neck
Cancerous cell line HeLa cell Proliferations cooperate with after being combined with cis-platinum (5 μ g/mL) and inhibit cell Proliferation (P ＜ 0.05), and apoptosis rate is bright
It showing and increases (P ＜ 0.05), molecular mechanism is expressed with up-regulation mammary gland silk suppression albumen (Maspin), and downward Caspase-3,
The protein expression of COX-2 is related.Cucoline (0.625mmol/L) is with carboplatin (40 μ g/mL) use in conjunction to human cervical carcinoma cell
Be HeLa cells proliferation equally have coordinate repression, mechanism of action may with joint effect cell cycle distribution, promote
Into apoptosis-related.In addition, the study found that anti-human transferrin receptor monoclonal antibody (TfR mAb) (100 μ g/mL) or
Sinomenine (100 μ g/mL) can induce human hepatoma cell line HepG2's Apoptosis, inhibits its proliferation, the cell cycle is made to stop
It is stagnant in the S phases, and the effect of Isodose use in conjunction is stronger.
Detection method includes the following steps to pancreatic carcinoma SW1990 Proliferation and apoptosis for the cucoline：
Step 1, SIN are dissolved with DMSO first, configuration storing liquid, a concentration of 100mmol/L, then will with serum-free DMEM
Storing liquid is diluted to the working solution of 10mmol/L；
Step 2, pancreatin pancreas digested cancer SW1990 cells, neutralization prepare cell suspension, count, are diluted to 1 × 105A/
Ml, per hole repopulating cell 3000 in 96 orifice plates, 5 SIN concentration gradients are arranged in every group of 5 multiple holes, experimental group, and control group is not done
Processing；Pancreatic cancer cell SW1990 is planted in three 96 orifice plates, respectively at three time points i.e. for 24 hours, 48h, 72h detect 96 orifice plates
Absorbance；Culture medium in hole is discarded when detection, the mixed liquor of CCK-810ul and serum-free DMEM 100ul is added per hole, carefully
It is incubated 2h in born of the same parents' incubator, the absorbance under microplate reader at Detection wavelength 450nm；
Step 3, plate clone Cell colony formation assay pancreatin pancreas digested cancer cell SW1990, prepares cell suspension,
Cell count 1 × 104A/mL gives by 1000 cell seedings in 6cm culture dishes after steril cell incubator culture 6-10h
Medicine；Experiment packet is same as above, and the medication amount of DMEM in high glucose culture medium and addition reaches 5mL, cultivates 10~14d, and 4% paraformaldehyde is solid
Determine 20-30min, several times, baking oven drying and processing is taken pictures for 1% violet staining 20-30min, PBS cleaning；
Step 4, pancreatin digestion prepare SW1990 cell suspensions, cell count 1 × 105A/ml, inoculating cell is in 6 orifice plates
It is interior, 5 × 10 are planted per hole4A cell, makes cell be uniformly distributed in 6 orifice plates, cultivates 24-30h, agent-feeding treatment, experiment packet
Ibid, continue culture for 24 hours, collect supernatant fluid in each hole and cleaned 1 time in streaming pipe, PBS, collect, 1ml0.05% is added to be free of
The trypsin digestion and cell of EDTA is collected in streaming pipe, and 800rpm, 5min are discarded supernatant, and 3ml PBS clean dry powder,
800rpm, 5min, abandon supernatant, every group of addition 150ul 1 × Binding Buffer, and suspension cell about reaches 1 × 105It is a,
2.5ulAnnexinV-FITC, 5ul PI, mixing are added in cell suspension；4 DEG C are protected from light 15min；Machine on flow cytometer
Detection records result；
Step 5, by the cell suspension prepared, uniformly in 6cm culture dishes, cell concentration reaches 1 × 10 for plantation5It is a,
Experiment packet and processing are same as above；After administration for 24 hours, supernatant cell is collected, cell dry powder is made, added per hole according to the amount of dry powder
Enter corresponding lysate 100-200ul, ultrasound fully cracking extraction albumen prepares standard protein curve using BCA kits, right
Each group sample is quantified, and calculates albumen concentration, loading volume is calculated according to 50ug applied sample amounts, is passed through electrophoresis successively, is turned
Film washes film, 4 DEG C of shaking tables incubation 12-16h of primary antibody, secondary antibody incubation at room temperature 2h, washes film, chemical luminescence reagent kit ECL configurations, Yu Ning
It is exposed under glue imaging system, using GAPDH as internal reference；
Step 6 is analyzed data using 22.0 softwares of SPSS, comparison among groups χ2It examines, is poor with P ＜ 0.05
It is different statistically significant.
Further, experimental group, that is, dosing group in the step 1, setting SIN concentration gradients 0.125,0.25,0.5,1.0,
2.0mmol/L, control group are not dealt with.
Further, in the step 2 experimental group be arranged 5 SIN concentration gradients, i.e., 0.125,0.25,0.5,1.0,
2.0mmol/L。
Another object of the present invention is to provide one kind by the cucoline to pancreatic carcinoma SW1990 Proliferation and apoptosis
Detection method obtain inhibition pancreatic cancer cell growth drug.
In conclusion advantages of the present invention and good effect are：Cucoline is inquired into cancer of pancreas by various experiment in vitro
The change of cell Proliferation apoptosis；CCK-8 methods detect various concentration gradient, and cucoline is thin to cancer of pancreas under different action time
The influence of born of the same parents' survival；The influence that plate clone experiment detection cucoline forms cell colony；Flow cytometry and immunoblotting
Influence of the experiment detection cucoline to Apoptosis.0,0.125,0.25,0.5,1.0,2.0mmol/L cucoline acts on
After SW1990 cells, cell proliferation rate reduces (P ＜ 0.05) as cucoline dosage increases, and cell colony forming quantity is gradual
It reduces (P ＜ 0.05), apoptosis rate gradually increases (P ＜ 0.05)；Westernblot results show PARP protein expression levels
Gradually increase (P ＜ 0.05).Cucoline can effectively inhibit the proliferation of cancer of pancreas to a certain degree, and can effectively induce
The generation of Apoptosis shows that cucoline may play an important roll in the treatment of cancer of pancreas.
Description of the drawings
Fig. 1 is detection method stream of the cucoline provided in an embodiment of the present invention to pancreatic carcinoma SW1990 Proliferation and apoptosis
Cheng Tu.
Fig. 2 is the chemical structural formula of cucoline provided in an embodiment of the present invention.
Fig. 3 is influence (CCK- of the various concentration cucoline provided in an embodiment of the present invention to SW1990 ability of cell proliferation
8) schematic diagram.
Fig. 4 is that influence of the cucoline of various concentration provided in an embodiment of the present invention to SW1990 cell clonal formations is (flat
Plate is cloned) schematic diagram.
Fig. 5 is influence schematic diagram of the cucoline of various concentration provided in an embodiment of the present invention to SW1990 Apoptosis.
Fig. 6 is influence signal of the cucoline provided in an embodiment of the present invention without drug concentration to PARP protein expressions
Figure.
Specific implementation mode
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
Cucoline can inhibit Several Kinds of Malignancy cell, to cervical carcinoma, osteosarcoma, lung cancer, breast cancer, liver cancer, gastric cancer
Etc. kinds of tumors have apparent inhibiting effect.
As shown in Figure 1, detection of the cucoline provided in an embodiment of the present invention to pancreatic carcinoma SW1990 Proliferation and apoptosis
Method includes the following steps：
S101：Experiment packet SIN is dissolved with DMSO first, configures storing liquid, a concentration of 100mmol/L, then use serum-free
Storing liquid is diluted to the working solution of 10mmol/L by DMEM.Experimental group, that is, dosing group, setting SIN concentration gradients 0.125,0.25,
0.5,1.0,2.0mmol/L, control group are not dealt with；
S102：CCK-8 cell proliferation experiment pancreatin pancreas digested cancer SW1990 cells, neutralization prepare cell suspension, count,
It is diluted to 1 × 105A/ml, per hole repopulating cell 3000 in 96 orifice plates, 5 SIN concentration are arranged in every group of 5 multiple holes, experimental group
Gradient, i.e., 0.125,0.25,0.5,1.0,2.0mmol/L, SIN waits for the adherent addition of SW1990 cells after cell inoculation 6h, right
It is not processed according to group.According to said method plantation pancreatic cancer cell SW1990 is in three 96 orifice plates, respectively at three time points i.e. for 24 hours,
48h, 72h detect the absorbance (OD values) of 96 orifice plates；Culture medium in hole is discarded when detection, CCK-810ul is added per hole and without blood
The mixed liquor of clear DMEM 100ul, is incubated 2h in cell incubator, the absorbance (OD under microplate reader at Detection wavelength 450nm
Value)；
S103：Plate clone Cell colony formation assay pancreatin pancreas digested cancer cell SW1990, prepares cell suspension, carefully
Born of the same parents count 1 × 104A/mL gives by 1000 cell seedings in 6cm culture dishes after steril cell incubator culture 6-10h
Medicine.Experiment packet is same as above, and is noticed that the medication amount of DMEM in high glucose culture medium and addition reaches 5mL, is cultivated 10~14d, 4% poly first
Aldehyde fixes 20-30min, and several times, baking oven drying and processing is taken pictures for 1% violet staining 20-30min, PBS cleaning；
S104：The digestion of Apoptosis by Flow Cytometry pancreatin prepares SW1990 cell suspensions, cell count 1 × 105
A/ml, inoculating cell plant 5 × 10 per hole in 6 orifice plates4A cell, makes cell be uniformly distributed in 6 orifice plates, cultivates 24-
30h, agent-feeding treatment, experiment packet are same as above, and continue culture for 24 hours, are collected supernatant fluid in each hole and are cleaned 1 time in streaming pipe, PBS,
It collecting, adds 1ml0.05% to be free of the trypsin digestion and cell of EDTA, be collected in streaming pipe, 800rpm, 5min are discarded supernatant,
3ml PBS clean dry powder, and 800rpm, 5min abandon supernatant, and every group of addition 150ul 1 × Binding Buffer, suspension cell is about
Reach 1 × 105It is a, 2.5ulAnnexinV-FITC, 5ul PI, mixing are added in cell suspension；4 DEG C are protected from light 15min；
Machine testing on flow cytometer records result；
S105：Westernblot experiments will the cell suspension that prepare uniformly plantation is in 6cm culture dishes, cell concentration
Reach 1 × 105A, experiment packet and processing are same as above.After administration for 24 hours, supernatant cell is collected, cell dry powder is made, according to
Corresponding lysate 100-200ul is added per hole for the amount of dry powder, and ultrasound fully cracking extraction albumen is prepared using BCA kits
Standard protein curve quantifies each group sample, and then calculates albumen concentration, and loading body is calculated according to 50ug applied sample amounts
Product passes through electrophoresis, transferring film, washes film, 4 DEG C of shaking tables incubation 12-16h of primary antibody, secondary antibody incubation at room temperature 2h, washes film, chemiluminescence successively
Kit ECL (A liquid：B liquid 1：1) it configures, is exposed under gel imaging system, using GAPDH as internal reference；
S106：22.0 softwares of statistical analysis application SPSS analyze data, comparison among groups χ2It examines.With P ＜
0.05 is statistically significant for difference.
The application principle of the present invention is further described with reference to experiment.
1 materials and methods
1.1 cells are Wuhan City, Hubei Province hospital of Tongji University courage pancreas surgery with reagent experiment pancreatic carcinoma SW1990 used
Laboratory give.Cucoline (Sinomenine, SIN) purity 99.88% is purchased from SELLECK companies.Lot number：S235901.
DMSO is bought from Sigma companies.DMEM high glucose mediums, 10% fetal calf serum, 0.25% trypsase, 0.05% is free of EDTA
Trypsase from Gibco companies buy.AnnexinV-FITC/PI apoptosis detection kit article No. 70-AP101-100,
100T is bought from Hangzhou Lian Ke Biotechnology Ltd..Cell Counting kit-8 (CCK-8) kit, 500
It is secondary, it is purchased from green skies company.GAPDH, PARP are bought from CST companies of the U.S..Sheep anti mouse (1:2000), goat-anti rabbit (1:2000) from
Doctor's moral biotech firm buys.
1.2 cell culture will have the DMEM culture mediums 4ml of serum to be added in the culture bottle of 25ml, 37 DEG C, the perseverance of 5%CO2
Warm incubator culture pancreatic cancer cell SW1990, makes the uniform adherent growth of cell, the cell in multiple growth period is taken to be tested.
1.3 experiment packet SIN are dissolved with DMSO first, configure storing liquid, a concentration of 100mmol/L, then use serum-free
Storing liquid is diluted to the working solution of 10mmol/L by DMEM.Experimental group, that is, dosing group, setting SIN concentration gradients 0.125,0.25,
0.5,1.0,2.0mmol/L, control group are not dealt with.
1.4CCK-8 cell proliferation experiment pancreatin pancreas digested cancer SW1990 cells, neutralization prepare cell suspension, count, dilute
It is interpreted into 1 × 105A/ml, per hole repopulating cell 3000 in 96 orifice plates, 5 SIN concentration ladders are arranged in every group of 5 multiple holes, experimental group
Degree, i.e., 0.125,0.25,0.5,1.0,2.0mmol/L, SIN waits for the adherent addition of SW1990 cells after cell inoculation 6h, compares
Group is not processed.According to said method plantation pancreatic cancer cell SW1990 is in three 96 orifice plates, respectively at three time points i.e. for 24 hours,
48h, 72h detect the absorbance (OD values) of 96 orifice plates.Culture medium in hole is discarded when detection, CCK-810ul is added per hole and without blood
The mixed liquor of clear DMEM 100ul, is incubated 2h in cell incubator, the absorbance (OD under microplate reader at Detection wavelength 450nm
Value).
1.5 plate clone Cell colony formation assay pancreatin pancreas digested cancer cell SW1990, prepare cell suspension, cell
Count 1 × 104A/mL is administered by 1000 cell seedings in 6cm culture dishes after steril cell incubator culture 6-10h.
Experiment packet is same as above, and is noticed that the medication amount of DMEM in high glucose culture medium and addition reaches 5mL, is cultivated 10~14d, 4% paraformaldehyde
Fixed 20-30min, several times, baking oven drying and processing is taken pictures for 1% violet staining 20-30min, PBS cleaning.
The digestion of 1.6 Apoptosis by Flow Cytometry pancreatin prepares SW1990 cell suspensions, cell count 1 × 105A/
Ml, inoculating cell plant 5 × 10 per hole in 6 orifice plates4A cell, makes cell be uniformly distributed in 6 orifice plates, cultivates 24-30h,
Agent-feeding treatment, experiment packet are same as above, and continue culture for 24 hours, are collected supernatant fluid in each hole and are cleaned 1 time in streaming pipe, PBS, are collected,
Add 1ml0.05% to be free of the trypsin digestion and cell of EDTA, is collected in streaming pipe, 800rpm, 5min are discarded supernatant, 3ml
PBS cleans dry powder, and 800rpm, 5min abandon supernatant, every group of addition 150ul 1 × Binding Buffer, and suspension cell about reaches
1×105It is a, 2.5ulAnnexinV-FITC, 5ul PI, mixing are added in cell suspension；4 DEG C are protected from light 15min；Streaming
Machine testing on cell instrument records result.
By the cell suspension prepared, uniformly in 6cm culture dishes, cell concentration reaches for plantation for 1.7Westernblot experiments
To 1 × 105A, experiment packet and processing are same as above.After administration for 24 hours, supernatant cell is collected, cell dry powder is made, according to dry
Corresponding lysate 100-200ul is added per hole for the amount of powder, and ultrasound fully cracking extraction albumen is prepared using BCA kits and marked
Quasi- albumen curve, quantifies each group sample, and then calculates albumen concentration, and loading body is calculated according to 50ug applied sample amounts
Product passes through electrophoresis, transferring film, washes film, 4 DEG C of shaking tables incubation 12-16h of primary antibody, secondary antibody incubation at room temperature 2h, washes film, chemiluminescence successively
Kit ECL (A liquid：B liquid 1：1) it configures, is exposed under gel imaging system, using GAPDH as internal reference.
1.8 statistical analysis application SPSS, 22.0 softwares analyze data, comparison among groups χ2It examines.With P ＜
0.05 is statistically significant for difference.
2 experimental results
2.1SIN is shown in Fig. 3 to the pancreatic cancer cell SW1990 influences being proliferated.Compared with the control group, each dosage of experimental group exists
For 24 hours, there is inhibiting effect to SW1990 cells in 48h and 72h, and with the cell activity of the increase SW1990 of SIN dosage by
It gradually reduces, difference all has statistical significance (P ＜ 0.05).Each dosage group is with the extension of action time, the cell of SW1990
Active gradually to lower, difference has statistical significance (P ＜ 0.05).
Fig. 4 is shown in influences of the 2.2SIN to pancreatic cancer cell SW1990 Colony formings.Compared with the control group, with sinomenium acutum alkali dense
The cell mass of the increase of degree, SW1990 individual cells Clone formations is substantially reduced, and the formation of cell colony quantity gradually decreases, difference
With statistical significance (P ＜ 0.05).
Fig. 5 is shown in influences of the 2.3SIN to SW1990 Apoptosis.Experimental result shows the increase with SIN concentration,
The ratio that Apoptosis occurs for SW1990 is significantly raised.Experimental group is in addition to 0.125mmol/L, and difference has system compared with the control group
Meter learns meaning (P ＜ 0.05).
Fig. 6 is shown in influences of the 2.4SIN to PARP protein expressions in SW1990 cells.Westernblot experimental results show with
The increase apoptosis-related protein PARP total proteins for SIN concentration gradually decrease, and spliced body expression gradually increases, and difference has statistics
Learn meaning (P ＜ 0.05).
This decimetre is in terms of a series of experiment in vitro inquire into cucoline to pancreatic cancer cell SW1990 proliferation and apoptosis
Influence.CCK-8 experiments prompt is with the increase of SIN concentration and the extension of action time, to pancreatic cancer cell SW1990's
Inhibiting effect gradually increases, and shows time and dosage correlation；In Colony forming experiment, with gradually increasing for SIN concentration,
Cell colony forming quantity and size reduce；Influences of the Flow cytometry SIN to SW1990 Apoptosis show with
The increase of SIN concentration, Apoptosis ratio significantly increases；Westernblot experimental results show the increasing with SIN concentration
Add, PARP total proteins gradually decrease, and shearing PARP is gradually increased, and SIN promotes the apoptosis of cancer of pancreas SW1990 cells.The above experiment
Show that SIN can inhibit the proliferation of cancer of pancreas SW1990 cells, promote apoptosis in vitro.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
All any modification, equivalent and improvement etc., should all be included in the protection scope of the present invention made by within refreshing and principle.
Claims (4)
1. a kind of cucoline is to the detection method of pancreatic carcinoma SW1990 Proliferation and apoptosis, which is characterized in that the cucoline
To pancreatic carcinoma SW1990 Proliferation and apoptosis, detection method includes the following steps：
Step 1, SIN are dissolved with DMSO first, configure storing liquid, a concentration of 100mmol/L, then will be stored with serum-free DMEM
Liquid is diluted to the working solution of 10mmol/L；
Step 2, pancreatin pancreas digested cancer SW1990 cells, neutralization prepare cell suspension, count, are diluted to 1 × 105A/ml, 96
Per hole repopulating cell 3000 in orifice plate, 5 SIN concentration gradients are arranged in every group of 5 multiple holes, experimental group, and control group is not processed；
Pancreatic cancer cell SW1990 is planted in three 96 orifice plates, respectively at three time points be for 24 hours, 48h, 72h detect the suctions of 96 orifice plates
Luminosity；Culture medium in hole is discarded when detection, and the mixed liquor of CCK-8 10ul and serum-free DMEM 100ul, cell training are added per hole
It supports and is incubated 2h in case, the absorbance under microplate reader at Detection wavelength 450nm；
Step 3, plate clone Cell colony formation assay pancreatin pancreas digested cancer cell SW1990, prepares cell suspension, cell
Count 1 × 104A/mL is administered by 1000 cell seedings in 6cm culture dishes after steril cell incubator culture 6-10h；
Experiment packet is same as above, and the medication amount of DMEM in high glucose culture medium and addition reaches 5mL, cultivates 10~14d, and 4% paraformaldehyde is fixed
20-30min, several times, baking oven drying and processing is taken pictures for 1% violet staining 20-30min, PBS cleaning；
Step 4, pancreatin digestion prepare SW1990 cell suspensions, cell count 1 × 105A/ml, inoculating cell is in 6 orifice plates, often
Hole plantation 5 × 104A cell, makes cell be uniformly distributed in 6 orifice plates, cultivates 24-30h, agent-feeding treatment, and experiment packet is same as above,
Continue culture for 24 hours, collects supernatant fluid in each hole and cleaned 1 time in streaming pipe, PBS, collect, 1ml0.05% is added to be free of EDTA's
Trypsin digestion and cell is collected in streaming pipe, and 800rpm, 5min are discarded supernatant, 3ml PBS cleaning dry powder, 800rpm,
5min, abandons supernatant, every group of addition 150ul 1 × Binding Buffer, and suspension cell about reaches 1 × 105It is a, in cell suspension
Middle addition 2.5ul Annexin V-FITC, 5ul PI, mixing；4 DEG C are protected from light 15min；Machine testing on flow cytometer, note
Record result；
Step 5, by the cell suspension prepared, uniformly in 6cm culture dishes, cell concentration reaches 1 × 10 for plantation5It is a, experiment point
Group and processing are same as above；After administration for 24 hours, supernatant cell is collected, cell dry powder is made, is added accordingly per hole according to the amount of dry powder
Lysate 100-200ul, ultrasound fully cracking extraction albumen, standard protein curve is prepared using BCA kits, to each group sample
Product are quantified, and calculate albumen concentration, loading volume is calculated according to 50ug applied sample amounts, are passed through electrophoresis successively, transferring film, are washed
4 DEG C of film, primary antibody shaking tables are incubated 12-16h, secondary antibody incubation at room temperature 2h, wash film, chemical luminescence reagent kit ECL configurations, in gel imaging
It is exposed under system, using GAPDH as internal reference；
Step 6 is analyzed data using 22.0 softwares of SPSS, comparison among groups χ2It examines, has for difference with P ＜ 0.05
Statistical significance.
2. cucoline as described in claim 1 exists to the detection method of pancreatic carcinoma SW1990 Proliferation and apoptosis, feature
In experimental group, that is, dosing group in the step 1, setting SIN concentration gradients 0.125,0.25,0.5,1.0,2.0mmol/L are right
It is not dealt with according to group.
3. cucoline as described in claim 1 exists to the detection method of pancreatic carcinoma SW1990 Proliferation and apoptosis, feature
In, in the step 2 experimental group be arranged 5 SIN concentration gradients, i.e., 0.125,0.25,0.5,1.0,2.0mmol/L.
4. a kind of detection of the cucoline to pancreatic carcinoma SW1990 Proliferation and apoptosis described in claims 1 to 3 any one
The drug for the inhibition pancreatic cancer cell growth that method obtains.
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| CN110940816A (en) * | 2019-12-19 | 2020-03-31 | 广西中医药大学附属瑞康医院 | Detection method for prevention and treatment of ulceration, nodule and canceration mechanism by spleen-tonifying, heat-clearing and blood-activating prescription |
| CN112924644A (en) * | 2021-01-28 | 2021-06-08 | 贵州医科大学附属医院 | Fluorine poisoning monitoring device for water health |
| CN113262211A (en) * | 2021-04-25 | 2021-08-17 | 永康市第一人民医院 | Method for detecting cell proliferation and apoptosis of cardamonin on pancreatic cancer cells |
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| CN110940816A (en) * | 2019-12-19 | 2020-03-31 | 广西中医药大学附属瑞康医院 | Detection method for prevention and treatment of ulceration, nodule and canceration mechanism by spleen-tonifying, heat-clearing and blood-activating prescription |
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Application publication date: 20180720 |