CN106432173B - A kind of breast cancer targeted drug potassium pepper of new chemical synthesis - Google Patents
A kind of breast cancer targeted drug potassium pepper of new chemical synthesis Download PDFInfo
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Abstract
The present invention relates to a kind of chemically synthesized drug potassium pepper and its it is used to prepare the application of development of target medicines for treatment of breast cancer.Breast cancer targeted drug potassium pepper (GBK), structural formula is as follows：Pass through the pharmacological effect assay certificate to breast cancer targeted drug potassium pepper, potassium pepper is inhibited to the proliferation of tumour cell, and the proliferation of normal cell is not influenced, therefore, it can be developed into a kind of novel antitumor clinical medicine, or with existing antitumor drug drug combination, further can also develop hypotoxicity, selective antitumour auxiliary drug.
Description
Technical field
The present invention relates to a kind of chemically synthesized drug potassium pepper and its it is used to prepare development of target medicines for treatment of breast cancer
Using more specifically to the chemical synthesis of drug potassium pepper, anticancer effect and answering for breast cancer targeted therapy
With research.
Background technology
Angiocardiopathy is mainly caused by the increase of Blood Cholesterol, about according to World Health Organization's estimation
20% apoplexy and 50% heart disease are related with the high cholesterol count in blood.Clinically widely used hypolipidemic
Object is statins (statins), such as Simvastatin (simvastatin), Atorvastatin (atorvastatin), Lip river
Cut down statin (lovastatin) etc..Suppression of the statins as 3-hydroxyl-3-methylglutaryl coenzyme A A (HMG-CoA) reductase
Preparation, inhibition HMG-CoA that can be reversible change to mevalonic acid, make the biosynthesis of intracellular cholesteryl and isoprenoid
It reduces.Fructus piperis longi (Piper longum L.) is common Piperaceae plant, is the traditional anaesthetic with effect for reducing fat.For
The active ingredient of fructus piperis longi is identified, pipering (piperine, GBO), fructus piperis longi ring alkali (pipernonaline), fructus piperis longi can be obtained
Rather (piperlonguminine, GBN) three kinds of active ingredients.It is total that these active ingredients can significantly reduce experimental rat serum
The content of cholesterol (TC), Triglycerides in Serum (TG) dramatically increases high-density lipoprotein cholesterol (high density
Lipoprotein cholesterol, HDL-C) content, the content (low of slight reduction low density lipoprotein cholesterol
Density lipoprotein cholesterol, LDL-C), the effect of lipid-loweringing is similar with lipid-loweringing Western medicine Simvastatin.With
Pipering is starting material, and successfully having synthesized has the active funing of fat biological, and in the synthesis process
Produce a series of derivatives with hypolipidemic activity, such as potassium pepper (potassium piperate, GBK).Pass through
Rat experiment in vivo, it was demonstrated that it is total that chemically synthesized funing and derivative potassium pepper can significantly reduce experimental rat serum
The content (LDL-C) of cholesterol (TC), Triglycerides in Serum (TG) and low density lipoprotein cholesterol increases high density lipoprotein level
The content (HDL-C) of white cholesterol, the effect of lipid-loweringing are similar with pipering and Simvastatin.
Statins also have the works such as antitumor, anti-inflammatory response, antiproliferative other than having the function of reducing blood lipid
With.Abnormal Lipid Metabolism is an important feature of cell carcinogenesis, because participating in Biosynthesis of cholesterol and protein iso-amylene
The mevalonate pathway of change takes part in many aspects of tumour formation.Oncogene, for example, Ras protein families Ras and Rho
The posttranslational modification of GTPases is also related with above-mentioned approach.Statins can pass through isoprenoid intermediary pyrophosphoric acid
Method Buddhist nun ester (farnesyl pyrophosphat, FPP) and geranyl pyrophosphate (geranyl pyrophosphate,
GPP) inhibit oncogene protein prenylation, this is extremely important to the modification of Ras protein families.Clinical research shows statins
Object can reduce marker of inflammation high-sensitive C-reactive protein (hypersensitive C-reactive protein, hs-CRP) expression
The effects that level, reduction inflammatory reaction.By reducing the synthesis of isoprenoid intermediary FPP and GPP, tumour can be inhibited thin
Born of the same parents' proliferation inhibits tumor blood vessels regeneration, metastases and infects.
It is antitumor whether anaesthetic fructus piperis longi active ingredient and its derivative also have the effect of, whether can be used as certain tumours
The ancillary drug for the treatment of is there is not yet relevant report.Based on above-mentioned principle, applicant explores anaesthetic fructus piperis longi active ingredient and its spreads out
The antitumor effect of biology, so as to the ancillary drug as certain oncotherapies.Applicant, which chooses chemically synthesized fructus piperis longi, to be had
Ingredient pipering (GBO), funing (GBN) and derivative potassium pepper (GBK) are imitated, its suppression to tumor cell proliferation is explored
Make use and molecule mechanism.It tests and finds in pipering (GBO), funing (GBN) and potassium pepper (GBK), only pepper
Sour potassium is inhibited to the proliferation of tumour cell, and is not influenced on the proliferation of normal cell.Further experiment is found
GBK has stronger inhibiting effect, GBK specificity to prevent the G1/S of breast tumor cell MCF-7 the proliferation of breast tumor cell
It transfers (transition).GBK antitumor machanisms are mainly the G1 phases by acting on the cell cycle, activate tumor suppressor
P53 promotes the expression of cyclin dependent kinase inhibitor p27, inhibits crucial compound in G1/S phase switching process
The expression of CyclinE/CDK2 activity and CDK2 inhibits the activity of S phase key compounds CyclinA/CDK2 and the table of CyclinA
It reaches, inhibits the activity of M phase key compounds CyclinB/CDK1 and the expression of CyclinB, finally inhibit cell cycle progression.And
By making the expression quantity of transcription factor E2F lower, turn that DNA replication dna originates protein component in the multienzyme complex MCM2-7 that untwists is influenced
Record and translation, inhibit DNA replication dna.GBK can also be by inducing cell for pressure response caused by oxygen reduction, induction breast simultaneously
Adenocarcinoma cell apoptosis.GBK may be by inhibiting DNA methylation and promoting the epigenetic modifications modes such as DNA methylase inhibitor
Inhibit the proliferation of breast cancer cell.It is tested by mice xenograft model, it was demonstrated that during acting in vivo, GBK processing
It can inhibit the further growth of breast cancer knurl in the case where not influencing mouse normal growth and development.GBK and Etoposide
Drug combination can reduce the dosage of Etoposide in the case where reaching identical inhibition rate of tumor cell, it is therefore possible to
It is used in combination with Etoposide in clinic, is used for the chemotherapy process of breast cancer.
The pharmacological effect of chemical synthesis compound, especially antitumor pharmacological effect are explored, common means are to measure
IC50 (drug concentration for capableing of inducing cell apoptosis 50%), cell proliferation experiment, Cell colony formation assay, cell cycle inspection
It surveys, mouse heteroplastic transplantation experiment.By above-mentioned experiment, it can detect whether drug can inhibit tumor cell proliferation, inhibit tumour
Cell cycle, if there is the function of inhibiting tumor cell proliferation similar with experiment in vitro in mouse experiment in vivo.
Invention content
The purpose of the present invention is inhibit to make to the specificity that breast tumor cell is proliferated by chemically synthesized potassium pepper
With with inherent molecular mechanism research, provide a kind of for treating breast cancer targeted drug potassium pepper, enter for potassium pepper and control
The clinical stage for treating breast cancer lays the foundation.
Meanwhile the purpose of the application also resides in and provides breast cancer targeted drug potassium pepper preparation method and breast cancer target
To the pharmacological effect identification method of drug potassium pepper.
What the purpose of the application was realized in：A kind of breast cancer targeted drug potassium pepper (GBK), structural formula is such as
Under：
Breast cancer targeted drug potassium pepper preparation method, it is characterised in that：Potassium pepper is closed by raw material of pipering
At water-soluble hypolipemic piperine derivate, specific synthesis step is as follows：First by 3.5kg potassium hydroxide in 15 liter of 95% second
It fully being dissolved in alcohol, 2kg piperings stirring fully dissolving is then added, back flow reaction 10h at this time controls temperature at 88 DEG C,
It filters to get to potassium pepper.
The pharmacological effect identification method of breast cancer targeted drug potassium pepper, it is characterised in that：Include the following steps：
(1) chemical synthesis:Potassium pepper is the water-soluble hypolipemic piperine derivate using pipering as Material synthesis, specifically
Synthesis step it is as follows：3.5kg potassium hydroxide is fully dissolved in 15 liter of 95% ethyl alcohol first, 2kg piperings are then added
Temperature is controlled at 88 DEG C, is filtered to get to piperic acid sylvite by stirring fully dissolving, back flow reaction 10h at this time；
(2) culture of tumor cell line and normal control cells system：
The title and training method of the tumor cell line and normal control cells system used in experimentation are as follows：
The heterogeneity of three kinds of culture mediums is purchased from different biotech firms, and wherein DMEM, RPMI-1640 is purchased from HyClone
Company, F12 purchased from gibco companies, mycillin mixed liquor Pen Strep (+10000Units/ml Penicillin ,+
10000 μ g/ml Streptomycin), glutamine L-Glu (100 × L-Glutamin), insulin (insulin) and ground plug
Meter Song (dexamethasone) is purchased from SIGMA companies；
Incubation step：Attached cell is placed in 37 DEG C in corresponding culture medium, 5% (v/v) CO2It is cultivated in incubator, when thin
When born of the same parents' density reaches the 80% of culture vessel floor space, according to 1：10 ratio carries out cell passage.First with suitable when passage
Trypsin digestion cell is measured, digestion is got up until most of cell is from culture dish bottom, complete with 9 times of pancreatin volumes later
Culture medium terminates digestion reaction.The passage of cell is carried out according to required cell concentration；
(3) potassium pepper is to normal cell and cancer cell toxicity test
By measuring potassium pepper to normal cell and cancer cell toxicity, with making for determining drug during subsequent experimental
With concentration, steps are as follows：
By a variety of normal cells (GES-1, L132, HSF, COS-7) and cancer cell (MCF-7, SUM159, HepG2, A549,
BGC-823, SGC-7901) from 37 DEG C, 5%CO2It is taken out in incubator, removes respective culture medium in super-clean bench, be added
1 × PBS of 10ml (Transgen Biotech) wash away remaining culture medium, and the pancreas of 1ml 0.05% is added in each culture dish
Enzyme is put back into cell incubator 37 DEG C, 5%CO2Middle digestion 3min.Culture dish is taken out after 3min, observation under the microscope is
It is no that cell dissociation gets up, appropriate corresponding culture medium is added in the Tissue Culture Dish that digestion is got up and terminates digestion, piping and druming is uniform
Afterwards, it takes 10 μ l on blood cell counting plate, the number of cell in 1ml culture mediums is counted with blood cell counting plate, adjust training later
Support base in number of cells, make its final concentration of 5 × 103/100μl.96 orifice plates are taken out, are added per hole in 96 orifice plate outermost layers
Then 100 μ 1 × PBS of l are arranged to the leftmost side one of 96 orifice plates and 100 μ l blank cultures are added, as negative control group, at it
The 100 corresponding cell suspensions of μ l are respectively added in Yu Kongzhong, 37 DEG C are placed back in incubator after shaking up, 5%CO2Culture for 24 hours, is used for
Cell adherent growth.For 24 hours afterwards in addition to negative control and positive control, 0.2 μ g/ml, 0.5 μ g/ml, 1 μ are sequentially added from left to right
G/ml, 10 μ g/ml, 50 μ g/ml, 100 μ g/ml, 200 μ g/ml and 400 μ g/ml concentration gradients GBK act on 48h in cell,
10 μ l CCK-8 solution are added to every hole (including negative control group and positive controls) after 48h, continue in cell culture
90min is cultivated, in microplate reader under OD=450nm wavelength, measures the survival rate of cell, GBK is to different cytosiies for prediction
IC50Value；
(4) the selective inhibitory experiment of potassium pepper
The inhibiting effect grown for different cancerous cell lines and normal cell system by measuring potassium pepper, determines recklessly
Whether green pepper acid potassium is to the selective inhibiting effect of the proliferation of certain cancer cells, and steps are as follows：
By adherent normal cell and cancer cell digestion get up, cell concentration is adjusted after cell count, make its a concentration of 5 ×
103100 μ 1 × PBS of l are added per hole in 96 orifice plate outermost layers by/100 μ l, and the corresponding blank training of each cell is added to first row
Base is supported, respectively plus 100 μ l cell suspensions into remaining each hole of 96 orifice plates, 37 DEG C, 5%CO2It is cultivated for 24 hours in cell incubator；
It is added into every hole according to the concentration of 0 μ g/ml, 50 μ g/ml, 75 μ g/ml, 100 μ g/ml, 290 μ g/ml, 400 μ g/ml after for 24 hours
The concentration gradient of this 6 GBK is all arranged in sterile no enzyme water or GBK solution, each cell respectively；Experiment same concentrations repeat every time
3 times, and independent experiment is repeated 3 times, each cell uses respective culture medium as 0 hole is adjusted, to every hole after arriving corresponding time point
10 μ l CCK-8 solution are added to continue to cultivate 90min, is read under OD=450nm wavelength with microplate reader, calculates cell activity；
(5) influence of the potassium pepper to tumour cell cycle
Antitumor drug for tumor cell proliferation inhibiting effect generally by generate cellular stress (cellular
), stress for example oxidative pressure, pressure, metabolism and protein toxic pressure and DNA damage are replicated；DNA replication dna pressure acts on
The different phase of cell cycle progression, block cell cycle progression are finally reached the purpose for inhibiting tumor cell proliferation；Therefore it examines
Influence of the potassium pepper to tumour cell cycle is surveyed, for confirming that it inhibits the mechanism of cancer cell multiplication.
MCF-7, HepG2, SGC-7901 are taken out, after vitellophag, cell is used into 6cm plates respectively, in cell incubator
37 DEG C, 5%CO2Culture is for 24 hours；With 0 μ g/ml, 1/2IC after for 24 hours50μg/ml、IC50μg/ml、2IC50It is molten that GBK is added in μ g/ml concentration
Liquid handles cell 48h；Cell is collected after 48h, uses flow cytomery：70% ethyl alcohol is prepared, -20 DEG C of precoolings are placed on；
Fall culture medium, a small amount of 1 × PBS is added into each 6cm plates, washes away remaining culture medium, 0.6ml is added into each 6cm plates
0.05% pancreatin is put back into 37 DEG C, 5%CO2Vitellophag 3min in cell incubator.After 3min, 6cm plates are taken out, to every
It is uniform that 1.4ml 1 × PBS piping and druming is added in a 6cm plates, terminates digestion, and be collected into 2ml centrifuge tubes；500rpm, from
Heart 10min.Centrifuge tube is taken out, supernatant is abandoned, 1ml 1 × PBS is rejoined and cell is resuspended, washes away residual pancreatin, 500rpm, from
Heart 10min.After 10min, centrifuge tube is taken out, supernatant is abandoned, 200 1 × PBS of μ l is added again, cell is resuspended；It is taken out from refrigerator
70% ethyl alcohol being pre-chilled in advance takes 800 μ l to be added in 1.5ml centrifuge tubes, is placed in and continues to be pre-chilled on ice；By 200 1 × PBS of μ l
The cell suspension of resuspension is added in 70% ethyl alcohol of precooling, is placed in and is fixed 30min on ice；RNase A are taken out from -20 DEG C, take 1
μ l 10mg/ml RNase+99 μ l deionized waters are configured to 100 μ g/ml Ribonuclease A (RNase A), spare；From 4
1mg/ml PI are taken out in DEG C refrigerator, take 62.5 μ l 1mg/ml PI+937.5 μ l deionized waters that PI is diluted to 62.5 μ g/ml,
It is spare；After 30min, from centrifuge tube is taken out on ice, it is placed in a centrifuge 500rpm, centrifuges 10min；Centrifuge tube is taken out after 10min,
Supernatant is abandoned, 500 1 × PBS of μ l is added, cell is resuspended, 500rpm centrifuges 10min, washes away remaining ethyl alcohol.Centrifuge tube is taken out,
Supernatant is abandoned, 100 μ l, 100 μ g/ml Ribonuclease A (RNase A) are respectively added into each centrifuge tube, at room temperature
It is incubated 5min；After 5min, the PI dyestuffs of 400 μ l, 62.5 μ g/ml are added into each centrifuge tube, working concentration is 50 μ g/ml,
It is placed on ice, is protected from light incubation dyeing 30min；After 30min, by the screen filtration of 200 mesh of cell suspension to fluidic cell loading
Guan Zhong, each loading Guan Douxu are placed on ice, sample detection, analyze data；
(6) influence of the potassium pepper to tumour cell signal path
Total serum IgE is extracted from the processed cancer cell of potassium pepper first, reverse transcription is complete at human cell is carried out after cDNA
Subgenomic transcription chip (global transcriptional microarray) screens, and filters out breast after potassium pepper processing
The differential expression base of adenoma cell (MCF-7) and stomach cancer cell (SGC-7901) relative to the corresponding tumor cell line not handled
Cause filters out the cellular pathways of GBK influences；
Extract the process and transcriptive process,reversed of total serum IgE：MCF-7 and SGC-7901 cells are taken out from cell incubator
Afterwards, got up with trypsin digestion, and with 2 × 106The cell concentration bed board of a/10ml, and be put in cell incubator and cultivate
For 24 hours, for 24 hours afterwards take out 2 10cm plates, according to 1.5IC50 μ g/ml concentration respectively into two culture dishes be added GBK solution and
The water (control group) of sterile no enzyme, is denoted as experimental group and control group, continues to be placed in incubator and cultivate 48h, extracted after 48h
Total serum IgE is as follows：2 culture dishes are taken out from incubator, remove culture medium, 1 × PBS is used in combination to wash once, wash away residual
The culture medium stayed；It is separately added into 1ml TransZol into 2 culture dishesTMUp jiggles culture dish, blows and beats cell, makes to split
Solution liquid energy is enough evenly distributed in culture dish surface, with cells into close contact.With the abundant spatula attached cell of cell spatula so that
Without obvious sediment in lysate；Cell pyrolysis liquid is added in the sterile no enzyme centrifuge tubes of 1.5ml with the pipette tips of sterile no enzyme；
Under conditions of sterile no enzyme, 5min is stood in room temperature；200 μ l chloroforms are added into the sterile no enzyme centrifuge tubes of 1.5ml, acutely vibrate
30s is placed at room temperature for 3min under the conditions of sterile no enzyme；Centrifuge tube is placed in 10000rpm in the refrigerated centrifuge being pre-chilled in advance, it is low
(4 DEG C) centrifugation 15min of temperature；It takes 500 μ l supernatants in the sterile no enzyme centrifuge tubes of a new 1.5ml, it is different that 500 μ l is then added
Propyl alcohol overturns mixing, places 10min at ambient temperature；10000rpm, low temperature (4 DEG C) centrifuge 10min；It is taken from centrifuge
Go out centrifuge tube, abandons supernatant；1ml is added into 1.5ml centrifuge tubes and shifts to an earlier date prepared 75% ethanol solution, vortex 1min；
7500rpm, (4 DEG C) centrifugation 5min of low temperature；Supernatant is abandoned, drying at room temperature under the conditions of sterile no enzyme makes remaining ethyl alcohol wave completely
Hair falls；50 μ l RNA lysates are added into 1.5ml centrifuge tubes；10min is placed in 60 DEG C of baking ovens, 3 μ are dispensed in super-clean bench
L is used for gel electrophoresis and Concentration Testing in small sterile no enzyme PCR pipe, remaining is stored in -80 DEG C of refrigerators for follow-up
qRT-PCR；Electrophoresis tank is handled with absolute ethyl alcohol, and reconfigures gel for electrophoresis and a small amount of sample is taken to run electrophoresis；The examination of reverse transcription
Agent is purchased from TransGen Biotech (article No. AT-311), is carried out according to kit specification reverse transcription；
Real-time fluorescence quantitative PCR is carried out to the difference expression gene that human cell's full-length genome transcription cDNA microarray goes out to test
Card：Fluorescent dye used in experiment is Takara companiesPremix Ex Taq II(Tli RNaseH Plus)
(article No. RR820A).
The application is as a kind of clinical medicine for treating breast cancer, it is characterised in that：The breast cancer targeted drug is recklessly
Green pepper acid potassium.
The application breast cancer targeted drug potassium pepper can prepare the application in treating breast cancer clinical medicine.
The application breast cancer targeted drug potassium pepper is preparing one of the application mode in treating breast cancer clinical medicine
It is, by breast cancer targeted drug potassium pepper and Etoposide drug combination.
Compared with the prior art, the present invention has the following advantages:
(1) specific inhibitory effect of the invention by being proliferated to breast tumor cell to chemical synthetic drug potassium pepper
With the research of inherent molecule mechanism, the clinical stage that treatment breast cancer is entered for potassium pepper lays the foundation.Potassium pepper is tool
There is the chemical synthetic drug of effect for reducing fat, therefore during hyperlipidemia patient takes for a long time, can be used to prevent breast simultaneously
The purpose of gland cancer morbidity.
(2) some antitumor drugs do not have cell-specific for the inhibiting effect of cell cycle, cause killing tumour
The cell cycle progression that normal cell is also inhibited while cell causes the body of patient bigger injury.To pepper
The research of sour potassium antitumor action and its mechanism, potassium pepper are inhibited to the proliferation of tumour cell, and to normal thin
The proliferation of born of the same parents does not influence, and therefore, has theory significance and potential applicability in clinical practice, can develop and novel antitumor face to be a kind of
Bed drug, or with existing antitumor drug drug combination, hypotoxicity, selective antitumor auxiliary may further be developed
Help drug.
Description of the drawings
Specific embodiments of the present invention will be described in further detail below in conjunction with the accompanying drawings.
Fig. 1 potassium pepper half lethal concentration in different cancerous cell lines and normal cell system measures.Potassium pepper pair
There is cytotoxicity in different cancer cells, do not have toxicity for normal cell.
The processing of Fig. 2 potassium pepper concentration gradients does not influence normal cell proliferation substantially.
Fig. 3 potassium pepper concentration gradients handle the proliferation of selective depression tumor cell proliferation, especially breast cancer cell.
Fig. 4 potassium pepper concentration gradients handle selective depression breast cancer cell cycle progression, to liver cancer and stomach cancer cell
Cell cycle progression without influence.
The processing of Fig. 5 potassium pepper influences the unlike signal access of breast cancer cell MCF-7 and stomach cancer cell SGC-7901.
Fig. 6 potassium pepper processing lower breast cancer cell in cell cycle and the relevant gene of DNA replication dna.
The processing of Fig. 7 potassium pepper can inhibit the growth of breast cancer cell xenograft tumours in NOD/SCID mouse bodies.
NOD/SCID Mouse mammary cells xenograft tumours histotomy pathological analysis after the processing of Fig. 8 potassium pepper, A are
HE dyeing shows that potassium pepper processing can reduce tissue malignization level, and B is that immunohistochemical staining shows that potassium pepper handles meeting
Reduce the expression of vascular endothelial growth factor.
The influence to MCF-7 cell activity is used in combination with Etoposide in Fig. 9 potassium pepper, by two kinds of drug collective effects
After MCF-7 cells, the dosage of Etoposide can be reduced while reaching similar cell inhibitory rate.
Specific implementation mode
The application example of the present invention is further illustrated in the following embodiments, but this is not intended to limit the model of the present invention
It encloses, these methods can be applied to other chemical synthetic drugs, the active constituent of natural products and other chronic drug anticancers
The research of drug effect.
Embodiment 1：Selective inhibitory of the potassium pepper to cancer cell
It is handled just with the GBK of 0 μ g/ml, 50 μ g/ml, 75 μ g/ml, 100 μ g/ml, 290 μ g/ml and 400 μ g/ml concentration
Normal cell (GES-1, HSF, L132, COS-7) and cancer cell (MCF-7, SUM159, A549, BGC-823, SGC-7901,
HepG2) 48h, is then added 10 μ l CCK-8 per hole, 37 DEG C, 5%CO2Handle cell 90min, the OD=450nm in microplate reader
Wavelength under measure absorbance value, and the formula in Excel tables：Cell activity=(absorbance value-feminine gender of dosing group
Control group absorbance value)/(absorbance value-negative control group absorbance value for adding water group), it obtains thin under different GBK concentration
Cytoactive ratio carries out 3 experiments independent of each other.It was found that as μ g/ml increase GBK concentration from 0 to 400, it is continuous in concentration
In uphill process, the inhibiting effect very little that the cell activity of normal cell is not suppressed or is subject to, and the activity of cancer cell
Constantly declining (referring to Fig. 2, Fig. 3).
Embodiment 2：Potassium pepper selective depression breast cancer cell cycle progression
GBK is to the inhibiting effect of cancer cell it is demonstrated experimentally that the proliferation of GBK selective depression cancer cells, especially breast cancer
Cell, and the proliferation of normal cell is not influenced substantially.Experimental result shows that GBK can specificity prevention breast cancer cell
The G1/S switchings (transition) of MCF-7, and the G1/S of other cancer cells (SGC-7901, HepG2) switchings are not influenced.
Cell is collected to handle breast cancer cell 48h, 48h with the GBK of 0 μ g/ml, 145 μ g/ml, 290 μ g/ml, 580 μ g/ml concentration,
After PI is protected from light dyeing, it is detected.With GBK concentration from 0 to 580 μ g/ml constantly rise during, in MCF-7 cell lines
Breast cancer cell in the G1 phases is being continuously increased, and the cell in the S phases is significantly reducing (referring to Fig. 4).
Embodiment 3：The potassium pepper processing breast cancer cell of influence after to(for) associated signal paths
Chip (global transcriptional microarray) technology is transcribed by human cell's full-length genome,
It is corresponding swollen relative to what is do not handled to filter out breast cancer cell (MCF-7) and stomach cancer cell (SGC-7901) after potassium pepper is handled
The differential expression of oncocyte system filters out the cellular pathways of GBK influences (referring to Fig. 5).It is thin from column it can be seen from the figure that MCF-7
The gene that expression quantity changes in born of the same parents is mainly concerned with the cell cycle, the tumor developments such as DNA replication dna and Apoptosis
Process；The gene majority to change in SGC-7901 cells is related to the response to organic matter, cell migration, with the hypertrophica heart
The diseases such as myopathy are related.
Embodiment 4：Influence after potassium pepper processing breast cancer cell to related gene expression level
It is found by the analysis of human cell's full-length genome chip, and in cell cycle and DNA replication dna associated signal paths
The gene of differential expression is most.Further the expression quantity to these related genes in GBK MCF-7 cells before and after the processing carries out
Real-time fluorescence quantitative PCR verification.
The RNA reverse transcriptions that the 290 processed MCF-7 cell extractions of μ g/ml GBK are arrived at cDNA and unused GBK at
The MCF-7 extractions gained RNA reverse transcriptions managed at cDNA be used as template, use the qRT-PCR primers of testing gene as reacting
Primer carries out qRT-PCR experiments (referring to Fig. 6).It can be seen that in the cell handled with GBK from block diagram, with cell week
Phase and the relevant gene of DNA replication dna are lowered compared with corresponding gene in the cell that unused GBK is handled.
Embodiment 5：Xenograft Tumor Models detection potassium pepper in vivo antitumor is established in NOD/SCID mouse
Effect
In NOD/SCID mouse selected by the foundation of heteroplastic transplantation model be in 3-4 week old 12 NOD/SCID it is female
Mouse, purchase are cultivated in Beijing Bo Ai zoo technicals Co., Ltd in SPF grades of mouse rooms, are raised all needed for NOD/SCID mouse
Feed, water, bedding and padding and animal need the implements etc. being in direct contact to be both needed to through 126 DEG C, 20min autoclavings.To
It needs to be handled as follows before NOD/SCID mouse internal injections cancer cell：MCF-7 cells in exponential phase are used
0.05% trypsin digestion is got up, and a certain amount of PBS is added and is collected in centrifuge tube, 1000rpm, low temperature after piping and druming uniformly
(4 DEG C) centrifuge 5min.Supernatant is abandoned, suitable PBS is added into centrifuge tube again, for cell to be resuspended.Cell count, three times
It is averaged, the numerical value as cell number.By Matrigel：PBS=1:1 ratio adjusts cell concentration, and it is 2 to make cell concentration
×106A/100 μ l.Prepared tumor cell suspension is subcutaneously injected to NOD/SCID mouse both hind legs in SPF grades of mouse rooms
100 μ l select 5 nude mices as a control group at random, and another 7 are only used as experimental group, wear ear tag.It is with two by NOD/SCID mouse
One unit, is placed in mouse cage and gives food and water daily, is cultivated 3 weeks in SPF grades of mouse rooms.It is drunk to mouse in experiment the previous day
The dual anti-water of Neomycin sulfate/Polymycin B sulfate, and change autoclaved litter box.In injection tumour
Weigh mouse weight, and statistical data within every 3 days after cell.8mm is grown in tumor mass3Afterwards, to every nude mice injection GBK drugs or
0.9% physiological saline.Experimental group injects GBK according to 10 μ g/g weight, and control group injects 0.9% physiological saline of respective volume,
Continuous injection 21 days.During administration, mouse is weighed and made a record every 3 days.Every 3 days with vernier caliper to tumor
The size of block measures record, and gross tumor volume is calculated according to following formula, gross tumor volume V=ab2/ 2, (wherein a is tumour
Longest diameter, b are tumour shortest diameters), and draw tumor growth curve and changes of weight curve.After 21 days, takes off neck and puts to death mice with tumor,
Tumor mass is won, removes lump weighing, and experimental group and control group placement are taken pictures together, record quality, Infiltrating and blood
For situation.It is placed in 10% formalin and impregnates, for subsequently doing paraffin embedding, slice, HE dyeing and immunohistochemistry, analysis knot
Fruit (participates in Fig. 7).
As can be seen that having injected control of the experimental group tumorous size than having injected physiological saline of GBK drugs from Fig. 7-A
Group tumorous size is obviously reduced, and there are significant differences.Before not injecting drug, there was no significant difference for the volume of mouse tumor
(p=0.239).We can find out from the line chart of Fig. 7-B tumorous sizes, since the 6th day, with injecting normal saline group
It compares, after mouse has injected GBK drugs, tumorous size can be significantly less than control group, and at the 21st day, the volume of control group was
The volume of 2.18225 ± 0.512813, GBK group is 0.994857 ± 0.384695, p=0.000487.
Embodiment 6：The immune NOD/SCID mouse tumor biopsy GAP-associated protein GAP tables that injection potassium pepper is detected with histochemistry
Up to horizontal variation.
By immunohistochemistry, compare in the processed tumor mass of GBK and untreated tumor mass with cell
The differential expression of period and cell death related protein matter in the tissue, further to prove that GBK drugs are to act on cell week
Phase is worked by blocking cell cycle and promotion Apoptosis.
Immunohistochemistry is as follows：Dewaxing：Glass slide is put into -100% alcohol of dimethylbenzene-dimethylbenzene-successively
- 70% alcohol of -80% alcohol of 100% alcohol -90% alcohol of -95% alcohol plays the mainly dimethylbenzene of dewaxing, foundation
It is the similar principle to mix.10min generally is put in each reagent, be warm to put a few minutes less, on the contrary, weather is colder
For words it is necessary to which the dewaxing time is appropriately extended, generally 12-15min.PBS washes 35 minutes each, addition 3%H2O210min is impregnated, from
And endogenic catalase is removed, then outwell H2O2It is washed in clear water twice, adds citrate buffer solution, be put into micro-
Boiling 3min (moderate heat) in wave stove, general to be cooled to room temperature just to boiling, then boiling is primary again, is cooled to room temperature, and steams
The purpose boiled is to make the site of antigen be exposed.Serum is closed：After being cooled to room temperature, citrate buffer solution is outwelled,
Washing 2 times, and glass slide is placed in 5min in PBS, it washes 2 times, dries the PBS liquid around tissue, 5%BSA closings, incubation at room temperature
10min.Add primary antibody：Serum is abandoned, primary antibody (1 is added dropwise：50 dilution antibody) 1%BSA dilutions, 4 DEG C are overnight.Add secondary antibody：By glass slide
It is taken out from refrigerator, is put into PBS and washes 3 times, each 5min, secondary antibody (goat antirabbit/anti-is added after drying the PBS around tissue
Mouse IgG-HRP), it is subsequently placed in 30~60min in 37 DEG C of incubators.PBS washes 3 times, each 5min, every slices plus 100 μ l newly match
PBS color development stoppings are added after the completion of colour developing in the DAB solution of system, microscopically observation 3~10 minutes, control dyeing.It redyes：It will show
It after slice, thin piece after color rinses a period of time with clear water, is soaked in hematoxylin and dyes, general animal tissue is half a minute, plant group
Knit 3-5min.Dehydration：After slice, thin piece after redying is placed in water flushing, glass slide is put into -80% alcohol of 70% alcohol-successively
- 100% -100% alcohol of alcohol of -95% alcohol of 90% alcohol-dimethylbenzene-dimethylbenzene.2min is placed in each reagent, is finally soaked
Bubble is moved in dimethylbenzene in vent cabinet.Mounting：It is dropped in beside tissue, then covered with coverslip, to be first laid flat with neutral gum
Then the other side is gently put down in side, in order to avoid generating bubble, seal slice, thin piece and be placed in vent cabinet and dry.
In order to further verify influence of the potassium pepper injection to tumor tissues form, HE dyeing and immune group have been carried out
Change analysis (referring to Fig. 8).As can be seen that having injected the mouse tumor compact structure of physiological saline group from Fig. 8-A, and tumour week
It is less to enclose necrosis, and the tumor tissues structure for having injected potassium pepper mouse is more loose, and downright bad more, the potassium pepper of surrounding
Injection can reduce the malignization degree of tumor tissues.Fig. 8-B are to detect vasculai endothelium growth factor VEGF by immunohistochemistry
In the front and back expression of potassium pepper injection, it can be seen from the figure that vegf expression is positive in injecting normal saline group, and
VEGF is presented negative in injecting potassium pepper group, illustrates that potassium pepper can inhibit the vascularization of near tumor cells.
Embodiment 7：Potassium pepper and influence of the Etoposide drug combination to cell activity
Etoposide is to be applied to clinical ripe cancer treatment drugs.Etoposide and tegafur, gimeracil and oteracil potassium capsules in clinic
It is used in combination, is used for the chemotherapy process of breast cancer.But since tegafur, gimeracil and oteracil potassium capsules sheet is as chemical synthesis Western medicine, there is certain poison negative
Effect.In order to further verify the drug effect of potassium pepper, we are total with Etoposide (VP-16) with potassium pepper (GBK)
Same-action measures individually and the influence after synergy to cell activity in MCF-7 cells.
After breast cancer cell MCF-7 digestion is got up, it is 5 × 10 that cell concentration is adjusted after cell count3/ 100 μ l, 96
100 μ 1 × PBS of l are added per hole for orifice plate outermost layer, blank cultures are added to first row, into remaining each hole of 96 orifice plates
Respectively plus 100 μ l cell suspensions, the concentration combination that 1+1,1+10,5+5,5+10 and 10+10 this 5 kinds of GBK+VP-16 are respectively set is surveyed
Determine cell activity, tests same concentrations every time and be repeated 3 times, and independent experiment is repeated 3 times, each cell uses respective culture medium
As 0 hole is adjusted, the GBK+VP-16 combinations of various concentration, at 37 DEG C, 5%CO are added for 24 hours248h, 48h are cultivated in cell incubator
The backward 10 μ l CCK-8 solution that are added per hole continue to cultivate 90min, are read, are calculated thin under OD=450nm wavelength with microplate reader
Cytoactive.
The results are shown in Figure 9, when Etoposide processing breast cancer cell MCF-7 is used alone, reaches 59.72%
Inhibiting rate need Etoposide concentration be 10 μ g/ml；When potassium pepper processing breast cancer cell MCF-7 is used alone
It waits, the Etoposide concentration for reaching 50.00% inhibiting rate needs is 290 μ g/ml；When by two kinds of drug collective effects in MCF-
After 7 cells, it is 5 μ g/ml+ potassium pepper of Etoposide, 5 μ g/ml to reach the combined concentration that 45.96% inhibiting rate needs, two kinds
The usage amount of drug all significantly reduces.So can obviously inhibit the growth of breast cancer cell after two kinds of drug collective effects, and
Substantially reduce the dosage of Etoposide.
A kind of pharmacological effect identification side of the breast cancer targeted drug potassium pepper of new chemical synthesis of the present invention
Method includes the following steps:
(7) chemical synthesis:Potassium pepper is the water-soluble hypolipemic piperine derivate using pipering as Material synthesis, specifically
Building-up process it is as follows：3.5kg potassium hydroxide is fully dissolved in 15 liter of 95% ethyl alcohol first, 2kg piperings are then added
Stirring fully dissolving, back flow reaction 10h, at this time by temperature control at 88 DEG C, filter, you can obtain piperic acid sylvite (in order to
Simplify, in further part the writing a Chinese character in simplified form using GBK as potassium pepper of specification).
(8) culture of tumor cell line and normal control cells system：
The title and training method of the tumor cell line and normal control cells system used in experimentation of the present invention are as follows：
The heterogeneity of three kinds of culture mediums is purchased from different biotech firms, and wherein DMEM, RPMI-1640 is purchased from HyClone
Company, F12 purchased from gibco companies, mycillin mixed liquor Pen Strep (+10000Units/ml Penicillin ,+
10000 μ g/ml Streptomycin), glutamine L-Glu (100 × L-Glutamin), insulin (insulin) and ground plug
Meter Song (dexamethasone) is purchased from SIGMA companies.
Attached cell is placed in 37 DEG C in corresponding culture medium, 5% (v/v) CO2It is cultivated in incubator, when cell density reaches
Culture vessel floor space 80% when, according to 1：10 ratio carries out cell passage.It is first digested with appropriate pancreatin when passage
Cell, until most of cell is from culture dish bottom, digestion is got up, and is terminated later with the complete medium of 9 times of pancreatin volumes
Digestion reaction.The passage of cell is carried out according to required cell concentration.
(9) potassium pepper is to normal cell and cancer cell toxicity test
By measuring potassium pepper to normal cell and cancer cell toxicity, to determine drug during subsequent experimental
Use concentration.
By a variety of normal cells (GES-1, L132, HSF, COS-7) and cancer cell (MCF-7, SUM159, HepG2, A549,
BGC-823, SGC-7901) from 37 DEG C, 5%CO2It is taken out in incubator, removes respective culture medium in super-clean bench, be added
1 × PBS of 10ml (Transgen Biotech) wash away remaining culture medium, and the pancreas of 1ml 0.05% is added in each culture dish
Enzyme is put back into cell incubator 37 DEG C, 5%CO2Middle digestion 3min.Culture dish is taken out after 3min, observation under the microscope is
It is no that cell dissociation gets up, appropriate corresponding culture medium is added in the Tissue Culture Dish that digestion is got up and terminates digestion, piping and druming is uniform
Afterwards, it takes 10 μ l on blood cell counting plate, the number of cell in 1ml culture mediums is counted with blood cell counting plate, adjust training later
Support base in number of cells, make its final concentration of 5 × 103/100μl.96 orifice plates are taken out, are added per hole in 96 orifice plate outermost layers
Then 100 μ 1 × PBS of l are arranged to the leftmost side one of 96 orifice plates and 100 μ l blank cultures are added, as negative control group, at it
The 100 corresponding cell suspensions of μ l are respectively added in Yu Kongzhong, 37 DEG C are placed back in incubator after shaking up, 5%CO2Culture for 24 hours, is used for
Cell adherent growth.For 24 hours afterwards in addition to negative control and positive control, 0.2 μ g/ml, 0.5 μ g/ml, 1 μ are sequentially added from left to right
G/ml, 10 μ g/ml, 50 μ g/ml, 100 μ g/ml, 200 μ g/ml and 400 μ g/ml concentration gradients GBK act on 48h in cell,
10 μ l CCK-8 solution are added to every hole (including negative control group and positive controls) after 48h, continue in cell culture
90min is cultivated, in microplate reader under OD=450nm wavelength, measures the survival rate of cell, GBK is to different cytosiies for prediction
IC50It is worth (referring to Fig. 1).
(10) the selective inhibitory experiment of potassium pepper
The inhibiting effect grown for different cancerous cell lines and normal cell system by measuring potassium pepper, determines recklessly
Whether green pepper acid potassium is to the selective inhibiting effect of the proliferation of certain cancer cells.
By adherent normal cell and cancer cell digestion get up, cell concentration is adjusted after cell count, make its a concentration of 5 ×
103100 μ 1 × PBS of l are added per hole in 96 orifice plate outermost layers by/100 μ l, and the corresponding blank training of each cell is added to first row
Base is supported, respectively plus 100 μ l cell suspensions into remaining each hole of 96 orifice plates, 37 DEG C, 5%CO2It is cultivated for 24 hours in cell incubator.
It is added into every hole according to the concentration of 0 μ g/ml, 50 μ g/ml, 75 μ g/ml, 100 μ g/ml, 290 μ g/ml, 400 μ g/ml after for 24 hours
The concentration gradient of this 6 GBK is all arranged in sterile no enzyme water or GBK solution, each cell respectively.Experiment same concentrations repeat every time
3 times, and independent experiment is repeated 3 times, each cell uses respective culture medium as 0 hole is adjusted, to every hole after arriving corresponding time point
10 μ l CCK-8 solution are added to continue to cultivate 90min, is read under OD=450nm wavelength with microplate reader, calculates cell activity.
(11) influence of the potassium pepper to tumour cell cycle
Antitumor drug for tumor cell proliferation inhibiting effect generally by generate cellular stress (cellular
), stress for example oxidative pressure, pressure, metabolism and protein toxic pressure and DNA damage are replicated.DNA replication dna pressure acts on
The different phase of cell cycle progression, block cell cycle progression are finally reached the purpose for inhibiting tumor cell proliferation.Therefore it examines
Survey influence of the potassium pepper to tumour cell cycle, it will help probe into its mechanism for inhibiting cancer cell multiplication.
MCF-7, HepG2, SGC-7901 are taken out, after vitellophag, cell is used into 6cm plates respectively, in cell incubator
37 DEG C, 5%CO2Culture is for 24 hours.With 0 μ g/ml, 1/2IC after for 24 hours50μg/ml、IC50μg/ml、2IC50It is molten that GBK is added in μ g/ml concentration
Liquid handles cell 48h.Cell is collected after 48h, uses flow cytomery：70% ethyl alcohol is prepared, -20 DEG C of precoolings are placed on.
Fall culture medium, a small amount of 1 × PBS is added into each 6cm plates, washes away remaining culture medium, 0.6ml is added into each 6cm plates
0.05% pancreatin is put back into 37 DEG C, 5%CO2Vitellophag 3min in cell incubator.After 3min, 6cm plates are taken out, to every
It is uniform that 1.4ml 1 × PBS piping and druming is added in a 6cm plates, terminates digestion, and be collected into 2ml centrifuge tubes.500rpm, from
Heart 10min.Centrifuge tube is taken out, supernatant is abandoned, 1ml 1 × PBS is rejoined and cell is resuspended, washes away residual pancreatin, 500rpm, from
Heart 10min.After 10min, centrifuge tube is taken out, supernatant is abandoned, 200 1 × PBS of μ l is added again, cell is resuspended.It is taken out from refrigerator
70% ethyl alcohol being pre-chilled in advance takes 800 μ l to be added in 1.5ml centrifuge tubes, is placed in and continues to be pre-chilled on ice.By 200 1 × PBS of μ l
The cell suspension of resuspension is added in 70% ethyl alcohol of precooling, is placed in and is fixed 30min on ice.RNase A are taken out from -20 DEG C, take 1
μ l 10mg/ml RNase+99 μ l deionized waters are configured to 100 μ g/ml Ribonuclease A (RNase A), spare.From 4
1mg/ml PI are taken out in DEG C refrigerator, take 62.5 μ l 1mg/ml PI+937.5 μ l deionized waters that PI is diluted to 62.5 μ g/ml,
It is spare.After 30min, from centrifuge tube is taken out on ice, it is placed in a centrifuge 500rpm, centrifuges 10min.Centrifuge tube is taken out after 10min,
Supernatant is abandoned, 500 1 × PBS of μ l is added, cell is resuspended, 500rpm centrifuges 10min, washes away remaining ethyl alcohol.Centrifuge tube is taken out,
Supernatant is abandoned, 100 μ l, 100 μ g/ml Ribonuclease A (RNase A) are respectively added into each centrifuge tube, at room temperature
It is incubated 5min.After 5min, the PI dyestuffs of 400 μ l, 62.5 μ g/ml are added into each centrifuge tube, working concentration is 50 μ g/ml,
It is placed on ice, is protected from light incubation dyeing 30min.After 30min, by the screen filtration of 200 mesh of cell suspension to fluidic cell loading
Guan Zhong, each loading Guan Douxu are placed on ice, sample detection, analyze data.
(12) influence of the potassium pepper to tumour cell signal path
Total serum IgE is extracted from the processed cancer cell of potassium pepper first, reverse transcription is complete at human cell is carried out after cDNA
Subgenomic transcription chip (global transcriptional microarray) screens, and filters out breast after potassium pepper processing
The differential expression base of adenoma cell (MCF-7) and stomach cancer cell (SGC-7901) relative to the corresponding tumor cell line not handled
Cause filters out the cellular pathways of GBK influences.
Extract the process and transcriptive process,reversed of total serum IgE：MCF-7 and SGC-7901 cells are taken out from cell incubator
Afterwards, got up with trypsin digestion, and with 2 × 106The cell concentration bed board of a/10ml, and be put in cell incubator and cultivate
For 24 hours, for 24 hours afterwards take out 2 10cm plates, according to 1.5IC50 μ g/ml concentration respectively into two culture dishes be added GBK solution and
The water (control group) of sterile no enzyme, is denoted as experimental group and control group, continues to be placed in incubator and cultivate 48h, extracted after 48h
Total serum IgE is as follows：2 culture dishes are taken out from incubator, remove culture medium, 1 × PBS is used in combination to wash once, wash away residual
The culture medium stayed.It is separately added into 1ml TransZol into 2 culture dishesTMUp jiggles culture dish, blows and beats cell, makes to split
Solution liquid energy is enough evenly distributed in culture dish surface, with cells into close contact.With the abundant spatula attached cell of cell spatula so that
Without obvious sediment in lysate.Cell pyrolysis liquid is added in the sterile no enzyme centrifuge tubes of 1.5ml with the pipette tips of sterile no enzyme.
Under conditions of sterile no enzyme, 5min is stood in room temperature.200 μ l chloroforms are added into the sterile no enzyme centrifuge tubes of 1.5ml, acutely vibrate
30s is placed at room temperature for 3min under the conditions of sterile no enzyme.Centrifuge tube is placed in 10000rpm in the refrigerated centrifuge being pre-chilled in advance, it is low
(4 DEG C) centrifugation 15min of temperature.It takes 500 μ l supernatants in the sterile no enzyme centrifuge tubes of a new 1.5ml, it is different that 500 μ l is then added
Propyl alcohol overturns mixing, places 10min at ambient temperature.10000rpm, low temperature (4 DEG C) centrifuge 10min.It is taken from centrifuge
Go out centrifuge tube, abandons supernatant.1ml is added into 1.5ml centrifuge tubes and shifts to an earlier date prepared 75% ethanol solution, vortex 1min.
7500rpm, (4 DEG C) centrifugation 5min of low temperature.Supernatant is abandoned, drying at room temperature under the conditions of sterile no enzyme makes remaining ethyl alcohol wave completely
Hair falls.50 μ l RNA lysates are added into 1.5ml centrifuge tubes.10min is placed in 60 DEG C of baking ovens, 3 μ are dispensed in super-clean bench
L is used for gel electrophoresis and Concentration Testing in small sterile no enzyme PCR pipe, remaining is stored in -80 DEG C of refrigerators for follow-up
qRT-PCR.Electrophoresis tank is handled with absolute ethyl alcohol, and reconfigures gel for electrophoresis and a small amount of sample is taken to run electrophoresis.The examination of reverse transcription
Agent is purchased from TransGen Biotech (article No. AT-311), is carried out according to kit specification reverse transcription.
Real-time fluorescence quantitative PCR is carried out to the difference expression gene that human cell's full-length genome transcription cDNA microarray goes out to test
Card：Fluorescent dye used in experiment is Takara companiesPremix Ex Taq II(Tli RNaseH Plus)
(article No. RR820A).
Described above, the present invention is by pressing down the specificity that breast tumor cell is proliferated chemical synthetic drug potassium pepper
It makes of the research with inherent molecule mechanism, the clinical stage that treatment breast cancer is entered for potassium pepper lays the foundation.Using this
The correlative study method of invention, is applicable not only to the research of chemical synthetic drug, is also applied for natural products activity extract
Research, while being also applied for the research of the active anticancer of other chronic disease medicines.
Claims (3)
1. potassium pepper GBK is preparing the application in treating breast cancer clinical medicine, the potassium pepper GBK, structural formula
It is as follows：
2. applications of the potassium pepper GBK according to claim 1 in preparing treatment breast cancer clinical medicine, feature exist
In：Drug potassium pepper GBK described in claim 1 and Etoposide drug combination.
3. the pharmacological effect identification method of breast cancer targeted drug potassium pepper described in claim 1, it is characterised in that：Including
Following steps：
(1) chemical synthesis:Potassium pepper is the water-soluble hypolipemic piperine derivate using pipering as Material synthesis, specific to close
At steps are as follows：3.5kg potassium hydroxide is fully dissolved in 15 liter of 95% ethyl alcohol first, the stirring of 2kg piperings is then added
Temperature is controlled at 88 DEG C, is filtered to get to piperic acid sylvite by fully dissolving, back flow reaction 10h at this time；
(2) culture of tumor cell line and normal control cells system：
The title and training method of the tumor cell line and normal control cells system used in experimentation are as follows：
Heterogeneity DMEM, RPMI-1640, F12, mycillin mixed liquor Pen Strep, the glutamine L- of three kinds of culture mediums
Glu, insulin and dexamethasone are purchased from different biotech firms；
Incubation step：Attached cell is placed in 37 DEG C in corresponding culture medium, 5% (v/v) CO2It is cultivated in incubator, works as cell density
When reaching the 80% of culture vessel floor space, according to 1：10 ratio carries out cell passage；First with appropriate pancreatin when passage
Vitellophag, until most of cell is from culture dish bottom, digestion is got up, later with the complete medium of 9 times of pancreatin volumes
Terminate digestion reaction；The passage of cell is carried out according to required cell concentration；
(3) potassium pepper is to normal cell and cancer cell toxicity test：
By measuring potassium pepper to normal cell and cancer cell toxicity, to determine, the use of drug is dense during subsequent experimental
Degree, steps are as follows：
By a variety of normal cell GES-1, L132, HSF, COS-7 and cancer cell MCF-7, SUM159, HepG2, A549, BGC-
823, SGC-7901 from 37 DEG C, 5%CO2It is taken out in incubator, removes respective culture medium in super-clean bench, 10ml 1 is added
× phosphate buffer PBS removes remaining culture medium, and the pancreatin of 1ml 0.05% is added in each culture dish, is put back into cell training
37 DEG C are supported in case, 5%CO2Middle digestion 3min；Culture dish is taken out after 3min, sees whether to play cell dissociation under the microscope
Come, the appropriate corresponding culture medium of addition terminates digestion in the Tissue Culture Dish that digestion is got up, and after blowing and beating uniformly, takes 10 μ l thin in blood
On born of the same parents' tally, the number of cell in 1ml culture mediums is counted with blood cell counting plate, adjusts the cell in culture medium later
Number, make its final concentration of 5 × 103/100μl；96 orifice plates are taken out, 100 μ l1 × PBS are added per hole in 96 orifice plate outermost layers, then
100 μ l blank cultures of addition are arranged to the leftmost side one of 96 orifice plates, and 100 μ l are respectively added in remaining hole as negative control group
Corresponding cell suspension places back in incubator 37 DEG C, 5%CO after shaking up2Culture for 24 hours, is used for cell adherent growth；24h
Afterwards in addition to negative control and positive control, 0.2 μ g/ml, 0.5 μ g/ml, 1 μ g/ml, 10 μ g/ml, 50 μ are sequentially added from left to right
G/ml, 100 μ g/ml, 200 μ g/ml and 400 μ g/ml concentration gradients GBK act on 48h in cell, to every hole after 48h, including
10 μ l CCK-8 solution are added in negative control group and positive controls, continue to cultivate 90min in cell culture, in enzyme mark
On instrument under OD=450nm wavelength, the survival rate of cell, ICs of the prediction GBK to different cytosiies are measured50Value；
(4) the selective inhibitory experiment of potassium pepper：
The inhibiting effect grown for different cancerous cell lines and normal cell system by measuring potassium pepper, determines piperic acid
Whether potassium is to the selective inhibiting effect of the proliferation of certain cancer cells, and steps are as follows：
By adherent normal cell and cancer cell digestion get up, cell concentration is adjusted after cell count, make its a concentration of 5 × 103/
100 μ 1 × PBS of l are added per hole in 96 orifice plate outermost layers, the corresponding blank culture of each cell is added to first row by 100 μ l
Base, respectively plus 100 μ l cell suspensions into remaining each hole of 96 orifice plates, 37 DEG C, 5%CO2It is cultivated for 24 hours in cell incubator；24h
Afterwards according to 0 μ g/ml, 50 μ g/ml, 75 μ g/ml, 100 μ g/ml, 290 μ g/ml, 400 μ g/ml concentration be added into every hole it is sterile
Without enzyme water or GBK solution, the concentration gradient of this 6 GBK is all arranged in each cell respectively；Experiment same concentrations are repeated 3 times every time,
And independent experiment is repeated 3 times, each cell uses respective culture medium as 0 hole is adjusted, and is added to every hole after arriving corresponding time point
10 μ l CCK-8 solution continue to cultivate 90min, are read under OD=450nm wavelength with microplate reader, calculate cell activity；
(5) influence of the potassium pepper to tumour cell cycle：
Antitumor drug for tumor cell proliferation inhibiting effect generally by generate oxidative pressure, replicate pressure, metabolism
With this kind of cellular stress of protein toxic pressure and DNA damage；DNA replication dna pressure acts on the not same order of cell cycle progression
Section, block cell cycle progression are finally reached the purpose for inhibiting tumor cell proliferation；Therefore detection potassium pepper is to tumour cell
The influence in period, for confirming that it inhibits the mechanism of cancer cell multiplication；
MCF-7, HepG2, SGC-7901 are taken out, after vitellophag, cell is used into 6cm plates respectively, 37 DEG C in cell incubator,
5%CO2Culture is for 24 hours；With 0 μ g/ml, 1/2IC after for 24 hours50μg/ml、IC50μg/ml、2IC50μ g/ml concentration is added at GBK solution
Manage cell 48h；Cell is collected after 48h, uses flow cytomery：70% ethyl alcohol is prepared, -20 DEG C of precoolings are placed on；Outwell training
Base is supported, a small amount of 1 × PBS is added into each 6cm plates, washes away remaining culture medium, 0.6ml is added into each 6cm plates
0.05% pancreatin is put back into 37 DEG C, 5%CO2Vitellophag 3min in cell incubator；After 3min, 6cm plates are taken out, to every
It is uniform that 1.4ml 1 × PBS piping and druming is added in a 6cm plates, terminates digestion, and be collected into 2ml centrifuge tubes；500rpm, from
Heart 10min；Centrifuge tube is taken out, supernatant is abandoned, 1ml1 × PBS is rejoined and cell is resuspended, wash away residual pancreatin, 500rpm, from
Heart 10min；After 10min, centrifuge tube is taken out, supernatant is abandoned, 200 1 × PBS of μ l is added again, cell is resuspended；It is taken out from refrigerator
70% ethyl alcohol being pre-chilled in advance takes 800 μ l to be added in 1.5ml centrifuge tubes, is placed in and continues to be pre-chilled on ice；By 200 1 × PBS of μ l
The cell suspension of resuspension is added in 70% ethyl alcohol of precooling, is placed in and is fixed 30min on ice；RNase A is taken out from -20 DEG C, takes 1 μ
L 10mg/ml RNase+99 μ l deionized waters are configured to 100 μ g/ml RNase A, spare；1mg/ml is taken out from 4 DEG C of refrigerators
PI takes 62.5 μ l 1mg/ml PI+937.5 μ l deionized waters that PI is diluted to 62.5 μ g/ml, spare；After 30min, from ice
Centrifuge tube is taken out, 500rpm is placed in a centrifuge, centrifuges 10min；Centrifuge tube is taken out after 10min, abandons supernatant, and 500 μ l are added
Cell is resuspended in 1 × PBS, and 500rpm centrifuges 10min, washes away remaining ethyl alcohol；Centrifuge tube is taken out, supernatant is abandoned, to each centrifugation
100 μ l, 100 μ g/mlRNA enzyme A are respectively added in pipe, are incubated 5min at room temperature；After 5min, 400 μ are added into each centrifuge tube
The PI dyestuffs of 62.5 μ g/ml of l, working concentration are 50 μ g/ml, are placed on ice, and incubation dyeing 30min is protected from light；It, will be thin after 30min
In the screen filtration to fluidic cell loading pipe of 200 mesh of born of the same parents' suspension, each loading Guan Douxu is placed on ice, sample detection, point
Analyse data；
(6) influence of the potassium pepper to tumour cell signal path：
Total serum IgE is extracted from the processed cancer cell of potassium pepper first, reverse transcription is at progress human cell's full genome after cDNA
Group transcription cDNA microarray, mammary tumour cell MCF-7 and stomach cancer cell SGC-7901 is not relative to after filtering out potassium pepper processing
The difference expression gene of the corresponding tumor cell line of processing filters out the cellular pathways of GBK influences；
Extract the process and transcriptive process,reversed of total serum IgE：By MCF-7 and SGC-7901 cells after being taken out in cell incubator, use
Trypsin digestion is got up, and with 2 × 106The cell concentration bed board of a/10ml, and be put in cell incubator and cultivate for 24 hours, for 24 hours
2 10cm plates are taken out afterwards, and GBK solution and sterile no enzyme is added into two culture dishes respectively according to the concentration of 1.5IC50 μ g/ml
Water, be denoted as experimental group and control group, continue to be placed in incubator and cultivate 48h, total serum IgE is extracted after 48h, specific steps are such as
Under：2 culture dishes are taken out from incubator, remove culture medium, 1 × PBS is used in combination to wash once, wash away remaining culture medium；To 2
1ml TransZol are separately added into culture dishTMUp jiggles culture dish, blows and beats cell, lysate is enable uniformly to divide
Cloth is on culture dish surface, with cells into close contact；With the abundant spatula attached cell of cell spatula so that without apparent heavy in lysate
It forms sediment；Cell pyrolysis liquid is added in the sterile no enzyme centrifuge tubes of 1.5ml with the pipette tips of sterile no enzyme；In the condition of sterile no enzyme
Under, 5min is stood in room temperature；200 μ l chloroforms are added into the sterile no enzyme centrifuge tubes of 1.5ml, acutely vibrate 30s, sterile no enzyme item
It is placed at room temperature for 3min under part；Centrifuge tube is placed in 10000rpm in the refrigerated centrifuge being pre-chilled in advance, 4 DEG C of centrifugation 15min of low temperature；
It takes 500 μ l supernatants in the sterile no enzyme centrifuge tubes of a new 1.5ml, 500 μ l isopropanols is then added, overturn mixing,
10min is placed under room temperature；10000rpm, 4 DEG C of low temperature centrifuge 10min；Centrifuge tube is taken out from centrifuge, abandons supernatant；To
1ml is added in 1.5ml centrifuge tubes and shifts to an earlier date prepared 75% ethanol solution, vortex 1min；7500rpm, 4 DEG C of centrifugations of low temperature
5min；Supernatant is abandoned, drying at room temperature under the conditions of sterile no enzyme makes remaining ethyl alcohol vapor away completely；Add into 1.5ml centrifuge tubes
Enter 50 μ l RNA lysates；10min is placed in 60 DEG C of baking ovens, 3 μ l are dispensed in super-clean bench in small sterile no enzyme PCR pipe
In, it is used for gel electrophoresis and Concentration Testing, remaining, which is stored in -80 DEG C of refrigerators, to be used for follow-up qRT-PCR；It is handled with absolute ethyl alcohol
Electrophoresis tank, and reconfigure gel for electrophoresis and a small amount of sample is taken to run electrophoresis；It is inverted according to the kit specification of reverse transcription
Record；
The difference expression gene gone out to human cell's full-length genome transcription cDNA microarray carries out real-time fluorescence quantitative PCR verification：It is real
Fluorescent dye is used in testingPremix Ex Taq II, Tli RNaseH Plus.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH06336417A (en) * | 1993-03-31 | 1994-12-06 | Shiseido Co Ltd | Ultraviolet light absorber and external preparation for skin mixed with the same |
| JP2005263638A (en) * | 2004-03-16 | 2005-09-29 | Kanebo Cosmetics Inc | Wrinkle remover |
| CN102283830A (en) * | 2011-08-12 | 2011-12-21 | 博日吉汗格日勒图 | Hypolipemic application of potassium piperonyl pentadienoate |
| CN102491966A (en) * | 2011-12-02 | 2012-06-13 | 博日吉汗格日勒图 | Piperonyl pentylene acid sodium and application on blood lipid reduction thereof |
| CN105348250A (en) * | 2015-10-20 | 2016-02-24 | 吐尔孙拜克·叶尔达 | Medicinal compound pepper acid hexamethylene diamine and preparation process thereof |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH06336417A (en) * | 1993-03-31 | 1994-12-06 | Shiseido Co Ltd | Ultraviolet light absorber and external preparation for skin mixed with the same |
| JP2005263638A (en) * | 2004-03-16 | 2005-09-29 | Kanebo Cosmetics Inc | Wrinkle remover |
| CN102283830A (en) * | 2011-08-12 | 2011-12-21 | 博日吉汗格日勒图 | Hypolipemic application of potassium piperonyl pentadienoate |
| CN102491966A (en) * | 2011-12-02 | 2012-06-13 | 博日吉汗格日勒图 | Piperonyl pentylene acid sodium and application on blood lipid reduction thereof |
| CN105348250A (en) * | 2015-10-20 | 2016-02-24 | 吐尔孙拜克·叶尔达 | Medicinal compound pepper acid hexamethylene diamine and preparation process thereof |
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