CN107529558A - The new opplication of the phosphatase of the kinases of polynucleotide 5 ' 3 ' - Google Patents
The new opplication of the phosphatase of the kinases of polynucleotide 5 ' 3 ' Download PDFInfo
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- CN107529558A CN107529558A CN201710757969.7A CN201710757969A CN107529558A CN 107529558 A CN107529558 A CN 107529558A CN 201710757969 A CN201710757969 A CN 201710757969A CN 107529558 A CN107529558 A CN 107529558A
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
- A61K31/585—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin containing lactone rings, e.g. oxandrolone, bufalin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
Abstract
The invention belongs to drug field, the new opplication of the open phosphatase of the kinases of polynucleotide 5 ' 3 ', the present invention proves that expression of the immunohistochemistry staining method in Pancreatic Adenocarcinoma is significantly lower than cancer beside organism by immunohistochemistry staining method, and PNKP expression is lower, cancer of pancreas grade malignancy is higher.Thus provide application of the phosphatase of the kinases of polynucleotide 5 ' 3 ' in the product of diagnosis of pancreatic cancer is prepared.Present invention also offers application of the triptonide as the inhibitors of phosphatases of the kinases of polynucleotide 5 ' 3 '.The present invention is further additionally provided using application of the phosphatase of the kinases of polynucleotide 5 ' 3 ' as target in the medicine for preparing treatment cancer of pancreas.Mark of the phosphatase of the kinases of polynucleotide 5 ' 3 ' as diagnosis of pancreatic cancer, make diagnosis of pancreatic cancer more accurate, quick, the target as treatment pancreatic cancer drug provides new therapy target and therapy approach for treatment cancer of pancreas.
Description
Technical field
The invention belongs to drug field, and in particular to the new opplication of the phosphatase of kinases -3 ' of polynucleotide -5 ', enters
One step be related to application and polynucleotide of the phosphatase of kinases -3 ' of polynucleotide -5 ' in the product of diagnosis of pancreatic cancer is prepared -
Application of the 5 ' phosphatases of kinases -3 ' in the medicine for preparing treatment cancer of pancreas.
Background technology
Cancer of pancreas is the malignant tumour in pancreatic cell and vessel cell source.Epidemiological study discloses, cancer of pancreas morbidity
Rate is in persistently to raise trend.Cancer of pancreas is the worst malignant tumour of prognosis, five year survival rate only 6%.In nearly 30 years, cancer of pancreas
Patient adds 6 times, and its incidence of disease raises year by year.New hair cancer of pancreas case load raises year by year, epidemiological specialist prediction,
Following 30 years, cancer of pancreas probably turns into the malignant tumour of the second lethal quantity in cancer, and the harm to the mankind will be more next
It is bigger.
In the last thirty years, although countries in the world medical worker and researcher have done insistent exert in anti-pancreatic cancer treatment
Power, but Pancreas cancer patients five year survival rate is only improved to 6% from 3%.Although surgical operation is to cure the most effective of solid tumor to control
Treatment means, but the Pancreas cancer patients more than 80% just spread when making a definite diagnosis in belly, or have multiple organ transfer, miss hand
The best opportunity of art, surgical operation have been willing and yet unable to help.In the Pancreas cancer patients that nearly 20% can perform the operation, even if receiving most preferable hand
Art treats (R0 excisions), and the life cycle of postoperative patient also only has the 15-19 months, and survival rate is only 20% within 5 years.John Hopkins etc.
Research team reports that standard pancreas and duodenum excision combine local lymph node dissection and can not improve patient in 1,3,5 year
Interior median survival interval (Hackert, et al., 2015；Krska,et al.,2015).Large clinical data shows, at present pancreas
5 years survival rates of gland cancer patient are 6% or so, and prognosis of the surgical operation for improving cancer of pancreas produces little effect (Hackert, et
al.,2015；Krska,et al.,2015).
Cancer of pancreas is insensitive to chemicotherapy, and is also easy to produce drug resistance.Research shows that gemcitabine (gemcitabine) is
5-Fu is substituted to turn into the first-line drug of pancreatic cancer drug treatment；But the existence middle position that gemcitabine list medicine can only treat 5-Fu
Time was extended to 5.6 months by 4.4 months, was only extended 1.2 months (Krska, et al., 2015).In recent years, using gemcitabine as
The Combination chemotherapy on basis is constantly applied to clinical test, including gemcitabine joint Tarceva or capecitabine or
Gemcitabine combined radio chemotherapy, as a result show that combined radio chemotherapy also can only extend Patients with Pancreatic Cancer short-term life cycle 2-4 months, but
Can not improve 5 year life cycle (Garrido-Laguna and Hidalgo, 2015；Rubinson and Wolpin,2015).
In addition, immunization therapy and antiangiogenic therapy etc. are also tried out in treatment cancer of pancreas, but curative effect is not good enough.
Treatment by Chinese herbs cancer of pancreas research at present is roughly divided into three aspects：(A) Chinese medicinal formulae:Have been reported by 7 taste Chinese medicines
The Qingyihuaji prescriptions (QYHJ) of composition are inhibited to pancreas cancer cell strain in vitro, but tumor suppression in animal body
Rate be only 50% or so (such as Pan Huijun, 2013), curative effect is not high.(B) traditional Chinese medicine monomer:It is Bufalin (such as Li Meiying, 2013), red
TanshinoneⅡA sulfonate (such as Chen Jing, 2013), PP2A inhibitor etc., can suppress pancreatic cancer cell growth in vitro,
But internal anti-pancreatic cancer effect is bad, so far there is not yet clinical treatment cancer of pancreas report.(C) Chinese medicine combined radio chemotherapy：In
Medicine compound preparation, which is combined, puts chemotherapy, can strengthen pancreatic cancer cell to gemcitabine sensitiveness, antitumous effect is slightly had institute
Improve, and side effects of chemotherapy (such as Zhu Xiaoyan, 2013) can be significantly reduced.But take it as a whole, at present Chinese medicine anti-pancreatic cancer curative effect
Also it is not high, not yet turn into the first-line drug of anti-pancreatic cancer so far.
As noted previously, as surgical operation is not suitable for 80% Patients with Pancreatic Cancer, and postoperative curative effect is bad, and chemicotherapy turns into
Treat cancer of pancreas Main Means (Hackert, et al., 2015；Krska,et al.,2015).Research shows, for early stage
Pancreas cancer patients, gemcitabine combined chemotherapy can improve Patients with Pancreatic Cancer reactivity and short-term survival rate, but can not extend patient
Long-term survival rate.In the combined radio chemotherapy of root value criterion, recent four months or so life cycle can be extended；But for Palliative
The patient for the treatment of, combined radio chemotherapy energy Partial controll tumour.For late period and metastatic Pancreas cancer patients, combined chemotherapy
FOLFIRINOX schemes and Albumin binding paclitaxel plus gemcitabine scheme, it can only also extend the short-term survival rate of patient
4 months or so, but 5 years survival rates of Patients with Pancreatic Cancer can not be improved.Because combined chemotherapy employs 4 kinds of anticarcinogens such as gemcitabine
Thing, it is larger to normal human's toxic side effect；Combined chemotherapy and radiotherapy are big to the toxic side effect of cancer of pancreas curative effect, can cause multiple dirty
Device dysfunction, cause some patients' over-treatment dead, anti-pancreatic cancer treatment faces huge challenge (Garrido-Laguna
and Hidalgo,2015；Rubinson and Wolpin,2015).
Polynueleotide kinase/phosphatase (polynucleotide kinase/phosphatase, PNKP) is a phase
To the dual-function dna end repair enzyme that molecular mass is 5.7 ten thousand dalton, it has the work of 5'- kinases and 3'- phosphatases simultaneously
Property, it can be catalyzed and repair 5'-P and 3'-OH ends, play the role of in DNA damage reparation important.Exist however, yet there are no PNKP
Correlative study report in terms of the diagnosis and treatment of cancer of pancreas.
The content of the invention
Present inventors studied the kinases -3 ' of polynucleotide -5 ' phosphatase (PNKP) in Pancreatic Adenocarcinoma and cancer beside organism
In expression, it was demonstrated that PNKP exceptions low expression in Patients with Pancreatic Cancer tumor tissues and cell, and PNKP expression it is lower, cancer of pancreas dislike
Property degree is higher.Therefore the invention provides application of the kinases -3 ' of the polynucleotide -5 ' phosphatase as pancreas carcinoma marker.
Further, the invention provides the phosphatase of kinases -3 ' of polynucleotide -5 ' in the product of diagnosis of pancreatic cancer is prepared
Application.
Wherein, the product of diagnosis of pancreatic cancer of the present invention includes but is not limited to RT-PCR diagnosis of pancreatic cancer kit, reality
When quantitative PCR diagnosis of pancreatic cancer kit, immune detection diagnosis of pancreatic cancer kit or protein chip diagnosis of pancreatic cancer kit.
Therefore present invention also offers a kind of RT-PCR diagnosis of pancreatic cancer kit, a kind of real-time quantitative PCR diagnosis of pancreatic cancer kit,
A kind of immune detection diagnosis of pancreatic cancer kit, a kind of protein chip diagnosis of pancreatic cancer kit.
RT-PCR diagnosis of pancreatic cancer kit of the present invention, swash including at least a specific amplified polynucleotide -5 '
The primer sets of the phosphatase gene of enzyme -3 '.
Real-time quantitative PCR diagnosis of pancreatic cancer kit of the present invention, including at least a specific amplified polynucleotide-
The primer sets of the 5 ' phosphatase genes of kinases -3 '.
Immune detection diagnosis of pancreatic cancer kit of the present invention includes but is not limited to ELISA kit, and printing and dyeing examination is immunized
Agent box, immunohistochemical kit, immunofluorescent reagent box.Immune detection diagnosis of pancreatic cancer kit of the present invention, including with
The antibody of the kinases -3 ' of polynucleotide -5 ' phosphatase specific binding.Protein chip diagnosis of pancreatic cancer reagent of the present invention
Box, including the antibody with the specific binding of the kinases -3 ' of polynucleotide -5 ' phosphatase.
The antibody with the specific binding of the kinases -3 ' of polynucleotide -5 ' phosphatase can be polyclonal antibody or list
Clonal antibody.Those skilled in the art can prepare pin and polynucleotide -5 ' using a series of methods known in the art
The special antibody of the phosphatase of kinases -3 '.For example, by the phosphatase gene of the kinases -3 ' product of polynucleotide -5 ' of purification or it
Antigen fragment be injected into animal body to produce polyclonal antibody.Equally, the phosphatase of kinases -3 ' of polynucleotide -5 ' is expressed
Or the cell of its antigen may also be used for causing animal immune and producing antibody.Antibody prepared in accordance with the present invention can be single
Clonal antibody, these monoclonal antibodies can be prepared in time with hybridoma.
Further, the kit of diagnosis of pancreatic cancer of the present invention also includes other components.
In certain embodiments, the kit of the immune detection diagnosis of pancreatic cancer also includes other groups of immune detection
Point, such as Enzymoimmune reagent kit also includes reaction buffer, the enzyme of labelled antibody, the enzyme of enzyme reaction substrate, wherein labelled antibody
There are (horseradish peroxidase), alkaline phosphorus enzyme (alkaline phosphatase) etc..
Tripterygium wilfordii is traditional Chinese medicine simply, triptonide (triptonide, TN) and triptolide
(triptolide) be Chinese herb triperygium wilfordii active component；On molecular structure, both belong to Diterpenes epoxides, its parent nucleus
Structure is similar, but TN C-14 positions are carbonyl, and the C-14 positions of triptolide are hydroxyl；The difference of this active group,
So that the function and toxicity between two compounds are far from each other.Larger (the ED50 0.83mg/ of toxicity of triptolide
kg)；And have no toxic action (Li, et al., 2015) using the TN of 20 times of effective doses in Mice Body.It has been reported that TN has
There are anti-inflammatory and Anti-fecundity, as male contraceptive pill, try out in the Contraception study of the animal such as mouse and rat, achieve one
Fixed contraceptive effect, the endotoxic very little of effective dosage ranges (such as Wang Lan, 2000；The such as Zhang Jianwei, 2001).Recently, He.L
Deng report, TN can promote the high expression of cell IL-37, enhancing immunological effect (He, et al., 2015).
Inventor has found that the triptonide (0-8nM) of nanomolar range can press down by suppressing PNKP promoter activities
The expression of pancreatic cancer cell PNKP genes and albumen processed, (A12B4C3 is by being attached to people with conventional PNKP inhibitor A12B4C3
The secondary structure of PNKP albumen is influenceed on PNKP albumen Trp402, so as to further suppress PNKP enzymatic activity, it is to PNKP
Expression have not significant impact) mechanism of action is different, and the PNKP inhibitor A12B4C3 than commonly using is strong more than 100 times.Therefore
Present invention also offers application of the triptonide as the inhibitors of phosphatases of kinases -3 ' of polynucleotide -5 '.
Present invention also offers the phosphorus of kinases -3 ' of polynucleotide -5 ' of a kind of medicine for treating cancer of pancreas, including effective dose
Sour enzyme inhibitor, its pharmaceutically acceptable salt or derivative.
The PNKP inhibitor or its pharmaceutically acceptable salt can directly or indirectly be added system by those skilled in the art
Required pharmaceutically acceptable various conventional auxiliary materials during standby different dosage forms, such as disintegrant, lubricant, emulsifying agent, adhesive,
In traditional drug formulations method, common formulations, including but not limited to capsule, micro-capsule, tablet, granule, dispersion powders, note are made
Penetrate agent, liposome, oral liquid, intravenous fluid, intramuscular injection or the pharmaceutical dosage form that may be directly applied to tumour.
PNKP inhibitor that can be containing 0.05wt%~90wt% in medicine of the present invention or its is pharmaceutically acceptable
Salt.In certain embodiments, PNKP inhibitor or its medicine that can be containing 15wt%~60wt% in medicine of the present invention
Acceptable salt on.
Medicine of the present invention can in the following manner in suitable mode be administered：Orally, rectally, nasal cavity are given
Medicine, local administration, oral administration, sublingual administration, parenterai administration as subcutaneously, vein, muscle, intraperitoneal, intrathecal, intra-ventricle,
In breastbone or intracranial injection or input, or the reservoir medication by a kind of explant, wherein it is preferred that intraperitoneal or intramuscular injection application method.
The dosage and application method of the medicine for the treatment of cancer of pancreas of the present invention depend on factors, including patient
Age, body weight, sex, natural health situation, nutrition condition, the activity intensity of compound, Time of Administration, metabolic rate, disease
The journey order of severity and the subjective judgement of diagnosis and treatment doctor.Those skilled in the art can be easily determined by making according to above-mentioned factor
With dosage and application method.General pharmaceutical preparation of the present invention can use according to the dosage of 0.005~10mg/kg/ days,
It can be used according to disease severity or the different dosages of formulation beyond this dosage range.
Preferably, the kinases -3 ' of the polynucleotide -5 ' inhibitors of phosphatases is triptonide, it pharmaceutically may be used
The salt or derivative of receiving.
Wherein, the triptonide has structure shown in Formulas I,
Triptonide of the present invention can also pass through known method by being extracted from tripterygium wilfordii to obtain
Be synthesized into.
As shown from the above technical solution, the present invention proves immunohistochemistry staining method by immunohistochemistry staining method
Expression in Pancreatic Adenocarcinoma is significantly lower than cancer beside organism, and PNKP expression is lower, and cancer of pancreas grade malignancy is higher.Therefore carry
Application of the phosphatase of kinases -3 ' of polynucleotide -5 ' in the product of diagnosis of pancreatic cancer is prepared is supplied.Present invention also offers thunder
Application of the public rattan lactone ketone as the inhibitors of phosphatases of kinases -3 ' of polynucleotide -5 '.The present invention further additionally provides poly
Application of the kinases -3 ' of the nucleotides -5 ' inhibitors of phosphatases in the medicine for preparing treatment cancer of pancreas.The kinases of polynucleotide -5 ' -
Mark of the 3 ' phosphatases as diagnosis of pancreatic cancer, make diagnosis of pancreatic cancer more accurate, quick, as treatment pancreatic cancer drug
Target provides new therapy target and therapy approach for treatment cancer of pancreas.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
There is the required accompanying drawing used in technology description to be briefly described.
Fig. 1 shows the Patients with Pancreatic Cancer tumor tissues PNKP exception low expressions of embodiment 1；Wherein scheme A PNKP specific antibodies
Expression of the PNKP albumen in Pancreatic Adenocarcinoma and cancer beside organism is analyzed with immunohistochemistry staining method；Scheme B：It is right
Expression of the PNKP albumen in Pancreatic Adenocarcinoma and cancer beside organism carries out statistical analysis；Scheme C：To PNKP albumen in pancreas
Expression in cancerous tissue (I-II phases, III-IV phases) and cancer beside organism carries out statistical analysis；
Fig. 2 shows the PNKP of embodiment 2 high expression in the normal tissue, and the low expression in pancreatic cancer cell；* P values are less than
0.01；
Fig. 3 shows that the PNKP of embodiment 3 is overexpressed and suppresses DNA of tumor cell damage, and arrow represents DNA damage in figure；
Fig. 4, which shows that the PNKP of embodiment 4 is overexpressed, reduces oncogenicity；Wherein scheme A：Equal number of cell is counted, with low melting point
After agarose nutrient solution is incubated 14 days, taken pictures with body formula microscope；Scheme B：The statistical chart of Clone formation number, * * P values are less than
0.01；
Fig. 5 shows that the PNKP inhibitor triptonide of embodiment 5 effectively suppresses PNKP genes table in pancreatic cancer cell
Reach；Scheme A：After human pancreatic cancer cell Patu8988 and Panc-1 and 0-8nM TN are incubated 3 days jointly, cell is collected, extracts RNA,
The influence that triptonide is expressed PNKP is detected from gene level by RT-PCR, * * P values are less than 0.01；Scheme B：It is right
The statistical analysis of Patu8988 cell results；Scheme C：To the statistical analysis of Panc-1 cell results；
Fig. 6 shows that the PNKP inhibitor triptonide of embodiment 6 effectively suppresses PNKP albumen table in pancreatic cancer cell
Reach；Scheme A：After human pancreatic cancer cell Patu8988 and Panc-1 and 0-8nM TN are incubated 3 days jointly, cell is collected, extracts albumen
Sample, the influence of expression of the triptonide to PNKP is detected by western-blot from protein level, and * * P values are less than
0.01；Scheme B：To the statistical analysis of Patu8988 cell results；Scheme C：To the statistical analysis of Panc-1 cell results；
Fig. 7 shows that the PNKP inhibitor triptonide of embodiment 7 suppresses the activity of PNKP promoters in pancreatic cancer cell；
Ordinate represents PNKP promoter activities in figure, and abscissa represents the concentration of triptonide；
Fig. 8 shows that DNA is damaged in the comet experimental verification PNKP inhibitor triptonide inducing pancreatic cancer cells of embodiment 8
Hinder, arrow represents DNA damage in figure；
Fig. 9 shows that the γ H2AX of embodiment 9 experiments prove PNKP inhibitor triptonide induced tumor DNA Damages；
Figure 10 shows the PNKP inhibitor triptonide inducing pancreatic cancer cell-apoptosis of embodiment 10；Ordinate table in figure
Show FITC-Annexin V staining power, abscissa represents PI staining power.
Embodiment
The invention discloses the new opplication of the phosphatase of kinases -3 ' of polynucleotide -5 '.Those skilled in the art can use for reference
Present disclosure, it is suitably modified technological parameter realization.In particular, all similar replacements and change are to this area skill
It is it will be apparent that they are considered as being included in the present invention for art personnel.The present invention method and product by compared with
Good embodiment is described, related personnel substantially can not depart from present invention, in spirit and scope to as described herein
Method is modified or suitably changed with combining, to realize and using the technology of the present invention.
For a further understanding of the present invention, below in conjunction with the embodiment of the present invention, to the technical side in the embodiment of the present invention
Case is clearly and completely described, it is clear that and described embodiment is only part of the embodiment of the present invention, rather than all
Embodiment.Based on the embodiment in the present invention, those of ordinary skill in the art institute under the premise of creative work is not made
The every other embodiment obtained, belongs to the scope of protection of the invention.
Unless otherwise specified, reagent and cell involved in the embodiment of the present invention are commercially available prod, can be passed through
Commercial channel purchase obtains.As immunohistochemical kit is purchased from Shanghai genome company.Cell culture medium RPMI 1640, DMEM are (high
Sugar) and hyclone be Hyclone Products.Alamarblue reagents are INVITROGEN Products.
Embodiment 1, the PNKP low expression in Pancreatic Adenocarcinoma
Post operation cancerous tissue and the cancer beside organism of 124 Patients with Pancreatic Cancer are have collected, with immunohistochemistry staining method point
Expression of the PNKP albumen in different tissues is analysed.
(1) experiment material
The Post operation cancerous tissue of 124 Patients with Pancreatic Cancer of collection and cancer beside organism are built into cancer of pancreas by commercial company
Organization chip；Immunohistochemical kit.
(2) experimental method
Before dewaxing, Pancreas cancer patients organization chip should be positioned at room temperature in 60 DEG C of insulating boxs and toast 30min, organized
Section, which is placed in dimethylbenzene, soaks 10min, soaks 10min again after changing dimethylbenzene, 5min, 95% ethanol are soaked in absolute ethyl alcohol
Middle immersion 5min, 5min is soaked in 85% ethanol, 5min is soaked in 75% ethanol, running water rinses, last distilled water flushing,
3min × 2 time；0.01M sodium citrates cushioning liquid (pH6.0) is heated in micro-wave oven to be put into section piece to after seething with excitement, and is broken
Electricity, interval 5min, 2 times repeatedly；PBS washes 3 each 5min；Normal Goat Serum confining liquid is added dropwise, room temperature 30min, it is unnecessary to get rid of
Liquid；The μ l of primary antibody 50 are added dropwise, 4 DEG C overnight；PBS washes 3 each 5min, and sheep anti mouse/rabbit secondary antibody 50 μ l, 37 DEG C of 1h is added dropwise；PBS washes 3
Secondary each 5min, add DAB colour developings 5-20min；PBS washes 3 each 5min, is positioned over drying in oven；Mounting, microscopy.
(3) experimental result
As a result show that PNKP (Adi.Normal tissue) tissues by the cancer of Patients with Pancreatic Cancer are expressed in high, in pancreas
It is low expression (Figure 1A and 1B) in carninomatosis human cancer tissue (PC).
Analysis of Stages is carried out using american cancer joint committee (AJCC) standard in 2007 to Patients with Pancreatic Cancer to show, I
Distinguish with II phases (early stage) and III and IV phases (late period) Patients with Pancreatic Cancer cancerous tissue PNKP gene expression doses than cancer beside organism
Low 3.5 times and 8.1 times (Fig. 1 C), show that Patients with Pancreatic Cancer cancerous tissue PNKP highly significants reduce (P<0.01).Clinical cancer of pancreas
Patients clinical Analysis of Stages discloses, and PNPK expressions are lower, and Patients with Pancreatic Cancer clinical stages is poorer, these results prompting PNKP
Expression height and pancreatic neoplasm grade malignancy height are closely related, and PNKP expression is lower, and cancer of pancreas grade malignancy is higher.
Embodiment 2, PNKP high expression in the normal tissue, and the low expression in pancreatic cancer cell
(1) experiment material
Human pancreatic cancer cell Patu-8988, Panc1, SW1990, CFPAC-1, ASPC-1 and BXPC-3 protect for this laboratory
Deposit；Cell culture medium DMEM (high sugar) is Hyclone Products.Trizol reagents：Purchased from Invitrogen companies of the U.S.；It is inverse
Transcript reagent box：Fermentas, the U.S.；PCR kit：ABI Products；Normal structure cDNA is purchased from commercial company；Primer
By Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
(2) experimental method
Take the logarithm the human pancreatic cancer cell in growth period, cell total rna, reverse transcription synthesis cDNA are extracted using Trizol.PCR
Reaction system is that 25 μ l, PCR conditions are 94 DEG C of pre-degeneration 3min；94 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 1min,
45 circulations, 72 DEG C of extension 8min.The data obtained passes through 2-ΔΔCTAnalyzed：Δ Ct=Ct (mRNA)-Ct (actin).Ct generations
PCR cycle number when the intensity level of fluorescence channel reaches given threshold in table reaction system.
(3) experimental result
Real-time quantitative PCR shows, PNKP mRNA six plants of human pancreas cancer cell strains (Patu-8988, Panc1, SW1990,
CFPAC-1, ASPC-1 and BXPC-3) in expression be substantially less than six kinds of health adult tissues (liver, pancreas, spleen, prostate,
Testis and ovary) in expression (Fig. 2), prompt PNKP high expression in the normal tissue, and the low expression in pancreatic cancer cell.
Embodiment 3, PNKP, which are overexpressed, suppresses DNA of tumor cell damage.
(1) experiment material
Human pancreatic cancer cell Patu-8988 is that this laboratory freezes.Cell culture medium DMEM (high sugar) is Hyclone companies
Product.Comet kits：Trevigen, the U.S..
(2) experimental method
People's PNKP gene clonings are entered into venus carriers, venus and venus-PNKP infects tumour cell respectively, and passes through
Screening turns into the tumor cell line of stable expression.Take the logarithm the above cell in growth period, be inoculated in culture dish, carry out UVB spokes
According to (2mJ/cm2), cell suspension is prepared, takes 30 thin L cell suspensions abundant in 200 outstanding L0.6% low melting-point agarose gel
Mix, mixed liquor is gently added dropwise in being covered with the slide of 1% plain agar sugar, after gelling is solid, placed in 4 DEG C of solution spinning liquids
20min, then electrophoresis 20min, the deionized water cleaning slide of precooling 3-5 times, then 70% ethanol, 4 DEG C of fixations with precooling
5min, after air-drying, SYBR Green I dyestuffs are added, room temperature places 10min.Finally, deionized water cleans up slide
Afterwards, taken pictures under fluorescence microscope.
(3) experimental result
Above result shows PNKP low expressions in tumor tissues and cell, because PNKP albumen is that a kind of DNA damage is repaiied
Recoverin.So, can PNKP reduce DNA damage after being overexpressedTherefore, we are observed using tail of a comet experiment (comet)
The effect for the pancreatic cancer cell DNA damage that UVB radiation is overexpressed and its compareed to PNKP.As a result show, can be significantly after UVB radiation
Ground promotes the human pancreatic cancer cell DNA damage of control, and the PNKP pancreatic cancer cell DNA damages being overexpressed then are influenceed less
(Fig. 3 A and 3B), prompt PNKP to be overexpressed and suppress DNA of tumor cell damage.
Embodiment 4, PNKP are overexpressed reduction oncogenicity.
(1) experiment material
Tumor cell line and reagent：Human pancreatic cancer cell Patu-8988 (venus-PNKP and venus) freezes for this laboratory
Deposit.Cell culture medium DMEM (high sugar) is Hyclone Products.
(2) experimental method
The pancreatic cancer cell that PNKP is overexpressed and its compareed is cultivated, cell is collected and simultaneously counts, each concentration takes 1000 work
Cell shakes to mix together with methylcellulose to be added in 30mm capsule.Cell culture was counted and taken pictures after 14 days, and
And statistical experiment result.
(3) experimental result
Colony formation result shows that the pancreatic cancer cell Colony-forming capacity that PNKP is overexpressed is noticeably greater than control group
Pancreatic cancer cell (Fig. 4 A and 4B), the oncogenicity of tumour cell can be significantly reduced by prompting PNKP to be overexpressed.
Embodiment 5, triptonide suppress the expression of pancreatic cancer cell PNKP genes
(1) experiment material
(1) tumor cell line and reagent：Human pancreatic cancer cell Patu-8988 and Panc1；Cell culture medium DMEM (high sugar)
For Hyclone Products.Trizol reagents：Purchased from Invitrogen companies of the U.S.；Reverse Transcriptase kit：Fermentas is beautiful
State；PCR kit：Dalian treasured bioengineering Co., Ltd；GelRed nucleic acid gel dyestuffs：Purchased from U.S. Boitium；Primer by
Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
(2) test medicine：Triptonide dimethyl sulfoxide (DMSO) (DMSO) hydrotropy, diluted with DMEM (high sugar) culture medium
Into required concentration.
(2) experimental method
Difference expression gene, DNA damage revision points are screened with Bioinformatic methods, then find out Triptolide ketolysis
Candidate gene, and determine triptonide gene expression dose in pancreatic cancer cell before and after the processing with PCR.Take the logarithm life
Long-term human pancreatic cancer cell, it is inoculated in T25 blake bottles, and is separately added into the complete training of the triptonide containing various concentrations
Base is supported, control group adds the culture medium containing 0.001%DMSO, and effect uses Trizol extraction cell total rnas, reverse transcription after 3 days
Synthesize cDNA.PCR reaction systems are that 25 μ l, PCR conditions are 94 DEG C of pre-degeneration 3min；94 DEG C of denaturation 30s, 55 DEG C of 30s that anneal, 72
DEG C extension 1min, 22-35 circulation, 72 DEG C extend 8min.PCR primer is clapped through 1.5% agarose gel electrophoresis, gel imager
According to.
(3) experimental result
RT-PCR shows that 8nM triptonides can make DNA damage reparation in cancer of pancreas Patu8988 and Panc1 cell
Gene PNKP mRNA level in-sites are readily apparent that to reduce (Fig. 5 A-5C), prompt very low dose of triptonide just can be effective
Ground suppresses PNKP gene expressions in pancreatic cancer cell.
The influence of embodiment 6, triptonide to pancreatic cancer cell PNKP protein expressions
(1) experiment material
(1) tumor cell line and reagent：Human pancreatic cancer cell Patu-8988 and Panc1；Cell culture medium DMEM (high sugar)
For Hyclone Products.
(2) test medicine：Triptonide dimethyl sulfoxide (DMSO) (DMSO) hydrotropy, diluted with DMEM (high sugar) culture medium
Into required concentration.
(2) experimental method
Influence of the triptonide to PNKP protein expressions in pancreatic cancer cell is determined using western-blot methods.
Take the logarithm the human pancreatic cancer cell in growth period, be inoculated in T25 blake bottles, and be separately added into triptonide containing various concentrations
Complete medium, control group adds the culture medium containing 0.001%DMSO, and effect collects cell and extracts the total egg of cell after 3 days
In vain.The poly- second propylene protein electrophoresis glue of SDS- is prepared, after gel polymerisation, each well adds the above-mentioned cell protein samples of 30mg
Product, electrophoresis is carried out with constant pressure 150V.After electrophoresis terminates, with constant current 250mA by the albumen on the poly- second propylene protein electrophoresis glue of SDS-
It is transferred on NC tunica fibrosas.After transferring film terminates, NC films are taken out, are closed 2 hours with 5% milk (1 × TBST preparations) confining liquid.Envelope
After closing end, NC films are washed 3 times with 1 × TBST, 10min/ times, incubate 4 DEG C of PNKP antibody overnight.After primary antibody is incubated, with 1 ×
TBST washes film 3 times, after 10min/ times, incubates the anti-rabbit IgG antibody of HRP horseradish peroxidases, is incubated at room temperature 2 hours.Two
After anti-incubation terminates, film is washed 3 times with 1 × TBST, after 10min/ times, ECL developer solutions is added, is shown with Koda gel imaging systems
Shadow.
(3) experimental result
As a result show, triptonide can significantly suppress PNKP eggs in human pancreas cancer Patu8988 and Panc1 cell
White expression, and dose dependent (Fig. 6 A-6C) is presented.Result above prompting triptonide can suppress human pancreatic cancer cell
The expression of interior PNKP albumen.
Embodiment 7, triptonide suppress the activity of pancreatic cancer cell PNKP promoters
Above result is shown, triptonide can suppress the expression of pancreatic cancer cell PNKP genes and albumen, its machine
Whether system is the then further detection tripterygium wilfordii by suppressing the activity of PNKP promoters and then the expression of suppressor and albumen
Influence of the lactone ketone to PNKP promoters.
(1) experiment material
(1) tumor cell line and reagent：Human pancreatic cancer cell Patu-8988 and Panc1；Cell culture medium DMEM (high sugar)
For Hyclone Products.
(2) test medicine：Triptonide dimethyl sulfoxide (DMSO) (DMSO) hydrotropy, diluted with DMEM (high sugar) culture medium
Into required concentration.
(2) experimental method
The positive cell containing PNKP promoter fragments is handled 48 hours with the medicine of various concentrations first.Then with double
Luciferase assay detects the activity of PNKP promoters, by the expression intensity for detecting intracellular luciferase reporter gene
To reflect the power of PNKP promoter activities.Concrete operations are：PNKP promoters are cloned on pGL4 carriers, then with the positive
Recon transfection tumor cell, screen to obtain the positive cell strain of stable transfection through G418.By the positive cell of exponential phase,
It is inoculated in 6 orifice plates, the drug-treated cell of various concentrations is then used respectively, in 37 DEG C, 5%CO2Incubator culture 48 hours.
Collect the cell after medicine effect and prepare protein lysate, take 20 μ L to carry out chemiluminescence detection experiment respectively, add fluorescence
Reacted after plain zymolyte 5 seconds, carry out fluoroscopic examination, record detection gained chemiluminescence fluorescent value with Chemiluminescence Apparatus immediately.With
Excel carries out finishing analysis to surveyed data, and carries out variance analysis to result with SPSS16.0 softwares.
(3) experimental result
As a result show that triptonide can substantially suppress PNKP promoter activities (Fig. 7).Show triptonide
Suppress the expression of pancreatic cancer cell PNKP genes and albumen by suppressing its promoter activity, with existing PNKP inhibitor
(suppressing PNKP activity) mechanism of action is different.
Embodiment 8, triptonide promote pancreatic cancer cell DNA damage by suppressing PNKP expression：Method one
Above result shows that triptonide is different from other PNKP inhibitor, and triptonide can pass through suppression
PNKP promoter activities suppress the expression of PNKP genes and albumen.It has also been found that PNKP albumen is a kind of DNA integrity scanning protein A,
In Pancreatic Adenocarcinoma low expression, and it is closely related with the grade malignancy of cancer of pancreas.Further for determination triptonide energy
It is no to be expressed by reducing PNKP come inducing pancreatic cancer cell DNA damage, test (comet) observation triptonide using the tail of a comet
Effect to pancreatic cancer cell DNA damage.
(1) experiment material
(1) tumor cell line and reagent：Human pancreatic cancer cell Patu-8988 and Panc1；Cell culture medium DMEM (high sugar)
For Hyclone Products.Comet kits：Trevigen, the U.S..
(2) test medicine：Triptonide dimethyl sulfoxide (DMSO) (DMSO) hydrotropy, diluted with DMEM (high sugar) culture medium
Into required concentration.
(2) experimental method
Take the logarithm the human pancreatic cancer cell in growth period, be inoculated in T25 blake bottles, and be separately added into Thunder God containing various concentrations
The complete medium of rattan lactone ketone, control group add the culture medium containing 0.001%DMSO, and effect prepares cell suspension after 3 days, taken
30 μ L cell suspensions fully mix in 200 μ L0.6% low melting-point agarose gel, mixed liquor are gently added dropwise in being covered with
On the slide of 1% plain agar sugar, after gelling is solid, 20min is placed in 4 DEG C of solution spinning liquids, then electrophoresis 20min, precooling
Deionized water cleaning slide 3-5 times, then the fixed 5min of 4 DEG C of 70% ethanol with precooling, after air-drying, add SYBR Green I
Dyestuff, room temperature place 10min.Finally, after deionized water cleans up slide, taken pictures under fluorescence microscope.
(3) experimental result
As a result show, human pancreatic cancer cell Patu-8988 and Panc1 DNA can be promoted after 8nM Triptolide ketolysises
Damage, but little (Fig. 8) is then influenceed on normal human liver cell Chang liver, prompt triptonide can selectivity
Ground promotes pancreatic cancer cell DNA damage.
Embodiment 9, triptonide promote pancreatic cancer cell DNA damage by suppressing PNKP expression：Method two
In addition to comet experiments can detect DNA damage, γ H2AX are also one of goldstandard for determining DNA damage.
The height of γ H2AX how much expression DNA damages in western-blot.Therefore, tripterygium wilfordii is determined using western-blot methods
Influence of the lactone ketone to γ H2AX protein expressions in pancreatic cancer cell.
(1) experiment material
(1) tumor cell line and reagent：Human pancreatic cancer cell Patu-8988 and Panc1；Cell culture medium DMEM (high sugar)
For Hyclone Products.
(2) test medicine：Triptonide dimethyl sulfoxide (DMSO) (DMSO) hydrotropy, diluted with DMEM (high sugar) culture medium
Into required concentration.
(2) experimental method
Take the logarithm the human pancreatic cancer cell in growth period, be inoculated in T25 blake bottles, and be separately added into Thunder God containing various concentrations
The complete medium of rattan lactone ketone, control group add the culture medium containing 0.001%DMSO, and effect is collected cell and extracted after 3 days
Total protein of cell.The poly- second propylene protein electrophoresis glue of SDS- is prepared, after gel polymerisation, each well adds the above-mentioned cells of 30mg
Protein sample, electrophoresis is carried out with constant pressure 150V.After electrophoresis terminates, with constant current 250mA by the poly- second propylene protein electrophoresis glue of SDS-
Protein delivery to NC tunica fibrosas on.After transferring film terminates, NC films are taken out, 2 are closed with 5% milk (1 × TBST preparations) confining liquid
Hour.After closing terminates, NC films are washed 3 times with 1 × TBST, 10min/ times, incubate 4 DEG C of γ H2AX antibody overnight.Primary antibody is incubated it
Afterwards, wash film 3 times with 1 × TBST, after 10min/ times, incubate the anti-rabbit IgG antibody of HRP horseradish peroxidases, incubation at room temperature 2
Hour.After secondary antibody incubation terminates, film is washed 3 times with 1 × TBST, after 10min/ times, ECL developer solutions are added, with Koda gel imagings
System is developed.
(3) experimental result
As a result show, triptonide can significantly induce γ H2AX in human pancreas cancer Patu8988 and Panc1 cell
The expression of albumen, and dose dependent (Fig. 9) is presented.γ H2AX experimental results also confirm that triptonide can be optionally
Promote pancreatic cancer cell DNA damage.
Embodiment 10, triptonide inducing apoptosis of tumour cell
(1) experiment material
(1) tumor cell line：Human pancreatic cancer cell PATU-8988；Cell culture medium DMEM (high sugar) is Hyclone companies
Product.
(2) test medicine：Triptonide dimethyl sulfoxide (DMSO) (DMSO) hydrotropy, diluted with DMEM (high sugar) culture medium
Into required concentration.
(2) experimental method
After the triptonide processing human pancreatic cancer cell PATU-8988 72h of various concentrations, collecting cell includes
Its supernatant, 1000rpm centrifugation 5min abandon supernatant, with ice PBS be resuspended cell, be transferred in 1.5ml EP pipes, 2000rpm from
Heart 5min, supernatant is abandoned, after adding the binding buffer of 300 μ L Annexin V resuspension cells, then be separately added into 4.5 μ L PI
And 3 after μ L Annexin V, flow cytometer detection Apoptosis after 4 DEG C of lucifuge 10min is put, experiment is independently repeated 3 times.
(3) experimental result
Flow cytomery result is shown：With increasing for triptonide concentration, the ratio of apoptosis of tumor cells
It is significantly raised and dose dependent (Figure 10) is presented.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. application of the kinases -3 ' of the polynucleotide -5 ' phosphatase as pancreas carcinoma marker.
2. application of the phosphatase of kinases -3 ' of polynucleotide -5 ' in the product of diagnosis of pancreatic cancer is prepared.
3. application as claimed in claim 2, it is characterised in that the product of the diagnosis of pancreatic cancer is RT-PCR diagnosis of pancreatic cancer
Kit, real-time quantitative PCR diagnosis of pancreatic cancer kit, immune detection diagnosis of pancreatic cancer kit or protein chip Diagnosis of Pancreatic
Cancer kit.
A kind of 4. immune detection diagnosis of pancreatic cancer kit, it is characterised in that including with the phosphoric acid of kinases -3 ' of polynucleotide -5 '
The antibody that enzyme spcificity combines.
A kind of 5. protein chip diagnosis of pancreatic cancer kit, it is characterised in that including with the phosphoric acid of kinases -3 ' of polynucleotide -5 '
The antibody that enzyme spcificity combines.
6. application of the kinases -3 ' of the polynucleotide -5 ' inhibitors of phosphatases in the medicine for preparing treatment cancer of pancreas.
A kind of 7. medicine for treating cancer of pancreas, it is characterised in that the phosphatase of kinases -3 ' of polynucleotide -5 ' including effective dose
Inhibitor or its pharmaceutically acceptable salt.
8. medicine according to claim 7, it is characterised in that the kinases -3 ' of the polynucleotide -5 ' inhibitors of phosphatases or
The content of its pharmaceutically acceptable salt is 0.05wt%~90wt%.
9. medicine according to claim 8, it is characterised in that the kinases -3 ' of the polynucleotide -5 ' inhibitors of phosphatases is
Triptonide, its pharmaceutically acceptable salt or derivative.
10. application of the triptonide as the inhibitors of phosphatases of kinases -3 ' of polynucleotide -5 '.
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| CN108309990A (en) * | 2018-02-05 | 2018-07-24 | 赖旭宇 | Triptonide is used to prepare Gli gene inhibitors and prevents the biomedical uses of liver-cancer medicine |
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|---|---|---|---|---|
| WO2010139069A1 (en) * | 2009-06-04 | 2010-12-09 | The Governors Of The University Of Alberta | Small molecule inhibitors of polynucleotide kinase/phosphatase. poly(adp-ribose) polymerase and uses thereof |
| WO2012058763A1 (en) * | 2010-11-05 | 2012-05-10 | The Governors Of The University Of Alberta | Synthetic lethality in cancer |
| CN103230401A (en) * | 2013-04-28 | 2013-08-07 | 苏州大学 | Application of triptonide to anti-angiogenesis drugs |
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| WO2010139069A1 (en) * | 2009-06-04 | 2010-12-09 | The Governors Of The University Of Alberta | Small molecule inhibitors of polynucleotide kinase/phosphatase. poly(adp-ribose) polymerase and uses thereof |
| WO2012058763A1 (en) * | 2010-11-05 | 2012-05-10 | The Governors Of The University Of Alberta | Synthetic lethality in cancer |
| CN103230401A (en) * | 2013-04-28 | 2013-08-07 | 苏州大学 | Application of triptonide to anti-angiogenesis drugs |
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| CN108309990A (en) * | 2018-02-05 | 2018-07-24 | 赖旭宇 | Triptonide is used to prepare Gli gene inhibitors and prevents the biomedical uses of liver-cancer medicine |
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