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CN108236722A - Purposes of the IDNK inhibitor in cancer treatment drug is prepared - Google Patents

Purposes of the IDNK inhibitor in cancer treatment drug is prepared Download PDF

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Publication number
CN108236722A
CN108236722A CN201810039649.2A CN201810039649A CN108236722A CN 108236722 A CN108236722 A CN 108236722A CN 201810039649 A CN201810039649 A CN 201810039649A CN 108236722 A CN108236722 A CN 108236722A
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idnk
inhibitor
cancer treatment
liver cancer
cell
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CN108236722B (en
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金成�
顾元龙
何友钊
刘敏丰
袁凤来
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Wuxi No 3 Peoples Hospital
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Wuxi No 3 Peoples Hospital
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088Compounds having three or more nucleosides or nucleotides
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

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  • Life Sciences & Earth Sciences (AREA)
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  • Chemical & Material Sciences (AREA)
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  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
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  • Molecular Biology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The invention belongs to biological medicine research fields, and in particular to purposes of the IDNK inhibitor in cancer treatment drug is prepared.After extensive and in-depth study, research for the first time finds to inhibit IDNK expression the present invention, can significantly affect the proliferation of 7404 cells of liver cancer cells BEL, influence the distribution of cell cycle, and promote the apoptosis of cell.It may be a key function gene of a promotion liver cancer genesis and development to show the gene, possibly as the potential treatment target spot of a liver cancer.

Description

Purposes of the IDNK inhibitor in cancer treatment drug is prepared
Technical field
The invention belongs to biological medicine research fields, and in particular to use of the IDNK inhibitor in cancer treatment drug is prepared On the way.
Background technology
At present, operation excision is the main treatment means of liver cancer, and still, the Postoperative recurrent rate of hepatocarcinoma patient is high, poor prognosis, About 80% liver cancer patient dies of relapse and metastasis according to statistics, and most of liver cancer patient has occurred and that remote organ turns when making a definite diagnosis It moves.Early diagnosis and treatment currently for liver cancer patient relapse and metastasis lack effective means, and basic reason is to liver The mechanism of cancer recurrence transfer is still not clear.
The research of mechanism of tumor metastasis is always the Disciplinary Frontiers in oncology studies.Numerous studies confirm that tumour turns It moves and nonrandom, but with selectivity, organ preferendum.Famous " the seed and soil " hypothesis of the propositions such as Paget:" seed " It is random behavior in itself to send out, but the microenvironment of specific tissue or organ is suitble to the survival and growth of certain " seeds ", is it " soil " of survival and development is provided, the transfer of " seed " is thus made to show tissue and organ specificity.Current research person is more Of tumour itself is paid close attention to, and the soil and microenvironment concern to tumour are less.It is special during its tangible hepatoma-targeting transfer Fixed " soil " is currently considered to played an important role during hepatoma Metastasis.Therefore seek tumour look for specific soil, New microenvironment is extremely urgent.
A kind of gluconokinase of IDNK gene codes is a kind of to need Mg2+Typical phosphotransferase, this kinases participates in D- gluconate degradation processes (PMID:23067238), which has in organs such as kidney, liver, small intestine, duodenums Higher expression (PMID 24309898).And the gene is located at the gene delection area of acute myeloid leukaemia del (9q).Mesh The characteristics of preceding kinases for finding IDNK gene codes is metabolized gluconic acid is similar with the effect in prokaryotes, thus it is speculated that the albumen The effect of the gluconic acid catabolism in human body may have been exercised.But do not have at present diligent for works of the IDNK in cell The research of energy.
Invention content
In order to overcome the problems of in the prior art, the purpose of the present invention is to provide IDNK inhibitor to prepare liver Purposes in cancer medicine.
To achieve these goals and other related purposes, the present invention adopt the following technical scheme that:
The first aspect of the present invention provides the purposes that IDNK inhibitor is used to prepare cancer treatment drug.
Further, the cancer treatment drug at least has one of following function:
Inhibit hepatoma cell proliferation, reduce liver cancer cells vigor, raising G1 phases liver cancer cells and G2/M phase liver cancer cells Ratio, reduction S phase liver cancer cells ratio, liver cancer cells is caused to occur, and the G1 phases block and the G2/M phases block, promote liver cancer thin Born of the same parents' apoptosis inhibits hepatic carcinoma growth.
Further, the IDNK inhibitor refers to the molecule for having inhibition to IDNK.
IDNK is included but not limited to inhibition:Inhibit IDNK activity or inhibit IDNK genetic transcriptions or Expression.
The IDNK inhibitor can be siRNA, shRNA, antibody, micromolecular compound.
As the embodiment of the present invention is enumerated, the IDNK inhibitor can be siRNA or shRNA.
The cancer treatment drug necessarily include IDNK inhibitor, and using IDNK inhibitor as aforementioned function it is effective into Point.
In the cancer treatment drug, the active ingredient for playing aforementioned function can be only IDNK inhibitor, also may include it He can play the molecule of similar function.
Also that is, IDNK inhibitor is one of the sole active ingredient of the cancer treatment drug or active ingredient.
The cancer treatment drug can be single composition substance, also can be multi-component compound.
The form of the cancer treatment drug can be that solid, liquid, gel, semi-fluid, aerosol etc. are each without specifically limited Kind material form.
The cancer treatment drug mainly for object for mammal, such as rodent, primate.
The second aspect of the present invention provides a kind of method for treating liver cancer, is to apply IDNK inhibitor to object.
The object can be the liver cancer cells of mammal or mammal.The mammal is preferably Rodentia Animal, artiodactylous animals, Perissodactyla animal, Lagomorph, primate etc..The primate be preferably monkey, Ape or people.The liver cancer cells can be isolated liver cancer.
The object can be the individual of the patient of suffering from hepatic cancer or the liver cancer of Waiting treatment.Or the object is liver The liver cancer cells of the individual of cancer patient or Waiting treatment liver cancer.
The IDNK inhibitor can be applied before, during and after liver cancer treatment is received to object.
The third aspect of the present invention provides a kind of cancer treatment drug, the IDNK inhibitor including effective dose.
Further, the cancer treatment drug, IDNK inhibitor and pharmaceutical carrier including effective dose.
The cancer treatment drug necessarily include IDNK inhibitor, and using IDNK inhibitor as aforementioned function it is effective into Point.
In the cancer treatment drug, the active ingredient for playing aforementioned function can be only IDNK inhibitor, also may include it He can play the molecule of similar function.
Also that is, IDNK inhibitor is one of the sole active ingredient of the cancer treatment drug or active ingredient.
The cancer treatment drug can be single composition substance, also can be multi-component compound.
The form of the cancer treatment drug can be that solid, liquid, gel, semi-fluid, aerosol etc. are each without specifically limited Kind material form.
The cancer treatment drug mainly for object for mammal, such as rodent, primate.
The fourth aspect of the present invention provides a kind of liver cancer combination therapy pharmaceutical composition, including a effective amount of IDNK inhibitor With other at least one cancer treatment drugs.
The combination therapy pharmaceutical composition can be any one in following form:
One) independent preparation is respectively prepared in IDNK inhibitor and other cancer treatment drugs, the dosage form of preparation can be identical Or it is different, administration route also may be the same or different.
When other cancer treatment drugs are antibody, parenteral type is generally used.When other cancer treatment drugs are During chemicals, form of medication can be relatively abundant, can be gastrointestinal administration can also be parenteral administration.Generally push away The known administration route recommended for each chemicals is administered.
Two) IDNK inhibitor and other cancer treatment drugs are configured to compound preparation, by IDNK inhibitor and other When cancer treatment drug is simultaneously applied simultaneously using the administration of identical administration route, the shape that the two is configured to compound preparation can be used Formula.
The fifth aspect of the present invention provides a kind of method for treating liver cancer, is to inhibit to object using a effective amount of IDNK It agent and applies other a effective amount of cancer treatment drugs to object and/or to object implements other liver cancer treatment means.
A effective amount of IDNK inhibitor can concurrently or sequentially be given and other at least one a effective amount of liver cancer are controlled Treat drug.
Be based on IDNK present invention firstly discovers that liver cancer treatment target spot, controlled with other liver cancer other than IDNK inhibitor It treats in combination therapies, can at least play the effect of curative effect addition, further enhance the therapeutic effect for liver cancer.
Other cancer treatment drugs include but is not limited to:Antibody drug, chemicals or target medicinal etc..
The IDNK inhibitor can be gastrointestinal administration or parenteral.Other described cancer treatment drugs can be with It is gastrointestinal administration or parenteral.For antibody drug, generally using parenteral.
The sixth aspect of the present invention provides IDNK inhibitor and is preparing in any one of following or multinomial effect drug Purposes:Inhibit hepatoma cell proliferation, reduce liver cancer cells vigor, raising G1 phases liver cancer cells and G2/M phase liver cancer cells Ratio, reduction S phase liver cancer cells ratio, liver cancer cells is caused to occur, and the G1 phases block and the G2/M phases block, promote liver cancer cells Apoptosis inhibits hepatic carcinoma growth.
Compared with prior art, the present invention has the advantages that:
After extensive and in-depth study, research for the first time finds to inhibit IDNK expression the present invention, and it is thin can to significantly affect liver cancer The proliferation of born of the same parents' BEL-7404 cells, influences the distribution of cell cycle, and promotes the apoptosis of cell.It may be one to show the gene An a key function gene for promoting liver cancer genesis and development, possibly as the potential treatment target spot of a liver cancer.
Description of the drawings
Fig. 1:Slow-virus infection BEL-7404 cell fluorescence pictures, top picture is infects shCtrl slow virus groups, lower part Slow virus group is interfered for infection shIDNK, left side is photograph via bright field, and right side is fluorescence photo.
Fig. 2A:ShIDNK, which strikes, subtracts Efficiency testing, and shIDNK slow virus significantly inhibits IDNK genes in BEL-7404 cells and exists The expression quantity of mRNA level in-site, column result show that * * represent p with average value ± SD<0.01.
Fig. 2 B:ShIDNK, which strikes, subtracts Efficiency testing, and Western Blot diagrams, disturbance target point exists to the heterogenous expression of IDNK Protein level, which strikes, subtracts effect.
Fig. 3 A:Celigo cell countings verification IDNK gene on cell proliferation influences, continuous 5 days record cells of Celigo Picture.
Fig. 3 B:Celigo cell countings verification IDNK gene on cell proliferation influences, shRNA slow-virus infections BEL- 7404 cells, the curve that shIDNK groups are changed over time with shCtrl cellular control unit numbers.
Fig. 3 C:Celigo cell countings verification IDNK gene on cell proliferation influences, shRNA slow-virus infections BEL- The comparison that the variation multiple of 7404 cells, shIDNK groups and shCtrl cellular control unit quantity changes over time.
Fig. 4 A:Mtt assay detects the influence of IDNK gene on cell proliferation vigor, and shRNA slow-virus infections BEL-7404 is thin Born of the same parents after culture 5 days, are handled 4 hours through MTT, and shIDNK groups and shCtrl control groups are in microplate reader to the light of wavelength 490nm The comparison that absorptivity changes over time.OD490 reflects the quantity for having great-hearted cell herein.
Fig. 4 B:Mtt assay detects the influence of IDNK gene on cell proliferation vigor, and shRNA slow-virus infections BEL-7404 is thin Born of the same parents after culture 5 days, are handled 4 hours through MTT, and shIDNK groups and shCtrl control groups are in microplate reader to the light of wavelength 490nm The comparison that absorption variations multiple changes over time.OD490 reflects the quantity for having great-hearted cell herein.
Fig. 5 A:Influences of the fluidic cell cycle detection shIDNK to BEL-7404 cell cycles is tied for the cell cycle Fruit schematic diagram.
Fig. 5 B:Influences of the fluidic cell cycle detection shIDNK to BEL-7404 cell cycles, column result is with thin Born of the same parents' percentage average value ± SD shows that * * represent p<0.01.
Fig. 6 A:Annexin V-APC fluidic cells apoptosis detects influences of the shIDNK to BEL-7404 Apoptosis, is Fluidic cell apoptosis schematic diagram.
Fig. 6 B:Annexin V-APC fluidic cells apoptosis detects influences of the shIDNK to BEL-7404 Apoptosis, column Shape result shows that * * represent p with cell percentages average value ± SD<0.01.
Specific embodiment
For the present inventor the study found that design is directed to the interfered target sequence of IDNK genes, packaging builds slow virus, After IDNK genes strike subtracting, the proliferation of liver cancer cells BEL-7404 cells can be significantly affected, influences point of cell cycle Cloth, and promote the apoptosis of cell.It may be a key function gene of a promotion liver cancer genesis and development to show the gene, Possibly as the potential treatment target spot of a liver cancer.
IDNK
It is a kind of to need Mg for a kind of gluconokinase2+Typical phosphotransferase, this kinases takes part in d- gluconic acids Ester degradation process (PMID:23067238), which has higher expression in organs such as kidney, liver, small intestine, duodenums (PMID 24309898)。
IDNK inhibitor
Refer to the molecule that there is inhibition for IDNK.IDNK is included but not limited to inhibition:Inhibit IDNK activity inhibits IDNK genetic transcriptions or expression.The IDNK inhibitor include but not limited to siRNA, shRNA, Antibody, micromolecular compound.
It is that IDNK vigor is instigated to decline to inhibit IDNK activity.Preferably, before compared to inhibition, IDNK vigor declines at least 10%, at least 30%, then good reduction at least 50% are preferably reduced, at least 70% is more preferably reduced, more preferably reduces at least 80%, best reduction at least 90%.
IDNK genetic transcriptions or expression is inhibited to refer to:Make IDNK gene do not transcribe or reduce IDNK gene transcription Activity or make IDNK gene do not express or reduce IDNK gene expression activity.
Those skilled in the art can be adjusted the genetic transcription or expression of IDNK using conventional method, such as clpp gene It removes, homologous recombination, RNA interfering etc..
The inhibition of genetic transcription or the expression of IDNK can detect expression quantity by PCR and Western Blot and verify.
Preferably, compared with wild type, IDNK genetic transcriptions or expression reduce at least 10%, preferably reduce at least 30%, then good reduction at least 50%, more preferably reduce at least 70%, and good reduction at least 90%, most preferably IDNK genes It does not express completely.
Micromolecular compound
Middle finger of the present invention is made of several or tens atoms, compound of the molecular mass below 1000.
IDNK inhibitor prepares cancer treatment drug
Cancer treatment drug is prepared using IDNK inhibitor as one of main active or main active.In general, medicine In object other than active ingredient, according to the needs of different dosage forms, will also include one or more pharmaceutically acceptable carriers or Auxiliary material.
" pharmaceutically acceptable " refers to that they will not be produced when biomolecule ontology and composition suitably give animal or people Raw unfavorable, allergy or other adverse reactions.
" pharmaceutically acceptable carrier or auxiliary material " should be compatible with IDNK inhibitor, can be blended without logical The effect of pharmaceutical composition is greatly lowered in the case of often.It can be as some of pharmaceutically acceptable carrier or auxiliary material substances Specific example be carbohydrate, such as lactose, dextrose and saccharose;Starch, such as cornstarch and potato starch;Cellulose and its spread out Biology, such as sodium carboxymethylcellulose pyce, ethyl cellulose and methylcellulose;Tragacanth powder;Malt;Gelatin;Talcum;Solid Lubricant, such as stearic acid and magnesium stearate;Calcium sulfate;Vegetable oil, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil And cupu oil;Polyalcohol, such as the third two liquor-saturated, glycerine, D-sorbite, mannitol and polyethylene glycol;Alginic acid;Emulsifier, such as Tween;Wetting agent, such as NaLS;Colorant;Flavoring agent;Tablet agent, stabilizer;Antioxidant;Preservative;Without heat Raw water;Isotonic salting liquid;With phosphate buffer etc..These substances are used to help the stability of formula or help as needed Acceptable mouthfeel or smell are generated in raising activity or its biological effectiveness or in the case of oral.
In the present invention, unless stated otherwise, pharmaceutical dosage form is not particularly limited, and can be made into injection, oral liquid, piece The dosage forms such as agent, capsule, dripping pill, spray can be prepared by conventional method.The selection of pharmaceutical dosage form should be with administering mode phase Matching.
Combination therapy pharmaceutical composition and method of administration
The combination therapy pharmaceutical composition can be any one in following form:
One) independent preparation is respectively prepared in IDNK inhibitor and other cancer treatment drugs, the dosage form of preparation can be identical Or it is different, administration route also may be the same or different.In use, can several medicines use simultaneously, also can several medicines successively use.First After when being administered, should formerly with drug still to body it is effective during in body apply other drugs.
Two) IDNK inhibitor and other cancer treatment drugs are configured to compound preparation, by IDNK inhibitor and other When cancer treatment drug is simultaneously applied simultaneously using the administration of identical administration route, the shape that the two is configured to compound preparation can be used Formula.
The common administrated method of antibody is intravenous injection, intravenous drip or arterial perfusion.Its usage and dosage can refer to existing Technology.
The common administrated method of micromolecular compound can be gastrointestinal administration either parenteral.siRNA、 ShRNA, antibody then generally use parenteral.Can be local administration can also be Formulations for systemic administration.
A effective amount of IDNK inhibitor can concurrently or sequentially be given and other at least one a effective amount of liver cancer are controlled Treat drug.
In use, can use a effective amount of IDNK inhibitor and other a effective amount of cancer treatment drugs simultaneously, also may be used A effective amount of IDNK inhibitor and other a effective amount of cancer treatment drugs are successively used.During consecutive administration, should formerly it use Drug still applies other drugs in effective period to organism to organism.
Before further describing the specific embodiments of the present invention, it should be appreciated that protection scope of the present invention is not limited to down State specific specific embodiment;It is also understood that the term used in the embodiment of the present invention is specific specific in order to describe Embodiment, the protection domain being not intended to be limiting of the invention.The experiment side of actual conditions is not specified in the following example Method, usually according to normal condition or the condition proposed by according to each manufacturer.
When embodiment provides numberical range, it should be appreciated that except non-present invention is otherwise noted, two ends of each numberical range Any one numerical value can be selected between point and two endpoints.Unless otherwise defined, in the present invention all technologies for using and Scientific terminology is identical with the normally understood meaning of those skilled in the art of the present technique.Except the specific method used in embodiment, set Outside standby, material, according to record of the those skilled in the art to the grasp of the prior art and the present invention, it can also use With described in the embodiment of the present invention method, any method, equipment and the material of the similar or equivalent prior art of equipment, material Expect to realize the present invention.
Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method using this technology lead Molecular biology, biochemistry, chromatin Structure and the analysis of domain routine, analytical chemistry, cell culture, recombinant DNA technology and The routine techniques of related field.These technologies existing perfect explanation in the prior art, for details, reference can be made to Sambrook etc. MOLECULAR CLONING:A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989and Third edition, 2001;Ausubel etc., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley&Sons, New York, 1987 and periodic updates;the Series METHODS IN ENZYMOLOGY, Academic Press, San Diego;Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998;METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999;With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
Embodiment 1
First, experimental method:
1st, prepared by IDNK genes interference slow virus
For IDNK genes, specific target spot interference sequence is devised, that is, target sequence is AAACAGAACTAAGC ATAAA, SEQ ID NO.1), and with TTCTCCGAACGTGTCACGT (SEQ ID NO.2) for negative control sequence, according to target Point sequence designs shRNA interference sequences, is built respectively into hU6-MCS-CMV-EGFP plasmid vectors, is carried with pHelper 1.0 2.0 vector plasmid of body and pHelper, cotransfection 293T cells, after transfection 48-72h obtain unpurified cell conditioned medium, and The slow virus that purifying concentration obtains high titre is carried out to supernatant.Specifically, it is for the sequence of the shRNA of IDNK genes (SEQ ID NO.3, are denoted as CCGGCCAAACAGAACTAAGCATAAA CTCGAGTTTATGCTTAGTTCTGTTTGGTTTTT shIDNK).Sequence for the siRNA of IDNK genes is AAACAGAACUAAGCAUAAA, SEQ ID NO.4).Negative control ShRNA is denoted as shCtrl.
2nd, RT-PCR testing goals clpp gene decreasing effect rate
Design of primers is carried out for IDNK genes.Extract control group shCtrl and shIDNK interference slow-virus infection group Cell, extracting RNA, reverse transcription acquisition cDNA using GAPDH as internal reference, pass through the mRNA expression feelings that RT-PCR detects IDNK respectively Condition.
3rd, Western Blot external sources verification target spot validity
The IDNK expression clonings plasmid containing fusion FLAG labels and the RNAi viruses for IDKN disturbance target points will be built Vector plasmid, in cotransfection to 293T cells, after 48h, it is simultaneously quantitative to collect albumen, and Western is carried out with flag antibody Blot detection albumen strikes decreasing effect rate.
4th, Celigo cell countings detection cell growth
After carrying out slow-virus infection 4 days to BEL-7404 cells, passage is inoculated in 96 orifice plates, and bed board leads to after 24 hours It crosses celigo instruments to read the cell of expression EGFP fluorescence and take pictures, is handled by software and calculate the thin of different groups in orifice plate Born of the same parents' number after continuously detecting 5 days, draws the growth curve chart of cell, analyzes cell growth condition.
5th, MTT detects cell viability
After carrying out slow-virus infection 4 days to BEL-7404 cells, passage is inoculated in 96 orifice plates, and after bed board 24, culture is eventually Only preceding 4h adds in the MTT solution of 20 μ L 5mg/mL, and 100 μ LDMSO solution are added in after 4h, carries out microplate reader detection.
6th, PI-FACS fluidic cells cycle detection
After carrying out slow-virus infection 4 days to BEL-7404 cells, bed board is passed on, after cell fusion degree up to after 85%, digestion Collect cell, carry out ethyl alcohol fix and wash, add in PI dye liquors cell is dyed, using flow cytometer to cell into Row detects and carries out data analysis with ModFit softwares.
7th, Annexin V-APC fluidic cells apoptosis detects
After carrying out slow-virus infection 4 days to BEL-7404 cells, bed board is passed on, after cell fusion degree up to after 85%, digestion Cell is collected, Annexin V-APC dyeing processing is added in, cell is detected using flow cytometer, and with guava ICtrlyte flow cytometry analysis softwares are analyzed.
2nd, experimental result
1st, IDNK genes interfere slow-virus infection and strike the verification of decreasing effect rate
BEL-7404 is infected with MOI 20 with the shIDNK interference slow virus and shCtrl control slow virus that build Cell in fluorescence microscopy Microscopic observation EGFP fluorescence and is taken pictures after 72 hours, as a result show cell infection efficiency reach 80% with On, cell state is good (Fig. 1).
After confirming infectious effect, pass through the jamming effectiveness of qPCR experimental verification shIDNK slow virus.QPCR results are shown ShIDNK slow virus significantly suppresses in BEL-7404 cells IDNK genes in the expression quantity (p of mRNA level in-site<0.05) it, strikes and subtracts Efficiency is up to 84.5% (Fig. 2A).It is total to by the way that the IDNK for containing fusion FLAG labels is overexpressed plasmid with shIDNK interference plasmids Transfect 293T cells, Western Blot detection FLAG labels, as a result display interference target spot to the heterogenous expression of IDNK in albumen Level, which is struck, subtracts effect, thus is Effective target site (Fig. 2 B).
2nd, IDNK genes AF panel BEL-7404 cell Proliferations
ShIDNK interference group and shCtrl control group BEL-7404 cells are connected by celigo cell countings 5 days continuous photographs to record, the results show that the interference of IDNK genes can significantly affect the growing multiplication (Fig. 3 A) of BEL-7404 cells. 2nd day, the 3rd day, the 4th day and the 5th day proliferation times compared with first day after shCtrl cellular control unit adherent growths Respectively 1.35 ± 0.02,3.55 ± 0.13,5.66 ± 0.21,8.92 ± 0.26 (Fig. 3 B), and shIDNK interference groups cell pastes The 2nd day, the 3rd day, the 4th day and the 5th day is respectively 1.08 ± 0.06 compared to the proliferation times with first day after wall growth, 1.86 ± 0.11,2.15 ± 0.1,2.69 ± 0.21, growth multiple is significantly suppressed (Fig. 3 C).
In addition, the influence of IDNK gene pairs cell viabilities is equally demonstrated with MTT detection methods.MTT experiment result is shown Show, the 2nd day, the 3rd day, the 4th day and the 5th day cell compared with first day is lived after shCtrl cellular control unit adherent growths Power multiple is respectively 1.357 ± 0.0134,1.888 ± 0.0862,2.55 ± 0.031,3.959 ± 0.0119, and shIDNK is done The 2nd day, the 3rd day, the 4th day and the 5th day cell viability multiple compared with first day is distinguished after disturbing group cell adherent growth It is 1.182 ± 0.0179,1.552 ± 0.0407,1.924 ± 0.0611,2.267 ± 0.0125, display shIDNK groups cell increases It grows and significantly slows down (Fig. 4).
3rd, the IDNK genes interference effect BEL-7404 cell cycles redistribute
Further, we interfere the influence to the BEL-7404 cell cycles with Flow cytometry IDNK genes. Streaming is the results show that shIDNK makes G1 phases cell and G2/M phase cell proportions significantly increase (p<0.01), S phase cell proportions Significantly reduce (p<0.01) it, may lead to that the generation G1 phases of cell block and the G2/M phases block (Fig. 5 A and 5B).
4th, IDNK genes interference promotes BEL-7404 Apoptosis
Cell-cycle arrest is usually along with the generation of Apoptosis, therefore next we pass through flow cytometer detection shIDNK Influence to Apoptosis.The cell results of flow cytometer detection Annexin V labels show that the apoptosis rate of shIDNK groups is shown Writing increases, and 34.03 ± 0.0629% (p are risen to by the 4.24 ± 0.1697% of shCtrl control groups<0.01), show IDNK Gene interferes the apoptosis (Fig. 6 A and 6B) for significantly promoting BEL-7404 cells.
The above, only presently preferred embodiments of the present invention, not to the present invention in any form with substantial limitation, It should be pointed out that for those skilled in the art, under the premise of the method for the present invention is not departed from, can also do Go out several improvement and supplement, these are improved and supplement also should be regarded as protection scope of the present invention.All technology people for being familiar with this profession Member, without departing from the spirit and scope of the present invention, makes a little when using disclosed above technology contents Perhaps the equivalent variations of variation, modification and evolution are the equivalent embodiment of the present invention;Meanwhile all substantive skills according to the present invention The variation, modification and evolution of any equivalent variations that art makees above-described embodiment, still fall within technical scheme of the present invention In the range of.
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Claims (10)

1.IDNK inhibitor is used to prepare the purposes of cancer treatment drug.
2. purposes according to claim 1, which is characterized in that the cancer treatment drug at least have following function it One:Inhibition hepatoma cell proliferation, reduction liver cancer cells vigor, raising G1 phases liver cancer cells and G2/M phase liver cancer cells ratio, Reduce S phase liver cancer cells ratios, cause liver cancer cells occur the G1 phases block and the G2/M phases block, promote hepatoma cell apoptosis, Inhibit hepatic carcinoma growth.
3. purposes according to claim 1, which is characterized in that the IDNK inhibitor refers to there is inhibition to IDNK Molecule.
4. purposes according to claim 1, which is characterized in that the IDNK inhibitor be selected from siRNA, shRNA, antibody, Micromolecular compound.
5. purposes according to claim 1, which is characterized in that the IDNK inhibitor for the cancer treatment drug only One of one active ingredient or active ingredient.
6. a kind of cancer treatment drug, the IDNK inhibitor including effective dose.
7. cancer treatment drug according to claim 6, which is characterized in that IDNK inhibitor is the cancer treatment drug Sole active ingredient or one of active ingredient.
8. a kind of cancer treatment drug combination, including a effective amount of IDNK inhibitor and other at least one cancer treatment drugs.
9. pharmaceutical composition according to claim 8, which is characterized in that the combination therapy pharmaceutical composition is selected from following form In any one:
One) IDNK inhibitor and other cancer treatment drugs are respectively prepared independent preparation, the dosage form of preparation can be identical or not Together, administration route also may be the same or different;
Two) IDNK inhibitor and other cancer treatment drugs are configured to compound preparation, by IDNK inhibitor and other liver cancer When medicine is simultaneously applied simultaneously using the administration of identical administration route, using the form that the two is configured to compound preparation.
10.IDNK inhibitor is being prepared with the purposes in any one of following or multinomial effect drug:Liver cancer cells are reduced to live Rate, inhibits liver cancer at the size inhibit liver cancer cells clonality, increase hepatoma cell apoptosis ratio, inhibiting hepatic carcinoma Tumour growth.
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