CN107099576A - A kind of applications of cell-cycle arrest agent KR in human breast cancer cell - Google Patents
A kind of applications of cell-cycle arrest agent KR in human breast cancer cell Download PDFInfo
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- CN107099576A CN107099576A CN201710449603.3A CN201710449603A CN107099576A CN 107099576 A CN107099576 A CN 107099576A CN 201710449603 A CN201710449603 A CN 201710449603A CN 107099576 A CN107099576 A CN 107099576A
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Abstract
The invention provides a kind of applications of cell-cycle arrest agent KR in human breast cancer cell, belong to Biochemistry and Molecular Biology technical field.A kind of applications of cell-cycle arrest agent KR in human breast cancer cell, step is as follows：(1) cell recovery；(2) cell culture；(3) medicine is prepared；(4) cell is handled；(5) cell proliferation inhibition rate is detected；(6) cell cycle is detected.Beneficial effects of the present invention：The data foundation of the present invention can raise CDKN1A in KR and cause ATP to exhaust, cause DNA biosynthesis blocks, and cell-cycle arrest has on the basis of in-vitro multiplication inhibitory activity of highly significant in the S phases.
Description
Technical field
The present invention relates to basic element of cell division kinetin riboside (KR) as a kind of cell-cycle arrest agent to mammary gland
JEG-3 antitumor action, belongs to Biochemistry and Molecular Biology technical field.
Background technology
Breast cancer is that occur the malignant tumour in mammary gland galandular epithelium tissue, according to IARC of the World Health Organization
(International Agency for Research on Cancer, IARC) is counted, and global women with breast cancer are new within 2015
Example of falling ill accounts for 25% or so of whole female malignant morbidities up to 1,550,000 or so；Because of breast cancer deaths, to account for all women pernicious
15% or so of tumor mortality, accounts for the 2% of all women dies.As other most countries, during breast cancer has become
The most common cancer of state women, in the East Coastal city such as Shanghai, breast cancer has surmounted lung cancer as women incidence of disease highest
Cancer；Annual Chinese Breast Cancer newly sends out quantity and The dead quantity and accounts for global 12.2% and 9.6% respectively at present.Clinically use
Although being played an important role in the conventional medicine for the treatment of breast cancer to treatment, obvious toxic side effect is there is.Through a large amount of
It was verified that with modern technologies, the active component of the natural active matter obtained from natural products can treat breast cancer, together
When mitigate chemicotherapy toxic side effect.Therefore, find and effectively improve therapeutic efficiency, the small natural drug of toxic side effect treats breast
Gland cancer is very necessary.
Basic element of cell division CK (cytokinin) is the class plant hormone that is found of year nineteen fifties, it
Played a decisive role in the growing of plant, such as：The division, tissue differentiation, lateral bud of cell is promoted to grow, suppression adventitious root,
Lateral root is formed, and suppresses aging, adjusts the apical dominance of plant, the transmission of nutrition quotation marks etc., the naturally occurring basic element of cell division is
The derivative of adenine, is that the H on its purine N6 positions is formed by other substituent groups.Length of the activity because of side chain, insatiable hunger
There is very big difference with spending different with other properties.The existence form of the basic element of cell division includes：Free, nucleosides, nucleosides -5 ' phosphorus
Acid, 3-, 7-, 9- and O-glycosides and amino conjugates.Common naturally occurring cell division have：Zeatin (Zeatin,
ZT), ribosylzeatin (Zeatin riboside, ZR), dihydro zeatin (dihydrozeatin, dHZT), isopentenyl gland
Glycosides (isopentenyl adenosine, i6A).Increasing research finds that CK not only adjusts growing for plant at present
The increment and differentiation to zooblast also function to critically important influence simultaneously.
KR (kinetin riboside) is a kind of nucleoside form for the basic element of cell division CK being present in plant, and popular name is
Kinetin nucleosides, molecular formula is C15H17N5O5, molecular weight is 347.3.The common structural feature of nucleoside compound is by glycosyl
Constituted with base, chemically in structure from the point of view of natural nucleus glycoside and nucleoside compound there is different degrees of similarity so that
Conjecture KR can also disturb or directly act on the biosynthesis of protein, nucleic acid, and disturb tumour cell and virus replication,
Played an important role in antitumor and anti-virus aspect.Factually checking reality KR is a kind of some cell line apoptosis of energy induction
Antiproliferative, its principle is can to raise CDKN1A to cause ATP to exhaust, causes cell-cycle arrest in the G2/M phases.It is right in vitro
Cancer cell is with very strong toxicity and apoptosis-promoting effect.Cause after breast cancer cell is handled with KR the cell cycle different degrees of
Be obstructed and (or) apoptosis.
The content of the invention
The present invention is directed to above-mentioned prior art, antineoplastic application in Breast cancer lines the invention provides KR.
Technical scheme：
A kind of applications of cell-cycle arrest agent KR in human breast cancer cell, step is as follows：
(1) cell recovery：The cryopreservation tube that will be equipped with freezing Breast cancer lines MCF7 takes out from -80 DEG C of refrigerators, fast
Speed is put into 37 DEG C of water-baths, gentle agitation, treats that liquid melts；Breast cancer lines MCF7 in cryopreservation tube is suspended in and contained
Have in the centrifuge tube of sterile RPMI 1640 culture mediums of 10% inactivated fetal bovine serum, 1000rpm, 5min centrifugations, centrifugation terminates
Afterwards, supernatant is abandoned, cell of gently upspringing enters sterile RPMI 1640 culture mediums with centrifuge tube, cell being suspended, turned again
Move on to left and right in blake bottle to gently shake, be uniformly distributed the cell in blake bottle, the breast carcinoma cell strain MCF7 recovered；
(2) cell culture：The Breast cancer lines MCF7 of recovery is placed in concentration for 5%CO2, relative humidity be
90%th, temperature observes cell to be cultivated in 37 DEG C of incubator after 10h, if adherent, and nutrient solution is all suctioned out, added again
Enter the RPMI 1640 culture mediums of equivalent；Treat cell length to blake bottle floor space 70%~80% pass on 1 time；
(3) medicine is prepared：KR powder is dissolved with the sterile culture mediums of RPMI 1640 without serum, makes the mother of KR solution
Liquid concentration reaches 1.2mM, continues that the mother liquor of the KR solution dissolved is diluted into concentration for 2 μ with the sterile culture mediums of RPMI 1640
M-200 μM, filtration sterilization, room temperature preservation；
(4) cell is handled：Cell in exponential phase is digested with pancreatin, after cell count, dilution, diluting cells
Density is 3 × 104~5 × 104Individual/ml, piping and druming is mixed, and cell is inoculated in 96 orifice plates, takes 100 μ l human breast carcinomas thin per hole
After born of the same parents' strain MCF7,12h, after after cell attachment, 7 groups of experiments, every group of 4 multiple holes, the 1st group are set：Blank control group；2nd group：
120 μ Μ KR groups；3rd group：20 μ Μ KR groups；4th group：10 μ Μ KR groups；5th group：2 μ Μ KR groups；6th group：1 μ Μ KR groups；7th
Group：0.2 μ Μ KR groups；The corresponding medicines of 10 μ l are added into each group respectively, after gently mixing, concentration are placed in for 5%CO2, it is relative
Humidity is to be incubated 2 days in the incubator that 90%, temperature is 37 DEG C, detection cell inhibitory effect activity；
(5) cell proliferation inhibition rate is detected：By the cell after step (4) processing, concentration is placed in for 5%CO2, relative humidity
To be incubated 48h, each 4 multiple holes in incubator that 90%, temperature is 37 DEG C；Detected, hanged per hole cell using MTT kits
Add 20 μ l MTT solution in liquid, be placed in 37 DEG C, CO23h is incubated in incubator, the absorbance of each group cell at 490nm, note is determined
Record data；
(6) cell cycle is detected：Cell in exponential phase is digested with pancreatin, after cell count, taken appropriate thin
Born of the same parents are diluted, and diluting cells density is 1 × 105~1.5 × 105Individual/ml, piping and druming is mixed, and cell is inoculated in 6 orifice plates, often
Hole is taken after 2ml Breast cancer lines MCF7,12h, after after cell attachment, blank control group and 10 μ Μ KR groups is set, to 10 μ
KR is added in Μ KR groups, the concentration for making KR is 10 μ Μ, after gently mixing, be placed in concentration for 5%CO2, relative humidity be 90%,
Temperature is distributed into two samples with 15ml centrifuge tubes to be incubated 36h in 37 DEG C of incubator, is detected according to cell cycle and apoptosis
Kit specification is fixed cell and prepared after the dye solution of iodate third, 12h, and PBS is added in each sample and is washed repeatedly, is resuspended
Cell, 37 DEG C of lucifuges of the third dye solution of iodate are incubated 30min needed for each sample is added, and are finally washed twice with PBS；300 mesh
With machine testing on BD FACSCalibur flow cytometers after screen filtration.
Beneficial effects of the present invention：The data foundation of the present invention can raise CDKN1A in KR and cause ATP to exhaust, cause
DNA biosynthesis blocks, cell-cycle arrest has on the basis of in-vitro multiplication inhibitory activity of highly significant in the S phases.
Brief description of the drawings
Fig. 1 is the inhibitory action that various concentrations KR breeds to human breast cancer cell.
Fig. 2 is influences of the KR to the human breast cancer cell cell cycle.2 (a) control group.After 2 (b) 10 μ Μ KR processing 36h
The cell cycle of MCF7 cells.
Embodiment
Below in conjunction with accompanying drawing and technical scheme, the embodiment of the present invention is further illustrated.
Cell, kit and reagent that the present invention is used：Human breast cancer MCF7 cell strain, hyclone, dual anti-(mould
Element/streptomysin), the culture mediums of RPMI 1640, MTT kits, cell cycle and apoptosis staining kit, KR.
Embodiment 1
In-vitro multiplication inhibitory activity of the KR to human breast cancer cell：By the human breast cancer cell MCF7 of recovery be incubated at containing
In the sterile RPMI1640 nutrient solutions of the streptomysin (dual anti-) of 10%FBS inactivated fetal bovine serums, 1% penicillin/1%, suspension is placed in
In cell bottle, in 37 DEG C, 5%CO2And cultivated in the incubator under saturated humidity, cell length to 70%-80% or so passage 1
It is secondary.Continue to cultivate and obtain the active human breast cancer cell of growth conditions, collect cell.By 5 × 10 in exponential phase3It is individual
Cell is inoculated in 96 orifice plates that 100 μ lRPMI 1640 culture mediums are contained in every hole, and according to experiment demand, treatment group is separately added into
10 μ l 120 μM, 200 μM, 100 μM, 20 μM, 10 μM, 2 μM of concentration KR, are put into incubator and are incubated 2 days, each 4 multiple holes.Adopt
Detected with MTT kits, add 20 μ l MTT solution in the cell of every hole, be placed in 37 DEG C, CO23h is incubated in incubator.Determine
The absorbance of each group cell at 490nm, record data analyzes each group number using the variance analysis method of Repeated Measurement Data
According to as a result as shown in Figure 1.Fig. 1 is visible, significantly low to Breast cancer lines MCF7 multiplication rate using the KR of various concentrations
In control group, show that KR can suppress Breast cancer lines MCF7 multiplication rate.
Embodiment 2
KR induces MCF7 Apoptosis：Cell in the above-mentioned culture of exponential phase is digested with pancreatin, cell count
Afterwards, appropriate cell is taken to be diluted, diluting cells density is 1 × 105~1.5 × 105Individual/ml, piping and druming is mixed, and cell is inoculated with
In 6 orifice plates, taken per hole after 2ml Breast cancer lines MCF7,12h, after after cell attachment, blank control group and 10 μ are set
Μ KR groups, 200 μ l100 μ Μ KR (working concentration is 10 μ Μ KR) are added into 10 μ Μ KR groups, after gently mixing, concentration are placed in
For 5%CO2, relative humidity be to be incubated 36h in the incubator that 90%, temperature is 37 DEG C, two samples are distributed into 15ml centrifuge tubes
Product, fix cell according to cell cycle and apoptosis detection kit specification and prepare after the dye solution of iodate third, 12h, often
10mlPBS is added in individual sample to wash twice, cell is resuspended, and each sample adds 500 μ l iodate 37 DEG C of lucifuges of the third dye solution
30min is incubated, is finally washed twice with PBS；With machine testing on BD FACSCalibur flow cytometers after 300 mesh sieve net filtrations；
As a result as shown in Fig. 2 Fig. 2 is visible, compared with control group, application KR can raise CDKN1A and cause ATP to exhaust, cause DNA to close
Into being obstructed, cell-cycle arrest is in the S phases, so as to have very strong toxicity to Breast cancer lines MCF7 in vitro and promote apoptosis
Effect.When with KR handle breast cancer cell MCF7 after cause the cell cycle be obstructed and (or) apoptosis.
Claims (1)
1. applications of a kind of cell-cycle arrest agent KR in human breast cancer cell, it is characterised in that step is as follows：
(1) cell recovery：The cryopreservation tube that will be equipped with freezing Breast cancer lines MCF7 takes out from -80 DEG C of refrigerators, puts rapidly
Enter in 37 DEG C of water-baths, gentle agitation, treat that liquid melts；By the Breast cancer lines MCF7 in cryopreservation tube be suspended in containing
In the centrifuge tube of the sterile RPMI 1640 culture mediums of 10% inactivated fetal bovine serum, 1000rpm, 5min centrifugations, after centrifugation terminates,
Supernatant is abandoned, cell of gently upspringing enters sterile RPMI 1640 culture mediums with centrifuge tube, cell being suspended, is transferred to again
Left and right gently shakes in blake bottle, is uniformly distributed the cell in blake bottle, the breast carcinoma cell strain MCF7 recovered；
(2) cell culture：The Breast cancer lines MCF7 of recovery is placed in concentration for 5%CO2, relative humidity be 90%, temperature
To be cultivated in 37 DEG C of incubators, cell is observed after 10h, if adherent, nutrient solution is all suctioned out, equivalent is added again
RPMI 1640 culture mediums；Treat cell length to blake bottle floor space 70%~80% pass on 1 time；
(3) medicine is prepared：KR powder is dissolved with the sterile culture mediums of RPMI 1640 without serum, makes the mother liquor of KR solution dense
Degree reaches 1.2mM, continue with the sterile culture mediums of RPMI 1640 by the mother liquor of KR solution dissolve be diluted to concentration be 2 μM-
200 μM, filtration sterilization, room temperature preservation；
(4) cell is handled：Cell in exponential phase is digested with pancreatin, after cell count, dilution, diluting cells density
For 3 × 104~5 × 104Individual/ml, piping and druming is mixed, and cell is inoculated in 96 orifice plates, and 100 μ l Breast cancer lines are taken per hole
After MCF7,12h, after after cell attachment, 7 groups of experiments, every group of 4 multiple holes, the 1st group are set：Blank control group；2nd group：120μΜ
KR groups；3rd group：20 μ Μ KR groups；4th group：10 μ Μ KR groups；5th group：2 μ Μ KR groups；6th group：1 μ Μ KR groups；7th group：
0.2 μ Μ KR groups；The corresponding medicines of 10 μ l are added into each group respectively, after gently mixing, concentration are placed in for 5%CO2, it is relatively wet
Degree is to be incubated 2 days in the incubator that 90%, temperature is 37 DEG C, detection cell inhibitory effect activity；
(5) cell proliferation inhibition rate is detected：By the cell after step (4) processing, concentration is placed in for 5%CO2, relative humidity be
90%th, temperature is is incubated 48h, each 4 multiple holes in 37 DEG C of incubator；Detected using MTT kits, per hole cell suspension
In plus 20 μ l MTT solution, be placed in 37 DEG C, CO23h is incubated in incubator, the absorbance of each group cell at 490nm, record is determined
Data；
(6) cell cycle is detected：Cell in exponential phase is digested with pancreatin, after cell count, takes appropriate cell to enter
Row dilution, diluting cells density is 1 × 105~1.5 × 105Individual/ml, piping and druming is mixed, and cell is inoculated in 6 orifice plates, is taken per hole
After 2ml Breast cancer lines MCF7,12h, after after cell attachment, blank control group and 10 μ Μ KR groups are set, to 10 μ Μ
KR is added in KR groups, the concentration for making KR is 10 μ Μ, after gently mixing, be placed in concentration for 5%CO2, relative humidity be 90%, temperature
Spend in the incubator for 37 DEG C and be incubated 36h, two samples are distributed into 15ml centrifuge tubes, detect and try according to cell cycle and apoptosis
Agent box specification is fixed cell and prepared after the dye solution of iodate third, 12h, and PBS is added in each sample and is washed repeatedly, is resuspended thin
Born of the same parents, 37 DEG C of lucifuges of the third dye solution of iodate are incubated 30min needed for each sample is added, and are finally washed twice with PBS；300 mesh sieves
With machine testing on BD FACSCalibur flow cytometers after net filtration.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108865999A (en) * | 2018-07-13 | 2018-11-23 | 大连理工大学 | Application of one kind induction differentiation agents in human muscle creatine kinase M2 type cell |
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|---|---|---|---|---|
| US20080139496A1 (en) * | 2001-03-06 | 2008-06-12 | Prendergast Patrick T | Combination therapy for reduction of toxicity of chemotherapeutic agents |
| WO2008045955A8 (en) * | 2006-10-10 | 2008-07-31 | Mayo Foundation | Inhibiting cyclin d polypeptides |
| US20140100174A1 (en) * | 2012-10-04 | 2014-04-10 | Kingsley Yianomah Quartey | Treatment for Cancer |
| CN104870442A (en) * | 2012-10-19 | 2015-08-26 | 阿斯特克斯治疗有限公司 | Bicyclic heterocycle compounds and their uses in therapy |
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2017
- 2017-06-15 CN CN201710449603.3A patent/CN107099576A/en not_active Withdrawn
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080139496A1 (en) * | 2001-03-06 | 2008-06-12 | Prendergast Patrick T | Combination therapy for reduction of toxicity of chemotherapeutic agents |
| WO2008045955A8 (en) * | 2006-10-10 | 2008-07-31 | Mayo Foundation | Inhibiting cyclin d polypeptides |
| US20140100174A1 (en) * | 2012-10-04 | 2014-04-10 | Kingsley Yianomah Quartey | Treatment for Cancer |
| CN104870442A (en) * | 2012-10-19 | 2015-08-26 | 阿斯特克斯治疗有限公司 | Bicyclic heterocycle compounds and their uses in therapy |
Non-Patent Citations (2)
| Title |
|---|
| PAULINA DUDZIK等: "effects of kinetin riboside on proliferation and proapoptotic activities in human normal and cancer cell lines", 《JOURNAL OF CELLULAR BIOCHEMISTRY》 * |
| 杨泽成: "布洛芬对人乳腺癌细胞系MCF-7的作用及其机制的实验研究", 《中国博士学位论文全文数据库》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108865999A (en) * | 2018-07-13 | 2018-11-23 | 大连理工大学 | Application of one kind induction differentiation agents in human muscle creatine kinase M2 type cell |
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Application publication date: 20170829 |