CN108159424A - A kind of method for cooperateing with All-trans retinoic acid plus medication and its application in the early young grain leukocytic leukemia for the treatment of - Google Patents
A kind of method for cooperateing with All-trans retinoic acid plus medication and its application in the early young grain leukocytic leukemia for the treatment of Download PDFInfo
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- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a kind of methods for cooperateing with all-trans retinoic acid (ATRA) drug combination, the expression that this method passes through scaffolding protein RACK1 in targeted inhibition acute promyelocytic leukemia cell, and all-trans retinoic acid is used in combination, so as to play the role of drug combination.It is a potential preferable novel targets in APL treatments that inventor discloses RACK1 for the first time in the present invention.The present invention also clearly proposes the expression of inhibition targeting proteins RACK1, and all-trans retinoic acid ATRA inducing cell apoptosis is used in combination, and treats the new treatment of APL.Treatment for acute promyelocytic leukemia provides important clinical data and reference value.
Description
Technical field
The present invention relates to biomedical, clinical technology fields, and in particular to a kind of collaboration All-trans retinoic acid plus medication
Method and its application in the early young grain leukocytic leukemia for the treatment of.
Background technology
Acute promyelocytic leukemia (Acute Promyelocytic Leukemia, APL) is that acute myeloid is white
A kind of specific type of blood disease, its main feature is that have a large amount of leukaemia cells unlimitedly hyperplasia in marrow and other hematopoietic tissues,
And into peripheral blood, and the manufacture of normal plasma cell is significantly inhibited.The tool statistics disease occupies the head in young man's malignant disease
Position, the cause of disease are still imperfectly understood so far.Chinese scholar in 1986 uses all-trans retinoic acid (all-trans for the first time in the world
Retinoic acid, ATRA) induction-differential therapy APL, and success is obtained, it is corrected by promoting the differentiation of APL cells
The exception of coagulation avoids the bone marrow suppression caused by chemotherapy and induces disseminated intravascular coagulation (disseminated
Intravascular coagulation, DIC) possibility, the treatment of leukaemia is made great breakthrough occur.But
There are still some problems in the therapeutic process of ATRA, and particularly occur in has very sternly in the treatment of acute promyelocytic leukemic
The complication of weight.Wherein leukocytosis is its major side effects, and clinical manifestation is fever, uncomfortable in chest, expiratory dyspnea, work of breathing
Energy failure, acute renal failure etc..Although having obtained higher complete remission rate (CR) using ATRA treatments, with
The combination of chemotherapeutic is still essential to the long-term remission of APL or even healing.Chemotherapeutic is clinically used extensively at present
Daunorubicin (Idarubicin) Combined with ATRA treats APL, still, anthracene nucleus medicament easily attractive cardiac toxic and marrow
Inhibit, in addition can also cause the adverse reactions such as digestive system Nausea and vomiting.Therefore urgently need to develop effective replacement therapy or
Person's scheme of combination drug therapy overcomes the use limitation of ATRA.
The protein kinase C receptor 1 (Receptor forActivated C Kinase 1, RACK1) of activation, is a kind of
Frame albumen is made of, molecular weight 36kDa 7 highly conserved 40 structural domains of tryptophan-aspartic acid (Trp-Asp, WD).
Research shows that RACK1 high expression in kinds of tumors tissue, such as breast cancer, lung cancer, liver cancer, and adjust the pernicious increasing of tumour
It grows.But currently without targeted inhibition RACK1 for the report of the early young grain leukocytic leukemia of joint Treated with All-trans Retinoic Acid.
Invention content
The purpose of the present invention is to provide a kind of methods of the early young grain leukocytic leukemia of new treatment, that is, cooperate with alltrans
The method of vitamin A acid drug combination.
The present invention to achieve the above object, after the present invention strikes low RACK1 using the special shRNA for targeting RACK1, combines
ATRA can promote APL apoptosis, while not influence the differentiation of cell.The induction of this and daunorubicin Combined with ATRA
The mode of APL meronecrosises is different.Cell " necrosis " usually can in vivo be caused excessively by what previous multinomial research confirmed
Strong inflammatory reaction, and cell " apoptosis " is then a kind of clean death pathways of comparison.
Primarily, the present invention provides a kind of method for cooperateing with All-trans retinoic acid plus medication, this method passes through targeting
Inhibit the expression of scaffolding protein RACK1 in acute promyelocytic leukemia cell, and all-trans retinoic acid is used in combination, so as to play
The effect of drug combination.
Preferably, the expression of scaffolding protein RACK1 is to pass through target in the targeted inhibition acute promyelocytic leukemia cell
Low RACK1 is struck to the special shRNA of RACK1.
Further, the present invention also provides a kind of progranulocyte leukemia drug, the drug includes active ingredient
All-trans retinoic acid and RACK1 inhibitor.
Preferably, the RACK1 inhibitor is the antisense oligonucleotides of RACK1 nucleic acid, siRNA, shRNA and RACK1
The inhibitor of albumen.
Preferably, the drug further includes the medicine of pharmaceutically acceptable carrier or other treatment progranulocyte leukemia
Object.
Carrier of the present invention is pharmaceutically acceptable carrier, is referred to：One or more biocompatible solids
Or liquid filler or gelatinous mass.They are suitable for people's use and it is necessary to have enough purity and sufficiently low toxicity." phase
In capacitive " referred to herein as composition the active constituent of each component energy and the present invention and they between mutually admix, it is and unknown
The aobvious drug effect for reducing active constituent.
Preferably, the carrier includes but not limited to：Diluent, buffer, suspension, emulsion, granule, encapsulation agents,
Excipient, adhesive, spray, cutaneous permeable agent, wetting agent, disintegrant, sorbefacient, surfactant, filler
Toner, corrigent or absorption carrier.
Preferably, the drug can be made including but not limited to microinjection agent, the dosage form suitable for transfection, parenteral solution,
Tablet, pulvis, granula, capsule.The drug of above-mentioned various dosage forms can be prepared according to the conventional method of pharmaceutical field.
Further, the answering in the early young grain leukocytic leukemia for the treatment of the present invention also provides the above method or drug
With.
Preferably, the application is the apoptosis that RACK1 strikes low induction APL cells, and it is thin to APL not influence all-trans retinoic acid
Born of the same parents induce the treatment of differentiation.
Preferably, RACK1 strikes low induction APL Apoptosis and is to rely on lysosome-autophagy approach.
Further, the present invention also provides a kind of RACK1 inhibitor, the inhibitor includes：The antisense of RACK1 nucleic acid
The inhibitor of oligonucleotides, siRNA, shRNA and RACK1 albumen.
Preferably, the inhibitor of the RACK1 is shRNA, the nucleotide sequence such as SEQ ID of the positive-sense strand of the shRNA
NO:Shown in 1, the nucleotide sequence such as SEQ IDNO of the antisense strand of the shRNA:Shown in 2.
Advantageous effect
It is a potential preferable novel targets in APL treatments that inventor discloses RACK1 for the first time in the present invention.The present invention is also
It clearly proposes the expression of inhibition targeting proteins RACK1, and all-trans retinoic acid ATRA inducing cell apoptosis is used in combination, treat
The new treatment of APL.Treatment for acute promyelocytic leukemia provides important clinical data and reference value.
Description of the drawings
Fig. 1：Daunorubicin Combined with ATRA induction APL meronecrosis situations are detected in NB4 cells；
Fig. 2：Daunorubicin Combined with ATRA induction APL meronecrosis situations are detected in HL-60 cells；
Fig. 3：Immunoblotting (WB) detection targeting RACK1shRNA strikes low RACK1 albumen situation in APL cells；
Fig. 4：ATRA inducing cell apoptosis is cooperateed with after low RACK1 is struck in NB4 cells；
Fig. 5：ATRA inducing cell apoptosis is cooperateed with after low RACK1 is struck in HL-60 cells；
Fig. 6：Low RACK1 is struck in NB4 cells does not influence the cell differentiation of ATRA inductions；
Fig. 7：Low RACK1 is struck in HL-60 cells does not influence the cell differentiation of ATRA inductions；
Fig. 8：Inflammatory cytokine secreting, expressing declines after low RACK1 is struck in HL-60 cells；
Fig. 9:Autophagy GAP-associated protein GAP LC3B expression declines after low RACK1 is struck in HL-60；
Figure 10:Autophagy inhibitor can reverse in NB4 cells strike low RACK1 caused by LC3B protein expressions decline；
Figure 11:Autophagy inhibitor can be reversed partly in NB4 cells strike low RACK1 caused by early apoptosis of cells increase
Add.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
Used in the conventional means that are well known to those skilled in the art of technological means.
The preparation of reagent, material
Experimental cell strain
APL cell line HL-60s cell in people source used in this subject is purchased from American Type Culture collection warehousing (American
Type culture collection, ATCC：CCL-240), NB4 cells are purchased from Su Zhoubei Na Chuanlian Bioisystech Co., Ltd
(BNCC337678).Experiment cell strain, which is used, is added to 10% fetal calf serum and the penicillin and streptomysin of 100 μ g/mL,
37 DEG C are placed on, 5%CO2It is cultivated in incubator.
Major experimental reagent
NC (non-targeting control) shRNA for targeting RACK1shRNA slow virus and non-targeted control is slow
Virus is purchased from Shanghai JiKai Gene Chemical Technology Co., Ltd, other common chemical reagent be domestic analysis it is pure or into
Mouth product.
Major experimental instrument and equipment
The preparation of common test reagent
(1) 1640 cell culture mediums of RPMI prepare (1L)：One bag of 1640 dry powder (Gibco), NaHCO32.0g/L, three steam
Water dissolution, room temperature are slowly stirred 4 hours, and it is 7.2 then to adjust pH value with concentrated hydrochloric acid, is settled to 1L, first with filter paper coarse filtration, then
Again with 0.22 μm of filtering with microporous membrane degerming (pH value generally rises 0.1-0.3 after filtering), it is sub-packed in 500mL vials,
It is sealed with sealed membrane, 4 DEG C of preservations are (final concentration of using preceding addition penicillin (final concentration of 100U/mL), streptomycin sulphate
100U/mL) and fetal calf serum (total volume 10%)；
(2) 4 × SDS sample-loading buffers prepare (50mL)：4g SDS, 0.2g bromophenol blues, 20mL glycerine, 20mL 1M
Tris-HCL pH 6.8, magnetic stirring apparatus are stirred at room temperature to being completely dissolved, and are then settled to 50mL with distilled water, packing, 900 μ
L/ manages (1.5mL EP pipes), and minus 20 DEG C of refrigerators preserve, and 100 μ L beta -mercaptoethanols are added in preceding often pipe；
(3) cells frozen storing liquid：Fetal calf serum presses 9 with DMSO：1 volume ratio mixed preparing forms, and matching while using；
(4) 5 × Tris- glycine running buffers prepare (2L)：Glycine 188g, Tris base 30.2g, SDS
10g is dissolved with distilled water, is stirred at room temperature to being completely dissolved, is settled to 1L, is placed at room temperature for spare, application method：Directly steamed with double
5 × Tris- glycine running buffers are diluted to 1 by water ×；
(5) 5 × albumen transfer buffer (2L)：Glycine 29g, SDS 3.7g, Tris base 58g, with double steamings
Water dissolution is stirred at room temperature to being completely dissolved, and is settled to 2L, and room temperature is spare.Application method：It is slow to take out the transfer of 5 × albumen of 200mL
Fliud flushing, 200mL absolute methanols, distilled water are settled to 1L, mixing；
(6) 10 × TBS buffers (2L)：Tris-base 48.46g, NaCl 160.12g, are dissolved with distilled water,
It is stirred at room temperature to being completely dissolved, is settled to 2L, room temperature is spare.Application method：5 × Tris- glycine electrophoresis is delayed with distilled water
Fliud flushing is diluted to 1 ×, by 1：1000 volume ratio adds in polysorbas20 (final concentration 0.1%), and mixing room temperature is spare；
(7) 10 × PBS prepare (1L)：NaCl 80g, KCl 2g, Na2HPO4.12H2O 36g, KH2PO42.4g, with double steamings
Water dissolution is stirred at room temperature to being completely dissolved, 1L is settled to concentrated hydrochloric acid adjustment pH=7.4 distilled waters；
The preparation (100mL) of (8) 5% bovine serum albumin(BSA) (BSA)：BSA 5g are weighed, are dissolved with 1 × TBST of 60mL,
It is stirred at room temperature to being completely dissolved, is then settled to 100mL with 1 × TBST, be sub-packed in 2mL EP pipes, often pipe 2mL, is stored in minus 20
DEG C refrigerator is spare；
The preparation (100mL) of (9) 5% skim milk：Skimmed milk power 5g is weighed, is dissolved with 1 × TBST of 60mL, room temperature
Then stirring is settled to 100mL to being completely dissolved with 1 × TBST, be contained in suitable reagent bottle (closing is used), be stored in negative
20 DEG C of refrigerators are spare；
(10) FACS washing lotions are prepared：1 × PBS, fetal calf serum (3%), sodium azide (0.01%).
1 ATRA of embodiment joint daunorubicins can induce the necrosis of APL cells
1st, cell recovery
A. the preparation before recovery cell：1640 cell culture mediums of RPMI are prepared, preheating；
B. cell is taken：The cell suspension frozen is transferred in the centrifuge tube containing 1640 culture mediums of RPMI；
C. it centrifuges, be resuspended, and be transferred in culture bottle, cultivated, while observe cell growth status.If cell density
It is excessively high, it passes in time, in case subsequent experimental uses.
2nd, cell secondary culture
A. cell growth status is observed：Cell is taken out from incubator, is placed on inverted micro- Microscopic observation cellular morphology
And cell number, observation cell whether need passage and cell passage required for diluted multiple；Then it is reentered into culture
In case.
B. cell is taken：Cell is taken out from incubator, after alcolhol burner flame is crossed at bottle cap, twist-off closure, bottleneck mistake again
Alcolhol burner flame gently blows afloat the cell sunk to the bottom with pipette, and is transferred in test tube, covers rubber plug；
C. it centrifuges, be resuspended：Centrifuge tube is put into centrifuge, is then centrifuged 4 minutes, discarded with the rotating speed of 1200r/min
Old culture medium supernatant, addition is fresh added with dual anti-and fetal calf serum RPMI1640 culture mediums, is then dispensed in suitable ratio
Into each culture bottle, every bottle of culture medium final volume is 8mL.3rd, cell freezes
A. frozen stock solution is by DMSO and fetal calf serum (HL-60 regular grades；NB4 top grades) by 1：9 ratio is formulated；
B. being taken out from incubator has the culture bottle in exponential phase cell, and be transferred into centrifuge tube, with
The rotating speed of 1200r/min, room temperature centrifuge 4 minutes；
C. old culture solution supernatant is discarded, the cells frozen storing liquid being configured in right amount is added in, cell is gently resuspended with pipette,
Using plate count method, to adjust the final densities of cell in frozen stock solution 3 × 106~1 × 107Between/mL；
D. cell suspension is sub-packed in cryopreservation tube to freeze.
4th, cell kind plate adds medicine irritation detection Apoptosis and necrosis
The cell that the present invention uses all is the cell that can reach stable growth cultivated after recovering 5-7 days.By ATRA dry powder
Being dissolved in suitable DMSO makes it store a concentration of 100mM, dispenses and is stored in minus 70 DEG C of refrigerators, faces 1 part of used time taking-up, and
Be diluted to 5mM uses liquid.
Take the logarithm NB4 the and HL-60 cells in growth period, be resuspended after centrifugation with fresh complete RPMI1640 culture mediums and according to
5×104The density of a cells/well is inoculated in 24 orifice plates, while handles NB4 and HL-60 cells respectively with ATRA (5 μM of final concentration)
3 days, final volume 1mL.Then 24 orifice plates are put into and are placed in saturated humidity, 5%CO2And it is cultivated in 37 DEG C of incubator.3
After it, then cell is handled with the daunorubicin of 0 μ g/L, 10 μ g/L, 50 μ g/L, 100 μ g/L concentration for 24 hours respectively.Daily observation is thin
Intracellular growth situation, as needed point hole, change liquid and add ATRA, after 24 hours, receive sample.
A. cell is received
Cell is gently blown afloat, cell suspension is transferred in a clean 10mL centrifuge tubes with pipettor (date has been marked,
Cell type, processing time and reagent treatment), 2000 revs/min centrifuge 5 minutes.Supernatant is abandoned, then uses AnnexinV
(1mL/ pipes) is resuspended in Buffer, and 2000 revs/min centrifuge 5 minutes, abandon supernatant again.
B. apoptosis dyeing and detection
It is operated according to the specification of AnnexinV/PI apoptosis detection kits.The every solencyte collected in previous step is added
Enter 100 μ L FACS washing lotions, be then gently resuspended in cell to 1.5mL centrifuge tubes with pipettor.With Annexin V Buffer weights
Outstanding every solencyte is separately added into the AnnexinV-APC antibody and 5 μ L PI dyestuffs of 2 μ L, covers EP pipe lids, flicks tube bottom, mixes
It is even, it is then protected from light and is placed at room temperature for 10 minutes.After ten minutes, it often manages and adds in 200 μ L Annexin V Buffer again, streaming is thin
Born of the same parents' instrument is analyzed.5th, flow cytometer detection daunorubicin main inducing cell necrosis in APL cells
Daunorubicin induction APL meronecrosises are as shown in Figure 1 and Figure 2.In Fig. 1, NB4 cells are with ATRA (final concentration of 5 μM)
Or DMSO is handled 2 days, and Flow cytometry is used after then being handled 24 hours, 24 hours with the daunorubicin of various concentration respectively
The representative figure (A) of apoptosis and statistical result (B).As shown in the figure, the NB4 cells that daunorubicin is handled, the necrosis of cell is applied alone
Rate significantly increases, and has the dependence of dosage i.e. with the increase of daunorubicin concentration, and necrosis rate also increases；And with ATRA with it is soft
The NB4 cells of erythromycin combination processing, necrosis rate are remarkably decreased, and without apparent dose dependent.Illustrate daunorubicin
Effect mainly induction NB4 cell deaths and by necrosis in a manner of based on, and be combined ATRA can not inducing cell necrosis.It is soft
When erythromycin concentration for the treatment of is 50 μ g/L and 100 μ g/L, add ATRA significant difference (* * P compared with not adding ATRA groups<
0.01)。
In Fig. 2, HL-60 cells ATRA (final concentration of 5 μM) or DMSO is handled 2 days, then respectively with various concentration
Daunorubicin uses the representative figure (A) of Flow cytometry apoptosis and statistical result (B) after handling 24 hours, 24 hours.Such as
Shown in figure, HL-60 cells that daunorubicin is applied alone to handle, the necrosis rate of cell significantly increases, and have the dependence of dosage i.e. with
The increase of daunorubicin concentration, necrosis rate also increases；And the HL-60 cells handled with ATRA and daunorubicin, necrosis rate are shown
It writes and declines, and illustrate that daunorubicin is mainly the necrosis of HL-60 cells cell without apparent dose dependent, daunorubicin
It can promote the differentiation of cell with ATRA drug combinations, it is suppressed that the necrosis of cell.Daunorubicin concentration for the treatment of is 50 μ g/L and 100
During μ g/L, add ATRA significant difference (* * P compared with not adding ATRA groups<0.01).
It these results suggest that daunorubicin is applied alone main inducing cell to necrose, and mainly induced after adding in ATRA combinations
Necrosis is less likely to occur for cell differentiation, cell.
2 WB of embodiment detection RACK1 albumen strikes inefficient fruit in APL cells
1st, cell infection slow virus：
A. cell prepares：According to the speed of growth of HL-60 and NB4 cells by HL60 and NB4 cell inoculations in good condition
To 24 orifice plates, per hole kind 1 × 105A cell, per hole final volume 1mL, is inoculated with two holes, transfect respectively NC shRNA slow virus and
RACK1shRNA slow virus；
B. virus infection：Cell is observed after inoculation 18 hours, under inverted microscope and has sunk to the bottom about covering bottom hole 60%, gently
By 24 orifice plates move into Biohazard Safety Equipment (ultraviolet to have irradiated 1h), 24 orifice plate of shallow decline with 1mL micropipettors along hole wall gently
600 μ L culture mediums (attention tries not to siphon away cell) are siphoned away, minus 70 DEG C of refrigerators take out viral juxtaposition and melt on ice, 4 DEG C of centrifugations
2 μ L NC shRNA slow virus and 5 μ L RACK1shRNA slow virus once, according to virus titer are sucked out in machine, brief centrifugation respectively
It adds in corresponding hole, gently shakes, be put into the wet box in incubator；
C. fluid infusion：Fresh 1640 complete mediums of RPMI are supplemented after 24 hours, and are determined whether according to cell density
Need a point hole；
D. cell growth status is then observed daily, and a point hole is determined the need for according to cell density, until phase
Until pass experiment terminates.
2nd, WB detects RACK1 Protein Assavs：
A. the RIPA lysates being pre-chilled in right amount are drawn in 2mL EP pipes, add in the 4 kinds of protease melted on ice in advance suppressions
The factor processed makes its final concentration of (1mM Na3VO4, 10mM PNPP, 1mMDTT, 10 μ g/mL Aprotinin), mixing is put on ice
For use.Prepare 2 1.5mL EP pipes, and respectively after the upper Cell Name of label, stimulating factor title, processing number of days and date received
It inserts spare on ice；
B. it takes out to add HL-60 the and NB4 cells of slow virus and place it on trash ice in incubator and be operated, use 1mL
Micropipettor, which is gently blown afloat, to be sunk to the bottom and because breaks up and adherent cell, and be collected in corresponding EP pipes, then in microscope
Lower observation residual cell, when necessary with each hole of normal saline flushing of precooling, 4 DEG C, 6000r/min is centrifuged 1 minute, is discarded
Culture medium supernatant, add in 1mL precooling physiological saline, blown with pipettor it is even, to wash away culture medium, 4 DEG C again, 6000r/min
Centrifugation 1 minute, discards supernatant, and goes as possible clean；
C. the RIPA lysates for preparing and being pre-chilled in right amount are added in accordingly according to the cell precipitation amount per bottom of the tube, use liquid relief
After device rifle point mixing, insert and crack 15 minutes on ice.4 DEG C of supercentrifuges, 12500r/min are centrifuged 15 minutes, are gently sucked out
Clearly and it is transferred in the 1.5mL EP pipes that preprepared is totally pre-chilled；
D. take that the addition of 2 μ L above-mentioned cell lysate supernatants is ready for respectively corresponding fills 600 μ L Coomassie brilliant blues
In the EP pipes of dye liquor, after mixing, according to Bradford methods, it is corresponding in ultraviolet specrophotometer λ=595nm that it is measured respectively
Relative protein concentration between absorbance value, i.e. each sample is proportionally added into appropriate 4 × SDS-PAGE sample-loading buffers, with egg
On the basis of the white minimum sample of concentration, calculate the supernatant requirement of other each sample with reference to total sample preparation gauge and required add
RIPA lysates are so that each protein sample is tuned into same concentrations；
E. the protein sample boiling water bath made is boiled 10 minutes, so that protein is fully denaturalized, SDS-PAGE electricity can be carried out
Swimming or jelly are saved backup in minus 20 DEG C of refrigerators.
SDS-PAGE electrophoresis：
A. it takes out and freezes in protein sample, 1 × SDS-PAGE sample-loading buffers and the albumen Marker of minus 20 DEG C of refrigerators, on ice
After thawing, sample boiling water bath is boiled 10 minutes, then 6000r/min, centrifuges 1 minute, takes out EP pipes, and flick even sample, then
Secondary 6000r/min centrifuges 1 minute, treats loading；
B. appropriate 5 × Tris- glycine running buffers is taken to be configured to 1 × Tris- glycine electrophoresis with deionized water to delay
Fliud flushing, for use.It takes out and shifts to an earlier date prepared gel (according to second edition molecular cloning formula：First match 10% lower floor's separation gel, it is different
5% upper strata concentration glue after abundant polymerization, is prepared, after it fully polymerize, tap water rinses, and is placed in dry in propyl alcohol sealing liquid face
In net plastic bag, it is spare to be placed in 4 DEG C of refrigerators), and glass offset plate is installed according to Bio-RAD companies specification, it adds in suitable
1 × Tris- glycine running buffers are measured, careful vertical extract matches glue comb, add completely interior liquid；
C. each protein sample is sequentially added in corresponding aperture, and according to predetermined requirement and designed sequence suitable
Albumen Marker is added in hole to mark, needs to add in 1 × SDS-PAGE loading Buffer of equivalent in the hole not being loaded, add
The hole of albumen Marker should also add suitable 1 × SDS-PAGE loadings Buffer, to avoid edge effect；
D. it sets every piece of glue and carries out electrophoresis according to constant current 15mA, electrophoresis is until that band of 15KDa is gone on albumen Marker
Gel end, you can stop electrophoresis.
Albumen transfers：
A. transfer groove is cleaned up, is put into the filter paper of suitable size that transfer is cut out with sponge and in advance, taking-up shifts to an earlier date
It prepares and 1 × albumen transfer buffer solution of 4 DEG C of precoolings is poured into slot, impregnate filter paper and sponge.Suitable size is cut as needed
Pvdf membrane, immerse and fill in the glass dish of absolute methanol；
B. unload offset plate, carefully gel be transferred on filter paper, then according to negative plate → sponge → filter paper → gel →
The sequence of pvdf membrane → filter paper → sponge → positive plate stacks successively, pays attention to not making occur bubble between pvdf membrane and gel, press
According to anode to anode, cathode will transfer clamping plate to the sequence of cathode and be inserted on transfer shelf, and then put in transfer groove, fill it up with transfer
Liquid, transfer groove are placed in mixture of ice and water, connect power supply, and constant pressure 60V is transferred 3 hours.Take out minus 20 DEG C of refrigerators preservation 5% is de-
Fat milk puts room temperature thawing.
Closing：
After transfer, melted 5% skim milk confining liquid in advance is poured into clean glass dish, removes PVDF
Then film is put into confining liquid rapidly, and place it in shaking table and slowly shake, and room temperature is closed 1 hour.
Primary antibody is incubated：
According to primary antibody operation instructions, draw a certain amount of primary antibody according to proper ratio and add in the advance melted WB mono- of room temperature
Anti- dilution (5%BSA), mixing after sealing film and antibody, are pasted on the rotary mixer being placed in 4 DEG C of refrigerators slowly
Rotation is incubated overnight.
Secondary antibody is incubated：
Next day takes out the pvdf membrane of overnight incubation, washes film 3 times with appropriate 1 × TBST, shaking table slowly shakes, 10 points every time
Clock.According to secondary antibody operation instructions, it is dilute to draw the advance melted WB secondary antibodies of room temperature of a certain amount of secondary antibody addition according to proper ratio
Liquid (5% skim milk) is released, mixing after sealing film and antibody, is pasted on rotary mixer and slowly rotates, incubation at room temperature 1
Hour.
Development：
After 1 hour, pvdf membrane is removed, washes film 3 times with appropriate 1 × TBST, shaking table slowly shakes, 10 minutes every time.It cuts
The smooth preservative film covering pvdf membrane (face-up) of suitable size is simultaneously put into development magazine, is gently driven away on film with toilet paper
Liquid；ECL luminescent solution A, B liquid of draws equal amounts in 2mL EP pipes and mixing, is then equably coated in band on pvdf membrane
Approximate location gently drives ECL mixed liquors extra on film away with toilet paper.Into darkroom, will shift to an earlier date prepared developer solution and
Fixing solution is poured into respectively in specified vinyl disc.The X-ray for cutting suitable size is put into magazine and covers on pvdf membrane, clamps dark
Box, rule of thumb, beginning time for exposure are generally 1 minute or so, immerse and development is slowly shaken in developer solution, be then placed in originally
It is rinsed in water, then is placed in fixing solution and terminates development.Then it is appropriately extended or shortens according to the power of first piece subsignal
Developing time, to show ideal slice, thin piece.Development finishes, and after fully cleaning remaining fixing solution with tap water, X-ray is placed in
It is dried up with hair-dryer or dried at room temperature on shelf, finally retouch upper Marker according to the cue mark in magazine, and in piece
On son mark the loading name of an article claim and loading sequence, primary antibody title, date and operator's name etc..
3rd, detection RACK1 struck in APL cells it is low after RACK1 albumen
As shown in figure 3, RACK1 struck in APL cells it is low after RACK1 protein decreaseds, illustrate to strike low with obvious effects.Specifically
Ground, (A) NB4 cell (B) HL-60 cells are to be handled 5 days with NC shRNA and RACK1shRNA respectively, are then detected and sent out with WB
The shRNA now designed can significantly strike low RACK1 expressing quantities.
ATRA inducing cell apoptosis is cooperateed with after striking low RACK1 in the detection cell of embodiment 3
The slow virus NB4 and HL-60 cells of 3 days have been infected in taking-up, add NC shRNA groups respectively plus DMSO and ATRA is (dense eventually
Spend is 5 μM), equally, RACK1shRNA groups respectively plus DMSO and ATRA (final concentration of 5 μM), after mixing, are positioned over suitable wet
Degree, 5% CO2, in 37 DEG C of incubators, cultivate 3 days, during which observe cell state daily and time-division hole add simultaneously DMSO and
ATRA.After three days, the cell of processing is taken out, gently blows afloat cell, cell suspension is transferred to a clean EP with pipettor manages
In (marked date, cell type, processing time and reagent treatment), 2000 revs/min, centrifuge 5 minutes.Supernatant is abandoned, then
(1mL/ pipes) is resuspended with Annexin V Buffer, 2000 revs/min of refrigerated centrifuge centrifuges 5 minutes, abandons supernatant again.So
100 μ L AnnexinV Buffer are added in per solencyte afterwards, after the resuspension cell of pipettor gently, are separately added into per solencyte
The AnnexinV-APC antibody of 5 μ L and 2 μ L PI dyestuffs, cover EP pipe lids, flick tube bottom, then mixing is protected from light and is placed at room temperature for 10
Minute.After ten minutes, the AnnexinVBuffer of 200 μ L of often pipe addition.After mixing, then flow cytomery is analyzed whole
Manage data.
The apoptosis situation that ATRA inducing cells are cooperateed with after low RACK1 is struck in NB4 cells, as shown in Figure 4.NB4 cells point
Not Yong NC shRNA and RACK1shRNA handle 5 days, then use Apoptosis by Flow Cytometry.(A) it is flow cytometer detection generation
Table figure, (B) are statistical result.The background apoptosis rate of the NB4 cells of RACK1shRNA and NC shRNA processing is very low, without notable
Sex differernce, and with after combination ATRA processing, NB4 apoptosis rates significantly increase, and NC shRNA and RACK1shRNA group phases
Difference more significant than apoptosis rate illustrates significantly induce NB4 Apoptosis with RACK1shRNA and ATRA combinations.
The cell of RACK1shRNA processing compares significant difference with the NCshRNA cells handled, while adds ATRA groups with being not added with
ATRA groups are compared to also significant difference (* P<0.05, * * P<0.01).
Cooperate with the apoptosis situation of ATRA inducing cells as shown in Figure 5 after striking low RACK1 in HL-60 cells.(A) it is streaming
Detection represents figure, and (B) is statistical result.Specifically, HL-60 cells are to handle 5 with NC shRNA and RACK1shRNA respectively
My god, then use Apoptosis by Flow Cytometry.It is handled with NC shRNA and thin with the HL-60 that NC shRNA and ATRA are handled
The background apoptosis rate of born of the same parents is all very low, and the two compares no significant difference.And after being combined ATRA processing, HL-60 cells wither
Dying rate significantly increases, and RACK1shRNA groups significantly increase compared to NC shRNA group HL-60 apoptosis rates, illustrate to use
RACK1shRNA and ATRA combinations can notable HL-60 cells Apoptosis.At the cell and NC shRNA that RACK1shRNA is handled
The cell of reason compares significant difference, while adds ATRA groups also significant difference (* P compared with being not added with ATRA groups<
0.05, * * P<0.01).
It these results suggest that striking low RACK1 with RACK1shRNA slow virus can cooperate with ATRA to promote HL-60 and NB4 cells
Apoptosis occurs.
Embodiment 4 detects the influence that low RACK1 is struck in APL cells to the ATRA cell differentiations induced
ATRA can induce NB4 and HL-60 cells to break up to granulocyte, detect the expression feelings of cell surface CD11b molecules
Condition can judge the differentiation situation of cell, the cell CD11b test positive of differentiation, and undifferentiated cell detection is feminine gender.Tool
Gymnastics is made as follows：
The slow virus NB4 and HL-60 cells of 3 days have been infected in taking-up, add NC shRNA groups respectively plus DMSO and ATRA is (dense eventually
Spend is 5 μM), equally, RACK1shRNA groups respectively plus DMSO and ATRA (final concentration of 5 μM), after mixing, are positioned over suitable wet
Degree, 5% CO2, in 37 DEG C of incubators, cultivate 3 days, during which observe cell state daily and time-division hole add simultaneously DMSO and
ATRA.After three days, the cell of processing is taken out, gently blows afloat cell, cell suspension is transferred to a clean EP with pipettor manages
In (marked date, cell type, processing time and reagent treatment), 2000 revs/min, centrifuge 5 minutes.Supernatant is abandoned, then
(1mL/ pipes) is resuspended with FACS washing lotions, 2000 revs/min, centrifuges 5 minutes, abandons supernatant again.Then 100 μ L are added in per solencyte
FACS washing lotions after cell gently are resuspended with pipettor, the Anti-human CD11b PE antibody of 2 μ L are added in per solencyte, is covered
EP pipe lids, flick tube bottom, then mixing is protected from light and is placed at room temperature for 20 minutes.After twenty minutes, addition 1mL FACS again are often managed to wash
Liquid, centrifuges 5 minutes, abandons supernatant again by 2000 revs/min.Then 1% paraformaldehyde of 200 μ L is added in per solencyte.Mixing
Afterwards, flow cytomery, then analysis and arrangement data.
As shown in fig. 6, do not influence cell differentiation after low RACK1 is struck in NB4 cells.(A) to represent streaming figure, (B) is
Statistical result.Specifically, NB4 cells are handled 5 days respectively with NC shRNA and RACK1shRNA, then examined with flow cytometry
Survey the expression of cell CD11b.The results show that the NB4 cell CD11b Positive rates with NC shRNA combination ATRA processing
It is 44.8%, and is 44% with the NB4 cell CD11b Positive rates of RACK1shRNA combination ATRA processing, the two is not aobvious
Sex differernce is write, illustrates not influence the cell differentiation that ATRA is induced after striking low RACK in NB4 cells.
As shown in fig. 7, nor affect on cell differentiation after low RACK1 is struck in HL-60 cells.(A) to represent streaming figure,
(B) it is statistical result.Specifically, HL-60 cells are handled 5 days respectively with NC shRNA and RACK1shRNA, streaming is then used
The expression of cell art detection cell CD11b.The results show that the HL-60 cells CD11b inspections of NC shRNA combination ATRA processing
It is 77.9% to survey positive rate, and is with the HL-60 cell CD11b Positive rates of RACK1shRNA combination ATRA processing
77.2%, the two does not have significant difference, illustrates do not have after low RACK1 is struck in HL-60 cells to the ATRA cell differentiations induced
Have an impact.
It these results suggest that striking low RACK1 with RACK1shRNA slow virus can cooperate with ATRA to promote apoptosis,
But do not influence cell differentiation.
The influence after low RACK1 to expression of inflammatory cytokines is struck in 5 ELISA of embodiment detections in APL cells.
ATRA can induce APL cells to be broken up to granulocyte, and granulocyte is the main source of internal inflammatory factor secretion.
Therefore clinically " leukocytosis " of ATRA inductions can cause body to generate too strong inflammatory reaction, this is the most main of ATRA
Want one of adverse reaction.Here we select tumor necrosis factor (TNF-α) and leucocyte (IL-6) as measurement inflammatory reaction
Leading indicator.APL cells are stimulated using lipopolysaccharides (LPS), the situation of cytokine secretion are detected, to judge RACK1 right
The effect of PAL cells.
1st, Supernatant samples are prepared
Taking-up has infected slow virus 3 days and has added DMSO and ATRA NB4 the and HL-60 cells of two days, will at third day
The method of control group and the previously described tally of experimental group cell counts, and identical cell number is then taken to rejoin 24 holes
Plate, while add LPS (final concentration of 1 μ g/mL), final volume is 500 μ L.After LPS is added to stimulate 4 hours, the cell of processing is taken out, gently
Featheriness plays cell, and cell suspension is transferred in clean EP pipes with pipettor and (has marked date, cell type, processing time
And reagent treatment), it 6000 revs/min, centrifuges 1 minute.Cell conditioned medium is transferred in other clean EP pipes and (has marked day
Phase, cell type, processing time and reagent treatment), it is frozen immediately in -20 DEG C of refrigerators, it is spare.
2nd, ELISA is tested
A. wrapper sheet：The evening before that day of ELISA experiments is done, takes out elisa plate item, on specific plank, Ran Houyong
10 × coating buffer are diluted to 1 by tri-distilled water ×, then with 1 × coatingBuffer respectively by TNF-α and
IL-6,250 × Capture antibody is diluted to 1 ×, 50 μ L/ holes of elisa plate are added in pipettor, are marked, with preservative film packet
It is good, four degree of refrigerator overnights.
B. overnight after, take 100mL 10 × PBS be diluted to 1L with distilled water, then add in 1mL polysorbas20 (final concentration
For 0.1%), after mixing, 250 μ L/ holes are washed 5 times.
C. with tri-distilled water by 5 × Assay Dilute be diluted to 1 ×, then 100 μ L/ holes, are placed at room temperature for 1 hour.
D. it is washed one time with PBST, 250 μ L/ holes.
E. the TNF-α and IL-6 standard items frozen in -70 DEG C of refrigerators is taken out, is placed on ice to melt, then with 1 × Assay
Dilute gradient dilutions are 8 concentration.The sample frozen in -20 DEG C is taken out, is placed on after melting on ice, distinguishes together with standard items
It is added in corresponding hole, 50 μ L/ holes.Incubation at room temperature 2 hours.
F. it is washed five times with PBST, 250 μ L/ holes.
G. with 1 × Assay Dilute respectively by 250 × TNF-α and IL-6Detection antibody be diluted to 1 ×,
Then 50 μ L/ holes are incubated at room temperature 1 hour.
H. it is washed five times with PBST, 250 μ L/ holes.It adds after PBST is placed at room temperature for 1-2 minutes, is discarding supernatant every time
I. with 1 × Assay Dilute by 250 × Avindin HRP horseradish enzyme antibodies be diluted to 1 ×, 50 μ L/ holes.
Room temperature is protected from light incubation 30 minutes.
J. it is washed seven times with PBST, 250 μ L/ holes.It adds after PBST is placed at room temperature for 1-2 minutes, discards supernatant every time
K. plus developing solution TMB, 50 μ L/ holes, room temperature are protected from light colour developing, according to the color of standard items to determine whether terminating aobvious
Color.
Plus 1N H l.2SO4Terminate liquid, 50 μ L/ holes, color development stopping.
M. upper machine testing, analyzes data.
3rd, testing result
As shown in figure 8, the situation of cytokine-expressing is detected after low RACK1 is struck in HL-60 cells.(A) it is surveyed for IL-6
Amount is as a result, (B) is TNF-α measurement result.HL-60 cells use NC shRNA and RACK1shRNA slow virus processing 5 days respectively, so
Afterwards plus ATRA or DMSO is handled 3 days, after LPS stimulates 4 hours again, receives supernatant, ELISA detections.The results show that added with ATRA's
In the case of, for RACK1shRNA groups compared with NC shRNA group cells, two kinds of cytokine TNF-α and IL-6 secretions are significant
Difference (* * P<0.01).The secretion of inflammatory factor can be substantially reduced after low RACK1 by illustrating to strike, and reduce inflammation reaction.This is facing
It is possible that improving the survival rate of patient on bed.
Low RACK1 is struck in 6 APL cells of embodiment by inhibiting cell autophagy that ATRA is cooperateed with to promote Apoptosis
The situation of change of autophagosome molecule L C3B after low RACK1 is struck in 6.1 detections in APL cells
Work (the RACK1Promotes Autophagy by Enhancing the Atg14L- that we deliver early period
Beclin 1-Vps34-Vps15Complex Formation upon Phosphorylation by AMPK.Cell
Rep.2015,13(7):1407-1417.) find that RACK1 molecules can be adjusted by regulating and controlling the assembling of autophagosome compound
Cell autophagy.Therefore it is contemplated that whether RACK1 is also to adjust Apoptosis by regulating and controlling autophagy approach in APL cells.
And LC3B albumen is the significant molecule for detecting autophagy, so we strike tables of the low RACK1 to LC3B by WB detections first
Up to the situation of influence.
After aforementioned method recovery cell and infection slow virus, the slow virus HL-60 cells of 3 days, NC have been infected in taking-up
After shRNA groups and RACK1shRNA groups give DMSO or ATRA (final concentration of 5 μM) mixing respectively, it is positioned over proper moisture, 5%
CO2, in 37 DEG C of incubators, cultivate 3 days, WB detection LC3B expressions.
The results are shown in Figure 9, and after low RACK1 is struck in NB4 cells, LC3B protein expressions are decreased obviously.Illustrate thin in APL
RACK1 can regulate and control autophagy approach really in born of the same parents.
Whether 6.2 detection autophagy inhibitors can reverse RACK1 in NB4 cells to strike low caused LC3B albumen reduction.
Further to prove that RACK1 is to regulate and control APL cell survivals by the approach that autophagy relies on.We are with special autophagy
Inhibitor E64d and pepstatinA come inhibit APL cells occur autophagy, observe its can reverse RACK1 strike it is low caused by LC3B
The reduction of albumen.
After aforementioned method recovery cell and infection slow virus, taking out the NB4 cell identical with above-mentioned processing method is
It is (final concentration of to add in autophagy inhibitor E64d and pepstatinA after 3 days for NC shRNA or the RACK1shRNA slow virus of infection
10 μ g/ml) or DMSO preact 30 minutes, being subsequently added into DMSO or (final concentration of 5 μM) of ATRA stimulates three days, then receives thin
Born of the same parents, sample preparation and WB detection LC3B albumen, method are consistent with the above method.
The results are shown in Figure 10, and LC3B expression declines after low RACK1 is struck in NB4 cells, while under ATRA stimulations, strikes
LC3B albumen also significantly reduces low RACK1 groups compared with the control group, however after adding in autophagy inhibitor, strike low RACK1 groups
LC3B protein levels are restored.Experimental result further explanation strikes low RACK1 inducing cell apoptosis and is to rely on autophagy approach.
6.3 detection lysosomal pathways in RACK1 struck in NB4 cells it is low after apoptosis situation
Equally, by aforementioned method recovery cell and infection slow virus after, add in autophagy inhibitor E64d and
PepstatinA (final concentration of 10 μ g/ml) pretreatment cell 30 minutes, then it is thin with DMSO or (final concentration of 5 μM) effects of ATRA
After born of the same parents three days, cell is received also according to above-mentioned method, contaminates apoptosis, upper machine flow measurement formula analyzes data.
As a result as shown in figure 11, Combined with ATRA can induce cell generation early apoptosis after low RACK1 is struck in NB4 cells,
And apoptosis rate significant difference compared with the control group.And after having added autophagy inhibitor, the early stage that RACK1 strikes low rear induction withers
Rate is died compared with the control group without significant difference.(**P<0.01).Illustrate to strike low RACK1 joints in NB4 cells
ATRA induced apoptosis is strictly the mechanism relied on by autophagy.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>Wu Huifang
<120>It is a kind of to cooperate with the method for All-trans retinoic acid plus medication and its in the early young grain leukocytic leukemia for the treatment of
Using
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA/RNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
ggatgagacc aactatgga 19
<210> 2
<211> 19
<212> DNA/RNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
tccatagttg gtctcatcc 19
Claims (10)
- A kind of 1. method for cooperateing with All-trans retinoic acid plus medication, which is characterized in that this method passes through targeted inhibition acute morning The expression of scaffolding protein RACK1 in young grain leukaemia cell, and all-trans retinoic acid is used in combination, so as to play drug combination Effect.
- 2. method as described in claim 1, which is characterized in that stent egg in the targeted inhibition acute promyelocytic leukemia cell The expression of white RACK1 is to strike low RACK1 by targeting the special shRNA of RACK1.
- 3. a kind of progranulocyte leukemia drug, which is characterized in that the drug includes the all-trans retinoic acid of active ingredient With RACK1 inhibitor.
- 4. drug as claimed in claim 3, which is characterized in that the RACK1 inhibitor is the antisense oligonucleotides of RACK1 nucleic acid The inhibitor of acid, siRNA, shRNA and RACK1 albumen.
- 5. the drug as described in claim 3 or 4, which is characterized in that the drug further include pharmaceutically acceptable carrier or its He treats the drug of progranulocyte leukemia.
- 6. drug described in method as claimed in claim 1 or 2 or claim 3~5 any one is in the early young grain leucocyte for the treatment of Application in leukaemia.
- 7. application as claimed in claim 6, which is characterized in that the application is the apoptosis that RACK1 strikes low induction APL cells, Without influencing treatment of the all-trans retinoic acid to the induction differentiation of APL cells.
- 8. application as claimed in claim 7, which is characterized in that the RACK1 strikes low induction APL Apoptosis and is to rely on lyase Body-autophagy approach.
- 9. a kind of RACK1 inhibitor, which is characterized in that the inhibitor includes：The antisense oligonucleotides of RACK1 nucleic acid, The inhibitor of siRNA, shRNA and RACK1 albumen.
- 10. inhibitor as claimed in claim 9, which is characterized in that the inhibitor of the RACK1 is shRNA, the shRNA's The nucleotide sequence of positive-sense strand such as SEQ ID NO:Shown in 1, the nucleotide sequence such as SEQ ID NO of the antisense strand of the shRNA: Shown in 2.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114432316A (en) * | 2022-01-17 | 2022-05-06 | 安徽医科大学第二附属医院 | Pharmaceutical composition containing all-trans retinoic acid and palbociclib and application thereof |
-
2018
- 2018-02-08 CN CN201810126393.9A patent/CN108159424A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| WEN-DIE WANG ETAL: "RACK1 expression contributes to JNK activity, but JNK activity does not enhance RACK1 expression in hepatocellular carcinoma SMMC-7721 cells", 《ONCOLOGY LETTERS》 * |
| WU HUIFANG ETAL: "RACK1 knockdown synergizes with All-Trans Retinoic Acid to induce apoptosis in Human Acute Myeloid Leukemia cells", 《十二届全国免疫学学术大会分会场交流报告集》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114432316A (en) * | 2022-01-17 | 2022-05-06 | 安徽医科大学第二附属医院 | Pharmaceutical composition containing all-trans retinoic acid and palbociclib and application thereof |
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