CN106282182A - MiR 6812 5p application in leukemic medicine is treated in preparation - Google Patents
MiR 6812 5p application in leukemic medicine is treated in preparation Download PDFInfo
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- CN106282182A CN106282182A CN201610634002.5A CN201610634002A CN106282182A CN 106282182 A CN106282182 A CN 106282182A CN 201610634002 A CN201610634002 A CN 201610634002A CN 106282182 A CN106282182 A CN 106282182A
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Abstract
The invention provides a kind of following 1) 4) in arbitrary described material have in preparation and promote the migration of tumor cell, spread and/or application in the product attacked: 1) miR 6812 5p；2) recombinant vector of the encoding gene containing miR 6812 5p；3) recombinant virus of the encoding gene containing miR 6812 5p；4) recombinant viral vector of the encoding gene containing miR 6812 5p.The experiment proves that, fail to affect the growth of Daudi cell after miR 6812 5p process LAN, but invasion and attack and the transfer ability of leukaemia can be obviously enhanced, may play an important role in leukemia transfer process, therefore illustrating, miR 6812 5p is relevant to leukemia transfer.
Description
Technical field
The present invention relates to biological technical field, concrete, relate to a kind of microRNA and treat leukemic medicine in preparation
In application.
Background technology
MicroRNA (miR), is the strand microRNA of a kind of size about 21-23 base, by with target gene mRNA
Base pairing guides silencing complex (RISC) degraded mRNA or the translation of suppression mRNA, generally at post-transcriptional level modulin
Express (finding that microRNA also can be in transcriptional level control gene expression recently).The deepest along with what microRNA was studied
Entering, increasing evidence shows that microRNA molecule plays proto-oncogene in the links of tumor development or presses down
The effect of oncogene.
Leukemia is a class hematopoietic stem cell malignant clone disease.Clonal leukaemia because proliferation out of control, point
The mechanism such as change obstacle, apoptosis are obstructed breed accumulation in bone marrow and other hemopoietic tissue in a large number, and infiltrate its hetero-organization and organ,
Normal hematopoiesis is suppressed simultaneously.Anemia, hemorrhage, infectious fever and liver in various degree seen from clinic, spleen, lymphadenectasis and
Skeleton pain.It is reported, the leukemic sickness rate in China each department accounts for the 6th in various tumors.Clinically, perform the operation or put
It is effective for treating for limitation leukemia, and still lacks effective medicine for transitivity leukemia treating.
Occur it is therefore desirable to find with leukemia, develop relevant miRNA, thus be the white blood of clinical treatment transitivity
A kind of effective means of sick offer.
Summary of the invention
It is an object of the present invention to provide a kind of following 1)-4) in arbitrary described material in preparation, there is promotion tumor merit
Application in the product of energy:
1)miR-6812-5p；
2) recombinant vector of the encoding gene containing miR-6812-5p；
3) recombinant virus of the encoding gene containing miR-6812-5p；
4) recombinant viral vector of the encoding gene containing miR-6812-5p.Described promotion tumor function is for promoting tumor
The migration of cell, spread and/or attack.Described tumor is leukemia.
Described product is medicine；
The sequence 1 that the nucleotides sequence of described microRNA-145 is classified as in sequence table.
Second object of the present invention is to provide a kind of reconstitution cell.
The reconstitution cell that the present invention provides, for by described miR-6812-5p, the described coding base containing miR-6812-5p
The recombinant vector of cause, the recombinant virus of the described encoding gene containing miR-6812-5p or the coding base containing miR-6812-5p
The recombinant viral vector of cause imports the reconstitution cell obtained in the cell that sets out.
The described cell that sets out is tumor cell；Described tumor cell is specially leukaemia.
Described reconstitution cell suppresses the migration of tumor cell in screening and/or preparation, spreads and/or attack in medicine
Application is also the scope of protection of the invention.
Third object of the present invention is to provide a kind of product having and promoting tumor function.
The product that the present invention provides, its active component is described miR-6812-5p, the described volume containing miR-6812-5p
The code recombinant vector of gene, the recombinant virus of the described encoding gene containing miR-6812-5p or containing microRNA-145
The recombinant viral vector of encoding gene.
Described promotion tumor is the migration of promotion tumor cell, spreads and/or attack.
Described tumor is leukemia；
Described product is medicine.
Suppression miR-6812-5p activity or the material expressed are preparing the application having in the product suppressing tumor function
Also it is the scope of protection of the invention.
Described suppression tumor is the migration of suppression tumor cell, spreads and/or attack, and described tumor is specially leukemia.
The experiment proves that, fail to affect the growth of Daudi cell after miR-6812-5p process LAN, but can show
Write invasion and attack and the transfer ability strengthening high transitivity leukaemia, may play an important role in leukemia transfer process, because of
This explanation, miR-6812-5p is relevant to leukemia transfer.
Accompanying drawing explanation
Fig. 1 is fluorescence microscopy Microscopic observation lentivirus-mediated GFP-miR-6812-5p expression in Daudi cell
The relative amount of miR-6812-5p in situation and RT-PCR detection cell；
Fig. 2 draws the growth curve of Daudi-miR-6812-5p, Daudi-nc for utilizing CCK-8；
Fig. 3 is the miR-6812-5p impact on Daudi cell migration ability；
Fig. 4 is the miR-6812-5p impact on Daudi cell invasion ability.
Detailed description of the invention
Experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
Embodiment 1, stablize the acquisition of the transitivity Leukemia Cell Lines of high expressed miR-6812-5p
One, the acquisition of Leukemia Cell Lines
Colorectal carcinoma cell line Daudi (ATCC), its culture medium is the hyclone (Hyclone) containing 10% and concentration is
1640 culture medium (Invitrogen, 31800-022) of the mycillin (Invitrogen) of 0.2%.
A, cell recovery
(1) take out the cryopreservation tube of Daudi (ATCC), being immediately placed in 37 DEG C of-40 DEG C of water-baths, quickly rocking until melting completely
Change.
(2) proceed to cell suspension, in the cold PBS of 10mL, put in 15mL centrifuge tube, use horizontal centrifuge centrifuge cell
Supernatant is abandoned after (1000 revs/min, 8 minutes).
(3) with proper amount of fresh culture medium suspension cell, cell suspension is proceeded in culture bottle, put 37 DEG C of carbon dioxide and cultivate
Case is cultivated.
B, cell are cultivated
Leukemia Cell Lines is put in culture medium, in 37 DEG C, 5%CO2Cell culture incubator in cultivate, raw according to cell
Long situation, every day or change fresh culture every other day, passes during cell concentration about culture bottle culture area 80%-90%
Generation.
C, cell cryopreservation
(1) choose and be in the cell of exponential phase of growth, within frozen first 1 day, change liquid 1 time to cell.
(2) cell culture fluid is collected, centrifugal (1000 revs/min, 5 minutes).
(3) abandon supernatant, use cryopreservation tube re-suspended cell, adjust cell concentration to 5 × 106/mL。
(4) cell suspension is moved into cryopreservation tube, tighten lid, indicate Cell Name and frozen date, put into freezing storing box, put
After-80 DEG C of refrigerators 24 hours, move to liquid nitrogen preserves.
D, passage
(1) cell culture fluid is collected, centrifugal (1000 revs/min, 5 minutes).
(2) remove supernatant, add fresh culture, with Dispette, cell is blown and beaten gently, make cell suspension.
(3) put down or culture dish by the cultivation that 1: 2 immigration is new, obtain passing on rear Daudi cell.
Two, cell transfecting
1, transfection
(wherein exogenous gene is miR-6812-5p to GFP-miR-6812-5p slow virus expression system, and No. Genebank is
NR_106870, is sequence 1, purchased from Shanghai JiKai Gene Chemical Technology Co., Ltd, for expressing microRNA-6812's
Lentiviral particle), carry out cell transfecting according to the operating guidance of the said firm:
1) the rear Daudi cell that passes on, well-grown step one obtained respectively is inoculated into 96 holes in day before transfection
Plate (is normally cultivated, 37 DEG C, 5%CO2Incubator), during transfection, cell density reaches 30%.
2), every hole discards original fluid, add 100 μ L culture medium and 1 μ L slow virus reagent (GFP-miR-6812-5p
Slow virus expression system), gentle mixing.
3), 37 DEG C cultivate after 24 hours, change normal incubation medium and terminate transfection.
4), then 37 DEG C cultivate after 72 hours, obtain Daudi-high cell line.
Use same method by NC slow virus expression system (purchased from the lucky triumphant limited public affairs of chemical gene technology in Shanghai, Shanghai
Department, it does not has exogenous gene miR-6812-5p compared with GFP-miR-6812-5p slow virus expression system) Daudi is thin in transfection
Born of the same parents, obtain Daudi-NC.
2, the expression of miR-6812-5p in detection cell
1), fluorescence microscope
The Daudi-high cell line of above-mentioned acquisition is placed under fluorescence inverted microscope, detects it and express miR-6812-
5p situation, result is as shown in Figure 1A and B, it can be seen that Daudi-high cell line stablizes high expressed miR-6812-5p
(having fluorescence reaction).Observe under fluorescence microscope that its transfection efficiency is higher than 90%.
2), RT-PCR detection
Utilize Trizol (Invitrogen, 15596-018) extract above-mentioned acquisition Daudi-high cell line and
Daudi-NC cell total rna, with propose total serum IgE as template, miR-6812-5p primer carries out RT-PCR, (draws with U6 as internal reference
Thing is bought from Qiagen, and article No. is MS00033740)；Daudi cell and Daudi (NC) cell with untransfected virus are right
According to.
Result is as shown in Figure 1 C: it is 2245 that the relative expression of the miR-6812-5p of Daudi-high cell line leads；Daudi-
It is 1 that the relative expression of the miR-6812-5p of NC leads；The relative expression of the miR-6812-5p in the Daudi cell of untransfected virus
Rate is 1；It can be seen that miR-6812-5p stablizes process LAN in Daudi-high cell line.
Embodiment 2, Daudi-high cell line stablize the functional study of high expressed miR-6812-5p
1, cell growth curve
(1) latter 24 hours of transfection is taken, the Daudi-high obtained by embodiment 1 that growth conditions is good, use general biography
Digest for method, make cell suspension.Counting, accurately by cell kind in 96 orifice plates, every porocyte sum requires one
Causing (2000/hole), the amount adding nutrient chemical also wants consistent, and arranges blank (culture medium of i.e. same volume), every hole
Final volume is 100 μ L.Put 37 DEG C, 5%CO2Incubator is hatched 24 hours.
(2) every hole adds the CCK-8 solution (purchased from Japan colleague's chemistry institute article No.: CK04) of 10ul.Can be according to tool
Body situation is increased or decreased the consumption of CCK-8 solution.
(3) regular culture conditions (37 DEG C, 5%CO2Incubator) under cultivate 4 hours (incubation time depend on being used
Cell type and cell concentration).
(4) horizontal jitter measures the OD value of 450nm wavelength by microplate reader after about 10 seconds, keeps data.
(5) every day of method successively measures, totally 7 days.The cell often cultivating no count to be given in 2 days changes liquid.
(6) with incubation time as transverse axis, OD value is the longitudinal axis, is depicted on semilogarithmic paper, after junction curve should
The growth curve of cell.
Test in triplicate, results averaged.
Result is shown in Fig. 2, it can be seen that Daudi-high is adding after CCK-8 solution the 96 of 24,48,72,96 hours
In orifice plate, the OD value of every porocyte is respectively 0.58,0.97,1.47,2.08.Blank add CCK-8 solution 24,48,
72, in 96 orifice plates of 96 hours, the sum of every porocyte is respectively 0.61,1.02,1.53,2.14；It can be seen that miR-6812-
Fail to affect the growth of Daudi cell after 5p process LAN.
2, Cell migration assay
(1) by Transwell cell (purchased from Corning company) with serum-free 1640 culture medium (Invitrogen,
31053036) balance at least one hour or overnight, contribute to more preferably attaching and growing.
(2) experiment the previous day, the Daudi-high cell hungry training in serum-free medium that will be obtained by embodiment 1
Support.
(3) Transwell is put into 24 orifice plates, room adds under Transwell the cultivation that 600 μ L contain 10% serum
Base is as chemotactic factor.
(4) first take transfection latter 24 hours and the cell of non-serum starved mistake, after digestion, carry out viable count, adjust cell
Concentration is 4 × 105/ mL, then in each Transwell cell, add the Daudi-high cell that 100 μ L are obtained by embodiment 1
Suspension (after hungry cultivation, 4 × 104Cell).Often group sets three parallel sampless.It is placed in incubator cultivation.
Suck room liquid after (5) 24 hours, moisten cotton swab with PBS and wipe and above film, do not pass through aperture on polycarbonate membrane
Cell, normal saline rinse, the most dry.
(6) 0.5mL methanol crystallization purple solution dyes 30 minutes, and flowing water rinses 3-5 time.
(7) carefully being cut by polycarbonate membrane with small blade, put film on microscope slide, film bottom surface upward, is used after dripping resin
Coverslip mounting, the cell number in 5 visuals field of random counter under microscope, summation, carry out statistical test, test in triplicate,
Results averaged.
With transfection NC Daudi-NC as negative control (NC).
Test in triplicate, results averaged.
Result is as it is shown on figure 3, miR-6812-5p group is Daudi-high, NC group is Daudi-NC, miR-6812-5p group
Cell number be 132, the cell number of NC group is 45, miR-6812-5p group be about NC group 3 times (P < 0.01), this explanation
MiR-6812-5p can promote the vertical transfer ability of cell.
3, cell invasion test
(1) rifle head, Eppendorf pipe, Matrigel glue (purchased from BD company), Transwell room, 24 orifice plates are placed in 4
DEG C overnight, embodiment 1 the Daudi-high cell obtained changes starved overnight in serum-free medium.Peptic cell, nothing
Blood serum medium is inhaled and is beaten, and adjusts cell concentration 1 × 106/mL。
(2) 4 DEG C dissolve Matrigel substrate overnight, are mixed by Matrigel glue with the rifle head of pre-cooling.
(3) being placed on ice by culture medium, pre-sniper's shot head takes upper in Transwell cell of 20 μ L Matrigel glue (1: 3)
Room, places 30 minutes for 37 DEG C.
(4) pre-temperature serum-free medium softly washs the Matrigel solidified, and adds 100 μ L cell suspension (1 × 105Carefully
Born of the same parents), often group sets three parallel holes.
(5) under, room adds the 600 μ L culture medium containing 10% hyclone.
(6) 37 DEG C, 5%CO it are placed in2Incubator in hatch 24 hours, it is thin that cotton swab wipes that filter membrane upper surface do not penetrates away
Born of the same parents.
(7) cell penetrated is fixed, violet staining, mounting, each sample standard deviation counting 5 high power lenses regard
Open country, takes its meansigma methods.The quantity of each group cell-penetrating filter membrane number as the index of its invasive ability of evaluation.
With the Daudi of transfection NC for negative control for comparison (NC).
Result as shown in Figure 4, miR-6812-5p group be Daudi-high, NC group be Daudi-NC, miR-6812-5p group
Cell number be 98, the cell number of NC group is 31, miR-6812-5p group relatively matched group (NC group) cell showed increased.
These external function preliminary experiments confirm that miR-6812-5p process LAN can be obviously enhanced high transitivity leukaemia's
Invasion and attack and transfer ability, highly point out it may play an important role in leukemia transfer process.
Claims (6)
- Following 1) arbitrary described material application in preparation has the product promoting tumor function in-4):1)miR-6812-5p；2) recombinant vector of the encoding gene containing miR-6812-5p；3) recombinant virus of the encoding gene containing miR-6812-5p；4) recombinant viral vector of the encoding gene containing miR-6812-5p；Described promotion tumor function is for promoting tumor cell Migration, spread and/or attack；Described tumor is leukemia；Described product is medicine；The sequence 1 that the nucleotides sequence of described miR-6812-5p is classified as in sequence table.
- 2. a reconstitution cell, for carrying the restructuring of described miR-6812-5p, the described encoding gene containing miR-6812-5p The restructuring of body, the recombinant virus of the described encoding gene containing miR-6812-5p or the encoding gene containing miR-6812-5p is sick Poisonous carrier imports the reconstitution cell obtained in the cell that sets out.
- Reconstitution cell the most according to claim 2, it is characterised in that: described cell is tumor cell；Described tumor cell It is specially leukaemia.
- 4. the reconstitution cell described in Claims 2 or 3 suppresses the migration of tumor cell in screening and/or preparation, spreads and/or invade Attack the application in medicine.
- 5. the reconstitution cell described in Claims 2 or 3 is used for the application screening in the model of suppression tumour medicine in preparation.
- 6. having the product promoting tumor function, its active component is described miR-6812-5p, described containing miR- The recombinant vector of the encoding gene of 6812-5p, the recombinant virus of the described encoding gene containing miR-6812-5p or containing miR- The recombinant viral vector of the encoding gene of 6812-5p.
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| CN110157739A (en) * | 2019-05-14 | 2019-08-23 | 天津市康婷生物工程集团有限公司 | A kind of method that can improve lymthoma Daudi cell transfecting marker representation amount proportion in cell mixing |
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