CN103233009A - Application of substance capable of reducing expression of zinc finger protein CTCF to preparation of drugs for treating leukemia - Google Patents
Application of substance capable of reducing expression of zinc finger protein CTCF to preparation of drugs for treating leukemia Download PDFInfo
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Abstract
The invention discloses application of a substance capable of reducing expression of a zinc finger protein CTCF to preparation of drugs for treating leukemia. In particular, the substance reducing expression of the zinc finger protein CTCF can be short hairpin RNA (shRNA) forming a stem-loop structure. The name of the shRNA is shRNA-3. One chain sequence of the stem in the stem-loop structure is SEQ ID No.1 and the other chain sequence of the stem in the stem-loop structure is SEQ ID No.2. The invention proves that the zinc finger protein CTCF is an anti-apoptosis factor and has the function of suppressing cell apoptosis in the ALL (acute lymphoblastic leukemia) cells. Apoptosis of tumor cells-leukemia cells can be promoted by interfering with expression of CTCF. The invention provides a new way for leukemia treatment. The zinc finger protein CTCF is expected to become a potential target for anti-leukemia treatment and has very extensive application prospect in the medical field.
Description
Technical field
The present invention relates to reduce the application of material in preparation treatment leukemia medicament that zinc finger protein CTCF expresses.
Background technology
Zinc finger protein CTCF claims the CCCTC binding factor again, many zinc fingerses albumen of the high conservative of being made up of 727 amino acid.It participates in the various biological regulatory function, comprises transcriptional activation or inhibition, chromatin insulation, the gene marking and x chromosome inactivation etc.
Discover that CTCF participates in the generation evolution of multiple human tumor.People have found the tumour-specific sudden change of CTCF zinc fingers in mammary cancer, prostate cancer and the nephroblastoma.This sudden change makes and the binding ability of CTCF forfeiture and downstream tumour target gene causes these abnormal gene expressions, causes tumour thus.In addition, CTCF can participate in the generation of tumour by epigenetics mechanism.The investigator finds that in the nephroblastoma, colorectal carcinoma and the rectum cancer there is the abnormal methylation phenomenon in the CTCF binding site, and infers and can cause the forfeiture of CTCF combined function thus, and then induced tumor.As seen the change of CTCF genetics and epigenetics plays a key effect in tumour takes place.
To be hematopoietic stem be obstructed and malignant proliferation and the hemopoietic system malignant disease that causes in different differentiation phases generation apoptosis obstacle, differentiation leukemia.This disease is modal malignant tumour of Childhood and the modal cause of death.About China's annual New Development leukemia of children 1.5 ten thousand examples, wherein (Acute Lymphoblastic Leukemia is modal type ALL) to children acute lymphoblastic leukaemia, accounts for 75% of leukemia of children.After the eighties in 20th century, because the combined reinforced treatment of multiple medicines and strong support treatment, the curative ratio of children ALL reaches about 80%, but the ALL curative ratio does not have obvious raising over nearly 30 years, and major cause is that leukemic pathogeny is not clear so far.In addition, leukemia relapse also is one of important factor.The recurrence rate of leukemia of children has become the most critical of the leukemia children rehabilitation factor that influences up to about 15% at present, not only gives infant itself but also has brought irremediable grievous injury humorous factor on good terms to family and society.
Therefore, the drug target of deeply inquire into leukemic pathogeny, exploring leukemia of children will help the diagnosis and treatment of leukemia of children.
Summary of the invention
A technical problem to be solved by this invention provides a kind of short hairpin RNA (shRNA, short hairpin RNA) and associated biomolecule material thereof that promotes apoptosis of leukemia.
The short hairpin RNA that promotes apoptosis of leukemia provided by the present invention, name is called shRNA-3, form stem ring (Stem-loop) structure, a chain-ordering of stem in the described loop-stem structure (Stem) is SEQ ID No.1, and another chain-ordering of stem is SEQ ID No.2 in the described loop-stem structure.
Wherein, SEQ ID No.1 is made up of 19 ribonucleotides, and SEQ ID No.2 is made up of 19 ribonucleotides.
Ring in the described short hairpin RNA (loop) makes described short hairpin RNA can produce the double-chain small disturbance RNA of being made up of the single stranded RNA shown in the single stranded RNA shown in the SEQ ID No.1 and the SEQ ID No.2 (siRNA) as long as satisfy.In one embodiment of the invention, the nucleotide sequence of described short hairpin RNA is SEQ ID No.3.
Wherein, SEQ ID No.3 is made up of 47 ribonucleotides, and the 1-19 position of SEQ ID No.3 is identical with SEQ ID No.1, and the 29-47 position of SEQ ID No.3 is identical with SEQ ID No.2.
The biomaterial that shRNA-3 provided by the present invention is relevant is 1) to 6) in any:
1) dna molecular of coding shRNA-3;
2) contain 1) expression cassette of described dna molecular;
3) contain 1) recombinant vectors of described dna molecular or contain 2) recombinant vectors of described expression cassette;
4) contain 1) recombinant microorganism of described dna molecular or contain 2) recombinant microorganism of described expression cassette or contain 3) recombinant microorganism of described recombinant vectors;
5) contain 1) transgenic cell line of described dna molecular or contain 2) transgenic cell line of described expression cassette or contain claim 3) transgenic cell line of described recombinant vectors;
6) siRNA, the nucleotide sequence of a chain such as SEQ ID No.1, the nucleotide sequence of another chain such as SEQ ID No.2.
In the relevant biomaterial of above-mentioned shRNA-3,1) dna molecular of described coding shRNA-3 specifically can be 1) to 3) in any double chain DNA molecule:
1) nucleotide sequence of a chain is SEQ ID No.4, another chain nucleotide sequence be SEQ ID No.5.
2) encoding sequence of described dna molecular is the 1-47 position of SEQ ID No.4;
3) encoding sequence of described dna molecular is the 12-58 position of SEQ ID No.5.
Wherein, SEQ ID No.4 is made up of 54 deoxyribonucleotides, and SEQ ID No.5 is made up of 58 deoxyribonucleotides.The 1-47 position of SEQ ID No.4 and the 12-58 position reverse complemental of SEQ ID No.5.
In the relevant biomaterial of above-mentioned shRNA-3,2) described expression cassette refers to express the DNA of shRNA-3 in host cell, this DNA not only can comprise the promotor that starts the shRNA-3 genetic transcription, also can comprise the terminator of termination shRNA-3 genetic transcription.Further, described expression cassette also can comprise enhancer sequence.In one embodiment of the invention, described expression cassette is made up of the dna molecular of U6 promotor (its sequence is shown in SEQ ID No.8), coding shRNA-3 and the Pol III terminator (sequence is 5 '-TTTTT-3 ') that stops the dna molecular of coding shRNA-3.3) described recombinant vectors specifically can be the recombinant expression vector pDsU6-sh-3 that replaces the expression shRNA-3 that fragment obtains between the Sac I of pDsU6 and the Hind III with the dna molecular of coding shRNA-3, or is the recombinant expression vector pDsU6-GFP-sh-3 of the expression shRNA-3 that obtains with fragment between the BamH I of the dna molecular replacement pDsU6-GFP of coding shRNA-3 and the Hind III.PDsU6-GFP uses the GFP(green fluorescent protein) gene replaces the recombinant expression vector of the expression GFP that fragment obtains between the Afl II of pDsU6 and the BamH I.4) described recombinant microorganism specifically can be bacterium, yeast, algae and fungi.5) described transgenic cell line does not comprise the reproductive material of animal and plant.
Another technical problem to be solved by this invention provides shRNA-3 and the associated biomolecule material is used at the face that treats and/or prevents leukemia side.
Provided by the present inventionly be applied as among the C1 to C4 any:
C1, treat and/or prevent leukemic product (as medicine), its activeconstituents comprises at least a among the b1-b3:
b1、shRNA-3；
The dna molecular of b2, coding shRNA-3;
The biomaterial that b3, above-mentioned shRNA-3 are relevant.
C2, promote the product (as medicine) of apoptosis of leukemia, its activeconstituents to comprise at least a among the described b1-b3;
At least a material treats and/or prevents application in the leukemic product (as medicine) in preparation among C3, the described b1-b3;
The application of at least a material in the product (as medicine) of preparation promotion apoptosis of leukemia among C4, the described b1-b3.
In the product of above-mentioned C1 and C2, activeconstituents also can only be at least a among the b1-b3.
Another technical problem to be solved by this invention provides the purposes of the material that reduces zinc finger protein CTCF genetic expression.
The purposes of the material of reduction zinc finger protein CTCF provided by the present invention genetic expression is a1 or a2:
A1, the material that reduces zinc finger protein CTCF genetic expression treat and/or prevent application in the leukemic product (as medicine) in preparation;
The application of material in the product (as medicine) of preparation promotion apoptosis of leukemia of a2, reduction zinc finger protein CTCF genetic expression.
Wherein, the material of described reduction zinc finger protein CTCF genetic expression is at least a among the described b1-b3.
In the such use, the aminoacid sequence of described zinc finger protein CTCF is shown in SEQ ID No.7, and the encoding sequence of described zinc finger protein CTCF gene is shown in the 445-2628 position of SEQ ID No.6.
Wherein, SEQ ID No.7 is made up of 727 amino-acid residues, and SEQ ID No.6 is made up of 3946 deoxyribonucleotides, and its encoding sequence is the 445-2628 position.
Above described leukemia cell can be the cell in the body, also can be clone, is NALM-6 as people's pre B cell acute lymphoblastic leukemia cell.
Experimental results show that, mRNA with zinc finger protein CTCF is target spot, RNA is disturbed recombinant expression vector---the recombinant expression vector pDsU6-GFP-sh-3 that expresses shRNA-3 changes the leukemia reconstitution cell that obtains among the leukemic clone NALM-6 of bone-marrow-derived lymphocyte over to, its zinc finger protein CTCF expression level is starkly lower than contrast, the overall apoptosis ratio of cell is 28%, is 3.8 times of contrast.The present invention proves that zinc finger protein CTCF is an anti-apoptosis factor, has the apoptotic effect of inhibition in the ALL cell, disturbs the expression of CTCF can promote the tumour cell leukemia cell that apoptosis takes place, and increases the leukemia cell to the susceptibility of chemotherapeutics.The present invention provides a new approach for leukemia treating, and zinc finger protein CTCF is expected to become a potential target spot of leukemia treatment, has very wide application prospect at medical field.
Description of drawings
Fig. 1 disturbs the Western blot result of CTCF among the reorganization leukemia cell for RNA.Wherein, last one arranges to being that primary antibodie detects zinc finger protein CTCF with the CTCF monoclonal antibody, and next row is for the GAPDH monoclonal antibody being primary antibodie detection confidential reference items GAPDH.
Fig. 2 is Annexin V (+)/PI (-) cell proportion among the flow cytometer detection RNA interference reorganization leukemia cell.
Fig. 3 is reorganization leukemia cell early apoptosis ratio bar graph.
Sh-luc among Fig. 1-3, sh-CTCF-1, sh-CTCF-2 and sh-CTCF-3 represent reorganization leukemia cell NALM-6/pDsU6-GFP-sh-luc, NALM-6/pDsU6-GFP-sh-1, NALM-6/pDsU6-GFP-sh-2 and NALM-6/pDsU6-GFP-sh-3 respectively.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
Experiment among the following embodiment all arranges three repetitions, results averaged.
Carrier pDsU6: be documented in the following document: Shilai Bao, Tao Lu, Xin Wang, Huyong Zheng, Li-E Wang, Qingyi Wei, Walter N Hittelman and Lei Li.Disruption of the Rad9/Rad1/Hus1 (9 –, 1 – 1) complex leads to checkpoint signaling and replication defects.Oncogene, 2004,23,5586 – 5593.The public can obtain from Beijing Children's Hospital, Capital Medical University, to repeat the application's experiment.
B-ALL clone NALM-6(pre B cell acute lymphoblastic leukemia cell is NALM-6) be documented in the following document: Gao Huifang, Zhang Han, Pei Pei, Liu Yi, Li Zhigang, Jiang Jin, Zhang Ruidong, superb, Qi Yu, Zheng Huyong. the expression of splicing factor SF2/ASF in the leukemia of children cell. " Capital University of Medical Sciences's journal ", the 30th the 03rd phase of volume of June in 2009.The public can obtain from Beijing Children's Hospital, Capital Medical University, to repeat the application's experiment.
The RNA of embodiment 1, zinc finger protein CTCF disturbs recombinant expression vector to make up
1, RNA disturbs the selection of target sequence
At the full length cDNA sequence (SEQ ID No.6) of zinc finger protein CTCF encoding gene CTCF, selecting following three segment DNA sequences is the target sequence that RNA disturbs:
The 809-827 position (i.e. 5 '-TGACTGTACCTGTTGCTAC-3 ') of sh-1:SEQ ID No.6
The 1103-1122 position (i.e. 5 '-ATGTAGATGTGTCTGTCTAC-3 ') of sh-2:SEQ ID No.6
The 1398-1416 position (i.e. 5 '-TACTCGTCCTCACAAGTGC-3 ') of sh-3:SEQ ID No.6
The 445-2628 position is open reading frame among the SEQ ID No.6, the zinc finger protein CTCF shown in the coding SEQ ID No.7.
2, siRNA (siRNA)
Design three kinds of siRNA(siRNA-1, siRNA-2 and siRNA-3 at three kinds of target sequences of step 1 zinc finger protein CTCF respectively).The target sequence of siRNA-1 is sh-1, and the target sequence of siRNA-2 is sh-2, and the target sequence of siRNA-3 is sh-3.
1）siRNA-1
siRNA-1-F：5’-ugacuguaccuguugcuac-3’；
siRNA-1-R：5’-guagcaacagguacaguca-3’；
2）siRNA-2
siRNA-2-F：5’-auguagaugugucugucuac-3’；
siRNA-2-R：5’-guagacagacacaucuacau-3’。
3）siRNA-3
siRNA-3-F：5’-uacucguccucacaagugc-3’（SEQ?ID?No.1）；
siRNA-3-R：5’-gcacuugugaggacgagua-3’（SEQ?ID?No.2）。
3, short hairpin RNA (shRNA)
According to three kinds of siRNA of step 2, design produces the shRNA-3 of the shRNA-1 of siRNA-1, the shRNA-2 that produces siRNA-2, generation siRNA-3.
The sequence of shRNA-1, shRNA-2 and shRNA-3 following (capitalization is the ring sequence, and all the other sequences are the stem sequence, form inverted repeats):
shRNA-1：
5’-ugacuguaccuguugcuacUUCAAGAGAguagcaacagguacaguca-3’；
shRNA-2：
5’-auguagaugugucugucuacUUCAAGAGAguagacagacacaucuacau-3’；
shRNA-3：
5’-uacucguccucacaagugcUUCAAGAGAgcacuugugaggacgagua-3’（SEQ?ID?No.3）。
Simultaneously, with the shRNA-luc of reticent luciferase gene in contrast:
shRNA-luc：5’-gaagcgcauccaauaccagcUUCAAGAGAgcugguauuggaugcgcuuc-3’
It is not have expressed proteins in the NALM-6 cell at preceding B acute lymphoblastic leukemia cell that luciferase (luciferase) is one, thus will be with the luciferase target spot shRNA-luc in contrast.
4, the design of the dna molecular of coding siRNA and shRNA is with synthetic
1) be that two single stranded DNA sequences of double chain DNA molecule (name be called sh-CTCF-1) of the expression shRNA-1 of target spot are as follows with sh-1:
CTCF-ssDNA809-1-F：
5’-tgactgtacctgttgctacTTCAAGAGAgtagcaacaggtacagtcacTTTTT
A-3’；
CTCF-ssDNA809-1-R：
5’-
AGCTTAAAAAgtgactgtacctgttgctacTCTCTTGAAgtagcaacaggtacagtca-3’。
2) be that two single stranded DNA sequences of double chain DNA molecule (name be called sh-CTCF-2) of the expression shRNA-2 of target spot are as follows with sh-2:
CTCF-ssDNA1103-2-F：
5’-atgtagatgtgtctgtctacTTCAAGAGAgtagacagacacatctacatcTTTTT
A-3’；
CTCF-ssDNA1103-2-R：
5’-
AGCTTAAAAAgatgtagatgtgtctgtctacTCTCTTGAAgtagacagacacatctacat-3’。
3) be that two single stranded DNA sequences of double chain DNA molecule (name be called sh-CTCF-3) of the expression shRNA-3 of target spot are as follows with sh-3:
CTCF-ssDNA1398-3-F（SEQ?ID?No.4）：
5’-tactcgtcctcacaagtgcTTCAAGAGAgcacttgtgaggacgagtacTTTTT
A-3’；
CTCF-ssDNA1398-3-R（SEQ?ID?No.5）：
5’-
AGCTTAAAAAgtactcgtcctcacaagtgcTCTCTTGAAgcacttgtgaggacgagta-3’。
4) be that two single stranded DNA sequences of double chain DNA molecule (name be called sh-luc) of the expression shRNA-luc of target spot are as follows with the luciferase:
lucDNA-F：
5’-gaagcgcatccaataccagcTTCAAGAGAgctggtattggatgcgcttccTTTTTTT
A-3’；
lucDNA-R：
5’-
AGCTTAAAAAAAggaagcgcatccaataccagcTCTCTTGAAgctggtattggatgcgcttc-3’。
Above-mentioned 8 single stranded DNAs are synthetic by Invitrogen company, and underscore partly is Hind III sticky end sequence.
Respectively get 5 μ g(10ng/ μ l) complementary single stranded DNA mixing, 100 ℃ of thermal treatments are after 5 minutes, and annealing at room temperature obtains double chain DNA molecule, get double chain DNA molecule 5 μ g and carry out the phosphorylation processing, obtain double-stranded DNA reactant sh-CTCF-1, sh-CTCF-2, sh-CTCF-3 and sh-luc.
Wherein, the reaction system (50 μ l) of above-mentioned phosphorylation processing: double-stranded DNA 5 μ g, 10 * T
4Kinase Buffer5 μ l, ATP(10mM) 5 μ l, T
4Polynucleotide Kinase(10U/ μ l) 2 μ l use ddH
2O is supplemented to 50 μ l.
The reaction conditions that above-mentioned phosphorylation is handled: 37 ℃ of water-baths 1 hour, 100 ℃ of cool to room temperature slowly after 10 minutes again obtain can be used for the double-stranded DNA reactant of construction recombination plasmid.
5, the RNA of zinc finger protein CTCF disturbs recombinant expression vector to make up
1) RNA disturbs the structure of recombinant expression vector pDsU6-sh-1, pDsU6-sh-2, pDsU6-sh-3 and pDsU6-sh-luc
After getting carrier pDsU6 and cutting with Sac I enzyme earlier, carrying out end-filling with T4Polymerase Klenow fragment handles, the linearizing fragment that gel reclaims is cut digestion with Hind III enzyme again, reclaim the skeleton fragment of pDsU6 carrier, be connected with four kinds of double-stranded DNA reactant that step 4 obtains respectively, obtain four kinds of RNA and disturb recombinant expression vector pDsU6-sh-1, pDsU6-sh-2, pDsU6-sh-3, pDsU6-sh-luc, confirm through order-checking, the recombinant expression vector of the expression shRNA-1 that pDsU6-sh-1 obtains for fragment between the Sac I of replacing pDsU6 with sh-CTCF-1 and the Hind III, the recombinant expression vector of the expression shRNA-2 that pDsU6-sh-2 obtains for fragment between the Sac I of replacing pDsU6 with sh-CTCF-2 and the Hind III, the recombinant expression vector of the expression shRNA-luc that the recombinant expression vector of the expression shRNA-3 that pDsU6-sh-3 obtains for fragment between the Sac I of replacing pDsU6 with sh-CTCF-3 and the Hind III, pDsU6-sh-luc obtain for fragment between the Sac I of replacing pDsU6 with sh-luc and the Hind III.
2) RNA of GFP mark disturbs the acquisition of recombinant expression vector
The construction process of carrier pDsU6-GFP-sh-1, pDsU6-GFP-sh-2, pDsU6-GFP-sh-3, pDsU6-GFP-sh-luc: the RNA that gets above-mentioned structure respectively disturbs the recombinant expression vector pDsU6-GFP of recombinant expression vector pDsU6-sh-1, pDsU6-sh-2, pDsU6-sh-3, pDsU6-sh-luc and expression GFP to carry out BamH I and Hind III double digestion, and postdigestive fragment reclaims through gel; Disturb the recovery fragment of recombinant expression vector to be connected with the pDsU6-GFP carrier segments respectively above-mentioned four kinds of RNA, thereby the RNA that to express GFP disturbs recombinant vectors pDsU6-GFP-sh-1, pDsU6-GFP-sh-2, pDsU6-GFP-sh-3, pDsU6-GFP-sh-luc, confirms fragment obtains between pDsU6-GFP-sh-1 is for the BamH I of replacing pDsU6-GFP with sh-CTCF-1 and Hind III expression GFP and the recombinant expression vector of shRNA-1 through order-checking; The expression GFP that pDsU6-GFP-sh-2 obtains for fragment between the BamH I of replacing pDsU6-GFP with sh-CTCF-2 and the Hind III and the recombinant expression vector of shRNA-2; The expression GFP that pDsU6-GFP-sh-3 obtains for fragment between the BamH I of replacing pDsU6-GFP with sh-CTCF-3 and the Hind III and the recombinant expression vector of shRNA-3; The expression GFP that pDsU6-GFP-sh-luc obtains for fragment between the BamH I of replacing pDsU6-GFP with sh-luc and the Hind III and the recombinant expression vector of shRNA-luc.
The construction process of above-mentioned carrier pDsU6-GFP is as follows: with pGFP-N1(available from Clontech) be template, at primer P5(5 '-AAGGATCCATTACCGCCATGCATTAG-3 ') and P6(5 '-CCTACGCCTTAAGATACATTG-3 ') guiding under pcr amplification GFP gene, amplified production is through BamH I single endonuclease digestion, and digestion fragment reclaims through gel; Get carrier pDsU6 and carry out Afl II single endonuclease digestion, carry out end-filling with T4Polymerase Klenow fragment and handle, the linearizing fragment was cut digestion with BamH I enzyme again after gel reclaimed, and digestion back fragment reclaims through gel; Reclaim fragments with these two kinds and connect, obtain carrier pDsU6-GFP, confirm that through order-checking pDsU6-GFP use the GFP(green fluorescent protein) recombinant expression vector of the expression GFP that fragment obtains between the Afl II of gene replacement pDsU6 and the BamH I.
1, the transfection cultivation of cell
24h before the transfection is with containing the RPMI-1640 substratum of 10% foetal calf serum (FBS) at 15ml culture dish, 37 ℃ and 5%CO
2Cultivating B-ALL clone NALM-6 under the condition reaches at 75%～90% o'clock and carries out transfection.
2, transfection
The RNA that has the GFP label that embodiment 1 is obtained disturbs recombinant expression vector pDsU6-GFP-sh-1, pDsU6-GFP-sh-2, pDsU6-GFP-sh-3 and carrier pDsU6-GFP-sh-luc(RNAi positive control, be used for reticent luciferase gene) difference transfection step 1 cultured cells, obtain to contain the reorganization leukemia cell NALM-6/pDsU6-GFP-sh-1 that RNA disturbs recombinant expression vector pDsU6-GFP-sh-1, contain the reorganization leukemia cell NALM-6/pDsU6-GFP-sh-2 that RNA disturbs recombinant expression vector pDsU6-GFP-sh-2, contain the reorganization leukemia cell NALM-6/pDsU6-GFP-sh-luc that RNA disturbs the reorganization leukemia cell NALM-6/pDsU6-GFP-sh-3 of recombinant expression vector pDsU6-GFP-sh-3 and contains carrier pDsU6-GFP-sh-luc.
The concrete grammar of above-mentioned transfection is as follows:
The NALM-6 cell density that counting step 1 is cultivated is got and is contained 2 * 10
6The nutrient solution of individual cell, centrifugal 5 minutes of 90g abandons substratum, adopts 100 μ l electricity to change liquid (Nucleofector Solution, Lonza) re-suspended cell; The transitional cell suspension adds 2 μ g DNA plasmids to 1.5ml Eppendorf pipe, refers to that abdomen flicks abundant mixing; Cell/DNA suspension is transferred in the electric revolving cup, avoids bubble, cover bowl cover; With electric revolving cup be positioned over electroporation electric swivel base (Nucleofector Device, Lonza) on, select electric carryover order C-005(specially to be applicable to NALM-6 clone); Get the RPMI-1640 substratum (10%FBS) of 2ml preheating and mix with cell suspension in the electric revolving cup, transfer in the Tissue Culture Dish, avoid cell suspension is aspirated repeatedly; Cells transfected is placed in the incubator, and 37 ℃, 5%CO
2Continue in the cell culture incubator to cultivate, be used for detection and the experiment of following embodiment.
Get the reorganization of four kinds behind transfection 72h leukemia cell NALM-6/pDsU6-GFP-sh-1 among the embodiment 2, NALM-6/pDsU6-GFP-sh-2, NALM-6/pDsU6-GFP-sh-3 and NALM-6/pDsU6-GFP-sh-luc, extract total protein respectively, with the GAPDH(glyceraldehyde-3-phosphate dehydrogenase) be confidential reference items, carrying out Western blot detects, the primary antibodie that detects zinc finger protein CTCF is CTCF (molecular weight 83KDa) monoclonal antibody (available from Millipore), the primary antibodie that detects confidential reference items GAPDH is GAPDH(molecular weight 34KDa) monoclonal antibody (available from Chinese Shanghai Kang Cheng company), the result as shown in Figure 1, the result shows: contain the reorganization leukemia cell NALM-6/pDsU6-GFP-sh-1 that RNA disturbs recombinant expression vector pDsU6-GFP-sh-1, containing RNA disturbs the reorganization leukemia cell NALM-6/pDsU6-GFP-sh-2 of recombinant expression vector pDsU6-GFP-sh-2 and contains among the reorganization leukemia cell NALM-6/pDsU6-GFP-sh-3 that RNA disturbs recombinant expression vector pDsU6-GFP-sh-3, zinc finger protein CTCF expression level all is lower than the reorganization leukemia cell NALM-6/pDsU6-GFP-sh-luc that contrast contains carrier pDsU6-GFP-sh-luc, and wherein RNA disturbs recombinant expression vector pDsU6-GFP-sh-1 and RNA to disturb the interference effect of recombinant expression vector pDsU6-GFP-sh-3 better.
The concrete grammar of said extracted total protein is as follows:
Centrifugal 5 minutes of 90g collects the reorganization leukemia cell, washes twice with the PBS of precooling, adds 150～300 μ l RIPA damping fluid [20mM Tris pH7.5,50mM NaCl, 2mM Na
3VO
4, 10mM NaF, 1mM EDTA, 0.1%TritonX-100 and proteinase inhibitor (Roche)], cracking 30min on ice, collecting cell.Ultrasonic 10 seconds * 2 times of 8V voltage, 40 seconds at interval; 4 ℃, the centrifugal 30min of 12000rpm; Draw supernatant, utilize the Brandford method to carry out protein quantification; Respectively get 20 μ g total proteins and carry out Western blot detection.
The concrete grammar that Western blot detects is as follows:
1) electrophoresis and change film: protein sample to be measured is carried out the SDS-PAGE electrophoresis, under voltage 80V electrophoresis 10-30 minute earlier, treat that the dyestuff forward position enters separation gel after, voltage is brought up to 160V continued electrophoresis about 1 hour, arrive the bottom of separation gel until tetrabromophenol sulfonphthalein.After electrophoresis finished, the electricity consumption transfer method was transferred to nitrocellulose filter (NC film) with the protein sample that separates, 400mA(100V), and 1 hour (or 350mA(95V), 1.5 hours).
2) closing membrane: washed (Nacl8.0g, 20ml1M Tris-Hcl, Tween-202ml, adding distil water 950ml demarcate pH value to 7.6, are settled to 1L again) NC film 10-15 minute with TBS-T solution.The NC film is put into the TBS-T solution that contains 5% skim-milk, room temperature sealing 1 hour (or 4 ℃ 12-24 hour).
3) immuning hybridization:
A, primary antibodie are hatched: be diluted in CTCF monoclonal antibody or GAPDH monoclonal antibody in the TBS-T solution that contains 5% skim-milk by 1:2000, hatch about 1 hour (40 minutes to 1 hour 20 minutes) with the NC film at decolorization swinging table under the room temperature, washed the NC film 10 minutes with TBS-T solution 50mL, repeat 3 times.
B, two anti-hatching: will be diluted in the TBS-T solution that contains 5% skim-milk by 1:3000 in conjunction with the goat anti-rabbit igg antibody of horseradish peroxidase, hatch about 45 minutes (40 minutes to 1 hour 20 minutes) with the NC film at decolorization swinging table under the room temperature, washed the NC film 10 minutes with TBS-T solution 50mL, repeat 3 times.
4) ECL reagent colour development: use ECL protein hybridization detection kit (Sweden Amersham company), carry out NC film color reaction with reference to operation instructions.
Embodiment 4, reorganization leukemia cell's apoptosis detects
Recombinant expression vector pDsU6-GFP-sh-1 and pDsU6-GFP-sh-3 carry out apoptosis and detect preferably to choose the RNA interference effect.Get the reorganization of three kinds behind transfection 72h leukemia cell NALM-6/pDsU6-GFP-sh-1, NALM-6/pDsU6-GFP-sh-3 and NALM-6/pDsU6-GFP-sh-luc among the embodiment 2, with flow cytometer percentage of cerebral apoptosis is carried out statistical study respectively, the result as shown in Figures 2 and 3.
The result shows: the total apoptosis ratio of cell of reorganization leukemia cell NALM-6/pDsU6-GFP-sh-1, NALM-6/pDsU6-GFP-sh-3 and contrast (reorganization leukemia cell NALM-6/pDsU6-GFP-sh-luc) is respectively 7.5%, 28% and 7.4%, illustrate that RNA disturbs recombinant expression vector pDsU6-GFP-sh-3 can effectively reduce/suppress the expression of CTCF in cell, the overall apoptosis ratio of NALM-6/pDsU6-GFP-sh-3 cell is 3.8 times of contrast (reorganization leukemia cell NALM-6/pDsU6-GFP-sh-luc).
The concrete grammar of above-mentioned flow cytometer statistics percentage of cerebral apoptosis is as follows:
Centrifugal 5 minutes of 300g collects the reorganization leukemia cell, washes twice with the PBS of precooling, uses Annexin V-APC/PI pair transfect cell apoptosis detection kit (available from U.S. company BD, BD Pharmingen556547) labeled cells.Adopt flow cytometer (U.S. company BD, FACSAria II) to collect 10000 GFP positive cells, the apoptosis per-cent of cell is carried out statistical study, observe the variation characteristics that the leukemia cell ties up to the apoptosis aspect.
Claims (10)
1. form the short hairpin RNA of loop-stem structure, it is characterized in that: stem chain-ordering is SEQ ID No.1 in the described loop-stem structure, and another chain-ordering of stem is SEQ ID No.2 in the described loop-stem structure.
2. short hairpin RNA according to claim 1, it is characterized in that: the nucleotide sequence of described short hairpin RNA is SEQ ID No.3.
3. the dna molecular of coding claim 1 or 2 described short hairpin RNAs.
4. dna molecular according to claim 3, it is characterized in that: described dna molecular is 1) to 3) in any:
1) nucleotide sequence of a chain is SEQ ID No.4, another chain nucleotide sequence be SEQ ID No.5.
3) encoding sequence of described dna molecular is the 1-47 position of SEQ ID No.4;
2) encoding sequence of described dna molecular is the 12-58 position of SEQ ID No.5.
5. with the associated biomolecule material of claim 1 or 2 described short hairpin RNAs, be 1)-5) in any:
1) contains the expression cassette of claim 3 or 4 described dna moleculars;
2) contain the recombinant vectors of claim 3 or 4 described dna moleculars or contain 1) recombinant vectors of described expression cassette;
3) contain the recombinant microorganism of claim 3 or 4 described dna moleculars or contain 1) recombinant microorganism of described expression cassette or contain 2) recombinant microorganism of described recombinant vectors;
4) contain the transgenic cell line of claim 3 or 4 described dna moleculars or contain 1) transgenic cell line of described expression cassette or contain claim 2) transgenic cell line of described recombinant vectors;
5) siRNA, the nucleotide sequence of a chain such as SEQ ID No.1, the nucleotide sequence of another chain such as SEQ ID No.2.
6. treat and/or prevent leukemic product, described product activity composition comprises at least a among the b1-b3:
B1, profit require 1 or 2 described short hairpin RNAs;
B2, claim 3 or 4 described dna moleculars;
B3, the described associated biomolecule material of claim 5.
7. promote the product of apoptosis of leukemia, described product activity composition comprises at least a among the b1-b3:
B1, profit require 1 or 2 described short hairpin RNAs;
B2, claim 3 or 4 described dna moleculars;
B3, the described associated biomolecule material of claim 5.
8. reduce the purposes of the material of zinc finger protein CTCF genetic expression, described purposes is a1 or a2:
A1, the material that reduces zinc finger protein CTCF genetic expression treat and/or prevent application in the leukemic product in preparation;
The application of material in the product of preparation promotion apoptosis of leukemia of a2, reduction zinc finger protein CTCF genetic expression.
9. purposes according to claim 8 is characterized in that: the material of described reduction zinc finger protein CTCF genetic expression is at least a among the b1-b3:
B1, profit require 1 or 2 described short hairpin RNAs;
B2, claim 3 or 4 described dna moleculars;
B3, the described associated biomolecule material of claim 5.
10. according to Claim 8 or 9 described purposes, it is characterized in that: the aminoacid sequence of described zinc finger protein CTCF is shown in SEQ ID No.7, and the encoding sequence of described zinc finger protein CTCF gene is shown in the 445-2628 position of SEQ ID No.6.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104278026B (en) * | 2013-07-12 | 2017-06-09 | 中国科学院上海生命科学研究院 | The foundation and application of zebra fish candidate stem cell defect model |
| CN106913861A (en) * | 2017-02-28 | 2017-07-04 | 上海交通大学医学院附属第九人民医院 | Application of the CTCF traps albumen in anti-uveal medicine is prepared |
| CN107119144A (en) * | 2017-07-05 | 2017-09-01 | 昆明医科大学第附属医院 | Multi-functional transcription regulatory factor CTCF DNA binding sites CTCF_55 application |
| CN107151708A (en) * | 2017-07-05 | 2017-09-12 | 昆明医科大学第附属医院 | Multi-functional transcription regulatory factor CTCF DNA binding sites CTCF_13 application |
| CN112094860A (en) * | 2020-09-09 | 2020-12-18 | 苏州大学 | CTCF-ETO2 blood disease fusion gene and detection primer and application thereof |
-
2013
- 2013-04-27 CN CN201310153407.3A patent/CN103233009B/en not_active Expired - Fee Related
Non-Patent Citations (4)
| Title |
|---|
| FRANCE等: "Heightened Expression of CTCF in Breast Cancer Cells Is Associated with Resistance to Apoptosis", 《CANCER RESEARCH》 * |
| HANCOCK等: "A CTCF-binding silencer regulates the imprinted genes AWT1 and WT1-AS and exhibits sequential epigenetic defects during Wilms’ tumourigenesis", 《HUMAN MOLECULAR GENETICS》 * |
| TORRANO等: "CTCF Regulates Growth and Erythroid Differentiation of Human Myeloid Leukemia Cells", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
| 刘秋英等: "CTCF/pEGFP-N1 过表达载体及CTCF-shRNA 真核表达载体的构建及鉴定", 《中国科技论文在线数据库》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104278026B (en) * | 2013-07-12 | 2017-06-09 | 中国科学院上海生命科学研究院 | The foundation and application of zebra fish candidate stem cell defect model |
| CN106913861A (en) * | 2017-02-28 | 2017-07-04 | 上海交通大学医学院附属第九人民医院 | Application of the CTCF traps albumen in anti-uveal medicine is prepared |
| CN106913861B (en) * | 2017-02-28 | 2020-02-21 | 上海交通大学医学院附属第九人民医院 | Application of CTCF trap protein in preparation of anti-uveal melanoma drugs |
| CN107119144A (en) * | 2017-07-05 | 2017-09-01 | 昆明医科大学第附属医院 | Multi-functional transcription regulatory factor CTCF DNA binding sites CTCF_55 application |
| CN107151708A (en) * | 2017-07-05 | 2017-09-12 | 昆明医科大学第附属医院 | Multi-functional transcription regulatory factor CTCF DNA binding sites CTCF_13 application |
| CN107151708B (en) * | 2017-07-05 | 2020-10-09 | 昆明医科大学第一附属医院 | Application of DNA binding site CTCF-13 of multifunctional transcription regulatory factor CTCF |
| CN107119144B (en) * | 2017-07-05 | 2020-10-09 | 昆明医科大学第一附属医院 | Application of DNA binding site CTCF-55 of multifunctional transcription regulatory factor CTCF |
| CN112094860A (en) * | 2020-09-09 | 2020-12-18 | 苏州大学 | CTCF-ETO2 blood disease fusion gene and detection primer and application thereof |
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| CN103233009B (en) | 2015-04-08 |
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