CN102747083B - Application of splicing factor oncoprotein SF2/ASF in preparation of medicines used for treating leukemia - Google Patents
Application of splicing factor oncoprotein SF2/ASF in preparation of medicines used for treating leukemia Download PDFInfo
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Abstract
The invention discloses application of the splicing factor oncoprotein SF2/ASF in preparation of medicines used for treating leukemia. The invention also provides small interfering RNA inhibiting the expression of the splicing factor oncoprotein SF2/ASF, and a coding gene and a vector thereof. According to results of experiments, the ratio of early apoptosis of leukemia cells can be obviously increased by inhibiting the expression of the splicing factor oncoprotein SF2/ASF represented by sequence 9 in a sequence table in the leukemia cells through utilization of the technology of RNA interference. The invention provides a novel target site for treatment of leukemia, and the target site has wide application prospects in the field of medicine.
Description
Technical field
The present invention relates to the application of shear factor cancer protein SF2/ASF in preparing medicament for treatment of leukemia.
Background technology
Alternative splicing is one of study hotspot of current oncology, and research thinks that it has participated in the generating process of tumour.SF2/ASF albumen is that a class is by SFRS1(Serine/Arginine-rich Splicing Factor1) genes encoding there is typical SR(Serine/Arginine-rich) splicing factor of constitutional features, and being a class, SR protein family is rich in the domain protein white matter of arginine and Serine sequence, not only participating in the constitutive splicing of Pre-mRNA, is also the important regulatory factor of alternative splicing.The alternative splicing of the adjustable many known proto-oncogenes of SR albumen and tumor suppressor gene, activates them in post-transcriptional level, thereby causes the Growth and reproduction of cell out of control.The reports such as Yea, the isomer KLF6-SV1 of transcription factor KLF6 obviously increases in hepatocellular carcinoma, makes cell infinite multiplication cause the generation of liver cancer.In addition, they also participate in event after a series of montages: the transportation of mRNA caryoplasm, mRNA decay and mRNA translation etc.The essence of SR albumen is the regulatory factor of mRNA metabolism, once function is damaged, will cause dysplasia and disease to occur.
SF2/ASF albumen plays an important role in the alternative splicing of work, inner cellular localization and transportation and Pre-mRNA mutually at protein-protein and protein-RNA.In different tissues, the expression of SF2/ASF is subject to strict regulation and control, and its expression amount has extremely important effect for cell and stable machine performance physiological function.The expression amount of SF2/ASF albumen can regulate and control the alternative splicing of proto-oncogene Ron, and when tumor-infiltrated, affects adhesion and the transport property of cell.
Research finds that SF2/ASF albumen existed expression phenomenon in many tumours.The discoveries such as Karni, SF2 is equal up-regulated in various human tumors, and part is because SFRS1 gene amplification causes; In the Human Lung Cancer clone of SF2/ASF up-regulated, recover the conversion conditions of SF2/ASF normal expression reversible clone; In inoblast, dystopy high expression level SF2/ASF albumen can transform by inducing cell, causes forming tumour in nude mouse, show that thus SF2/ASF is a proto-protein.
In addition, SF2/ASF is at mTOR(mammalian target of rapamycin) also play very important effect in signal path.Now clear and definite mTOR signal path is activated in many tumours, and the retarding agent of this path enters the clinical II phase as clinical cancer therapy drug and tests.Research discovery, SF2/ASF can change the activity of mTOR signal path, and specificity activates the mTORC1 branch in this path.Adopt RNAi perturbation technique to suppress mTOR path, can remove the tumorigenesis ability of SF2 overexpressing cell.In addition, the tumour of SF2 up-regulated has very high susceptibility to mTOR inhibitors.
Leukemia is modal malignant tumour of Childhood and the modal cause of death.The annual new leukemia of children of China 1.5 ten thousand example left and right, wherein children acute lymphoblastic leukaemia (Acute Lymphoblastic Leukemia, ALL) is modal type, accounts for 75% of leukemia of children.After the eighties in 20th century, due to the combined reinforced treatment of multiple medicines and strong support treatment, the curative ratio of children ALL has reached 80% left and right, but over nearly 30 years, ALL curative ratio is without obvious raising, and major cause is that leukemic pathogeny is not clear so far.In addition, leukemia relapse is also one of important factor.The recurrence rate of leukemia of children, up to 15% left and right, has become the most critical of the leukemia children rehabilitation factor that affects at present, not only gives infant itself but also has brought irremediable grievous injury humorous factor on good terms to family and society.
Summary of the invention
The object of this invention is to provide the application of a kind of shear factor cancer protein SF2/ASF in preparing medicament for treatment of leukemia.The full-length cDNA of described shear factor cancer protein SF2/ASF is as shown in sequence table sequence 10, and 210-956 position is open reading frame, the albumen shown in code sequence list sequence 9.
The invention provides the short hairpin RNA that suppresses or reduce the generated siRNA of shear factor cancer protein SF2/ASF expression, its nucleotide sequence is comprised of a stem ring sequence and two inverted repeats that lay respectively at described stem ring sequence both sides, and described two inverted repeats are respectively sequence shown in sequence table sequence 1 and sequence table sequence 2; Or, sequence shown in sequence table sequence 3 and sequence table sequence 4.
The present invention also provide by described short hairpin RNA, derived following 1) or 2) siRNA:
1) nucleotide sequence of a chain is as shown in sequence table sequence 1, and the nucleotide sequence of another chain is as shown in sequence table sequence 2;
2) nucleotide sequence of a chain is as shown in sequence table sequence 3, and the nucleotide sequence of another chain is as shown in sequence table sequence 4.
It is described short hairpin RNA and described siRNA that the present invention also provides described RNA() encoding gene.
The double chain DNA molecule that described gene is comprised of the single stranded DNA of sequence table sequence 5 and sequence table sequence 6, or the double chain DNA molecule being formed by the single stranded DNA of sequence table sequence 7 and sequence table sequence 8.
It is described short hairpin RNA and described siRNA that the present invention also provides the described RNA(of the expression that contains described gene) recombinant expression vector.
Described recombinant expression vector is the recombinant vectors obtaining between the multiple clone site of described gene insertion vector pDsU6 or GFP-pDsU6.
The construction process of described GFP-pDsU6 is as follows: take pEGFP-N1(purchased from Clontech) be template, at primer P1(5 '-AAGGATCCATTACCGCCATGCATTAG-3 ') and P2(5 '-CCTACGCCTTAAGATACATTG-3 ') guiding under pcr amplification GFP gene, amplified production, through BamH I single endonuclease digestion, is connected into the recombinant vectors obtaining between the Afl II of carrier pDsU6 and the site of BamH I.
The present invention also provides recombinant bacterium or the recombinant cell lines that contains described RNA or described gene.
The present invention also provides a kind of leukemic product (or medicine) that treats and/or prevents, and in the activeconstituents of described product (or medicine), contains described RNA, gene, recombinant expression vector and/or recombinant bacterium;
In the activeconstituents of described product (or medicine), also contain vincristine(VCR) and/or cytosine arabinoside.
The present invention protects albumen shown in sequence table sequence 9 in exploitation, to prevent and/or treat the application in leukemia product (or medicine) as target spot.
The present invention protects the material that suppresses protein expression shown in sequence table sequence 9 to prevent and/or treat the application in leukemia product (or medicine) in preparation.
The present invention protects arbitrary described RNA, gene, recombinant expression vector, recombinant bacterium or reconstitution cell to tie up to preparation and treats and/or prevents the application in leukemia product (or medicine).
Described treat and/or prevent leukemia product (or medicine) have following a) and b) at least one characteristic:
A) expression of albumen shown in sequence table sequence 9 in reduction or inhibition leukemia cell;
B) improve leukemia cell's early apoptosis ratio.
Described leukemia cell specifically can be bone-marrow-derived lymphocyte leukemia cell.
Experimental results show that, the mRNA of splicing factor cancer protein SF2/ASF of take is target spot, disturb recombinant expression vector GFP-pDsU6-sh-1 and GFP-pDsU6-sh-2 to proceed to the leukemia reconstitution cell obtaining in the leukemic clone Nalm-6 of bone-marrow-derived lymphocyte RNA, its splicing factor cancer protein SF2/ASF expression level is starkly lower than contrast; Early apoptosis of cells ratio is respectively 9.4% and 4.9%, is 3 times and 1.5 times of contrast; After 1 μ g/ml chemotherapeutics vinealeucoblastine(VLB) is processed, the early apoptosis ratio of restructuring leukemia cell Nalm-6/GFP-pDsU6-sh-1 and Nalm-6/pDsU6-sh-Luc is respectively 23.0% and 17.6%; After 50 μ g/ml cytosine arabinosides are processed, the early apoptosis ratio of restructuring leukemia cell Nalm-6/GFP-pDsU6-sh-1 and Nalm-6/pDsU6-sh-Luc is respectively 50.3% and 30.2%; After 45 μ g/ml physiological saline are processed, the early apoptosis ratio of restructuring leukemia cell Nalm-6/GFP-pDsU6-sh-1 and Nalm-6/pDsU6-sh-Luc is respectively 8.3% and 4.2%.
The present invention proves that splicing factor cancer protein SF2/ASF is an anti-apoptosis factor, in ALL cell, there is the effect of inhibited apoptosis, disturb the expression of SF2/ASF can promote tumour cell leukemia cell that apoptosis occurs, and increase the susceptibility of leukemia cell to chemotherapeutics.The present invention provides a new approach for leukemia treating, and splicing factor cancer protein SF2/ASF is expected to become a potential target spot of leukemia treatment, at medical field, has very wide application prospect.
Accompanying drawing explanation
Fig. 1 is the Western Blot result that RNA disturbs splicing factor cancer protein SF2/ASF in restructuring leukemia cell.Wherein, a upper row is for take SF2/ASF monoclonal antibody as primary antibodie detection splicing factor cancer protein SF2/ASF, and next row is for take GAPDH monoclonal antibody as primary antibodie detection internal reference GAPDH.
Fig. 2 is that flow cytometer detects Annexin V (+)/PI (-) cell proportion in RNA interference restructuring leukemia cell.
Fig. 3 is restructuring leukemia cell early apoptosis ratio bar graph.
Fig. 4 is that the RNA after flow cytometer detection of drugs is processed disturbs AnnexinV (+)/PI (-) cell proportion in restructuring leukemia cell.Wherein, NS is physiological saline, and VCR is chemotherapeutics vincristine(VCR), and Ara-c is chemotherapeutics cytosine arabinoside.
Fig. 5 is the restructuring leukemia cell early apoptosis ratio bar graph after drug treating.Wherein, NS is physiological saline, and VCR is chemotherapeutics vincristine(VCR), and Ara-c is chemotherapeutics cytosine arabinoside.
Sh-Luc in Fig. 1-5, sh-2 and sh-1 represent respectively restructuring leukemia cell Nalm-6/GFP-pDsU6-sh-Luc, Nalm-6/GFP-pDsU6-sh-2 and Nalm-6/GFP-pDsU6-sh-1.
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
Experiment in following embodiment all arranges three repetitions, results averaged.
Carrier pDsU6 and carrier Luc siRNA: Beijing Children's Hospital, Capital Medical University, Inst. of Genetics and Development Biology, CAS; Reference: 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 leukemic clone Nalm-6 of bone-marrow-derived lymphocyte: Beijing Children's Hospital, Capital Medical University, Inst. of Genetics and Development Biology, CAS; Reference: 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 leukemia of children cell. the journal > > of the < < Capital University of Medical Sciences the 03rd phase in 2009.
The RNA of embodiment 1, splicing factor cancer protein SF2/ASF disturbs recombinant expression vector to build
1, RNA disturbs the selection of target sequence
For the full length cDNA sequence (as shown in sequence table sequence 10) of splicing factor cancer protein SF2/ASF encoding gene SFRS1, selecting following two segment DNA sequences is the target sequence that RNA disturbs:
Sh-1: the 271-290 position of sequence table sequence 8 (i.e. 5 '-GTAACTTACCTCCAGACATC-3 ')
Sh-2: the 479-498 position of sequence table sequence 8 (i.e. 5 '-AAGCGGCCGTGGAACAGGCC-3 ')
In sequence table sequence 10,210-956 position is open reading frame, the splicing factor cancer protein SF2/ASF shown in code sequence list sequence 9.
2, siRNA (siRNA)
According to two of step 1 kinds of target sequences, two kinds of siRNA(siRNA-1 of splicing factor cancer protein SF2/ASF and siRNA-2) sequence as follows:
1）siRNA-1
S iRNA-1-F:5 '-guaacuuaccuccagacauc-3 ' (shown in sequence table sequence 1);
S iRNA-1-R:5 '-gaugucuggagguaaguuac-3 ' (shown in sequence table sequence 2);
2）siRNA-2
SiRNA-2-F:5 '-aagcggccguggaacaggcc-3 ' (shown in sequence table sequence 3);
SiRNA-2-R:5 '-ggccuguuccacggccgcuu-3 ' (shown in sequence table sequence 4).
3, short hairpin RNA (shRNA)
According to two of step 2 kinds of siRNA, two kinds of shRNA(shRNA-1 and shRNA-2) sequence following (capitalization is stem ring sequence, and all the other sequences are two short inverted repeats):
shRNA-1：
5’-guaacuuaccuccagacaucUUCAAGAGAgaugucuggagguaaguuac-3’；
shRNA-2：
5’-aagcggccguggaacaggccUUCAAGAGAggccuguuccacggccgcuu-3’。
4, the design of the DNA molecular of coding siRNA and shRNA is with synthetic
Two single stranded DNA sequences of the double chain DNA molecule of the expression shRNA-1 that the sh-1 of take is target spot are as follows:
Shown in SF2/ASF-ssDNA271-1-F(sequence table sequence 5):
5’-gtaacttacctccagacatcTTCAAGAGAgatgtctggaggtaagttacTTTTTTTA-3’；
Shown in SF2/ASF-ssDNA271-1-R(sequence table sequence 6):
5’-
AGCTTAAAAAAAgtaacttacctccagacatcTCTCTTGAAgatgtctggaggtaagttac-3’。
Two single stranded DNA sequences of the double chain DNA molecule of the expression shRNA-2 that the sh-2 of take is target spot are as follows:
Shown in SF2/ASF-ssDNA479-2-F(sequence table sequence 7):
5’-aagcggccgtggaacaggccTTCAAGAGAggcctgttccacggccgcttTTTTTTTA-3’；
Shown in SF2/ASF-ssDNA479-2-R(sequence table sequence 8):
5’-
AGCTTAAAAAAAaagcggccgtggaacaggccTCTCTTGAAggcctgttccacggccgctt-3’。
Above-mentioned 4 single stranded DNAs are synthesized by Invitrogen company, and underscore is partly Hind III sticky end sequence.
5, the RNA of splicing factor cancer protein SF2/ASF disturbs recombinant expression vector to build
1) acquisition of double-stranded DNA reactant
Get respectively 5 μ g(10ng/ μ l) complementary single stranded DNA synthetic in step 4 mixes, 100 ℃ of thermal treatments are after 5 minutes, and annealing at room temperature, obtains two kinds of double chain DNA molecules, get respectively these two kinds of double-stranded DNAs 5 μ g and carry out phosphatizing treatment, obtain two kinds of double-stranded DNA reactant.
The reaction system of above-mentioned phosphatizing treatment (50 μ l): 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 of above-mentioned phosphatizing treatment: 37 ℃ of water-baths 1 hour, then 100 ℃ of cool to room temperature slowly after 10 minutes, obtain can be used for the double-stranded DNA reactant of construction recombination plasmid.
2) RNA disturbs recombinant expression vector to build
After getting carrier pDsU6 and first cutting with Sac I enzyme, by T4Polymerase Klenow fragment, carry out end-filling processing, the linearizing fragment that gel reclaims is cut digestion with Hind III enzyme again, reclaim the skeleton fragment of pDsU6 carrier, two kinds of double-stranded DNA reactant that obtain with step 1) are connected, obtain two kinds of RNA and disturb recombinant expression vector pDsU6-sh-1 and pDsU6-sh-2, through order-checking, confirm, RNA disturbs recombinant expression vector pDsU6-sh-1 to insert the recombinant vectors that gene shown in sequence table sequence 5 obtains between the multiple clone site Sac I of carrier pDsU6 and Hind III; RNA disturbs recombinant expression vector pDsU6-sh-2 to insert the recombinant vectors that gene shown in sequence table sequence 7 obtains between the multiple clone site Sac I of carrier pDsU6 and Hind III.
3) RNA of GFP mark disturbs the acquisition of recombinant expression vector
By step 2) RNA that obtains disturbs recombinant expression vector pDsU6-sh-1 and pDsU6-sh-2 to transform respectively top10 competent cell, with LB substratum (containing 100ng/ μ l kantlex), cultivate in a small amount, a small amount of alkaline process upgrading grain, carry out respectively BamH I and Hind III double digestion, the fragment that reclaims 350bp is connected with the carrier GFP-pDsU6 skeleton fragment through BamH I and Hind III double digestion respectively, obtains two kinds of RNA with GFP label and disturbs recombinant expression vector GFP-pDsU6-sh-1 and GFP-pDsU6-sh-2.
The construction process of above-mentioned carrier GFP-pDsU6 is as follows: take pEGFP-N1(purchased from Clontech) be template, at primer P1(5 '-AAGGATCCATTACCGCCATGCATTAG-3 ') and P2(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, by T4 Polymerase Klenow fragment, carry out end-filling processing, after gel reclaims, linearizing fragment is cut digestion with BamH I enzyme again, and after digestion, fragment reclaims through gel; By these two kinds, reclaim fragment and connect, obtain carrier GFP-pDsU6, correct through sequence verification.
1, the cultivation of cell for transfection
24h before transfection, uses the RPMI-1640 substratum that contains 10% foetal calf serum (FBS) at 15ml culture dish, 37 ℃ and 5%CO
2under condition, cultivate and when the leukemic clone Nalm-6 of bone-marrow-derived lymphocyte reaches 75%～90%, carry out transfection.
2, transfection
The RNA with GFP label that embodiment 1 is obtained disturbs recombinant expression vector GFP-pDsU6-sh-1 and GFP-pDsU6-sh-2 and carrier GFP-pDsU6-sh-Luc(RNAi positive control, for reticent luciferase gene) difference transfection step 1 cultured cells, obtain the restructuring leukemia cell Nalm-6/GFP-pDsU6-sh-1 that disturbs recombinant expression vector GFP-pDsU6-sh-1 containing RNA, contain the restructuring leukemia cell Nalm-6/GFP-pDsU6-sh-Luc that RNA disturbs the restructuring leukemia cell Nalm-6/GFP-pDsU6-sh-2 of recombinant expression vector GFP-pDsU6-sh-2 and contains carrier GFP-pDsU6-sh-Luc.
The construction process of above-mentioned carrier GFP-pDsU6-sh-Luc is as follows: get carrier Luc siRNA and carry out BamH I and Hind III double digestion, reclaim small pieces segment DNA, with through BamH I, be connected with the skeleton fragment of the carrier GFP-pDsU6 of Hind III double digestion, obtain recombinant vectors GFP-pDsU6-sh-Luc.
The concrete grammar of above-mentioned transfection is as follows:
The Nalm-6 cell density that counting step 1 is cultivated, gets containing 2 * 10
6the nutrient solution of individual cell, centrifugal 5 minutes of 90g, abandons substratum, adopts 100 μ l electricity to turn liquid (Nucleofector Solution, Lonza) re-suspended cell; Transitional cell suspension, to 1.5ml Eppendorf pipe, adds 2 μ g DNA plasmids, refers to that abdomen flicks abundant mixing; Cell/DNA suspension is transferred in electric revolving cup, avoids bubble, cover bowl cover; The electric swivel base (Nucleofector Device, Lonza) that electric revolving cup is positioned over to electroporation is upper, selects electric carryover order C-005(to be specially applicable to Nalm-6 clone); The RPMI-1640 substratum (10%FBS) of getting 2ml preheating mixes with the cell suspension in electric revolving cup, transfers in Tissue Culture Dish, avoids cell suspension repeatedly to aspirate; The cell of transfection is placed in incubator, and 37 ℃, 5%CO
2in cell culture incubator, continue to cultivate, for detection and the experiment of following embodiment.
Embodiment 3, Western Blot detect the expression of splicing factor cancer protein SF2/ASF in restructuring leukemia cell
Get the restructuring of three kinds after transfection 72h leukemia cell Nalm-6/GFP-pDsU6-sh-1 in embodiment 2, Nalm-6/GFP-pDsU6-sh-2 and Nalm-6/GFP-pDsU6-sh-Luc, extract respectively total protein, take GAPDH(glyceraldehyde-3-phosphate dehydrogenase) be internal reference, carry out Western blot detection, the primary antibodie that detects splicing factor cancer protein SF2/ASF is SF2/ASF (molecular weight 34KDa) monoclonal antibody (purchased from Santa Cruz company), the primary antibodie that detects internal reference GAPDH is GAPDH monoclonal antibody (purchased from Chinese Shanghai Kang Cheng company), result as shown in Figure 1, result shows: containing RNA, disturb the restructuring leukemia cell Nalm-6/GFP-pDsU6-sh-1 of recombinant expression vector GFP-pDsU6-sh-1 and containing RNA, disturb in the restructuring leukemia cell Nalm-6/GFP-pDsU6-sh-2 of recombinant expression vector GFP-pDsU6-sh-2, splicing factor cancer protein SF2/ASF expression level is starkly lower than contrast containing the restructuring leukemia cell Nalm-6/GFP-pDsU6-sh-Luc of carrier GFP-pDsU6-sh-Luc.
The concrete grammar of said extracted total protein is as follows:
90g collects restructuring leukemia cell in centrifugal 5 minutes, with the PBS of precooling, washes twice, 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, interval; 4 ℃, the centrifugal 30min of 12000rpm; Draw supernatant, utilize Brandford method to carry out protein quantification; Respectively get 20 μ g total proteins and carry out Western blot detection.
The concrete grammar that above-mentioned Western blot detects is as follows:
1) electrophoresis and transferring film: protein sample to be measured is carried out to SDS-PAGE electrophoresis, and first electrophoresis 10-30 minute under voltage 80V, enters after separation gel until dyestuff forward position, bring up to 160V by voltage and continue electrophoresis approximately 1 hour, until tetrabromophenol sulfonphthalein arrives the bottom of separation gel.After electrophoresis finishes, electricity consumption transfer method is transferred to nitrocellulose filter (NC film) by separated protein sample, 400mA(100V), and 1 hour (or 350mA(95V), 1.5 hours).
2) closing membrane: wash (Nacl8.0g, 20ml1M Tris-Hcl, Tween-202ml, adding distil water 950ml demarcate pH value to 7.6, then be settled to 1L) NC film 10-15 minute with TBS-T solution.NC film is put into the TBS-T solution containing 5% skim-milk, 1 hour (or 4 ℃ of 12-24 hour) of room temperature sealing.
3) immuning hybridization A, primary antibodie are hatched: by SF2/ASF (molecular weight 34KDa) monoclonal antibody (or GAPDH monoclonal antibody) by 1:(2000-3000) be diluted in containing in the TBS-T solution of 5% skim-milk, under room temperature, hatch approximately 1 hour (40 minutes to 1 hour 20 minutes) together with NC film is on decolorization swinging table, with TBS-T solution 50mL, wash NC film 10 minutes, repeat 3 times.
B, two anti-hatching: in connection with the goat anti-rabbit igg antibody (or in conjunction with horseradish peroxidase sheep anti-mouse igg antibody) of horseradish peroxidase by 1:(3000-5000) be diluted in and contain in the TBS-T solution of 5% skim-milk, under room temperature, hatch approximately 45 minutes (40 minutes to 1 hour 20 minutes) together with NC film is on decolorization swinging table, with TBS-T solution 50mL, wash NC film 10 minutes, 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, restructuring leukemia cell's apoptosis detects
Get the restructuring of three kinds after transfection 72h leukemia cell Nalm-6/GFP-pDsU6-sh-1, Nalm-6/GFP-pDsU6-sh-2 and Nalm-6/GFP-pDsU6-sh-Luc in embodiment 2, with flow cytometer, percentage of cerebral apoptosis is carried out to statistical study respectively, result as shown in Figures 2 and 3.
Result shows: the early apoptosis of cells ratio of restructuring leukemia cell Nalm-6/GFP-pDsU6-sh-1, Nalm-6/GFP-pDsU6-sh-2 and contrast (restructuring leukemia cell Nalm-6/GFP-pDsU6-sh-Luc) is respectively 9.4%, 4.9% and 3.0%, illustrate that RNA disturbs recombinant expression vector GFP-pDsU6-sh-1 and GFP-pDsU6-sh-2 all can effectively reduce/suppress the expression of SF2/ASF in cell, wherein the interference effect of GFP-pDsU6-sh-1 is better.
The concrete grammar of above-mentioned flow cytometer statistics percentage of cerebral apoptosis is as follows:
300g collects restructuring leukemia cell in centrifugal 5 minutes, with the PBS of precooling, washes twice, uses two transfect cell apoptosis detection kit (purchased from U.S. company BD, the BD Pharmingen 556547) labeled cells of AnnexinV-APC/PI.Adopt flow cytometer (U.S. company BD, FACSAria II) to collect the cell of 10000 GFP positives, the apoptosis per-cent of cell is carried out to statistical study, observe the Variation Features that leukemia cell ties up to apoptosis aspect.
The restructuring leukemia cell's that embodiment 5, chemotherapeutics were processed apoptosis detects
Get restructuring leukemia cell Nalm-6/GFP-pDsU6-sh-1 and the Nalm-6/GFP-pDsU6-sh-Luc of transfection after 48 hours in embodiment 2, add respectively conventional clinically acute lymphoblastic leukemia (ALL) chemotherapeutics vincristine(VCR) (VCR) 1 μ g/ml and cytosine arabinoside (Ara-c) 50 μ g/ml and physiological saline (NS) 45 μ g/ml, harvested cell after 72 hours, adopt flow cytometer to carry out apoptosis detection to cell, method is with embodiment 4, and result as shown in Figure 4 and Figure 5.
Result shows: after VCR and Ara-c drug treating, the early apoptosis ratio of restructuring leukemia cell Nalm-6/GFP-pDsU6-sh-1 and Nalm-6/GFP-pDsU6-sh-Luc obviously increases.After VCR processes, the early apoptosis ratio of restructuring leukemia cell Nalm-6/GFP-pDsU6-sh-1 and Nalm-6/GFP-pDsU6-sh-Luc is respectively 23.0% and 17.6%; After Ara-c processes, the early apoptosis ratio of restructuring leukemia cell Nalm-6/GFP-pDsU6-sh-1 and Nalm-6/GFP-pDsU6-sh-Luc is respectively 50.3% and 30.2%; After NS processes, the early apoptosis ratio of restructuring leukemia cell Nalm-6/GFP-pDsU6-sh-1 and Nalm-6/pDsU6-sh-Luc is respectively 8.3% and 4.2%.
Embodiment 3-5 proves, splicing factor cancer protein SF2/ASF is an anti-apoptosis factor, in ALL cell, there is the effect of inhibited apoptosis, disturb the expression of SF2/ASF can promote that tumour cell---apoptosis occurs leukemia cell, and increase the susceptibility of leukemia cell to chemotherapeutics.
Claims (9)
1. short hairpin RNA, its nucleotide sequence is comprised of a stem ring sequence and two inverted repeats that lay respectively at described stem ring sequence both sides, and described two inverted repeats are respectively sequence shown in sequence table sequence 1 and sequence table sequence 2.
2. by the derivative following siRNA of short hairpin RNA described in claim 1:
Article one, the nucleotide sequence of chain is as shown in sequence table sequence 1, and the nucleotide sequence of another chain is as shown in sequence table sequence 2.
3. the encoding gene of siRNA described in short hairpin RNA or claim 2 described in claim 1, is characterized in that: the double chain DNA molecule that described encoding gene forms for the single stranded DNA by sequence table sequence 5 and sequence table sequence 6.
4. the recombinant expression vector that contains siRNA described in short hairpin RNA described in the expression claim 1 of encoding gene described in claim 3 or claim 2, described recombinant expression vector is the recombinant vectors obtaining between the multiple clone site of encoding gene insertion vector pDsU6 or GFP-pDsU6 described in claim 3.
5. the recombinant bacterium that contains siRNA described in short hairpin RNA described in claim 1, claim 2, encoding gene claimed in claim 3 or recombinant expression vector claimed in claim 4.
6. the recombinant cell lines that contains siRNA described in short hairpin RNA described in claim 1, claim 2, encoding gene claimed in claim 3 or recombinant expression vector claimed in claim 4.
7. treat and/or prevent a leukemic product, it is characterized in that: in the activeconstituents of described product, contain siRNA described in short hairpin RNA described in claim 1, claim 2, encoding gene claimed in claim 3, recombinant expression vector claimed in claim 4, recombinant bacterium claimed in claim 5 and/or recombinant cell lines claimed in claim 6.
8. product according to claim 7, is characterized in that: in the activeconstituents of described product, contain vincristine(VCR) and/or cytosine arabinoside.
9. described in claim 1, described in short hairpin RNA, claim 2, siRNA, encoding gene claimed in claim 3, recombinant expression vector claimed in claim 4, recombinant bacterium claimed in claim 5 or reconstitution cell claimed in claim 6 tie up to preparation and treat and/or prevent the application in leukemia product.
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