CN103585619A - Application of DJ-1 protein in preparation of product for diagnosis and therapy of osteosarcoma - Google Patents
Application of DJ-1 protein in preparation of product for diagnosis and therapy of osteosarcoma Download PDFInfo
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- CN103585619A CN103585619A CN201310528113.4A CN201310528113A CN103585619A CN 103585619 A CN103585619 A CN 103585619A CN 201310528113 A CN201310528113 A CN 201310528113A CN 103585619 A CN103585619 A CN 103585619A
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Abstract
The invention provides application of a DJ-1 protein in preparation of products for diagnosis and therapy of osteosarcoma, and the amino acid sequence of the DJ-1 protein is shown as SEQID No:1. Experiments show that the expression of the DJ-1 protein in an osteosarcoma tissue is significantly higher than that in a non tumor tissue, and over expression (knockout) of the DJ-1 protein in osteosarcoma cells can significantly reduce (increase) the sensitivity of the osteosarcoma cells to first-line treatment drugs, so that the DJ-1 protein can be used as a specific marker protein for diagnosis and therapy of the osteosarcoma, and can improve the accuracy of the diagnosis of the osteosarcoma and the effectiveness of the therapy of the osteosarcoma. The DJ-1 protein provides a new therapeutic target and effective new drugs for the prevention and therapy of the osteosarcoma.
Description
Technical field
The invention belongs to field of biological pharmacy, relate to the application of a kind of DJ-1 albumen in preparation diagnosis and treatment osteosarcoma product.
Background technology
Osteosarcoma is one of modal pernicious Boneforming tumor, and epidemiological study finds that this disease is apt to occur in the 15-25 teenager in year, and male's sickness rate is higher than women.Osteosarcoma predilection site is the metaphysis of long bone, and this malignancy is high, poor prognosis, and after simple amputation, 5 years survival rates of patient are lower than 20%.Therefore, efficient diagnosis is the Focal point and difficult point in current bone tumor research with preventing and treating osteosarcoma.
People DJ-1 albumen is by DJ-1 gene (Genebank No.NM 007262.4) coding, is one of Thij/PfPI family member.The function of this albumen is not yet verified completely at present, and early hair style parkinson disease are relevant to mankind's autosomal recessive inheritance, AR to studies have reported that it; Aspect tumor, having report DJ-1 unconventionality expression in kinds of tumors sexually transmitted disease (STD) becomes, and may participate in developing of malignant tumor, but can DJ-1 albumen there is no research report as labelled molecule and the treatment target spot of osteosarcoma diagnosis.
Summary of the invention
The object of this invention is to provide the application of a kind of DJ-1 albumen in the osteosarcomatous medicine of preparation treatment, the aminoacid sequence of this DJ-1 albumen is as shown in SEQ ID No:1.The osteosarcomatous medicine of described treatment comprises: by RNA, disturb the double stranded RNA that suppresses DJ-1 gene expression, or for suppressing small peptide and the micromolecular compound of DJ-1 protein function.
Another object of the present invention is to provide the application of a kind of DJ-1 albumen in preparation diagnosis osteosarcoma product, and the aminoacid sequence of this DJ-1 albumen is as shown in SEQ ID No:1.The osteosarcomatous product of described diagnosis comprises: with RT-PCR, real-time quantitative PCR, immune detection, in situ hybridization or the osteosarcomatous product of gene chip diagnosis.
Wherein saidly by immune detection, diagnose osteosarcomatous product to comprise: the antibody of being combined with DJ-1 protein-specific.
Describedly with RT-PCR, diagnose osteosarcomatous product at least to comprise the primer of a pair of specific amplification DJ-1 gene: forward primer: 5 '-AGACGGTCATCCCTGTAGAT-3 ' (SEQ ID No:2); Reverse primer: 5 '-GCTCCTTCAGTATCTCCTTCAC-3 ' (SEQ ID No:3).
Describedly with real-time quantitative PCR, diagnose osteosarcomatous product at least to comprise the primer of a pair of specific amplification DJ-1 gene: forward primer: 5 '-AGCCGTGATGTGGTCATTT-3 ' (SEQ ID No:4); Reverse primer: 5 '-GTCTTTAGCAAGAGGGTGTGT-3 ' (SEQ ID No:5).
Describedly with in situ hybridization, diagnose osteosarcomatous product to comprise: with the probe of the nucleic acid array hybridizing of DJ-1 gene: 5 '-TCATCCACATTACTGGAGGTAAGTAGT-3 ' (SEQ ID No:6).
Describedly with the osteosarcomatous product of gene chip diagnosis, comprise: with the probe of the nucleic acid array hybridizing of DJ-1 gene: 5 '-ACTCGGTTGTCTCGTCATCCTGGACGT-3 ' (SEQ ID No:7).
In the present invention, can prepare the specific antibody for DJ-1 albumen by a series of methods known in the art.For example, the people DJ-1 albumen of purification or its antigen fragment are injected in animal body to produce polyclonal antibody.Equally, the cell of expression people's DJ-1 albumen or its antigen fragment also can be used for animal to cause immunity and produce antibody.Antibody prepared in accordance with the present invention can be also monoclonal antibody, and these monoclonal antibodies can be prepared by hybridoma technology.Antibody of the present invention comprises the antibody that can prevent DJ-1 function, can be also the antibody that does not affect people DJ-1 protein function.Each antibody-like can produce by the segment of people DJ-1 albumen or functional domain are caused to immunity, and people DJ-1 protein product and fragment thereof can produce or synthesize with Peptide synthesizer with recombination method.With the protein bound antibody of DJ-1 of non-modified form, can utilize the gene outcome for example producing in E. coli at prokaryotic cell carry out immune animal and obtain.With the antibody that post translational modification form is combined as glycosylation or phosphorylation DJ-1 albumen or polypeptide, can utilize the gene outcome producing in as yeast or insect cell at eukaryotic cell carry out immune animal and obtain.
In the present invention, described probe can be DNA, RNA, DNA-RNA chimera, PNA or other derivants.The length of described probe is restriction not, if can complete specific hybrid, with object nucleotide sequence specific binding, any length can.The length of described probe may be as little to 25,20,15 or 10 base length.Equally, the length of described probe can be grown to 60,80,100,150,300 base pairs or longer, even whole gene.Because different probe length has different impacts to hybridization efficiency, signal specificity, the length of described probe is at least 14 base pairs conventionally, the longlyest generally be no more than 30 base pairs, best with 15-25 base pair with the length of object nucleotide sequence complementation.Described probe self complementary series is most preferably less than 4 base pairs, in order to avoid affect hybridization efficiency.
People DJ-1 albumen is to be encoded by DJ-1 gene (Genebank No.NM 007262.4), DJ-1 unconventionality expression in kinds of tumors sexually transmitted disease (STD) becomes is reported in research at present, and may participate in developing of malignant tumor, but can DJ-1 albumen there is no research report as labelled molecule and the treatment target spot of osteosarcoma diagnosis.The present invention experimental results show that the expression of DJ-1 albumen in osteosarcoma tissue is apparently higher than nonneoplastic tissue; And in osteosarcoma cell, cross expression (knocking out) DJ-1 albumen and can significantly reduce the sensitivity of (increase) osteosarcoma cell to first-line treatment medicine.Therefore, DJ-1 albumen can be used as the specificity marker protein of osteosarcoma diagnosis and treatment, improves the accuracy of osteosarcoma diagnosis and the effectiveness for the treatment of.DJ-1 albumen of the present invention provides new treatment target and effective new drug for preventing and treating osteosarcoma.
accompanying drawing explanation
Fig. 1 is that the present invention verifies the expression of DJ-1 in human osteosarcoma by Immunohistochemical Method.
Fig. 2 is that the present invention detects DJ-1 protein level in osteosarcoma by Immunohistochemical Method.
Fig. 3 adopts plasmid transfection method to cross in osteosarcoma cell KHOS to express the impact on external osteosarcoma cell drug susceptibility after DJ-1 albumen.
Fig. 4 adopts plasmid transfection method to cross in osteosarcoma U 2OS to express the impact on external osteosarcoma cell drug susceptibility after DJ-1 albumen.
Fig. 5 adopts SiRNA specificity to knock out the impact on external osteosarcoma cell drug susceptibility after the DJ-1 albumen in osteosarcoma cell KHOS.
Fig. 6 adopts SiRNA specificity to knock out the impact on external osteosarcoma cell drug susceptibility after the DJ-1 albumen in osteosarcoma U 2OS.
The specific embodiment
Below in conjunction with drawings and Examples, the present invention is further detailed explanation.Following examples are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in embodiment, conventionally according to normal condition, or the condition of advising according to manufacturer.
Embodiment 1:
Choose osteosarcoma clinical sample 51 examples, by 10% formalin, fix 7 days, prepare subsequently paraffin-embedded piece of tissue.It is the paraffin section of 8 microns that sample is prepared to thickness, and adopts Immunohistochemical Method to detect the expression of DJ-1 albumen (aminoacid sequence is as shown in SEQ ID No:1), meanwhile chooses another section and carries out H & E dyeing.At the expression of just putting micro-Microscopic observation DJ-1 albumen, and the situation of H & E dyeing.Referring to Fig. 1, in sample cancerous tissue, the expression of DJ-1 albumen is higher than nonneoplastic tissue in same slice, thin piece.
Embodiment 2:
Expression to DJ-1 albumen in 51 routine osteosarcoma clinical samples in previous embodiment 1 is added up, and finds that strong positive is 24 examples, and the positive is 15 examples, and the weak positive is 7 examples, and feminine gender is 5 examples (Fig. 2).
Transfection DJ-1 gene (DJ-1 group) respectively in go down to posterity the osteosarcoma cell KHOS that cultivates and U2OS, and blank (PCMV6 group) is set.Until transfection, after 24 hours, give respectively cisplatin, paclitaxel, amycin and methotrexate, the growth inhibited effect of detection of drugs to transfectional cell.Referring to Fig. 3 and Fig. 4, cross after expression DJ-1 gene, osteosarcoma cell KHOS and U2OS significantly reduce drug susceptibility.
Embodiment 4:
In go down to posterity the osteosarcoma cell KHOS that cultivates and U2OS, adopt respectively SiRNA specificity to knock out DJ-1 gene (SiRNA DJ-1 group), and blank (NC group) is set.Until transfection, after 24 hours, give respectively cisplatin, paclitaxel, amycin and methotrexate, the growth inhibited effect of detection of drugs to cell.Referring to Fig. 5 and Fig. 6, knock out after DJ-1 gene, osteosarcoma cell KHOS and U2OS significantly increase drug susceptibility.
<110> Zhejiang University
The application of <120> DJ-1 albumen in the diagnosis of preparation osteosarcoma and treatment product
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5’-AGACGGTCATCCCTGTAGAT-3’
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5'-?GCTCCTTCAGTATCTCCTTCAC-3'
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5'-ACTCGGTTGTCTCGTCATCCTGGACGT-3'
Claims (7)
1. the application of DJ-1 albumen in the osteosarcomatous medicine of preparation treatment, is characterized in that, the aminoacid sequence of this DJ-1 albumen is as shown in SEQ ID No:1.
2. a DJ-1 albumen is diagnosed the application in osteosarcoma product in preparation, it is characterized in that, the aminoacid sequence of this DJ-1 albumen is as shown in SEQ ID No:1, and the osteosarcomatous product of described diagnosis comprises: with RT-PCR, real-time quantitative PCR, immune detection, in situ hybridization or the osteosarcomatous product of gene chip diagnosis.
3. the application of a kind of DJ-1 albumen according to claim 2 in preparation diagnosis osteosarcoma product, is characterized in that, describedly by immune detection, diagnoses osteosarcomatous product to comprise the antibody of being combined with DJ-1 protein-specific.
4. the application of a kind of DJ-1 albumen according to claim 2 in preparation diagnosis osteosarcoma product, it is characterized in that, describedly with RT-PCR, diagnose osteosarcomatous product at least to comprise the primer of a pair of specific amplification DJ-1 gene: forward primer: 5 '-AGACGGTCATCCCTGTAGAT-3 ', reverse primer: 5 '-GCTCCTTCAGTATCTCCTTCAC-3 '.
5. the application of a kind of DJ-1 albumen according to claim 2 in preparation diagnosis osteosarcoma product, it is characterized in that, describedly with real-time quantitative PCR, diagnose osteosarcomatous product at least to comprise the primer of a pair of specific amplification DJ-1 gene: forward primer: 5 '-AGCCGTGATGTGGTCATTT-3 ', reverse primer: 5 '-GTCTTTAGCAAGAGGGTGTGT-3 '.
6. the application of a kind of DJ-1 albumen according to claim 2 in preparation diagnosis osteosarcoma product, it is characterized in that, describedly with in situ hybridization, diagnose osteosarcomatous product to comprise: with the probe of the nucleic acid array hybridizing of DJ-1 gene: 5 '-TCATCCACATTACTGGAGGTAAGTAGT-3 '.
7. the application of a kind of DJ-1 albumen according to claim 2 in preparation diagnosis osteosarcoma product, it is characterized in that, describedly with the osteosarcomatous product of gene chip diagnosis, comprise: with the probe of the nucleic acid array hybridizing of DJ-1 gene: 5 '-ACTCGGTTGTCTCGTCATCCTGGACGT-3 '.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102481269A (en) * | 2009-05-11 | 2012-05-30 | 博格生物系统有限责任公司 | Methods for treatment of disease using an epimetabolic shifter (coenzyme q10) |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102481269A (en) * | 2009-05-11 | 2012-05-30 | 博格生物系统有限责任公司 | Methods for treatment of disease using an epimetabolic shifter (coenzyme q10) |
Non-Patent Citations (3)
| Title |
|---|
| MITSUGI,H. ET AL: "NP_009193", 《NCBI》, 28 October 2013 (2013-10-28) * |
| SOPHIE VASSEUR ET AL: "DJ-1/PARK7 is an important mediator of hypoxia-induced cellular responses", 《PNAS》, 27 January 2009 (2009-01-27) * |
| 严金金等: "DJ-1与肿瘤的关系", 《广东医学》, 31 December 2011 (2011-12-31), pages 3147 - 3149 * |
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Application publication date: 20140219 |