WO2014124551A1 - Application of dkk4 gene and coding protein thereof in preparation of medicament - Google Patents

Application of dkk4 gene and coding protein thereof in preparation of medicament Download PDF

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WO2014124551A1
WO2014124551A1 PCT/CN2013/001192 CN2013001192W WO2014124551A1 WO 2014124551 A1 WO2014124551 A1 WO 2014124551A1 CN 2013001192 W CN2013001192 W CN 2013001192W WO 2014124551 A1 WO2014124551 A1 WO 2014124551A1
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dkk4
gastrointestinal stromal
gene
stromal tumors
drug
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PCT/CN2013/001192
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French (fr)
Chinese (zh)
Inventor
曹晖
汪明
张志刚
徐佳
屠霖
王超杰
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上海交通大学医学院附属仁济医院
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57446—Specifically defined cancers of stomach or intestine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention relates to a medicament, in particular to the application of the DKK4 gene and the encoded protein thereof for preparing a medicament, in particular to the application of the DKK4 gene and the encoded protein thereof for preparing a medicament for detecting or treating gastrointestinal stromal tumors.
  • Gastrointestinal stromal tumor is the most common mesenchymal origin of the gastrointestinal tract, mainly in the stomach (about 70%). In recent years, the incidence of GIST has increased rapidly year by year. In recent years, epidemiological statistics at home and abroad have shown that the incidence of GIST exceeds 20/million person/year. The 2009 epidemiological survey in China showed that the incidence rate reached about 30/million people/year. It is estimated that the number of new cases in China can reach about 4-5 million cases per year. GIST has become the most common malignant tumor in the digestive tract after gastric cancer and intestinal cancer. Most of GIST originates from the submucosal of the gastrointestinal tract and protrudes into and out of the cavity. Therefore, there are no special symptoms in the early stage.
  • Carcinoembryonic antigen has been successfully applied in the early diagnosis of colorectal cancer or the application of alpha-fetoprotein in the screening of liver cancer.
  • CCA Carcinoembryonic antigen
  • GIST which is difficult to diagnose and diagnose, requires a simple and feasible biology. Markers are used for clinical diagnosis. GIST's biological behavior is diverse, spanning benign, low-grade, moderately malignant, and highly malignant. The vast majority of small GISTs can be followed up for life without any progress. The prognosis of highly malignant cases is extremely poor, and the growth is extremely fast.
  • imatinib mesylate a small-molecule tyrosine kinase receptor inhibitor
  • GIST the most fundamental molecular abnormality (c-kit gene mutation) in the pathogenesis of GIST. Play an effective anti-tumor effect.
  • imatinib mesylate a small-molecule tyrosine kinase receptor inhibitor
  • GIST the most fundamental molecular abnormality (c-kit gene mutation) in the pathogenesis of GIST. Play an effective anti-tumor effect.
  • some problems have arisen in the clinical practice of targeted drug therapy applications: 1. About 5-10% of patients have primary resistance to imatinib, and most are effective for initial treatment with imatinib. Patients will eventually inevitably develop to stimulate drug resistance, and most of the drug resistance occurs within 6 months to 2 years after the initial treatment; 2.
  • the DKK gene family was first discovered by Glinka in amphibians from African cockroach embryonic cells in 1998. In the vertebrate, the DKK family has four members, encoding DKK1, DKK2, DKK3, and DKK4 proteins, which have high homology.
  • DKK4 (Dickkopf homolog 4) is located at 8pl l. 2- pl l. 1 , a secreted glycoprotein involved in the development of the body and the regulation of the Wnt signaling pathway, which corresponds to the Wnt signaling pathway.
  • the receptor binds to regulate cell differentiation, proliferation, migration or carcinogenesis and plays an important role in tumorigenesis. There has been no previous study on the relationship between DKK4 gene and gastrointestinal stromal tumors. Summary of the invention
  • the technical problem to be solved by the present invention is to overcome the above-mentioned deficiencies and to study the application of the DKK4 gene in the manufacture of medicine.
  • the invention provides the application of the DKK4 gene and its encoded protein in the preparation of a medicament.
  • the present invention discloses the use of the DKK4 gene and its encoded protein for the preparation of a medicament for detecting or treating gastrointestinal stromal tumors.
  • the DKK4 gene name of the present invention is a DKK4 gene name of the present invention.
  • the DKK4 gene sequence is shown in Sequence Listing 1 (SEQ ID NO. 1).
  • the term "DKK4" as used herein refers to a gene found in amphibian embryonic cells of the African cockroach.
  • the DKK4 gene referred to in the present invention includes its entire DNA coding sequence, its RNA sequence, a mutant thereof, and a functionally active fragment thereof.
  • the DKK4-encoded protein is a protein synthesized in the human body based on the CDS region sequence in the DKK4 gene messenger RNA. It is to be understood that the substitution of the nucleotides in the codon is acceptable when encoding the same amino acid. It is also to be understood that nucleotide substitutions are also acceptable when conservative amino acid substitutions result from nucleotide substitutions.
  • the medicament for detecting gastrointestinal stromal tumor comprises a drug or a kit for diagnosing gastrointestinal stromal tumor by RT-PCR, PCR, immunoassay, in situ hybridization, and gene chip.
  • the drug for detecting gastrointestinal stromal tumor by RT-PCR includes at least one pair of primers which specifically amplify the DKK4 gene.
  • the drug for detecting gastrointestinal stromal tumors by qPCR includes at least one pair of primers which specifically amplify the DKK4 gene.
  • the drug for immunodetecting gastrointestinal stromal tumors includes an antibody, a polyclonal antibody, and a monoclonal antibody that specifically bind to the DKK4 protein.
  • the drug for detecting gastrointestinal stromal tumors by in situ hybridization includes a probe that hybridizes to the DKK4 nucleic acid sequence.
  • the drug for detecting gastrointestinal stromal tumors using a gene chip includes a probe that hybridizes to the DKK4 nucleic acid sequence.
  • the kit for detecting gastrointestinal stromal tumors comprises purified reagents, labels, and the like for RNA isolation, RNA amplification, immunohistochemical staining, enzyme-linked immunosorbent assay, protein quantitative analysis.
  • the kit consists of DKK4 as an active ingredient and a pharmaceutically acceptable carrier.
  • the kit describes DKK4 as: ⁇ 4 Dickkopf homolog 4, dickkopf homolog 4), a gene found in embryonic cells of the first amphibian African cockroach, whose protein-encoding product is a secreted glycoprotein.
  • kits in the kit typically comprise at least one vial, test tube, flask, PET bottle, syringe or other container in which one component can be placed and, preferably, can be suitably aliquoted.
  • the kit will typically also contain a second, third or other additional container in which the additional components are placed separately.
  • the kit of the invention will typically also include a container for containing the reactants, sealed for commercial sale. Such containers may include injection molded or blow molded plastic containers in which the desired vials are retained.
  • a further object of the present invention is to provide a use of the DKK4 gene and its encoded protein for the preparation of a medicament for treating gastrointestinal stromal tumors.
  • the medicament for treating gastrointestinal stromal tumor of the present invention comprises: a double-stranded ribonucleic acid which inhibits DKK4 gene expression by RNA interference, a tumor vaccine based on DKK4 antigen protein or a protein which inhibits DKK4 protein activity.
  • the medicament for treating gastrointestinal stromal tumor of the present invention can be administered orally, cutaneously or parenterally.
  • the medicament for treating gastrointestinal stromal tumors of the present invention can be formulated into a dosage form such as oral, inhalation, injection or suppository by conventional techniques in the art.
  • the inventor first selected 12 fresh surgical resected specimens of gastric GIST, according to the current risk classification criteria (considering tumor size and mitotic phase), according to the increasing risk of tumor recurrence as the specimen sequence number (low risk 4 cases, medium 4 cases of danger, 4 cases of high risk).
  • Gene expression profiling microarray studies of 12 specimens were performed using Roche's NimbleGen chip, and genes that were significantly up-regulated or down-regulated with increasing risk of tumor among different risk groups were screened (p ⁇ 0.05).
  • the selected genes were verified by real-time quantitative PCR in another 24 GIST fresh tumor specimens (14 high-risk, 10 low-risk).
  • tissue microarray technology used tissue microarray technology to construct tissue microarrays containing 139 GIST tissue specimens (including tumor and paraneoplastic normal tissue, respectively), and used immunohistochemical techniques to detect the expression of DKK4 in these specimens and found DKK- 4
  • the inventors used ELISA to detect plasma samples from 20 patients with GIST and 25 patients with non-GIST (including 16 healthy people, 3 gastric cancers, 3 intestinal cancers, and 3 patients with abdominal mesenchymal tumors).
  • the content of DKK-4 protein was calculated based on the DKK4 standard gradient concentration to calculate the DKK4 content.
  • DKK4 has a particularly high specific expression in gastrointestinal stromal tumors, especially in highly dangerous gastrointestinal stromal tumors. It was confirmed that the DKK4 gene is associated with the biological characteristics of GIST and the prognosis of patients with GIST.
  • the protein-coding product DKK4 protein of this gene can be detected in serum as a secreted glycoprotein, and its level is specifically increased in GIST patients, providing a new means for early diagnosis and prognosis of GIST diseases. .
  • a DKK4-specific primer can be designed based on the nucleotide sequence.
  • the full-length nucleotide sequence or a fragment thereof can usually be obtained by a PCR amplification method, a recombinant method or a synthetic method.
  • primers can be designed in accordance with the disclosed nucleotide sequences, particularly open reading frame sequences, and can be prepared using commercially available cDNA libraries or conventional methods known to those skilled in the art.
  • the library is used as a template to amplify the relevant sequences.
  • the recombination method can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then separating the relevant sequence from the proliferated host cell by conventional methods.
  • synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short.
  • a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then connecting them.
  • DNA sequence encoding the protein of the present invention (or a fragment thereof, a derivative) by chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (e.g., vectors) and cells known in the art.
  • the DKK4 polynucleotide sequence can be inserted into a recombinant expression vector.
  • the host is replicable and stable in vivo, and any plasmid and vector can be used.
  • An important feature of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and a translational control element.
  • Methods well known to those skilled in the art can be used to construct expression vectors containing DKK4 DNA sequences and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, and the like.
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis.
  • the transformation vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • Vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences, can be used to transform appropriate host cells to enable expression of the protein.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples are: Escherichia coli, bacterial cells of the genus Streptomyces; fungal cells such as yeast; plant cells; insect cells; animal cells, and the like.
  • Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase, treated with CaC, and the procedures used are well known in the art.
  • Another method is to use MgCl 2 .
  • Conversion can also be carried out by electroporation if desired.
  • the host is a eukaryote
  • the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like.
  • the obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture may be selected from various conventional media depending on the host cell used.
  • the cultivation is carried out under conditions suitable for the growth of the host cell.
  • the selected promoter is induced by a suitable method (e.g., temperature conversion or chemical induction) and the cells are cultured for a further period of time.
  • the recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell.
  • the recombinant protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • An exemplary method of detecting the presence or absence of a DKK4 protein or nucleic acid in a biological sample comprises obtaining a biological sample of the test subject, the biological sample being contacted with a labeled nucleic acid probe capable of hybridizing to DKK4 mRNA or genomic DNA.
  • the nucleic acid probe may be, for example, a human nucleic acid or a portion thereof, such as a nucleic acid probe that is at least 15, 30, 50, 100 nucleotides in length and is capable of sufficiently hybridizing to DKK4 mRNA or genomic DNA under stringent conditions.
  • Other probes for use in the diagnostic assays of the invention are as described herein.
  • the nucleic acid probe is contacted with the amplified tag sequence.
  • the probe is preferably attached to a chromophore but can be labeled.
  • the probe is attached to a binding partner, such as an antibody or biotin, or another binding partner that carries a detectable domain.
  • detection can be performed by Southern blotting and hybridization to labeled probes.
  • Techniques involved in Southern blotting are well known to those skilled in the art (see Sambrook et al., 1989).
  • Conventional assays include biochips, fluorescence imaging techniques, cell flow counting, and the like.
  • the invention also encompasses polyclonal and monoclonal antibodies, particularly monoclonal antibodies, which are specific for a polypeptide encoded by DKK4 DNA or a fragment thereof.
  • specificity means that the antibody binds to the DKK4 gene product or fragment.
  • the antibodies of the present invention can be prepared by various techniques known to those skilled in the art.
  • the DKK4 antibody of the present invention can be detected by ELISA, Western blot analysis, or coupled to a detection group by chemiluminescence or isotope tracing.
  • DKK4 (Dickkopf homolog 4)
  • the chromosome is located at 8pll. 2- pl l. 1, a secreted glycoprotein involved in the development of the body and the regulation of the Wnt signaling pathway, which corresponds to the Wr t signal transduction pathway.
  • the receptor binds to regulate cell differentiation, proliferation, migration or carcinogenesis and plays an important role in tumorigenesis.
  • the inventors have experimentally proved that the DKK4 gene can be used for the preparation of a medicament for diagnosing or treating gastrointestinal stromal tumors.
  • the present invention provides for the first time to use the detection of DKK gene and its products in serum/tissue to achieve early diagnosis of GIST or prognosis of GIST patients undergoing surgery, and establish high-throughput chip screening + real-time quantitative PCR verification at the gene level. And large sample clinical samples at the protein level, the results can be Based on the experimental results, DKK4 can significantly distinguish between tumor and non-tumor, high-risk tumors and low-risk tumors at both the gene level and the protein level. The results are reproducible and their expression levels are different between different groups. The difference is huge and has good clinical application value.
  • Figure 7 Total survival curves of 326 patients with gastrointestinal stromal tumors by Kaplan-Meier method, in which DKK4 staining was negatively marked with ⁇ , weak positive was marked with Qin, positive was marked with ⁇ , and strong positive was marked with gardening.
  • Figure 8 Recurrence-free survival curve of 326 patients with gastrointestinal stromal tumors by Kaplan-Meier method, in which DKK4 staining was negative for labeling, weak positive was marked with Weng, positive was marked with ⁇ , and strong positive was marked with country.
  • Figure 9 The ordinate is the DKK4 plasma concentration, the left scatter is the plasma sample of patients with gastrointestinal stromal tumors, and the right side is the plasma sample of the non-gastrointestinal stromal tumor population.
  • RNA extraction reagent RNAiso Plus was purchased from Bao Bioengineering Co., Ltd.
  • the reverse transcription kit High Capacity cDNA Reverse Transcription Kits was purchased from Invitrogen
  • the fluorescence quantitative PCR kit Power SYBR Green PCR Master Mix was purchased from Invitrogen, DKK4 and housekeeping genes.
  • the tumor tissue of patients with gastrointestinal stromal tumors originated from Renji Hospital affiliated to Shanghai Jiao Tong University School of Medicine. Tissue specimens were immediately frozen in liquid nitrogen after surgery and then stored in an ultra-low temperature freezer at -80 °C.
  • RNA extraction sample was weighed and quickly transferred to a mortar precooled with liquid nitrogen, and the tissue was ground with a mortar while continuously adding liquid nitrogen until it was ground into a powder. Add the appropriate amount of RNAiso Plus to the mortar, completely cover the sample that has been ground into a powder, and then let it stand at room temperature until the sample is completely melted. Continue grinding until the lysate is transparent. The slurry was transferred to a centrifuge tube and allowed to stand at room temperature for 5 minutes. Centrifuge at 12,000 g for 5 minutes at 4 °C. Carefully pipette the supernatant and transfer to a new centrifuge tube.
  • RNAiso Plus 1/5 volume of chloroform of RNAiso Plus was added and shaken vigorously for 15 seconds. After the solution was fully emulsified, it was allowed to stand at room temperature for 5 minutes and centrifuged at 12 000 g for 4 minutes at 4 °C. Transfer the supernatant to another new centrifuge tube. An equal volume of isopropanol was added to the supernatant, and the tube was thoroughly mixed upside down and allowed to stand at 15 to 30 ° C for 10 minutes. Centrifuge at 12, 000 g for 10 minutes at 4 °C. Discard the supernatant, add 1 ml of 75% ethanol along the tube wall, wash the tube wall upside down, centrifuge at 12 000 g for 4 minutes at 4 ° C, and discard the ethanol. The precipitate was dried at room temperature for 2 to 5 minutes, and the precipitate was dissolved by adding an appropriate amount of RNase-free water, and the RNA concentration was measured with Nanodrop 2000 and stored at -80 °C.
  • the reaction system is as follows:
  • the reaction tube was placed in a PCR apparatus, and the thermal cycle system was stored at 25 ° C for 10 minutes - 37 ° 120 minutes • 85 ° C for 5 minutes - 4 ° C.
  • Realtime PCR assay A 20 ul reaction system was prepared in a 96-well plate. 3 replicate holes per reaction, Relatively quantitative detection of DKK4 gene expression by ⁇ -actin as an internal reference gene.
  • the Realtime amplification system is as follows:
  • the 96-well plate with the sample added is placed in the ABI9300 fluorescence quantitative PCR machine for reaction, under the thermal cycle.
  • DKK4 mAb (ab38589) and secondary antibody goat polyclonal anti-rabbit IgG (ab6721) were purchased from abeam.
  • the DAB developer and its substrate kit were purchased from Thermo Fisher. The remaining reagents were all domestically analyzed.
  • the tumor tissue of patients with gastrointestinal stromal tumors originated from Renji Hospital affiliated to Shanghai Jiao Tong University School of Medicine.
  • the chip array was constructed by Suzhou Xinxin Biotechnology Co., Ltd., with a dot matrix diameter of 1. 6mm and a layer thickness of 3mm.
  • Chip 1 included 139 gastrointestinal stromal tumor tissues and corresponding adjacent normal tissues, and chip 2 included 189 gastrointestinal stromal tumor tissues.
  • Antigen retrieval Electric furnace heating 0. 01M sodium citrate buffer solution (pH 6. 0) to about 95 ° C, put into the tissue chip and heat for 15 minutes, wash with PBS.
  • Eliminate endogenous enzymes Incubate with 3% hydrogen peroxide at 37 ° C for 30 minutes, wash with PBS.
  • Antigen blocking 10% goat serum was incubated for 1 hour at room temperature.
  • Primary antibody incubation Add primary antibody (DKK4 mAb) 150 ul, incubate overnight at 4 ° C, wash with PBS.
  • the cytoplasmic staining was used as the positive reaction site, and the positive intensity of the positive cells of each array point was 0, 1, 2, 3, respectively, without coloring, light yellow, brownish yellow and tan, and the coloring positive area was colorless and colored. ⁇ 1/3, 1/3 ⁇ 2/3, ⁇ 2/3 respectively score 0, 1, 2, 3, and then judge the result according to the sum of the two scores: 0 is negative, 1-2 is weak Positive, 3-4 is positive, 5-6 is strong positive (Note: Select a representative area for each slice, count in 400 times field of view, total 5 fields, take the average to avoid random Sex)
  • tissue microarray were input into SPSS20. 0 for analysis.
  • the correlation between DKK4 expression and clinical pathological data was analyzed by chi-square test.
  • the significance level was defined as p ⁇ 0.05.
  • Survival analysis was performed by Kaplan-Meier method, and the significance level was defined as p ⁇ 0.05.
  • Typical results of immunohistochemical staining are shown in Figure 2-6.
  • Survival/recurrence curves were plotted by Kaplan-Meier method, and the survival/recurrence curves of grouped patients based on DKK-4 expression intensity were also clearly distinguished, as shown in Figure 7-8.

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Abstract

The present invention provides application of a DKK4 gene and a coding protein thereof in preparation of a medicament, and in particular, application in preparation of a medicament for detecting or treating a gastrointestinal stromal tumor. The medicament is a detection medicament consisting of a DKK4 antibody serving as an active ingredient and a medicinal carrier or consisting of a DKK4 specific primer serving as an active ingredient and a medicinal carrier. The medicament is a detection kit prepared from a DKK4 antibody or a specific primer thereof serving as an active ingredient and a medicinal carrier.

Description

DKK4基因及其编码蛋白在制备药物中的应用 技术领域  Application of DKK4 gene and its encoded protein in preparation of medicine
本发明涉及药物， 具体涉及 DKK4基因及其编码蛋白在制备药物中的应用， 尤其涉及 DKK4基因及其编码蛋白在制备检测或治疗胃肠道间质瘤药物中的应 用。 背景技术  The invention relates to a medicament, in particular to the application of the DKK4 gene and the encoded protein thereof for preparing a medicament, in particular to the application of the DKK4 gene and the encoded protein thereof for preparing a medicament for detecting or treating gastrointestinal stromal tumors. Background technique
胃肠道间质瘤 (gastrointestinal stromal tumor, GIST) 是胃肠道最常见 的间叶来源肿瘤， 主要发生于胃 （约占 70%)。 近年来 GIST的发病率有逐年较快 增加的趋势， 近年来国内外流行病学统计显示 GIST发病率超过 20/百万人 /年。 2009年中国流行病学调査显示其发病率达到约 30/百万人 /年， 据推算， 中国每 年的新发病例数可达 4-5万例左右。 GIST 然已成为消化道内仅次于胃癌、 肠 癌的最常见的恶性肿瘤。 GIST大多起源于胃肠道粘膜下， 向腔内外突出生长， 因而早期并无特殊症状，大多数患者是在肿瘤生长到一定阶段， 出现消化道出血 或发现腹部肿块才来就诊，甚至有不少患者直到肿瘤破溃穿孔甚至腹腔内广泛破 散才能得到诊断， 导致错失最佳治疗时机。 由于 GIST起病部位隐匿， 给早期诊 断带来困难， 向腔外突出为主的胃 GIST在内镜检査中较易漏诊， 即使内镜下发 现病灶， 由于病灶深度的原因，大多不能像上皮源性肿瘤一样取得明确的病理诊 断； 小肠或胃肠道外 GIST的诊断更至今仍是困扰临床医师的一大难题， 几乎没 有可靠的影像学诊断手段， 甚至往往需要行剖腹探查才能实现诊断。 癌胚抗原 (CEA)应用于大肠癌的早期诊断或甲胎蛋白应用于肝癌的筛选已经取得了成功， 而相较于其他肿瘤， 起病隐匿诊断困难的 GIST更需要一种简单可行的生物学标 志物来应用于临床诊断。 GIST的生物学行为多样， 跨度覆盖良性、 低度恶性、 中度恶性、 高度恶性。 绝大多数体积很小的 GIST可以终生随访而无任何进展。 高度恶性病例预后极差， 生长极快， 在靶向治疗药物伊马替尼问世之前， 其中生 存期仅有 10-20个月， 5年生存率 <10%。 同时 GIST的生物学行为较难预测， 虽 然目前有普遍被接受的 NIH危险度分级（主要依据肿瘤大小、核***相以及肿瘤 原发部位）， 但是临床上也有不少病例的病程发展较难用上述分级标准来解释。 甚至有文献报道一些看似"良性的 GIST",即肿瘤直径 <2cm，核***相<5/50 ？ 的肿瘤患者，术后短期内发生复发和转移；也不乏肿瘤巨大的患者在接受手术治 疗后即使不接受药物治疗也能获得长期生存。因此，临床上亟待一种能够有效判 断 GIST预后的生物学指标。 Gastrointestinal stromal tumor (GIST) is the most common mesenchymal origin of the gastrointestinal tract, mainly in the stomach (about 70%). In recent years, the incidence of GIST has increased rapidly year by year. In recent years, epidemiological statistics at home and abroad have shown that the incidence of GIST exceeds 20/million person/year. The 2009 epidemiological survey in China showed that the incidence rate reached about 30/million people/year. It is estimated that the number of new cases in China can reach about 4-5 million cases per year. GIST has become the most common malignant tumor in the digestive tract after gastric cancer and intestinal cancer. Most of GIST originates from the submucosal of the gastrointestinal tract and protrudes into and out of the cavity. Therefore, there are no special symptoms in the early stage. Most patients have a certain stage of tumor growth, gastrointestinal bleeding or abdominal mass is found, and there are even many. The patient can not be diagnosed until the tumor is ruptured and even broken in the abdominal cavity, leading to missed optimal treatment. Due to the insidious site of GIST, it is difficult to diagnose early. The gastric GIST, which is mainly out of the cavity, is more likely to be missed in endoscopy. Even if the lesion is found under endoscopy, most of it cannot be like epithelium due to the depth of the lesion. A clear pathological diagnosis is obtained in the same way as a source tumor; the diagnosis of small intestine or extra-gastrointestinal GIST is still a major problem that plagues clinicians. There is almost no reliable imaging diagnostic method, and even a laparotomy is often required to achieve diagnosis. Carcinoembryonic antigen (CEA) has been successfully applied in the early diagnosis of colorectal cancer or the application of alpha-fetoprotein in the screening of liver cancer. Compared with other tumors, GIST, which is difficult to diagnose and diagnose, requires a simple and feasible biology. Markers are used for clinical diagnosis. GIST's biological behavior is diverse, spanning benign, low-grade, moderately malignant, and highly malignant. The vast majority of small GISTs can be followed up for life without any progress. The prognosis of highly malignant cases is extremely poor, and the growth is extremely fast. Before the advent of the targeted therapy drug imatinib, the survival period is only 10-20 months, and the 5-year survival rate is <10%. At the same time, the biological behavior of GIST is difficult to predict. Although there is a generally accepted NIH risk classification (mainly based on tumor size, mitotic phase and primary tumor site), there are many cases in which clinical development is difficult to use. Grading criteria to explain. There are even reports in the literature that some seemingly "benign GIST", ie tumor diameter <2cm, mitotic phase <5/50? In patients with cancer, recurrence and metastasis occur within a short period of time after surgery; and patients with large tumors can achieve long-term survival even after receiving medical treatment without receiving medical treatment. Therefore, there is a clinical need for a biological indicator that can effectively judge the prognosis of GIST.
小分子酪氨酸激酶受体抑制剂甲磺酸伊马替尼的问世极大地改变了 GIST的 治疗策略， 该药物以 GIST发病的最根本分子异常 （c-kit基因突变） 为治疗靶 点， 发挥有效的抗肿瘤的作用。 然而，在靶向药物治疗应用的临床实践中也出现 了一些问题： 1. 约有 5- 10%的患者对伊马替尼原发耐药， 而大多数对伊马替尼 初始治疗有效的患者最终会不可避免地发展为激发耐药，耐药多发生于初始治疗 开始后 6个月至 2年内； 2. 目前高复发风险 GIST患者手术后接受一定时间的伊 马替尼辅助治疗已经取得共识， 然而辅助治疗合适的人群选择始终存在争议，放 宽治疗适应症必然会带来医疗资源的浪费，增加耐药的发生；过度严苛的适应症 也会导致一部分患者错过理应接受的合理治疗， 如果能够有更客观合理判断 GIST患者复发风险的指标， 将能极大地有利于 GIST的防治。  The advent of imatinib mesylate, a small-molecule tyrosine kinase receptor inhibitor, has dramatically changed the therapeutic strategy of GIST, which is the most fundamental molecular abnormality (c-kit gene mutation) in the pathogenesis of GIST. Play an effective anti-tumor effect. However, some problems have arisen in the clinical practice of targeted drug therapy applications: 1. About 5-10% of patients have primary resistance to imatinib, and most are effective for initial treatment with imatinib. Patients will eventually inevitably develop to stimulate drug resistance, and most of the drug resistance occurs within 6 months to 2 years after the initial treatment; 2. The current high-recurrence risk of GIST patients after surgery for a certain period of time after the adjuvant treatment with imatinib has been obtained Consensus, however, the choice of appropriate treatment for patients is always controversial. Relaxing treatment indications will inevitably lead to waste of medical resources and increase the incidence of drug resistance; excessively harsh indications will also cause some patients to miss reasonable treatment. If there is a more objective and reasonable judgment of the risk of recurrence of GIST patients, it will be greatly beneficial to the prevention and treatment of GIST.
DKK基因家族由 Glinka于 1998年首次在两栖动物非洲蟾蜍胚胎细胞中发 现。 在脊椎动物中 DKK家族有四个成员， 分别编码 DKK1、 DKK2、 DKK3、 DKK4蛋 白，它们有较高的同源性。 DKK4(Dickkopf同源物 4)染色体定位于 8pl l. 2- pl l. 1 , 为一种分泌型糖蛋白， 参与机体发育及 Wnt信号转导途径的调控， 其通过与 Wnt 信号转导途径相应的受体结合来调控细胞的分化、 增殖、 迁移或癌变等特性，在 肿瘤发生方面发挥重要作用。 之前对于 DKK4基因与胃肠道间质瘤发生的关系没 有相应的研究。 发明内容  The DKK gene family was first discovered by Glinka in amphibians from African cockroach embryonic cells in 1998. In the vertebrate, the DKK family has four members, encoding DKK1, DKK2, DKK3, and DKK4 proteins, which have high homology. DKK4 (Dickkopf homolog 4) is located at 8pl l. 2- pl l. 1 , a secreted glycoprotein involved in the development of the body and the regulation of the Wnt signaling pathway, which corresponds to the Wnt signaling pathway. The receptor binds to regulate cell differentiation, proliferation, migration or carcinogenesis and plays an important role in tumorigenesis. There has been no previous study on the relationship between DKK4 gene and gastrointestinal stromal tumors. Summary of the invention
本发明所要解决的技术问题在于克服上述不足之处，研究设计 DKK4基因在制 药中的应用。  The technical problem to be solved by the present invention is to overcome the above-mentioned deficiencies and to study the application of the DKK4 gene in the manufacture of medicine.
本发明提供了 DKK4基因及其编码蛋白在制备药物中的应用。  The invention provides the application of the DKK4 gene and its encoded protein in the preparation of a medicament.
具体地， 本发明公开了 DKK4基因及其编码蛋白在制备检测或治疗胃肠道间质瘤 药物中的应用。 Specifically, the present invention discloses the use of the DKK4 gene and its encoded protein for the preparation of a medicament for detecting or treating gastrointestinal stromal tumors.
本发明所述 DKK4基因名称：  The DKK4 gene name of the present invention:
中文名： DKK4 (Dickkopf 同源物 4) 英文名： DKK4 ( dickkopf homolog 4) Chinese name: DKK4 (Dickkopf homolog 4) English name: DKK4 ( dickkopf homolog 4)
DKK4基因序列见序列表 1 (SEQ ID NO. 1 )。 The DKK4 gene sequence is shown in Sequence Listing 1 (SEQ ID NO. 1).
本文所用术语 "DKK4"指在两栖动物非洲蟾蜍胚胎细胞中发现的一个基因。 本发明所指的 DKK4基因包括其完整的 DNA编码序列， 其 RNA序列， 其突变体， 以及其功能上活性的片段。 DKK4编码蛋白为根据 DKK4基因信使 RNA中 CDS区域 序列在人体内翻译合成的蛋白质。需理解的是， 当编码相同的氨基酸时， 密码子 中的核苷酸的取代是可接受的。另外需理解的是， 由核苷酸取代而产生的保守的 氨基酸取代时， 核苷酸的变换也是可被接受的。  The term "DKK4" as used herein refers to a gene found in amphibian embryonic cells of the African cockroach. The DKK4 gene referred to in the present invention includes its entire DNA coding sequence, its RNA sequence, a mutant thereof, and a functionally active fragment thereof. The DKK4-encoded protein is a protein synthesized in the human body based on the CDS region sequence in the DKK4 gene messenger RNA. It is to be understood that the substitution of the nucleotides in the codon is acceptable when encoding the same amino acid. It is also to be understood that nucleotide substitutions are also acceptable when conservative amino acid substitutions result from nucleotide substitutions.
本发明所述检测胃肠道间质瘤药物包括用 RT- PCR、 PCR、 免疫检测、 原位杂 交、 基因芯片诊断胃肠道间质瘤的药物或试剂盒。  The medicament for detecting gastrointestinal stromal tumor according to the present invention comprises a drug or a kit for diagnosing gastrointestinal stromal tumor by RT-PCR, PCR, immunoassay, in situ hybridization, and gene chip.
所述用 RT-PCR检测胃肠道间质瘤的药物至少包括一对特异性扩增 DKK4基因 的引物。  The drug for detecting gastrointestinal stromal tumor by RT-PCR includes at least one pair of primers which specifically amplify the DKK4 gene.
所述用 qPCR检测胃肠道间质瘤的药物至少包括一对特异性扩增 DKK4基因的 引物。  The drug for detecting gastrointestinal stromal tumors by qPCR includes at least one pair of primers which specifically amplify the DKK4 gene.
所述用免疫检测胃肠道间质瘤的药物包括与 DKK4蛋白特异性结合的抗体、 多克隆抗体和单克隆抗体。  The drug for immunodetecting gastrointestinal stromal tumors includes an antibody, a polyclonal antibody, and a monoclonal antibody that specifically bind to the DKK4 protein.
所述用原位杂交检测胃肠道间质瘤的药物包括与 DKK4核酸序列杂交的探 针。  The drug for detecting gastrointestinal stromal tumors by in situ hybridization includes a probe that hybridizes to the DKK4 nucleic acid sequence.
所述用基因芯片检测胃肠道间质瘤的药物包括与 DKK4核酸序列杂交的探 针。  The drug for detecting gastrointestinal stromal tumors using a gene chip includes a probe that hybridizes to the DKK4 nucleic acid sequence.
所述用于检测胃肠道间质瘤的试剂盒包含用于 RNA分离、 RNA扩增、免疫组 织化学染色、酶联免疫反应、蛋白定量分析的纯化的试剂、标记等。所述试剂盒 由 DKK4作为活性成分与药用载体组成。  The kit for detecting gastrointestinal stromal tumors comprises purified reagents, labels, and the like for RNA isolation, RNA amplification, immunohistochemical staining, enzyme-linked immunosorbent assay, protein quantitative analysis. The kit consists of DKK4 as an active ingredient and a pharmaceutically acceptable carrier.
所述试剂盒对 DKK4的描述为： ΦΚΚ4 Dickkopf 同源物 4， dickkopf homolog 4)，一种首先两栖动物非洲蟾蜍胚胎细胞中发现的基因，其蛋白编码产物为一种 分泌性糖蛋白。  The kit describes DKK4 as: ΦΚΚ4 Dickkopf homolog 4, dickkopf homolog 4), a gene found in embryonic cells of the first amphibian African cockroach, whose protein-encoding product is a secreted glycoprotein.
试剂盒的组分可以以水介质的形式或以冻干的形式来包装。试剂盒中适当的 容器通常至少包括一种小瓶、试管、 长颈瓶、 宝特瓶、 针筒或其它容器， 其中可 放置一种组分， 并且优选地， 可进行适当地等分。在试剂盒中存在多于一种的组 分时， 试剂盒中通常也将包含第二、第三或其它附加的容器， 其中分离地放置附 加的组分。然而， 不同组合的组分可被包含在一个小瓶中。本发明的试剂盒通常 也将包括一种用于容纳反应物的容器，密封以用于商业销售。这种容器可包括注 模或吹模的塑料容器， 其中可保留所需的小瓶。 The components of the kit may be packaged in the form of an aqueous medium or in a lyophilized form. Suitable containers in the kit typically comprise at least one vial, test tube, flask, PET bottle, syringe or other container in which one component can be placed and, preferably, can be suitably aliquoted. There are more than one group in the kit In time sharing, the kit will typically also contain a second, third or other additional container in which the additional components are placed separately. However, different combinations of components can be included in one vial. The kit of the invention will typically also include a container for containing the reactants, sealed for commercial sale. Such containers may include injection molded or blow molded plastic containers in which the desired vials are retained.
本发明的又一目的是提供了 DKK4基因及其编码蛋白在制备治疗胃肠道间质 瘤药物中的应用。  A further object of the present invention is to provide a use of the DKK4 gene and its encoded protein for the preparation of a medicament for treating gastrointestinal stromal tumors.
本发明所述治疗胃肠道间质瘤药物包括： 通过 RNA干扰抑制 DKK4基因表达 的双链核糖核酸、基于 DKK4抗原蛋白的肿瘤疫苗或抑制 DKK4蛋白活性的蛋白质。  The medicament for treating gastrointestinal stromal tumor of the present invention comprises: a double-stranded ribonucleic acid which inhibits DKK4 gene expression by RNA interference, a tumor vaccine based on DKK4 antigen protein or a protein which inhibits DKK4 protein activity.
本发明所述治疗胃肠道间质瘤药物可以通过口服、 皮肤或肠胃外方式给药。 本发明所述治疗胃肠道间质瘤药物可以通过本领域常规技术制成口服、 吸 入、 注射或栓剂等剂型。  The medicament for treating gastrointestinal stromal tumor of the present invention can be administered orally, cutaneously or parenterally. The medicament for treating gastrointestinal stromal tumors of the present invention can be formulated into a dosage form such as oral, inhalation, injection or suppository by conventional techniques in the art.
本发明人通过下列试验： The inventors passed the following tests:
a. 发明人首先选取 12例胃 GIST的新鲜手术切除标本， 按照目前使用的危险度 分级标准 （综合考虑肿瘤大小及核***相）， 按照肿瘤复发风险递增为标本顺序 编号 （低危 4例、 中危 4例、 高危 4例）。 利用 Roche公司的 NimbleGen芯片对 该 12例标本进行基因表达谱微阵列研究， 筛选出在不同危险度分组间随肿瘤危 险度升高呈显著上调或下调的基因 （p<0. 05， fold change>2) c 之后对筛选出的 基因在另外 24例 GIST新鲜肿瘤标本 （14例高危、 10例低危） 中进行实时定量 PCR验证。 筛选出 DKK4基因无论是在表达谱芯片结果中 （p=0. 00024， fold change=12. 63 ) 还是定量 PCR验证结果中 (p=0. 0029, fold change=42. 16) 均 呈现出在高危组 GIST中显著高表达。 a. The inventor first selected 12 fresh surgical resected specimens of gastric GIST, according to the current risk classification criteria (considering tumor size and mitotic phase), according to the increasing risk of tumor recurrence as the specimen sequence number (low risk 4 cases, medium 4 cases of danger, 4 cases of high risk). Gene expression profiling microarray studies of 12 specimens were performed using Roche's NimbleGen chip, and genes that were significantly up-regulated or down-regulated with increasing risk of tumor among different risk groups were screened (p<0.05). 2) After c, the selected genes were verified by real-time quantitative PCR in another 24 GIST fresh tumor specimens (14 high-risk, 10 low-risk). The DKK4 gene was screened out in the expression microarray results (p=0. 00024, fold change=12. 63) or quantitative PCR results (p=0. 0029, fold change=42. 16). Significantly high expression in the high-risk group GIST.
b. 发明人利用组织微阵列技术构建了包含 139例 GIST组织标本（分别包含肿瘤 和瘤旁正常组织） 的组织芯片， 并利用免疫组织化学技术检测 DKK4在这些标本 中的表达情况，发现 DKK-4在瘤旁正常组织中阳性表达率远低于在肿瘤中阳性表 达率 （7. 2% vs 86. 5% )， 且 DKK-4表达强度与肿瘤直径 （p=0. 043 ) 肿瘤分级b. The inventors used tissue microarray technology to construct tissue microarrays containing 139 GIST tissue specimens (including tumor and paraneoplastic normal tissue, respectively), and used immunohistochemical techniques to detect the expression of DKK4 in these specimens and found DKK- 4 The positive expression rate in normal tissues adjacent to the tumor was much lower than that in the tumor (7.2% vs 86.5%), and the intensity of DKK-4 expression and tumor diameter (p=0.043) tumor grade
(p=0. 018) 呈正相关。 (p=0. 018) is positively correlated.
c.发明人扩大样本， 构建了包含另外 187例 GIST肿瘤组织标本的组织芯片， 进 一步分析 DKK-4表达与 GIST临床病理资料及预后的关系，不仅发现 DKK4的表达 与肿瘤直径（p<0. 001 )、 核***相 （p=0. 02 )、 和肿瘤分级 (p<0001 )呈正相关， 还与 GIST患者术后生存状态 （p=0. 031 ) 及复发或转移 （p=0. 041 ) 密切相关。 通过 Kaplan-Meier方法绘制生存 /复发曲线， 根据 DKK4表达强弱进行分组患者 的生存 /复发曲线也可得以明显区分。 c. The inventors expanded the sample and constructed a tissue microarray containing another 187 specimens of GIST tumor tissue, and further analyzed the relationship between DKK-4 expression and clinical pathological data and prognosis of GIST, and found not only DKK4 expression and tumor diameter (p<0. 001), mitotic phase (p=0. 02), and tumor grade (p<0001) were positively correlated, It was also closely related to postoperative survival status (p=0.031) and recurrence or metastasis (p=0.041) in patients with GIST. The survival/recurrence curve was plotted by the Kaplan-Meier method, and the survival/recurrence curves of the grouped patients according to the DKK4 expression intensity were also clearly distinguished.
d. 本发明人运用 ELISA方法检测 20例 GIST患者血浆标本及 25例非 GIST人群 (包括 16例健康人群、 3例胃癌、 3例肠癌及 3例腹腔间叶源性肿瘤患者）血清 标本中 DKK-4蛋白的含量， 根据 DKK4标准品梯度浓度绘制标准曲线计算 DKK4 含量。 结果显示 GIST患者平均血清 DKK- 4蛋白含量（359. 5 ± 156. 4pg/ml )明显 高于非 GIST人群 (54. 92±4. 705pg/ml )， 差异有统计学意义 (P=0. 0347 )0 e. 根据上述实验结果尤其是大样本量的临床资料分析， 发明人已经发现 DKK4 在胃肠道间质瘤尤其是高度危险的胃肠道间质瘤中有特别高的特异性表达，证实 了 DKK4基因与 GIST的生物学特性及 GIST患者预后存在一定联系。 该基因的蛋 白编码产物 DKK4蛋白作为一种分泌型糖蛋白， 可以在血清中得到检测， 且其水 平在 GIST患者中特异性升高，为 GIST疾病的早期诊断和预后判断提供一种新的 手段。 d. The inventors used ELISA to detect plasma samples from 20 patients with GIST and 25 patients with non-GIST (including 16 healthy people, 3 gastric cancers, 3 intestinal cancers, and 3 patients with abdominal mesenchymal tumors). The content of DKK-4 protein was calculated based on the DKK4 standard gradient concentration to calculate the DKK4 content. The results showed that the average serum DKK-4 protein content of GIST patients (359. 5 ± 156. 4pg/ml) was significantly higher than that of non-GIST population (54. 92±4. 705pg/ml), the difference was statistically significant (P=0. 0347 ) 0 e. Based on the above experimental results, especially large sample size clinical data analysis, the inventors have found that DKK4 has a particularly high specific expression in gastrointestinal stromal tumors, especially in highly dangerous gastrointestinal stromal tumors. It was confirmed that the DKK4 gene is associated with the biological characteristics of GIST and the prognosis of patients with GIST. The protein-coding product DKK4 protein of this gene can be detected in serum as a secreted glycoprotein, and its level is specifically increased in GIST patients, providing a new means for early diagnosis and prognosis of GIST diseases. .
在得到了 DKK4的核酸片段的情况下，可根据核苷酸序列来设计 DKK4特异性 引物 （探针）。 核苷酸全长序列或其片段通常可以用 PCR扩增法、 重组法或人工 合成的方法获得。 对于 PCR扩增法， 可根据本发明所公开的有关核苷酸序列，尤 其是开放阅读框序列来设计引物， 并用市售的 cDNA库或按本领域技术人员已知 的常规方法所制备的 cDNA库作为模板， 扩增而得有关序列。 当序列较长时， 常 常需要进行两次或多次 PCR扩增，然后再将各次扩增出的片段按正确次序拼接在 一起。一旦获得了有关的序列， 就可以用重组法来大批量地获得有关序列。这通 常是将其克隆入载体， 再转入细胞，然后通过常规方法从增殖后的宿主细胞中分 离得到有关序列。  In the case where a nucleic acid fragment of DKK4 is obtained, a DKK4-specific primer (probe) can be designed based on the nucleotide sequence. The full-length nucleotide sequence or a fragment thereof can usually be obtained by a PCR amplification method, a recombinant method or a synthetic method. For PCR amplification, primers can be designed in accordance with the disclosed nucleotide sequences, particularly open reading frame sequences, and can be prepared using commercially available cDNA libraries or conventional methods known to those skilled in the art. The library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the amplified fragments are spliced together in the correct order. Once the relevant sequences have been obtained, the recombination method can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then separating the relevant sequence from the proliferated host cell by conventional methods.
此外， 还可用人工合成的方法来合成有关序列， 尤其是片段长度较短时。通 常， 通过先合成多个小片段， 然后再进行连接可获得序列很长的片段。  In addition, synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short. Usually, a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then connecting them.
目前， 已经可以完全通过化学合成来得到编码本发明蛋白（或其片段， 衍生 物）的 DNA序列。 然后可将该 DNA序列引入本领域中已知的各种现有的 DNA分子 (或如载体)和细胞中。  At present, it has been possible to obtain a DNA sequence encoding the protein of the present invention (or a fragment thereof, a derivative) by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (e.g., vectors) and cells known in the art.
本发明中， DKK4多核苷酸序列可***到重组表达载体中。 总之， 只要能在 宿主体内复制和稳定，任何质粒和载体都可以用。表达载体的一个重要特征是通 常含有复制起点、 启动子、 标记基因和翻译控制元件。 In the present invention, the DKK4 polynucleotide sequence can be inserted into a recombinant expression vector. In short, as long as The host is replicable and stable in vivo, and any plasmid and vector can be used. An important feature of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and a translational control element.
本领域的技术人员熟知的方法能用于构建含 DKK4的 DNA序列和合适的转录 / 翻译控制信号的表达载体。 这些方法包括体外重组 DNA技术、 DNA合成技术、体 内重组技术等。所述的 DNA序列可有效连接到表达载体中的适当启动子上， 以指 导 mRNA合成。 转化载体还包括翻译起始用的核糖体结合位点和转录终止子。  Methods well known to those skilled in the art can be used to construct expression vectors containing DKK4 DNA sequences and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, and the like. The DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. The transformation vector also includes a ribosome binding site for translation initiation and a transcription terminator.
此外，表达载体优选地包含一个或多个选择性标记基因， 以提供用于选择转 化的宿主细胞的表型性状，如真核细胞培养用的二氢叶酸还原酶、新霉素抗性以 及绿色荧光蛋白 (GFP)， 或用于大肠杆菌的四环素或氨苄青霉素抗性。  Furthermore, the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
包含上述的适当 DNA序列以及适当启动子或者控制序列的载体，可以用于转 化适当的宿主细胞， 以使其能够表达蛋白质。  Vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences, can be used to transform appropriate host cells to enable expression of the protein.
宿主细胞可以是原核细胞， 如细菌细胞； 或是低等真核细胞， 如酵母细胞； 或是高等真核细胞， 如哺乳动物细胞。代表性例子有： 大肠杆菌， 链霉菌属的细 菌细胞； 真菌细胞如酵母； 植物细胞； 昆虫细胞； 动物细胞等。  The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: Escherichia coli, bacterial cells of the genus Streptomyces; fungal cells such as yeast; plant cells; insect cells; animal cells, and the like.
用重组 DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主 为原核生物如大肠杆菌时， 能吸收 DNA的感受态细胞可在指数生长期后收获，用 CaC 处理， 所用的步骤在本领域众所周知。 另一种方法是使用 MgCl2。 如果需 要， 转化也可用电穿孔的方法进行。 当宿主是真核生物， 可选用如下的 DNA转染 方法： 磷酸钙共沉淀法， 常规机械方法如显微注射、 电穿孔、 脂质体包装等。 获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用 的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长 的条件下进行培养。 当宿主细胞生长到适当的细胞密度后， 用合适的方法 (如温 度转换或化学诱导)诱导选择的启动子， 将细胞再培养一段时间。 Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E. coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase, treated with CaC, and the procedures used are well known in the art. Another method is to use MgCl 2 . Conversion can also be carried out by electroporation if desired. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like. The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The cultivation is carried out under conditions suitable for the growth of the host cell. After the host cell has grown to the appropriate cell density, the selected promoter is induced by a suitable method (e.g., temperature conversion or chemical induction) and the cells are cultured for a further period of time.
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞 外。如果需要， 可利用其物理的、化学的和其它特性通过各种分离方法分离和纯 化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并 不限于： 常规的复性处理、 用蛋白沉淀剂处理 (盐析方法)、 离心、 渗透破菌、超 处理、 超离心、 分子筛层析 (凝胶过滤）、 吸附层析、 离子交换层析、 高效液相层 析 (HPLC)和其它各种液相层析技术及这些方法的结合。 在获得了核酸序列后， 可根据核酸序列设计特异性核酸探针。设计探针的方 法是本领域常规的， 可见 Sambrook等人， 分子克隆实验室手册 (New York : Cold Spring Harbor Laboratory Press, 1989)中所述。检测生物样品中是否存在 DKK4 蛋白或核酸的示范性方法包括获得测试受试者的生物样品，使该生物样品接触能 与 DKK4 mRNA或基因组 DNA杂交的标记的核酸探针。 该核酸探针可以是， 例如人 的核酸或及一部分， 如长至少 15、 30、 50、 100个核苷酸并能在严谨条件下与 DKK4 mRNA或基因组 DNA充分杂交的核酸探针。 用于本发明诊断试验的其它探针 如本文所述。 The recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell. If desired, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods. After the nucleic acid sequence is obtained, a specific nucleic acid probe can be designed based on the nucleic acid sequence. Methods for designing probes are routine in the art and can be found in Sambrook et al., Molecular Cloning Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). An exemplary method of detecting the presence or absence of a DKK4 protein or nucleic acid in a biological sample comprises obtaining a biological sample of the test subject, the biological sample being contacted with a labeled nucleic acid probe capable of hybridizing to DKK4 mRNA or genomic DNA. The nucleic acid probe may be, for example, a human nucleic acid or a portion thereof, such as a nucleic acid probe that is at least 15, 30, 50, 100 nucleotides in length and is capable of sufficiently hybridizing to DKK4 mRNA or genomic DNA under stringent conditions. Other probes for use in the diagnostic assays of the invention are as described herein.
核酸探针与扩增的标记序列接触。该探针优选连接到一种发色团，但可被放 射标记。 在另一个实施例中， 探针连接到一种结合伴侣上， 如抗体或生物素，或 另一种携带可检测结构域的结合伴侣上。  The nucleic acid probe is contacted with the amplified tag sequence. The probe is preferably attached to a chromophore but can be labeled. In another embodiment, the probe is attached to a binding partner, such as an antibody or biotin, or another binding partner that carries a detectable domain.
在传统的方法中，检测可通过 Southern印迹以及与标记的探针杂交来进行。 Southern印迹所涉及的技术是本领域技术人员所熟知的（参见 Sambrook等， 1989) 常规的检测还有生物芯片、 荧光显影技术、 细胞流式计数等。  In a conventional method, detection can be performed by Southern blotting and hybridization to labeled probes. Techniques involved in Southern blotting are well known to those skilled in the art (see Sambrook et al., 1989). Conventional assays include biochips, fluorescence imaging techniques, cell flow counting, and the like.
另一方面， 本发明还包括对 DKK4 DNA或是其片段编码的多肽具有特异性的 多克隆抗体和单克隆抗体， 尤其是单克隆抗体。 这里， "特异性"是指抗体能结 合于 DKK4基因产物或片段。较佳地，指那些能与 DKK4基因产物或片段结合但不 识别和结合于其它非相关抗原分子的抗体。本发明的抗体可以通过本领域内技术 人员己知的各种技术进行制备。  In another aspect, the invention also encompasses polyclonal and monoclonal antibodies, particularly monoclonal antibodies, which are specific for a polypeptide encoded by DKK4 DNA or a fragment thereof. Here, "specificity" means that the antibody binds to the DKK4 gene product or fragment. Preferably, those antibodies which bind to the DKK4 gene product or fragment but which do not recognize and bind to other non-related antigen molecules. The antibodies of the present invention can be prepared by various techniques known to those skilled in the art.
本发明所述 DKK4抗体可以通过 ELISA、 Western印迹分析，或者与检测基团 偶联， 通过化学发光、 同位素示踪等方法来检测。  The DKK4 antibody of the present invention can be detected by ELISA, Western blot analysis, or coupled to a detection group by chemiluminescence or isotope tracing.
DKK4 (Dickkopf 同源物 4) 染色体定位于 8pll. 2- pl l. 1， 为一种分泌型糖 蛋白，参与机体发育及 Wnt信号转导途径的调控，其通过与 Wr t信号转导途径相 应的受体结合来调控细胞的分化、增殖、迁移或癌变等特性， 在肿瘤发生方面发 挥重要作用， 本发明人通过试验证明 DKK4基因可用于制备诊断或治疗胃肠道间 质瘤药物。  DKK4 (Dickkopf homolog 4) The chromosome is located at 8pll. 2- pl l. 1, a secreted glycoprotein involved in the development of the body and the regulation of the Wnt signaling pathway, which corresponds to the Wr t signal transduction pathway. The receptor binds to regulate cell differentiation, proliferation, migration or carcinogenesis and plays an important role in tumorigenesis. The inventors have experimentally proved that the DKK4 gene can be used for the preparation of a medicament for diagnosing or treating gastrointestinal stromal tumors.
本发明首次提出利用基于血清 /组织中 DKK基因及其产物的检测来达到早期 诊断 GIST或对接受手术治疗的 GIST患者进行预后判断，建立在基因水平上的高 通量芯片筛选 +实时定量 PCR验证和蛋白水平上的大样本临床样本验证， 结果可 靠， 且根据实验结果， DKK4无论是在基因水平还是蛋白水平上都能够显著区分 肿瘤与非肿瘤、 高危肿瘤与低危肿瘤， 结果的重复性好， 且其在不同组别间的表 达量的差异巨大， 具有较好的临床应用价值。 附图说明 The present invention provides for the first time to use the detection of DKK gene and its products in serum/tissue to achieve early diagnosis of GIST or prognosis of GIST patients undergoing surgery, and establish high-throughput chip screening + real-time quantitative PCR verification at the gene level. And large sample clinical samples at the protein level, the results can be Based on the experimental results, DKK4 can significantly distinguish between tumor and non-tumor, high-risk tumors and low-risk tumors at both the gene level and the protein level. The results are reproducible and their expression levels are different between different groups. The difference is huge and has good clinical application value. DRAWINGS
图 1 : 实时定量 PCR结果显示， 在 24例胃肠道间质瘤 （14例高危、 10例低危） 的新鲜肿瘤标本中， 低危组 （以 标注） 中 DKK4基因的表达量显著低于高危组 (以▲标注) 中的表达量 （p=0. 0029， fold change=42. 16)。 Figure 1: Real-time quantitative PCR results showed that in 24 fresh gastrointestinal stromal tumors (14 high-risk, 10 low-risk) fresh tumor specimens, the DKK4 gene expression was significantly lower in the low-risk group (labeled). The expression level in the high-risk group (marked with ▲) (p=0. 0029, fold change=42. 16).
图 2: 免疫组化染色结果 （瘤旁正常组织， 阴性反应） Figure 2: Immunohistochemical staining results (normal tissue next to the tumor, negative reaction)
图 3: 免疫组化染色结果 （肿瘤组织， 阴性反应） Figure 3: Immunohistochemical staining results (tumor tissue, negative reaction)
图 4: 免疫组化染色结果 （肿瘤组织， 弱阳性反应） Figure 4: Immunohistochemical staining results (tumor tissue, weak positive reaction)
图 5: 免疫组化染色结果 （肿瘤组织， 阳性反应） Figure 5: Immunohistochemical staining results (tumor tissue, positive reaction)
图 6: 免疫组化染色结果 （肿瘤组织， 强阳性反应） Figure 6: Results of immunohistochemical staining (tumor tissue, strong positive reaction)
图 7: 通过 Kaplan- Meier方法绘制的 326例胃肠道间质瘤患者的总生存曲线， 其中 DKK4染色阴性以♦标注， 弱阳性以秦标注， 阳性以▲标注， 强阳性以園标 注。 Figure 7: Total survival curves of 326 patients with gastrointestinal stromal tumors by Kaplan-Meier method, in which DKK4 staining was negatively marked with ♦, weak positive was marked with Qin, positive was marked with ▲, and strong positive was marked with gardening.
图 8: 通过 Kaplan-Meier方法绘制的 326例胃肠道间质瘤患者的无复发生存曲 线， 其中 DKK4染色阴性以令标注， 弱阳性以翁标注， 阳性以▲标注， 强阳性以 國标注。 Figure 8: Recurrence-free survival curve of 326 patients with gastrointestinal stromal tumors by Kaplan-Meier method, in which DKK4 staining was negative for labeling, weak positive was marked with Weng, positive was marked with ▲, and strong positive was marked with country.
图 9: 纵坐标为 DKK4血浆浓度， 左侧散点为胃肠道间质瘤患者血浆标本， 右侧 为非胃肠道间质瘤人群血浆标本。 具体实施方式 Figure 9: The ordinate is the DKK4 plasma concentration, the left scatter is the plasma sample of patients with gastrointestinal stromal tumors, and the right side is the plasma sample of the non-gastrointestinal stromal tumor population. detailed description
实施例 1. 实时定量 PCR分析：  Example 1. Real-time quantitative PCR analysis:
1. 1 主要试剂  1. 1 main reagent
RNA抽提试剂 RNAiso Plus购自宝生物工程有限公司， 逆转录试剂盒 High Capacity cDNA Reverse Transcription Kits购自 Invitrogen公司， 焚光定量 PCR试剂盒 Power SYBR Green PCR Master Mix购自 Invitrogen公司， DKK4 及管家基因 P -肌动蛋白的引物设计采用 Primer 3软件， 引物合成由上海生工生 物工程技术服务有限公司完成， 其余试剂均为分析纯。 The RNA extraction reagent RNAiso Plus was purchased from Bao Bioengineering Co., Ltd., the reverse transcription kit High Capacity cDNA Reverse Transcription Kits was purchased from Invitrogen, and the fluorescence quantitative PCR kit Power SYBR Green PCR Master Mix was purchased from Invitrogen, DKK4 and housekeeping genes. P-actin primer design using Primer 3 software, primer synthesis by Shanghai Biotech Engineering Engineering Services Co., Ltd. completed, the remaining reagents were of analytical grade.
1. 2 胃肠道间质瘤病人肿瘤组织样品的收集  1. 2 Collection of tumor tissue samples from patients with gastrointestinal stromal tumors
胃肠道间质瘤患者的肿瘤组织来源于上海交通大学医学院附属仁济医院。 手术后组织标本立即置液氮中冷冻， 随后保存于- 80°C超低温冰箱。  The tumor tissue of patients with gastrointestinal stromal tumors originated from Renji Hospital affiliated to Shanghai Jiao Tong University School of Medicine. Tissue specimens were immediately frozen in liquid nitrogen after surgery and then stored in an ultra-low temperature freezer at -80 °C.
1. 3 实时荧光定量分析 PCR分析  1. 3 real-time fluorescence quantitative analysis PCR analysis
总 RNA抽提： 将超低温冻结的 RNA提取样品称量后迅速转移至用液氮预冷 的研钵中， 用研杵研磨组织， 其间不断加入液氮， 直至研磨成粉末状。 向研钵中 加入适量的 RNAiso Plus, 将研磨成粉末状的样品完全覆盖， 然后室温静置， 直 至样品完全融化，再用研杵继续研磨至裂解液呈透明状。将匀桨液转移至离心管 中， 室温静置 5分钟。 12，000 g 4°C离心 5分钟。 小心吸取上清液， 移入新的离 心管中。 加入 RNAiso Plus的 1/5体积量的氯仿， 剧烈振荡 15秒， 待溶液充分 乳化后， 再室温静置 5分钟， 12, 000 g 4°C离心 15分钟。 吸取上清液转移至另 一新的离心管中。 向上清中加入等体积的异丙醇， 上下颠倒离心管充分混匀后， 在 15〜30°C下静置 10分钟。 12, 000 g 4°C离心 10分钟。 弃去上清， 沿管壁加 入 75%的乙醇 1 ml， 上下颠倒洗涤离心管管壁， 12, 000 g 4°C离心 5分钟后弃 去乙醇。 室温干燥沉淀 2〜5分钟， 加入适量的 RNase-free水溶解沉淀后用 Nanodrop2000测定 RNA浓度后于- 80 °C保存。  Total RNA extraction: The ultra-low temperature frozen RNA extraction sample was weighed and quickly transferred to a mortar precooled with liquid nitrogen, and the tissue was ground with a mortar while continuously adding liquid nitrogen until it was ground into a powder. Add the appropriate amount of RNAiso Plus to the mortar, completely cover the sample that has been ground into a powder, and then let it stand at room temperature until the sample is completely melted. Continue grinding until the lysate is transparent. The slurry was transferred to a centrifuge tube and allowed to stand at room temperature for 5 minutes. Centrifuge at 12,000 g for 5 minutes at 4 °C. Carefully pipette the supernatant and transfer to a new centrifuge tube. 1/5 volume of chloroform of RNAiso Plus was added and shaken vigorously for 15 seconds. After the solution was fully emulsified, it was allowed to stand at room temperature for 5 minutes and centrifuged at 12 000 g for 4 minutes at 4 °C. Transfer the supernatant to another new centrifuge tube. An equal volume of isopropanol was added to the supernatant, and the tube was thoroughly mixed upside down and allowed to stand at 15 to 30 ° C for 10 minutes. Centrifuge at 12, 000 g for 10 minutes at 4 °C. Discard the supernatant, add 1 ml of 75% ethanol along the tube wall, wash the tube wall upside down, centrifuge at 12 000 g for 4 minutes at 4 ° C, and discard the ethanol. The precipitate was dried at room temperature for 2 to 5 minutes, and the precipitate was dissolved by adding an appropriate amount of RNase-free water, and the RNA concentration was measured with Nanodrop 2000 and stored at -80 °C.
逆转录合成 cDNA: RNA解冻后在 0. 2mlPCR管中配置反应溶液， 反应体系如 下表：  Reverse transcription synthesis cDNA: RNA was thawed and the reaction solution was placed in a 0.2 ml PCR tube. The reaction system is as follows:
Figure imgf000011_0001
Figure imgf000011_0001
加样后将反应管置于 PCR仪， 热循环体系为 25°C 10分钟—— 37Γ 120分钟 •85°C5分钟—— 4°C保存。  After the sample was applied, the reaction tube was placed in a PCR apparatus, and the thermal cycle system was stored at 25 ° C for 10 minutes - 37 ° 120 minutes • 85 ° C for 5 minutes - 4 ° C.
Realtime PCR检测： 在 96孔板中配制 20ul反应体系。每反应设 3个复孔， 以 Ρ -肌动蛋白为内参基因进行相对定量检测 DKK4基因表达量。 Realtime PCR assay: A 20 ul reaction system was prepared in a 96-well plate. 3 replicate holes per reaction, Relatively quantitative detection of DKK4 gene expression by Ρ-actin as an internal reference gene.
Realtime扩增体系如下表:  The Realtime amplification system is as follows:
Figure imgf000012_0001
Figure imgf000012_0001
将加好样品的 96孔板放在 ABI9300荧光定量 PCR仪中进行反应，热循环体 下-.  The 96-well plate with the sample added is placed in the ABI9300 fluorescence quantitative PCR machine for reaction, under the thermal cycle.
Figure imgf000012_0002
Figure imgf000012_0002
Realtime PCR结果用 2-ΔΔσΤ法进行分析。 实验结果如图 1所示， 在 24 例胃肠道间质瘤 （14例高危、 10例低危） 的新鲜肿瘤标本中， 低危组中 DKK4 基因的表达量显著低于高危组中的表达量 （p=0. 0029， fold change=42. 16 )„ 实施例 2. DKK4在胃肠道间质瘤病人组织中的表达情况  Realtime PCR results were analyzed by the 2-ΔΔσΤ method. The results of the experiment are shown in Figure 1. In 24 cases of gastrointestinal stromal tumors (14 high-risk, 10 low-risk) fresh tumor specimens, the expression level of DKK4 gene in the low-risk group was significantly lower than that in the high-risk group. Amount (p=0. 0029, fold change=42. 16 ) „ Example 2. Expression of DKK4 in tissues of patients with gastrointestinal stromal tumors
2. 1 主要试剂  2. 1 main reagent
DKK4单抗（ab38589)及二抗山羊多克隆抗兔 IgG (ab6721 )购自 abeam公 司。 DAB显色剂及其底物试剂盒购自赛默飞公司。 其余试剂均为国产分析纯。  DKK4 mAb (ab38589) and secondary antibody goat polyclonal anti-rabbit IgG (ab6721) were purchased from abeam. The DAB developer and its substrate kit were purchased from Thermo Fisher. The remaining reagents were all domestically analyzed.
2. 2 胃肠道间质瘤组织芯片阵列的构建  2. 2 Construction of gastrointestinal stromal tumor tissue chip array
胃肠道间质瘤患者的肿瘤组织来源于上海交通大学医学院附属仁济医院。 芯片阵列构建由苏州新芯生物技术有限公司完成， 点阵直径 1. 6mm, 层厚 3mm。 芯片一包括 139例胃肠道间质瘤肿瘤组织及相应邻近正常组织， 芯片二包括 189 例胃肠道间质瘤肿瘤组织。  The tumor tissue of patients with gastrointestinal stromal tumors originated from Renji Hospital affiliated to Shanghai Jiao Tong University School of Medicine. The chip array was constructed by Suzhou Xinxin Biotechnology Co., Ltd., with a dot matrix diameter of 1. 6mm and a layer thickness of 3mm. Chip 1 included 139 gastrointestinal stromal tumor tissues and corresponding adjacent normal tissues, and chip 2 included 189 gastrointestinal stromal tumor tissues.
2. 3 免疫组织化学染色  2. 3 Immunohistochemical staining
脱蜡水化： 二甲苯 10分钟——二甲苯 1/2 10分钟——无水乙醇 5分钟一 一 95%乙醇 5分钟—— 70%乙醇 10分钟—— PBS洗。 Dewaxing hydration: xylene 10 minutes - xylene 1/2 10 minutes - anhydrous ethanol 5 minutes A 95% ethanol for 5 minutes - 70% ethanol for 10 minutes - washed with PBS.
抗原修复： 电炉加热 0. 01M枸橼酸钠缓冲溶液 （pH6. 0) 至 95°C左右， 放 入组织芯片加热 15分钟， PBS洗。  Antigen retrieval: Electric furnace heating 0. 01M sodium citrate buffer solution (pH 6. 0) to about 95 ° C, put into the tissue chip and heat for 15 minutes, wash with PBS.
消除内源性酶： 3%过氧化氢 37°C孵育 30分钟， PBS洗。  Eliminate endogenous enzymes: Incubate with 3% hydrogen peroxide at 37 ° C for 30 minutes, wash with PBS.
抗原封闭： 10%山羊血清室温孵育 1小时。  Antigen blocking: 10% goat serum was incubated for 1 hour at room temperature.
一抗孵育： 滴加一抗 （DKK4单抗） 150ul， 4°C孵育过夜， PBS洗。  Primary antibody incubation: Add primary antibody (DKK4 mAb) 150 ul, incubate overnight at 4 ° C, wash with PBS.
二抗孵育： 滴加二抗 （羊抗兔多抗） 150ul， 室温孵育 1小时， PBS洗。 发色： DAB显色 5-10分钟， 显微镜下控制发色程度， PBS洗。  Incubation of secondary antibody: Add secondary antibody (goat anti-rabbit polyclonal antibody) 150 ul, incubate for 1 hour at room temperature, and wash with PBS. Hair color: DAB color development 5-10 minutes, control the degree of coloration under the microscope, wash with PBS.
苏木精复染 1分钟， 自来水冲洗 15分钟。  Hematoxylin counterstained for 1 minute, rinsed with tap water for 15 minutes.
脱水、 封片、 镜检。  Dehydration, sealing, microscopic examination.
2. 4 组织芯片结果判断  2. 4 organization chip results judgment
以胞浆着色为阳性反应部位，对每个阵列点阳性细胞的阳性强度按无着色、 淡黄色、 棕黄色和棕褐色分别打 0、 1、 2、 3分， 着色阳性面积按无着色、 着色 <1/3、 1/3〜2/3、 〉2/3分别打 0、 1、 2、 3分， 然后根据两项打分之和判断其结 果： 0分为阴性， 1-2分为弱阳性， 3-4分为阳性， 5-6分者为强阳性 (注： 每张 切片选择有代表性的区域， 在 400倍视野下进行计数， 共计 5个视野， 取其平均 值以避免随意性）  The cytoplasmic staining was used as the positive reaction site, and the positive intensity of the positive cells of each array point was 0, 1, 2, 3, respectively, without coloring, light yellow, brownish yellow and tan, and the coloring positive area was colorless and colored. <1/3, 1/3~2/3, 〉2/3 respectively score 0, 1, 2, 3, and then judge the result according to the sum of the two scores: 0 is negative, 1-2 is weak Positive, 3-4 is positive, 5-6 is strong positive (Note: Select a representative area for each slice, count in 400 times field of view, total 5 fields, take the average to avoid random Sex)
2. 5 统计学分析  2. 5 Statistical analysis
将组织芯片结果输入 SPSS20. 0中进行分析， 以卡方检验分析 DKK4表达与 临床病理资料相关性， 显著性水平定义为 p〈0. 05。 以 Kaplan-Meier法进行生存 分析， 显著性水平定义为 p<0. 05。  The results of tissue microarray were input into SPSS20. 0 for analysis. The correlation between DKK4 expression and clinical pathological data was analyzed by chi-square test. The significance level was defined as p<0.05. Survival analysis was performed by Kaplan-Meier method, and the significance level was defined as p<0.05.
免疫组化染色典型结果见图 2-图 6。 分析结果显示 DKK-4在瘤旁正常组织 中阳性表达率远低于在肿瘤中阳性表达率 （7. 2% vs 86. 5%)， 且 DKK4的表达与 肿瘤直径 （p<0. 001 )、 核***相 （p=0. 02 )、 和肿瘤分级 （p〈0001 ) 呈正相关， 还与 GIST患者术后生存状态 （p=0. 031 ) 及复发或转移 （p=0. 041 ) 密切相关。 通过 Kaplan-Meier方法绘制生存 /复发曲线，根据 DKK- 4表达强弱进行分组患者 的生存 /复发曲线也可得以明显区分， 见图 7-图 8。  Typical results of immunohistochemical staining are shown in Figure 2-6. The results showed that the positive expression rate of DKK-4 in normal tissues adjacent to the tumor was much lower than that in the tumor (7.2% vs 86.5%), and the expression of DKK4 and tumor diameter (p<0.001) , mitotic phase (p=0. 02), and tumor grade (p<0001) were positively correlated, and were also closely related to postoperative survival status (p=0.031) and recurrence or metastasis (p=0.041) in patients with GIST. . Survival/recurrence curves were plotted by Kaplan-Meier method, and the survival/recurrence curves of grouped patients based on DKK-4 expression intensity were also clearly distinguished, as shown in Figure 7-8.

Claims

权利 要求 Rights request
1、 DKK4基因在制备药物中的应用， 其特征在于， 所述 DKK4基因序列为 SEQ ID NO. 1-2。 1. The application of DKK4 gene in the preparation of medicines, characterized in that the DKK4 gene sequence is SEQ ID NO. 1-2.
2、 DKK4基因在制备检测胃肠道间质瘤药物中的应用，其特征在于，所述 DKK4 基因序列为 SEQ ID NO. 1-2。 2. The application of DKK4 gene in the preparation and detection of drugs for gastrointestinal stromal tumors, characterized in that the DKK4 gene sequence is SEQ ID NO. 1-2.
3、 根据权利要求 2所述的应用， 其特征在于， 所述检测胃肠道间质瘤药物 为用 RT-PCR、 qPCR、 免疫检测、 原位杂交、 基因芯片检测胃肠道间质瘤的药物 或试齐 IJ盒。 3. Application according to claim 2, characterized in that the drug for detecting gastrointestinal stromal tumors is to detect gastrointestinal stromal tumors using RT-PCR, qPCR, immune detection, in situ hybridization, and gene chips. Medication or test kit.
4、根据权利要求 3所述的应用， 其特征在于，所述用 RT- PCR检测胃肠道间 质瘤的药物为至少一对特异性扩增 DKK4基因的引物。 4. The application according to claim 3, characterized in that the drug for detecting gastrointestinal stromal tumors using RT-PCR is at least one pair of primers that specifically amplify the DKK4 gene.
5、 根据权利要求 3所述的应用， 其特征在于， 所述用 qPCR检测胃肠道间 质瘤的药物为至少一对特异性扩增 DKK4基因的引物。 5. The application according to claim 3, characterized in that the drug for detecting gastrointestinal stromal tumors by qPCR is at least one pair of primers that specifically amplify the DKK4 gene.
6、 根据权利要求 3所述的应用， 其特征在于， 所述用免疫检测胃肠道间质 瘤的药物为与 DKK4蛋白特异性结合的抗体、 多克隆抗体和单克隆抗体。 6. The application according to claim 3, characterized in that the drugs for immunological detection of gastrointestinal stromal tumors are antibodies, polyclonal antibodies and monoclonal antibodies that specifically bind to DKK4 protein.
7、 根据权利要求 3所述的应用， 其特征在于， 所述用原位杂交检测胃肠道 间质瘤的药物包括与 DKK4核酸序列杂交的探针； 所述用基因芯片检测胃肠道间 质瘤的药物为与 DKK4核酸序列杂交的探针。 7. The application according to claim 3, wherein the drug for detecting gastrointestinal stromal tumors using in situ hybridization includes a probe that hybridizes with the DKK4 nucleic acid sequence; and the use of gene chips for detecting gastrointestinal stromal tumors. The drug for tumors is a probe that hybridizes to the DKK4 nucleic acid sequence.
8、 根据权利要求 3所述的应用， 其特征在于， 所述用于检测胃肠道间质瘤 的试剂盒为用于 RNA分离、 扩增细胞中 RNA的纯化的试剂或标记。 8. The application according to claim 3, characterized in that the kit for detecting gastrointestinal stromal tumors is a reagent or label used for RNA isolation and purification of RNA in amplified cells.
9、 DKK4基因在制备治疗胃肠道间质瘤药物中的应用，其特征在于，所述 DKK4 基因序列为 SEQ ID NO. 1-2。 9. The application of DKK4 gene in the preparation of drugs for the treatment of gastrointestinal stromal tumors, characterized in that the DKK4 gene sequence is SEQ ID NO. 1-2.
10、根据权利要求 9所述的应用， 其特征在于， 所述治疗胃肠道间质瘤药物 为：通过 RNA干扰抑制 DKK4基因表达的双链核糖核酸、基于 DKK4抗原蛋白的肿 瘤疫苗或抑制 DKK4蛋白活性的蛋白质。 10. The application according to claim 9, characterized in that the drug for treating gastrointestinal stromal tumors is: double-stranded ribonucleic acid that inhibits DKK4 gene expression through RNA interference, a tumor vaccine based on DKK4 antigen protein, or a drug that inhibits DKK4 protein active protein.
PCT/CN2013/001192 2013-02-18 2013-10-08 Application of dkk4 gene and coding protein thereof in preparation of medicament WO2014124551A1 (en)

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