WO2016065877A1 - Cd166作为肝癌诊断血清标志物的应用及其试剂盒 - Google Patents
Cd166作为肝癌诊断血清标志物的应用及其试剂盒 Download PDFInfo
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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
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- the invention relates to the technical field of clinical diagnosis, in particular to the application of CD166 as a serum marker for diagnosis of liver cancer and a kit thereof.
- Liver cancer a liver malignant tumor
- Liver malignant tumors can be divided into two major categories: primary and secondary.
- Primary liver malignant tumor originates from the epithelial or mesenchymal tissue of the liver. The former is called primary liver cancer. It is a high-risk malignant tumor in China. The latter is called sarcoma and compared with primary liver cancer. Rare.
- Secondary or metastatic liver cancer refers to the invasion of the malignant tumors of multiple organs of the body into the liver. It is usually found in liver metastases of malignant tumors such as stomach, biliary tract, pancreas, colorectal, ovary, uterus, lung and breast.
- liver Since the liver is the largest substantial organ of the human body and bears all kinds of important metabolic functions of the human body, the occurrence of malignant tumors in the liver will lead to serious life-threatening consequences. Because the liver has a rich supply of blood flow, the important structure of the human body is closely related to the vena cava, portal vein, biliary system, etc., and the pathogenesis of liver malignant tumors is concealed, and the invasive growth is rapid, which makes the treatment difficult. Therefore, early detection, early diagnosis, and early treatment are essential for the treatment of liver cancer.
- liver cancer mainly relies on imaging examination, liver biopsy and other methods.
- these detection methods have certain limitations. For example, even a good fine needle puncture has a limited false negative rate and has the risk of spreading the tumor and implanting the needle.
- Serological detection techniques for cancer have been the focus of research.
- serum markers for cancer such as HCC
- serum markers for serum detection or prognosis of liver cancer are currently no satisfactory.
- Serum CD166 also known as activated leukocyte cell adhesion molecule, is a transmembrane glycoprotein receptor widely distributed in human tissues and organs, and exerts a wide range of physiological and pathological functions.
- the CD166-encoding gene is located on the long arm of human chromosome 3 and contains about 16 kb base pairs in 16 exons.
- the gene promoter is located nearby and is rich in a GC cassette, contains DNA binding sequences that bind to the transcription factors kappa B and AP-1, and additionally contains abundant negative or positive regulatory sequences that specifically participate in the regulation of CD166 in tissues. expression.
- CD166 belongs to the immunoglobulin superfamily (IgSF) member transmembrane recognition molecule, and the extracellular portion includes five functional domains (VVC2C2C2), three of which are constant domains (C2 domain) and two are variable domains (V type).
- the natural molecular protein of CD166 has a molecular weight of about 69 kD and is glycosylated to form a mature protein having a molecular weight of about 105 kD, which is a ligand for the lymphocyte antigen CD6.
- CD166 is widely distributed in activated leukocytes, monocytes, endothelial cells, fibroblasts, neurons, Mesenchymal stem cells, hematopoietic stem cells, myeloid progenitor cells, bone marrow stromal cells, and some glandular cells are mainly expressed on leukocytes and thymic epithelial cells. CD166 is involved in the body's inflammatory response, cell adhesion, hematopoiesis, peripheral and central nervous system development, and tumor invasion.
- CD166 may be one of the brain tumor stem cell markers, and can be used together with the currently recognized brain tumor stem cell marker CD133 as a marker for cancer stem cells. 4.
- the recurrence rate of CD166 high expression group was higher, and CD166 was associated with recurrence of meningioma.
- a primary object of the present invention is to provide a serum specific marker for detecting liver cancer.
- the present invention provides the use of a CD166 protein or a specific antibody thereof for preparing a diagnostic reagent or kit for detecting the prognosis of liver cancer or liver cancer.
- the CD166 protein or a specific antibody thereof is conjugated with or with a detectable label.
- the detectable label is selected from the group consisting of a chromophore, a chemiluminescent group, a fluorophore, an isotope or an enzyme.
- the test is a serum test.
- the serum test is an ELISA method or a double-antibody sandwich time-resolved immunofluorescence method (TRFIA method).
- the invention also provides the use of the CD166 protein or a specific antibody thereof for detecting the prognosis of liver cancer or liver cancer.
- the test is a serum test.
- GenBank serial number of the CD166 protein on NCBI is GENE. ID 214.
- the invention also provides a diagnostic kit for detecting the prognosis of liver cancer or liver cancer, the kit comprising an agent for determining the content of CD166 protein in serum, and a label or a description, the label or the specification indicating the reagent
- the cassette is used to detect the prognosis of liver cancer or liver cancer.
- the reagent for determining the content of CD166 protein in serum is specific for CD166 protein.
- sexual antibodies are also preferred.
- the specific antibody to the CD166 protein is conjugated with or with a detectable label.
- the detectable label is selected from the group consisting of a chromophore, a chemiluminescent group, a fluorophore, an isotope or an enzyme.
- the label or the specification states that if the serum CD166 concentration of the test subject is ⁇ 211 ng/ml (preferably ⁇ 261 ng/ml, more preferably ⁇ 311 ng/ml), then The probability of developing liver cancer in the subject is greater than that of the normal population.
- the invention also provides the use of a CD166 protein which is used as a marker for detecting liver cancer.
- the test is a serum test.
- the invention also provides a method for detecting the prognosis of liver cancer or liver cancer, wherein the method is to detect the content of CD166 protein in a serum sample, thereby determining the probability of liver cancer in a subject from which the serum sample is derived or the stage of liver cancer.
- the present invention also provides the use of an antagonist of CD166 protein for the preparation of a medicament for treating/preventing liver cancer.
- the antagonist comprises an siRNA against a CD166 protein, an antisense RNA, an antibody, or a combination thereof.
- the present invention proves for the first time that CD166 protein can be used as a marker for diagnosis of liver cancer, and can detect and predict the occurrence and development of liver cancer diseases by detecting the concentration of CD166 protein in serum, and has the advantages of convenient sampling, easy detection, specificity and sensitivity. . Since CD166 is a cell surface molecule, it can be used not only to diagnose liver cancer, but also to be a molecular target for the treatment of liver cancer.
- FIG 1 Western Blotting detects the concentration of CD166 in the serum of two groups of normal and eight groups of liver cancer patients (HI is Healthy individual, H is HCC).
- Figure 2 Serum CD166 levels in different disease populations. Box plots show the concentration of CD166 in the serum of normal, colon, liver, hepatitis B, hepatitis C, cirrhosis, gastric, breast and lung cancer patients.
- FIG. 3 Paired T-test results show changes in CD166 concentrations in pre- and post-operative serum of patients with liver cancer.
- Figure 4 Chi-square test results show that the concentration of CD166 is related to the clinical pathological stage of liver cancer patients.
- Figure 5 ROC curve showing the specificity and sensitivity of CD166 and AFP in the diagnosis of liver cancer.
- CD166 can be used as a new serum marker for liver cancer for the diagnosis and prognosis of liver cancer.
- the present invention has been completed on this basis.
- the human gene CD166 (GeneBank accession number: GENE.ID 214) has a full length of 4884 bp and encodes 570 amino acid residues.
- CD166 protein and “CD166 polypeptide” are used interchangeably and refer to a protein or polypeptide having the amino acid sequence of human CD166 protein (GeneBank Accession No.: GENE. ID 214). In addition, the term also includes full length CD166 and fragments thereof.
- the CD166 protein referred to in the present invention includes its entire amino acid sequence, a mutant thereof, and a functionally active fragment thereof.
- CD166 protein referred to in the present invention also includes glycosylated or non-glycosylated proteins.
- the invention includes polyclonal and monoclonal antibodies, particularly monoclonal antibodies, that are specific for human CD166 polypeptides.
- specificity means that the antibody binds to a human CD166 gene product or fragment.
- the antibodies of the present invention include those capable of binding to and inhibiting the human CD166 protein, as well as those which do not affect the function of the human CD166 protein.
- the invention also includes those antibodies that bind to a modified or unmodified form of the human CD166 gene product.
- the invention encompasses not only intact monoclonal or polyclonal antibodies, but also immunologically active antibody fragments, such as Fab' or (Fab) 2 fragments; antibody heavy chains; antibody light chains; genetically engineered single-chain Fv molecules ( Ladner et al., U.S. Patent No. 4,946,778); or chimeric antibodies, such as antibodies that have murine antibody binding specificity but still retain antibody portions from humans.
- immunologically active antibody fragments such as Fab' or (Fab) 2 fragments
- antibody heavy chains such as antibody heavy chains; antibody light chains; genetically engineered single-chain Fv molecules ( Ladner et al., U.S. Patent No. 4,946,778); or chimeric antibodies, such as antibodies that have murine antibody binding specificity but still retain antibody portions from humans.
- Antibodies of the invention can be prepared by a variety of techniques known to those skilled in the art. For example, a purified human CD166 gene product or a fragment thereof that is antigenic can be administered to an animal to induce polyclonal antibody production. Similarly, cells expressing human CD166 protein or antigenic fragments thereof can be used to immunize animals to produce resistance. body.
- the antibody of the invention may also be a monoclonal antibody. Such monoclonal antibodies can be prepared using hybridoma technology (see Kohler et al, Nature 256; 495, 1975; Kohler et al, Eur. J. Immunol. 6: 511, 1976; Kohler et al, Eur. J. Immunol).
- the antibody of the present invention includes an antibody which blocks the function of the human CD166 protein and an antibody which does not affect the function of the human CD166 protein.
- the various antibodies of the invention can be obtained by conventional immunological techniques using fragments or functional regions of the human CD166 gene product. These fragments or functional regions can be prepared by recombinant methods or synthesized using a polypeptide synthesizer.
- An antibody that binds to an unmodified form of the human CD166 gene product can be produced by immunizing an animal with a gene product produced in a prokaryotic cell (eg, E.
- an antibody that binds to a post-translationally modified form eg, glycosylated or phosphorylated
- a protein or polypeptide can be obtained by immunizing an animal with a gene product produced in a eukaryotic cell, such as a yeast or insect cell.
- Antibodies against human CD166 protein can be used in immunohistochemistry to detect human CD166 protein in specimens, particularly serum samples.
- the present invention also provides a method for detecting serum or liver cancer prognosis, especially serological detection methods.
- the invention provides an ELISA method for detecting serum CD166.
- the invention also provides a kit for detecting liver cancer serum or prognosis of liver cancer, which comprises the anti-CD166 immunoglobulin or immunoconjugate of the invention, or an active fragment thereof.
- the present invention also provides a pharmaceutical composition comprising the above antagonist of CD166, and a pharmaceutically acceptable carrier.
- the pharmaceutical composition can be used to increase the survival of a liver cancer patient.
- the antagonist includes siRNA against CD166, antisense RNA, antibody, or a combination thereof.
- the antagonists also include small molecule compounds that reduce CD166 expression or activity.
- these materials can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium wherein the pH is usually from about 5 to about 8, preferably from about 6 to about 8, although the pH may be The nature of the formulation and the condition to be treated vary.
- the formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, intraperitoneal, intravenous, or topical administration.
- the pharmaceutical composition of the present invention can be directly used for inhibiting the occurrence or metastasis of liver cancer cells. In addition, it can be combined with other tumor therapeutics.
- the pharmaceutical composition of the present invention contains a safe and effective amount of the above-mentioned CD166 antagonist of the present invention and is pharmaceutically acceptable Carrier or excipient.
- Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
- the pharmaceutical preparation should be matched to the mode of administration.
- the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably prepared under sterile conditions.
- the amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram body weight to about 5 milligrams per kilogram body weight per day.
- the polypeptides of the invention may also be used with other therapeutic agents.
- a safe and effective amount of a CD166 antagonist of the invention is administered to a mammal, wherein the safe and effective amount is usually at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 8 milligrams per kilogram.
- the body weight preferably the dose is from about 10 micrograms per kilogram of body weight to about 1 milligram per kilogram of body weight.
- specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
- the kit was purchased from Shanghai Lichen Biotechnology Co., Ltd., the full name of the kit: human CD166 ELISA KIT, article number: LC9012, composition: microporous microplate 12 wells*8, standard (4ng/ml) 0.6ml, standard dilution 6 ml of liquid, 6 ml of sample diluent, detection antibody-HRP, washing buffer 25 ml, substrate A, substrate B, stop solution, sealing film, instructions, and ziplock bag.
- the detection method of CD166 in serum samples is: double-antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA). Specific steps: (1) Wash buffer was prepared before the experiment, and the ELISA plate was equilibrated at room temperature for 20 minutes. (2) Set standard holes, sample holes, blank holes (nothing added). Standard wells were added with 50 ⁇ l of different concentrations of standard; the sample wells were explored in advance, and the serum samples were diluted 5 times (sample wells plus 40 ⁇ l serum sample dilution, 10 ⁇ l serum sample to be tested). (3) 100 ⁇ l of horseradish peroxidase (HRP)-labeled detection antibody was added to the standard well and the sample well, and incubated at 37 ° C for 60 minutes in an incubator.
- HRP horseradish peroxidase
- Liver cancer was confirmed by enhanced CT, serum AFP and postoperative pathological sections.
- 51 patients with liver cancer were collected from Ruijin Hospital.
- 48 patients were randomly selected and randomly divided into 8 groups, 6 in each group.
- Eighty-five patients with normal physical examination were collected from Shanghai Tenth People's Hospital (normal population was defined as AFP as normal, radiographic liver-free lesions) and 12 patients were randomly selected and randomly divided into 2 groups, 6 in each group.
- the serum of each group of subjects was collected, and 6 serum samples of each group were mixed in equal amounts into serum samples to be tested.
- the concentration of CD166 in each group of serum samples was determined by conventional Western Blotting method using SDS-PAGE gel.
- the concentration of CD166 in the serum of all the samples was measured by the ELISA kit of Shanghai Lichen Biotechnology Co., Ltd. (for the specific procedure, the detection method of CD166 in the above serum samples).
- the concentration of CD166 in the serum was determined by ELISA method (see the method for detecting CD166 in the above serum samples), and the quartile was classified according to the concentration, corresponding to the corresponding pathological stage, and the X list was prepared.
- the experimental results were statistically analyzed by STATA. The chi-square test in the software is analyzed.
- Figure 1 shows the results of Western Blotting for detecting the concentration of CD166 in the serum of two groups of normal and eight groups of liver cancer patients, wherein HI is Healthy individual and H is HCC. The results showed that the serum levels of CD166 in the serum of 8 groups of liver cancer patients (each group of 8 patients with liver cancer patients mixed) were significantly higher than those of the normal group (8 groups of normal patients in each group of serum mixed in equal amounts).
- Alpha-fetoprotein is a classic serological marker for early diagnosis of liver cancer.
- the receiver operating curve (ROC) of AFP and CD166 is shown in Figure 5, showing the sensitivity and specificity of CD166. All are better.
- CD166 was almost undetectable in normal human serum, but the expression of CD166 in serum of liver cancer patients was significantly increased (Fig. 1).
- the average concentration of CD166 was 216.8 ⁇ 577.1 ng/ml.
- the average concentration of 89 cases of intestinal cancer was 636.2 ⁇ 488.5ng/ml, male/female 0.82/1.
- the average concentration of 51 cases of liver cancer was 7138.89 ⁇ 5434.68ng/ml, male/female 18/1.
- the average concentration of 48 cases of hepatitis B was 883.66. ⁇ 1139.32ng/ml, male/female 1.4/1.
- the average concentration of hepatitis C in 40 cases was 327.5 ⁇ 145.3ng/ml, male/female 1.35/1.
- the average concentration of 41 cases of cirrhosis was 964.99 ⁇ 1275.136ng/ml, male / Female 2.73/1.
- the average concentration of 21 cases of gastric cancer was 156.8 ⁇ 55.47ng/ml, male/female 6/1.
- the average concentration of breast cancer was 369.3 ⁇ 638.48ng/ml and the average concentration of 21 cases of lung cancer was 332 ⁇ 94.59ng. /ml
- the concentration of CD166 in the serum of male/female patients such as 1.63/1 (Fig. 2), it was found that the increase of CD166 in the serum of patients with liver cancer was more specific.
- CD166 decreased significantly after surgery.
- concentration of AFP was simultaneously measured in the serum of normal human and liver cancer patients, and the ROC curve was plotted with CD166.
- the area under the CD166 curve was 0.9860, and the area under the AFP curve was 0.9142 (Fig. 5). The specificity and sensitivity of CD166 were found. They are very good.
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Abstract
本发明涉及CD166作为肝癌诊断血清标志物的应用及其试剂盒,可以通过检测血清中的CD166浓度来诊断和预示肝癌疾病。
Description
本发明涉及临床诊断技术领域,具体地说,涉及CD166作为肝癌诊断血清标志物的应用及其试剂盒。
肝癌即肝脏恶性肿瘤,是外科疾病中的常见病和多发病。肝脏恶性肿瘤可分为原发性和继发性两大类。原发性肝脏恶性肿瘤起源于肝脏的上皮或间叶组织,前者称为原发性肝癌,是我国高发的、危害极大的恶性肿瘤;后者称为肉瘤,与原发性肝癌相比较较为少见。继发性或称转移性肝癌,系指全身多个器官起源的恶性肿瘤侵犯至肝脏,一般多见于胃、胆道、胰腺、结直肠、卵巢、子宫、肺、乳腺等器官恶性肿瘤的肝转移。由于肝脏是人体最大的实质性器官,承担人体的各类重要代谢功能,因此肝脏一旦出现恶性肿瘤将导致危及生命的严重后果。又由于肝脏具有丰富的血流供应,与人体的重要结构如下腔静脉、门静脉、胆道***等关系密切,加上肝脏恶性肿瘤发病隐匿,侵袭性生长快速,导致其治疗甚为困难。因此,早期发现、早期诊断、早期治疗,对于肝癌的治疗至关重要。
目前肝癌的诊断主要依靠影像学检查、肝穿刺组织学检查等方法,然而这些检测方法均具有一定的局限性。例如即使是很好的细针穿刺仍取材有限有较高的假阴性率,并且有使肿瘤扩散和针道种植的危险。癌症的血清学检测技术一直是研究的重点,然而,目前现有的针对癌症(如HCC)检测尚缺乏令人满意的血清标志物,更缺乏可用于血清检测或肝癌预后检测的血清标志物。
血清CD166又称为白细胞活化黏附因子(activated leukocyte cell adhesion molecule),是一种广泛存在于人体组织器官的跨膜糖蛋白受体,发挥广泛生理病理功能。CD166编码基因位于人3号染色体长臂上,包含16个外显子约150kb碱基对。基因启动子位于其附近且富含GC盒,包含可结合转录因子kappa B和AP-1的DNA结合序列,另外包含丰富的负性或正性调节序列,在组织中特异性的参与调节CD166的表达。CD166属于免疫球蛋白超家族(IgSF)成员跨膜识别分子,胞外部分包括5个功能结构域(VVC2C2C2),其中三个为恒定结构域(C2域),两个为可变结构域(V型)。CD166的天然分子蛋白分子量约为69kD,糖基化后形成分子量约为105kD的成熟蛋白,其是淋巴细胞抗原CD6的配基。CD166广泛分布于活化的白细胞、单核细胞、内皮细胞、纤维母细胞、神经元、
间叶组织干细胞、造血干细胞、骨髓祖细胞、骨髓基质细胞以及一些腺体细胞,主要在白细胞和胸腺上皮细胞上表达。CD166参与机体炎症反应、细胞粘附、造血、外周及中枢神经***发育以及肿瘤的侵袭。
中国期刊《南方医科大学》2013年刊出的论文“CD133及CD166在脑膜瘤中的表达及临床意义”,作者通过一系列实验研究得出以下结论:1、CD133与CD166在不同级别的脑膜瘤均有表达,CD133及CD166阳性细胞表达水平随脑膜瘤病理级别增高而增高。并且在不同级别组脑膜瘤中CD133阳性细胞(p=0.001)与CD166阳性细胞(p=0.000)IOD值差异均有显著性。2、CD133及CD166表达成正相关关系。经Spearman直线相关分析表明,CD133阳性细胞与CD166阳性细胞IOD值呈显著正相关(p=0.000)。3、切片染色可观察到CD133与CD166共定位表达情况;CD166可能为脑肿瘤干细胞标记物之一,与目前公认的脑肿瘤干细胞标记物CD133可共同作为肿瘤干细胞的标记物。4、CD166高表达组复发率较高,CD166与脑膜瘤复发相关。
然而,目前关于CD166作为肝癌诊断标志物还未见报道。
发明内容
本发明的主要目的在于提供一种用于检测肝癌的血清特异标志物。
本发明提供了CD166蛋白或其特异性抗体的用途,用于制备检测肝癌或肝癌预后的诊断试剂或试剂盒。
在另一优选例中,所述的CD166蛋白或其特异性抗体偶联有或带有可检测标记。
在另一优选例中,所述的可检测标记选自下组:生色团、化学发光基团、荧光团、同位素或酶。
在另一优选例中,所述的检测是血清检测。
在另一优选例中,所述的血清检测是ELISA法、或双抗夹心时间分辨免疫荧光法(TRFIA法)。
本发明还提供了CD166蛋白或其特异性抗体的用途,用于检测肝癌或肝癌预后。
在另一优选例中,所述的检测是血清检测。
在另一优选例中,所述的CD166蛋白在NCBI上的GenBank序列号是GENE.ID 214。
本发明还提供了一种用于检测肝癌或肝癌预后的诊断试剂盒,所述的试剂盒包含测定血清中CD166蛋白含量的试剂,以及标签或说明书,所述的标签或说明书注明所述试剂盒用于检测肝癌或肝癌预后。
在另一优选例中,所述的测定血清中CD166蛋白含量的试剂为CD166蛋白的特异
性抗体。
在另一优选例中,所述的CD166蛋白的特异性抗体偶联有或带有可检测标记。
在另一优选例中,所述的可检测标记选自下组:生色团、化学发光基团、荧光团、同位素或酶。
在另一优选例中,所述的标签或说明书中注明以下内容:如果检测对象的血清CD166浓度≥211ng/ml(较佳地≥261ng/ml,更佳地≥311ng/ml),则该对象发生肝癌的几率大于正常人群。
本发明还提供了一种CD166蛋白的用途,它被用作检测肝癌的标志物。
在另一优选例中,所述的检测是血清检测。
本发明还提供了一种检测肝癌或肝癌预后的方法,所述的方法是检测血清样品中CD166蛋白的含量,以此判断所述的血清样品来源的对象患肝癌的概率或肝癌的分期。
本发明还提供了一种CD166蛋白的拮抗剂的用途,被用于制备治疗/预防肝癌的药物。
在另一优选例中,所述的拮抗剂包括针对CD166蛋白的siRNA、反义RNA、抗体、或其组合。
应理解,在本发明范围内,本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合,从而构成新的或优选的技术方案。限于篇幅,在此不再一一累述。
本发明优点在于:
(1)本发明首次证实了CD166蛋白可以作为肝癌诊断的标志物,可以通过检测血清中的CD166蛋白浓度来诊断和预示肝癌疾病的发生发展,具有取样方便、易于检测、特异性和灵敏度高等优点。由于CD166是细胞表面分子,因此不仅可以用来诊断肝癌,而且有望成为治疗肝癌的分子靶点。
(2)血清检测方法更方便快速,更容易为病人接受。
(3)便于动态监察HCC患者的病情进展。
附图1:免疫印迹(Western Blotting)检测2组正常人和8组肝癌患者血清中CD166的浓度(HI是Healthy individual,H是HCC)。
附图2:不同疾病人群血清中CD166的含量,箱式图显示正常人、肠癌、肝癌、乙型肝炎、丙型肝炎、肝硬化、胃癌、乳腺癌和肺癌患者血清中CD166的浓度。
附图3:配对T检验结果显示肝癌患者术前和术后血清中CD166浓度的变化。
附图4:卡方检验结果显示CD166的浓度和肝癌患者的临床病理分期有关。
附图5:ROC曲线显示CD166和AFP诊断肝癌的特异性和灵敏度。
本发明人经过广泛而深入的研究,首次发现血清中CD166浓度或水平在肝癌患者和正常人群中存在差异,且血清中CD166浓度或水平与肝癌患者的分期有明显的相关性。因此CD166可作为新的肝癌血清标志物,用于肝癌的诊断与预后判断。在此基础上完成了本发明。
CD166蛋白和基因
人基因CD166(GeneBank登录号:GENE.ID 214)全长4884bp,编码570个氨基酸残基。
在本发明中,术语“CD166蛋白”、“CD166多肽”可互换使用,都指具有人CD166蛋白的氨基酸序列(GeneBank登录号:GENE.ID 214)的蛋白或多肽。此外,该术语还包括全长的CD166及其片段。本发明所指的CD166蛋白包括其完整的氨基酸序列、其突变体以及其功能上活性的片段。
此外,本发明所指的CD166蛋白还包括糖基化或非糖基化的蛋白。
特异性抗体
在本发明中,术语“抗CD166的特异性抗体”、“CD166的特异性抗体”可互换使用。
本发明包括对人CD166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”是指抗体能结合于人CD166基因产物或片段。较佳地,指那些能与人CD166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。本发明中抗体包括那些能够结合并抑制人CD166蛋白的分子,也包括那些并不影响人CD166蛋白功能的抗体。本发明还包括那些能与修饰或未经修饰形式的人CD166基因产物结合的抗体。
本发明不仅包括完整的单克隆或多克隆抗体,而且还包括具有免疫活性的抗体片段,如Fab’或(Fab)2片段;抗体重链;抗体轻链;遗传工程改造的单链Fv分子(Ladner等人,美国专利No.4,946,778);或嵌合抗体,如具有鼠抗体结合特异性但仍保留来自人的抗体部分的抗体。
本发明的抗体可以通过本领域内技术人员已知的各种技术进行制备。例如,纯化的人CD166基因产物或者其具有抗原性的片段,可被施用于动物以诱导多克隆抗体的产生。与之相似的,表达人CD166蛋白或其具有抗原性的片段的细胞可用来免疫动物来生产抗
体。本发明的抗体也可以是单克隆抗体。此类单克隆抗体可以利用杂交瘤技术来制备(见Kohler等人,Nature 256;495,1975;Kohler等人,Eur.J.Immunol.6:511,1976;Kohler等人,Eur.J.Immunol.6:292,1976;Hammerling等人,In Monoclonal Antibodies and T Cell Hybridomas,Elsevier,N.Y.,1981)。本发明的抗体包括能阻断人CD166蛋白功能的抗体以及不影响人CD166蛋白功能的抗体。本发明的各类抗体可以利用人CD166基因产物的片段或功能区,通过常规免疫技术获得。这些片段或功能区可以利用重组方法制备或利用多肽合成仪合成。与人CD166基因产物的未修饰形式结合的抗体可以用原核细胞(例如E.Coli)中生产的基因产物来免疫动物而产生;与翻译后修饰形式结合的抗体(如糖基化或磷酸化的蛋白或多肽),可以用真核细胞(例如酵母或昆虫细胞)中产生的基因产物来免疫动物而获得。
抗人CD166蛋白的抗体可用于免疫组织化学技术中,以检测标本(尤其是血清样本)中的人CD166蛋白。
检测方法
利用CD166存在于血清中,且与肝癌发生及预后密切相关这一特点,本发明还提供了血清检测或肝癌预后检测的方法,尤其是血清学检测方法。
在本发明的一个优选例中,本发明提供一种检测血清CD166的ELISA法。
检测试剂盒
本发明还提供了一种肝癌血清检测或肝癌预后检测的试剂盒,它含有本发明的抗CD166的免疫球蛋白或免疫偶联物,或其活性片段。
药物组合物
本发明还提供了一种药物组合物,它含有上述的CD166的拮抗剂,以及药学上可接受的载体。所述的药物组合物可用于提高肝癌患者的生存期。
在本发明中,所述的拮抗剂包括针对CD166的siRNA、反义RNA、抗体、或其组合。此外,所述的拮抗剂还包括可以降低CD166表达或活性的小分子化合物。
通常,可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中,其中pH通常约为5-8,较佳地pH约为6-8,尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药,其中包括(但并不限于):腹膜内、静脉内、或局部给药。
本发明的药物组合物可直接用于抑制肝癌细胞的发生或转移。此外,还可与其他肿瘤治疗剂联用。
本发明的药物组合物含有安全有效量的本发明上述的CD166拮抗剂以及药学上可接
受的载体或赋形剂。这类载体包括(但并不限于):盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式,例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量,例如每天约1微克/千克体重-约5毫克/千克体重。此外,本发明的多肽还可与其他治疗剂一起使用。
使用药物组合物时,是将安全有效量的本发明的CD166拮抗剂施用于哺乳动物,其中该安全有效量通常至少约10微克/千克体重,而且在大多数情况下不超过约8毫克/千克体重,较佳地该剂量是约10微克/千克体重-约1毫克/千克体重。当然,具体剂量还应考虑给药途径、病人健康状况等因素,这些都是熟练医师技能范围之内的。
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件,例如Sambrook等人,分子克隆:实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件,或按照制造厂商所建议的条件。除非另外说明,否则百分比和份数是重量百分比和重量份数。
实施例1
1、实验材料
试剂盒买自上海丽臣生物科技有限公司,试剂盒全称:human CD166ELISA KIT,货号:LC9012,组成:微孔酶标板12孔*8条、标准品(4ng/ml)0.6ml、标准品稀释液6ml、样本稀释液6ml、检测抗体-HRP、洗涤缓冲液25ml、底物A、底物B、终止液、封板膜、说明书、自封袋。
2、实验方法
血清样本中CD166的检测方法为:双抗一步夹心法酶联免疫吸附(ELISA)法。具体步骤:(1)实验前准备洗涤缓冲液,ELISA板在室温平衡20分钟。(2)设置标准孔、样品孔、空白孔(什么也不加)。标准孔分别加入不同浓度的标准品50μl;样品孔提前摸索条件,血清样品稀释5倍(样品孔加40μl血清样本稀释液,10μl待测血清样本)。(3)标准孔和样品孔加入辣根过氧化物酶(HRP)标记的检测抗体100μl,37℃恒温箱孵育60分钟。(4)弃去液体,吸水纸上拍干,每孔加洗涤液,洗涤5次。(5)标准孔、样品孔和空白孔都加入底物A和B各50μl,37度避光孵育15分钟。(6)所有孔加入终止液50μl,15分钟内在450nm波长处测定各孔的吸光值。结果判定:首先标准孔和样品孔的吸光值都要减去空白孔的吸光值;其次根据标准孔的吸光值和浓度,绘制标准品的线性回归曲线;根据标准曲线和样品孔的吸光值计算出待测血清中CD166的浓度,再乘以稀释倍数
5倍,得出血清样品CD166的最终浓度,单位是ng/ml。
2.1 Western Blotting检测2组正常人和8组肝癌患者血清中CD166的浓度
通过增强CT、血清AFP以及术后病理切片确诊肝癌,从瑞金医院采集51例肝癌患者,随机抽取48人份血清并随机分为8组,每组6人。从上海第十人民医院采集85例体检正常人群(正常人群定义为AFP为正常值,影像学无肝占位病变)并随机抽取12人份血清并随机分为2组,每组6人。采集每组受试者血清,每组取6人份受试者血清等量混合为待测血清样品。使用SDS-PAGE胶通过传统Western Blotting方法检测每组血清样品中CD166的浓度。
2.2比较不同疾病人群血清中CD166的浓度
将所有标本稀释10倍后用上海丽臣生物科技有限公司的ELISA试剂盒检测所有标本血清中CD166的浓度(具体步骤见上述血清样本中CD166的检测方法)。实验结果用STATA统计学软件中单因素方差分析,及多重比较Bonferroni法比较肝癌患者和其他肝脏疾病、其他***癌症及正常人血清中CD166的浓度。
2.3比较肝癌患者术前和术后血清中CD166的浓度
将所有标本稀释10倍后用上海丽臣生物科技有限公司的ELISA试剂盒检测所有标本血清中CD166的浓度(具体步骤见上述血清样本中CD166的检测方法)。实验结果用STATA统计学软件,采用配对样本T检验比较同一肝癌患者肝癌切除术前和术后血清中CD166的变化情况。
2.4检测不同肝癌分期患者血清中CD166的浓度
利用ELISA方法(具体步骤见上述血清样本中CD166的检测方法)检测血清中CD166的浓度,并且根据浓度大小进行四分位数分级,对应相应的病理分期,制作行X列表,实验结果用STATA统计学软件中卡方检验进行分析。
2.5制作受试者工作曲线
分别利用ELISA法(具体步骤见上述血清样本中CD166的检测方法)检测85例正常人、51例肝癌患者血清中AFP和CD166的浓度。实验结果用STATA统计学软件制作受试者工作曲线(ROC曲线),并计算曲线下面积。
3、实验结果
(1)图1为免疫印迹(Western Blotting)检测2组正常人和8组肝癌患者血清中CD166的浓度的结果,其中HI是Healthy individual,H是HCC。结果表明8组肝癌患者血清(每组为8人份肝癌病人血清等量混合)中CD166的浓度明显高于2组正常人(每组为8人份正常病人血清等量混合)。
(2)分别在85例正常人、89例肠癌、51例肝癌、48例乙型肝炎、40例丙型肝炎、41例肝硬化、21例胃癌、25例乳腺癌和21例肺癌患者血清中使用试剂盒检测CD166的浓度,结果如图2所示,发现肝癌患者血清中CD166的浓度显著高于正常人、其他肝脏疾病和其他***癌症。
(3)检测肝癌患者(7例)肝癌切除术前后血清中CD166浓度,结果如图3所示,发现术后患者血清CD166含量下降明显。
(4)检测不同肝癌分期患者血清中CD166浓度,结果如图4所示,行X列表卡方检验显示CD166的浓度和肝癌患者的分期有关系,分期越高CD166的浓度越大。
(5)甲胎蛋白(α-fetoprotein,AFP)是经典的早期辅助诊断肝癌的血清学指标,AFP和CD166的受试者工作曲线(ROC)如图5所示,显示CD166的灵敏度和特异性都较好。
4、结果分析
正常人血清中几乎检测不到CD166,但是肝癌患者血清中CD166的表达明显升高(图1)。为了证明肝癌患者血清中CD166的升高是特异性的,我们分别检测了85例正常人,CD166平均浓度是216.8±577.1ng/ml。89例肠癌平均浓度是636.2±488.5ng/ml,男/女0.82/1。51例肝癌平均浓度是7138.89±5434.68ng/ml,男/女18/1。48例乙型肝炎平均浓度是883.66±1139.32ng/ml,男/女1.4/1。40例丙型肝炎平均浓度是327.5±145.3ng/ml,男/女1.35/1。41例肝硬化平均浓度是964.99±1275.136ng/ml,男/女2.73/1。21例胃癌平均浓度是156.8±55.47ng/ml,男/女6/1。25例乳腺癌平均浓度是369.3±638.48ng/ml和21例肺癌平均浓度是332±94.59ng/ml,男/女1.63/1等病人血清中CD166的浓度(图2),发现肝癌患者血清中CD166的升高是比较特异的。同时我们也分析了同一个肝癌患者肝癌切除术前和术后血清中CD166的表达情况,发现术后CD166明显下降(图3)。最后,在上述正常人和肝癌患者血清中同时检测AFP的浓度,并且和CD166绘制了ROC曲线,CD166曲线下面积是0.9860,AFP曲线下面积是0.9142(图5),发现CD166的特异性和灵敏度都非常好。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员,在不脱离本发明方法的前提下,还可以做出若干改进和补充,这些改进和补充也应视为本发明的保护范围。
Claims (10)
- CD166蛋白或其特异性抗体的用途,其特征在于,用于制备检测肝癌或肝癌预后的诊断试剂或试剂盒。
- 根据权利要求1所述的用途,其特征在于,所述的CD166蛋白或其特异性抗体偶联有或带有可检测标记。
- 根据权利要求2所述的用途,其特征在于,所述的可检测标记选自下组:生色团、化学发光基团、荧光团、同位素或酶。
- 根据权利要求1所述的用途,其特征在于,所述的检测是血清检测。
- CD166蛋白或其特异性抗体的用途,其特征在于,用于检测肝癌或肝癌预后。
- 根据权利要求5所述的用途,其特征在于,所述的检测是血清检测。
- 一种用于检测肝癌或肝癌预后的诊断试剂盒,其特征在于,所述的试剂盒包含测定血清中CD166蛋白含量的试剂,以及标签或说明书,所述的标签或说明书注明所述试剂盒用于检测肝癌或肝癌预后。
- 根据权利要求7所述的诊断试剂盒,其特征在于,所述的测定血清中CD166蛋白含量的试剂为CD166蛋白的特异性抗体。
- 一种CD166蛋白的用途,其特征在于,它被用作检测肝癌的标志物。
- 一种检测肝癌或肝癌预后的方法,其特征在于,所述的方法是检测血清样品中CD166蛋白的含量,以此判断所述的血清样品来源的对象患肝癌的概率或肝癌的分期。
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2015
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