WO2016206596A1 - Cthrc1在肝硬化诊断和治疗中的应用 - Google Patents

Cthrc1在肝硬化诊断和治疗中的应用 Download PDF

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WO2016206596A1
WO2016206596A1 PCT/CN2016/086854 CN2016086854W WO2016206596A1 WO 2016206596 A1 WO2016206596 A1 WO 2016206596A1 CN 2016086854 W CN2016086854 W CN 2016086854W WO 2016206596 A1 WO2016206596 A1 WO 2016206596A1
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cthrc1
protein
liver
nucleic acid
antibody
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张志刚
李军
王亚辉
马铭泽
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上海市肿瘤研究所
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    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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    • 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
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6806—Determination of free amino acids
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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

Definitions

  • the invention relates to the field of molecular biology, especially gene diagnosis and treatment, in particular to the application of CTHRC1 gene in the diagnosis and treatment of liver cirrhosis.
  • Cirrhosis the precancerous lesions manifested as liver fibrosis, is a very common chronic liver disease [1] . Most patients with liver fibrosis will turn into irreversible cirrhosis after 15 to 20 years, and even further develop into liver cancer [2] .
  • the pathological manifestation of liver fibrosis is the excessive deposition of fibrous connective tissue in the liver (such as Collagen), which is the result of imbalance between Fibrogenesis and Fibrolysis. Activation of hepatic stellate cells is a central event in liver fibrosis during liver fibrosis. Unlike liver cancer, early diagnosis of cirrhosis is very difficult. There are no clear molecular markers at present.
  • Clinical detection methods mainly use liver biopsy for diagnosis, followed by hepatosplenomegaly, ascites and cirrhosis complications. These clinicopathological features such as gastrointestinal hemorrhage, hepatic encephalopathy, liver and kidney syndrome, etc. are diagnosed [3] .
  • the existing detection methods have certain defects.
  • the biopsy has certain trauma to the patient, and it is difficult to diagnose the early cirrhosis based on the complications. Therefore, it is of great significance to find molecular markers that are simple and easy to accurately monitor the condition.
  • CTHRC1 is a secreted glycoprotein with a molecular weight of approximately 26 kDa, which was first discovered in a rat model of balloon-induced arterial injury [4] .
  • CTHRC1 is involved in vascular remodeling and is highly expressed in pancreatic cancer tissues [5] .
  • CTHRC1 is highly expressed in breast cancer, liver cancer, gastric cancer, melanoma, and colon cancer, and promotes invasion and migration of the above tumors [6-8] .
  • CTHRC1 inhibits the TGF- ⁇ pathway, but does not inhibit the TGF- ⁇ pathway of endothelial cells [9] .
  • the promoter region of the CTHRC1 gene has a Smad binding site, suggesting that CTHRC1 can be regulated by TGF- ⁇ [8] .
  • TGF- ⁇ 1 TGF- ⁇ 1
  • CTHRC1 can activate Wnt/PCP signaling pathway by interacting with Wnt5a and ROR2, and inhibit Wnt/ ⁇ -catenin signaling pathway, thereby regulating cytoskeletal rearrangement and cell movement [10] . Therefore, CTHRC1 has a very important regulatory effect on TGF- ⁇ and Wnt signaling pathways.
  • CTHRC1 gene is up-regulated in human cirrhosis tissue by immunohistochemistry and may be involved in the regulation of liver fibrosis.
  • CTHRC1 has the effect of aggravating the progression of liver fibrosis and accelerating liver function damage.
  • CTHRC1 can accelerate the activation of hepatic stellate cells and promote the movement and contraction of stellate cells, and the CTHRC1 neutralizing antibody can reverse the effect of CTHRC1 after blocking CTHRC1.
  • the use of CTHRC1 neutralizing antibodies in animals can greatly slow the progression of liver fibrotic lesions.
  • An aspect of the present invention provides a use of a CTHRC1 nucleic acid sequence or protein as an agent for detecting liver cirrhosis or liver fibrosis in a sample, wherein the nucleic acid sequence of CTHRC1 is represented by SEQ ID NO: 1, and the CTHRC1 protein is represented by SEQ ID. NO: 2 is shown.
  • the aforementioned reagent is an antibody for recognizing an epitope of a CTHRC1 protein.
  • the aforementioned reagent may also be a nucleic acid probe for recognizing the CTHRC1 nucleic acid sequence.
  • Another aspect of the present invention provides a CTHRC1 nucleic acid sequence or protein for use as a medicament for the preparation of a medicament for preventing or treating liver cirrhosis or liver fibrosis, wherein the nucleic acid sequence of CTHRC1 is as shown in SEQ ID NO: 1, and the CTHRC1 protein is as SEQ ID NO: 2 is shown.
  • the aforementioned drug is an antibody for recognizing an epitope of a CTHRC1 protein.
  • the aforementioned drugs include antisense nucleic acids of the CTHRC1 nucleic acid sequence.
  • Another aspect of the invention provides a CTHRC1 nucleic acid sequence or protein as an in vitro sieve
  • a drug target for the preparation and preparation of a liver cirrhosis or liver fibrosis drug wherein the nucleic acid sequence of CTHRC1 is shown in SEQ ID NO: 1, and the CTHRC1 protein is shown in SEQ ID NO: 2.
  • kits for cirrhosis or liver fibrosis which comprises a container containing a CTHRC1 antibody, that is, an antibody of a protein epitope.
  • the container may include a CTHRC1 antibody as a primary antibody and an HRP-labeled goat anti-rabbit antibody as a secondary antibody.
  • the container contains a CTHRC1-specific probe.
  • CTHRC1 as described in the text of the present invention may correspond to a CTHRC1 protein or nucleic acid sequence in different contexts.
  • CTHRC1 protein can be prepared by various conventional preparation methods. Such as genetic engineering methods or synthetic methods.
  • a CTHRC1 nucleic acid sequence can be expressed as a protein.
  • a recombinant expression plasmid containing the CTHRC1 gene sequence is obtained, it can be transformed into the corresponding host for protein expression.
  • the "CTHRC1 protein” of the present invention also includes a natural or induced genetic variant of the protein as set forth in SEQ ID NO: 2.
  • the induced variants can be obtained by various techniques, such as random mutations produced by radiation or mutagens, and can also be obtained by techniques such as site-directed mutagenesis or other known molecular biology techniques.
  • the "CTHRC1 protein” also includes analogs having residues of natural L-form amino acids (such as D-type amino acids), and analogs having non-naturally occurring or synthetic amino acids (such as amino acids, Y-amino acids, etc.).
  • the CTHRC1 nucleic acid sequence can be prepared by various conventional preparation methods.
  • the CTHRC1 nucleic acid sequence of the present invention can usually be obtained by a PCR amplification method, a recombinant method or a synthetic method.
  • primers can be designed according to the nucleic acid sequences of the present invention, particularly the open reading frame sequences, and the cDNA library prepared by a commercially available cDNA library or a conventional method known to those skilled in the art can be used as a template. Increased related sequences.
  • the CTHRC1 sequence can be inserted into an appropriate expression vector to obtain a recombinant expression plasmid.
  • any variant of the polynucleotide of SEQ NO: 1 is, as long as it has more than 70% homology with the polynucleotide, and is within the scope of the present invention.
  • the change of the polynucleotide A body refers to a polynucleotide sequence having one or more nucleotide changes. Variants of this polynucleotide may result in a living allelic variant or a non-biotic variant, including substitution variants, deletion variants, and insertion variants.
  • allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion or insertion of a plurality of nucleotides, but does not substantially alter the function of the polypeptide encoded thereby.
  • the epitope of CTHRC1 (Epitope), also known as the antigenic determinant, is an immunologically active region on the antigen molecule and is responsible for binding to the antigenic receptor on the surface of the antibody molecule or immune cell.
  • the antibody against the epitope of CTHRC1 described in the present invention refers to any CTHRC1 antibody known in the art, and includes any antibody prepared by various techniques known to those skilled in the art.
  • CTHRC1 antibody can be used in immunohistochemistry to detect CTHRC1 protein in biopsy specimens and as a specific therapeutic agent for preventing liver fibrosis or cirrhosis.
  • the antibody can be detected by ELISA, Western blotting, or coupled to a detection group by chemiluminescence, isotope tracing, and the like.
  • a CTHRC1 nucleic acid probe refers to contact with an amplified tag sequence.
  • the probe is preferably attached to a chromophore but can be radiolabeled; or the probe is attached to a binding partner, such as an antibody or biotin, or another binding partner that carries a detectable domain. .
  • the present invention firstly proposes the use of the CTHRC1 gene and its products in the field of cirrhosis/fibrosis to achieve early detection and treatment effects.
  • CTHRC1 has potential applications as a good biomarker.
  • Figure 1a-b is a statistical diagram of immunohistochemical staining and expression levels of CTHRC1 in tissues
  • Figure 2 is a tissue staining diagram of the degree of liver fibrosis in mice in CCl 4 and TAA chemically induced model mice;
  • Figure 3 is a tissue staining diagram of the degree of liver fibrosis in mice subjected to CTHRC1 neutralizing antibody treatment and IgG-treated CCl 4 and TAA chemically induced model mice;
  • Figure 4 is a graph showing the degree of liver injury in mice in CCl 4 and TAA chemically induced model mice;
  • Figure 5 is a graph showing the degree of liver injury in mice induced by CTHRC1 neutralizing antibody treatment and IgG treatment in CCl 4 and TAA chemically induced mice, respectively;
  • Figure 6 is a schematic diagram showing the contraction test of hepatic stellate cell gel treated by CTHRC1 neutralizing antibody treatment and IgG or mAb, respectively;
  • Figure 7 is a schematic diagram of liver stellate cell migration assays treated with CTHRC1 neutralizing antibody and IgG or mAb, respectively.
  • CTHRC1 The expression of CTHRC1 in human liver cirrhosis tissue microarray was examined and it was found that CTHRC1 was significantly up-regulated in cirrhotic tissues.
  • the expression plasmid of CTHRC1 was constructed and transfected into 293T cells to purify the recombinant CTHRC1 protein. Through gel shrinkage and Transwell experiments, it was observed that recombinant CTHRC1 protein can promote hepatic stellate cell contraction and migration, and this effect can be reversed by using CTHRC1 neutralizing antibody.
  • CTHRC1 neutralizing antibodies in wild-type mice alleviate liver fibrosis and liver damage induced by CCl4 and TAA.
  • CTHRC1 primary antibody was purchased from Abcam, HRP secondary antibody was purchased from Abmart; DAB chromogenic solution was purchased from Thermo; neutral gum was purchased from Shanghai Shenggong Biotechnology Co., Ltd.; cirrhosis tissue chip was purchased from Xi'an Alina Biotech Ltd., product number: LV805.
  • the tissue chip contains 30 normal liver tissues, 10 hepatitis tissues, and 40 cirrhotic tissues.
  • antigen heat repair boiling water heating 0.01M sodium citrate antigen repair solution (pH6.0) to about 95 ° C, and then carefully placed in the tissue chip or tissue section water bath for 15 minutes, after removal, can be naturally cooled to room temperature; ⁇ -SMA Antibody staining does not require an antigen thermal repair step;
  • secondary antibody incubation add secondary antibody (goat anti-rabbit-HRP labeled secondary antibody 1:500 dilution, rabbit anti-mouse-HRP labeled secondary antibody 1:200 dilution) 150-200ul, incubate for 1 hour at room temperature, set the humid chamber Internally, after washing, wash with PBS 3 times;
  • DAB coloring solution currently used
  • DAB coloring generally lasts 1-5 minutes. Control the degree of color development under the microscope, stop the reaction with tap water, and then rinse with PBS;
  • the CTHRC1 gene and the expression vector are all stored in the laboratory, the CTHRC1 gene sequence is shown in SEQ ID NO: 1, and the expression vector is V152, V162 plasmid.
  • CTHRC1-V152 transfected 293 cell culture medium was collected and purified by protein affinity chromatography to obtain recombinant CTHRC1-StrepII tag fusion protein. Specific steps are as follows.
  • the column consists of a chromatography tube, a gasket, an outlet plug, a cap, and agarose beads in the tube.
  • the affinity Beads used for the CTHRC1-StrepII tagged fusion protein is a Strep-Tactin Sepharose magnetic bead manufactured by the German company iba (Goettingen, Germany).
  • Strep Tag II is a novel fusion protein tag containing only 8 amino acids.
  • the V152 vector is a complete fusion protein eukaryotic expression system based on this tag sequence.
  • the principle of Strep Tag purification technology is based on the specific binding of biotin/streptavidin.
  • Strep Tag II can be incorporated into Streptavidin's biotin pocket.
  • Strep-Tactin is a modified Streptavidin.
  • Strep-Tactin has a affinity with Strep Tag II that is about 100 times greater than that of ordinary Streptavidin. It is a very efficient affinity chromatography molecule.
  • 2 ml of Strep-Tactin Sepharose was added to the column.
  • CTHRC1-/-C57BL/6 mouse SPF grade CTHRC1 knockout C57 mice (CTHRC1-/-C57BL/6 mouse) were purchased from Hiroshi Sasaki (Lab for Embryonic Induction, Kobe, Japan).
  • CCl 4 -induced liver fibrosis model 6-week-old C57 wild-type and CTHRC1-/- mice were intraperitoneally injected with CCl 4 twice a week, and CCl 4 was injected in a ratio of 1:3. The oil was diluted and injected at a dose of 0.5 ⁇ L/g (body weight) per mouse. A total of 16 injections were required, and all mice were sacrificed 48 hours after the last injection, and the liver and serum were taken for the experiment.
  • TAA thioacetamide chemically induced liver fibrosis experiment: 6-week-old male C57 wild-type and CTHRC1-/- mice were injected intraperitoneally with TAA (ultra-pure water, protected from light) three times a week. Injection was administered at a dose of 0.2 mg/g (body weight) per mouse. A total of 8 weeks of injection is required. All mice were sacrificed 48 h after the last injection, and the liver and serum were taken for the experiment.
  • TAA thioacetamide
  • the chemical induction experiment includes the CCl 4 and TAA induction models as above.
  • the experimental animals were selected from 6-week-old male C57 wild-type mice and divided into CTHRC1 neutralizing antibody-treated group and IgG-treated group.
  • the CTHRC1 treatment group and the IgG treatment group were respectively given neutralizing antibody (customized by Hangzhou Huaan Company) or IgG 1 mg/mouse/time, intraperitoneal injection three times a week, and were treated at intervals of chemical injection for 8 weeks. All mice were sacrificed 48 h after the last injection, and the liver and serum were taken for the experiment.
  • the liver tissue obtained in Experiment 3.1 was stained, and the picric acid red staining of picric acid could make the collagen stain for a long time. Under the polarized light microscope, it can enhance the birefringence of the collagen fiber, so that the collagen tissue can be specifically displayed. Under polarized light microscopy, type I collagen appears yellow or red, type III collagen appears green, and type II collagen appears blue-green or gray-blue. 0.1% picric acid Sirius red solution preparation method: Sirius red 0.1g, saturated picric acid solution 100ml, picric acid saturated liquid (1.22%).
  • Tissue sections need to be dewaxed to hydration, then immersed in 0.1% picric acid Sirius red for at least 1 hour, then rinsed with tap water for 4 min; hematoxylin counterstained for 3 min (not required), dehydrated with gradient ethanol, dehydration time should be short, dry , neutral gum seal.
  • the experimental results are shown in Figures 2 and 3.
  • the test kit for the mouse serum obtained in Experiment 3.1, ALT and AST was purchased from Shanghai Shenshou Youfu Medical Diagnostic Products Co., Ltd. Including alanine aminotransferase (ALT) assay kit, aspartate aminotransferase (AST) assay kit.
  • the specific procedure is as follows: serum sample 7.5 ⁇ l + R-1 150 ⁇ l, 5 min after R-2 50 ⁇ l, and optical density value after 53 seconds (A340 nm) After 247 seconds, the second optical density value (A340nm) is measured.
  • the AST test method is the same as ALT. The experimental results are shown in Figures 4 and 5.
  • the hepatic stellate LX-2 cell line was preserved in this room and purchased from Millipore (Example 5).
  • LX-2 cells were cultured overnight in serum-free DMEM culture medium; the cells were digested with trypsin, centrifuged, and counted; then a collagen crystal lattice mixture was prepared and performed on ice.
  • the mixed liquid is prepared in the total volume required. Then, 2 mL of the mixed solution was added to each 3.5 cm dish, shaken, and the culture dish was placed in a 37 ° C cell culture incubator;
  • the collagen crystal lattice mixture was prepared and divided into at least 7 treatment groups, and the recombinant CTHRC1 protein obtained in Example 2, 0 nM, 20 nM, 50 nM, and the mAb protein 20 nM, 50 nM, and the IgG protein 20 nM, 50 nM, were respectively added.
  • Each dish contains three wells, and each dish needs to add 2ml of mixed liquid. Calculate the total amount. The specific operations are all carried out on ice.
  • Cell preparation Digest the LX-2 cells filled with two 10cm dishes, centrifuge, add 11ml of cell culture medium containing 10% serum, resuspend the cells, remove 10ml of cell suspension and add to the prepared collagen mixture. The electric pipette was repeatedly blown and mixed, then the cell/collagen mixture was aspirated in 7 ml groups and added to four 50 ml sterilized centrifuge tubes, and the recombinant CTHRC1 protein was added to the 7 centrifuge tubes to achieve the protein concentration. 0nM, 20nM, 50nM, simultaneously adding mAb protein to 20nM, 50nM, and IgG protein reached 20nM, 50nM.
  • each centrifuge tube was thoroughly mixed with an electric pipette, and the mixture was separately added to each 3.5 cm culture dish in an amount of 2 ml/dish, and 2 ml was added in two portions. Incubator culture. After 1 hour, perform the “shake” operation and take a picture every 4 hours. Note that the photo should turn off the flash off light to avoid reflection of the liquid surface.
  • the area ratio of the gel to the culture dish was calculated using IPR software, and the results shown in Fig. 6 were obtained by statistical analysis of the TTEST method carried out by EXEL.
  • the effect of protein treatment on the migration ability of LX-2 cells should be noted.
  • the recombinant CTHRC1 protein obtained in Example 2 was added to the culture medium of the lower chamber to achieve protein concentration: 0 nM, 20 nM, 50 nM, and mAb protein 20 nM, 50 nM. And IgG protein 20 nM, 50 nM.
  • the lower chamber was incubated with low serum medium (DMEM medium containing 1% to 2% serum) and resuspended in serum in serum-free DMEM.
  • the Transwell chamber is taken out, the chamber is washed twice with PBS, the chamber is slightly dried, and then the chamber is stained in 0.1% crystal violet staining solution for 1 hour. Note that bubbles are avoided between the small outdoor membrane and the crystal violet dye solution. .
  • CTHRC1 is involved in the regulation of liver fibrosis.
  • CTHRC can aggravate the progression of liver fibrosis and accelerate liver function damage in animals.
  • CTHRC1 accelerates the activation of hepatic stellate cells and promotes the movement and contraction of stellate cells.
  • CTHRC1 neutralizing antibodies After blocking CTHRC1 with a CTHRC1 neutralizing antibody, the effect of CTHRC1 can be reversed.
  • the use of CTHRC1 neutralizing antibodies in animals can greatly slow the progression of liver fibrotic lesions. The above results indicate that the gene or its probe can be the latest target for the diagnosis and treatment of cirrhosis.
  • Tan F, Liu F, Liu H, et al. CTHRC1 is associated with peritoneal carcinomatosis in colorectal cancer: a new predictor for prognosis. Med Oncol, 2013, 30(1): 473.

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Abstract

CTHRC1核酸序列或蛋白在制备检测肝硬化或肝纤维的试剂中的应用、在制备预防或治疗肝硬化或肝纤维化的药物中的应用、以及作为体外筛选和制备肝硬化或肝纤维化药物的药靶的应用。其中CTHRC1的核酸序列如SEQ ID NO：1所示，CTHRC1蛋白如SEQ ID NO:2所示。

Description

CTHRC1在肝硬化诊断和治疗中的应用 技术领域
本发明涉及分子生物学特别是基因诊断和治疗领域，尤其是CTHRC1基因在肝硬化诊断和治疗中的应用。
背景技术
肝硬化，其前期病变表现为肝纤维化，是十分常见的肝脏慢性疾病[1]。多数肝纤维化患者经过15到20年的时间后会转变为不可逆转的肝硬化，甚至会进一步发展为肝癌[2]。肝纤维化的的病理表现为肝脏内纤维结缔组织的过度沉积(如Collagen)，是纤维增生(Fibrogenesis)和纤维分解(Fibrolysis)不平衡的结果。在肝纤维化进程中，肝星状细胞的活化是肝纤维化的中心事件。不同于肝癌，肝硬化的早期诊断十分困难，目前没有明确的分子标志物，临床检测手段主要是采用肝穿刺活检来进行诊断，其次是根据肝脾肿大、腹腔积液以及肝硬化并发症(如上消化道出血、肝性脑病、肝肾综合症等)这些临床病理特征来进行诊断[3]。但已有的检测手段存在一定缺陷，活检对于病人来说有一定创伤，而以并发症为依据又很难诊断出早期肝硬化。因此，寻找简便易行且能够准确监测病情的分子标志物的意义重大。
CTHRC1是一种分子量大约为26KDa的分泌性糖蛋白，最初发现于球囊扩张动脉损伤大鼠模型研究中[4]。CTHRC1参与了血管重构，并在胰腺癌组织中高表达[5]。此外，CTHRC1在乳腺癌、肝癌、胃癌、黑色素瘤以及结肠癌中呈高表达，并且促进上述肿瘤的侵袭迁移[6-8]。在血管平滑肌细胞中，CTHRC1抑制TGF-β通路，但是对内皮细胞的TGF-β通路没有抑制作用[9]。CTHRC1基因的启动子结合区域有Smad的结合位点，这提示了CTHRC1可以受到TGF-β的调节[8]。在胃癌的研究中发现，加TGF-β1处理后，胃癌 细胞的CTHRC1表达水平逐渐上调，伴随着TGF-β下游Smad信号通路的活化[8]。CTHRC1可以通过与Wnt5a和ROR2相互作用来激活Wnt/PCP信号通路，抑制Wnt/β-catenin信号通路，从而发挥调节细胞骨架重排和细胞运动的功能[10]。所以，CTHRC1对于TGF-β和Wnt信号通路具有非常重要的调节作用。
我们通过免疫组化实验发现，CTHRC1基因在人肝硬化组织中表达上调，可能参与调节肝纤维化的进展。我们进一步通过体内实验证实CTHRC1具有加剧肝纤维化进展，加速肝功能损伤的作用。之后通过体外实验发现CTHRC1可以加速肝星状细胞的活化，促进星状细胞的运动和收缩，而使用CTHRC1中和抗体针对CTHRC1进行阻断后，可逆转CTHRC1的作用。在动物体内使用CTHRC1中和抗体后，可以很大程度上减缓肝纤维化病变的进展。
发明内容
本发明的目的是提供与CTHRC1基因或产物相关的肝硬化/纤维化检测和治疗药物以及方法。
本发明的一方面在于提供一种CTHRC1核酸序列或蛋白作为在样品中检测肝硬化或肝纤维化的试剂中的应用，其中CTHRC1的核酸序列如SEQ ID NO：1所示，CTHRC1蛋白如SEQ ID NO：2所示。
其中，前述试剂是用于识别CTHRC1蛋白抗原表位的抗体。
或者，前述试剂也可是用于识别CTHRC1核酸序列的核酸探针。
本发明的另一方面在于提供一种CTHRC1核酸序列或蛋白作为在制备预防或治疗肝硬化或肝纤维化的药物中的应用，其中CTHRC1的核酸序列如SEQ ID NO：1所示，CTHRC1蛋白如SEQ ID NO：2所示。
其中，前述药物是用于识别CTHRC1蛋白抗原表位的抗体。
或者，前述药物包括CTHRC1核酸序列的反义核酸。
本发明的另一方面在于提供一种CTHRC1核酸序列或蛋白作为体外筛 选和制备肝硬化或肝纤维化药物的药靶的应用，其中CTHRC1的核酸序列如SEQ ID NO：1所示，CTHRC1蛋白如SEQ ID NO：2所示。
本发明的又一方面在于提供一种肝硬化或肝纤维化的试剂盒，该试剂盒中包括一容器，容器中含有CTHRC1抗体，即蛋白抗原表位的抗体。更进一步地，该容器中可包括CTHRC1抗体作为一抗以及HRP标记的山羊抗兔抗体作为二抗。
或，该容器中含有CTHRC1特异性探针。
术语：
本发明文本中所述的CTHRC1，在不同的语境下可能对应CTHRC1蛋白或核酸序列。
其中，CTHRC1蛋白可以采用各种常规的制备方法制备。如基因工程方法或者人工合成方法等。例如，可将CTHRC1核酸序列表达为蛋白而得。一旦获得了含有CTHRC1基因序列的重组表达质粒，就可将其转化到相应宿主中进行蛋白表达。本发明所述“CTHRC1蛋白”也包括如SEQ ID NO：2所示蛋白的天然或诱导的遗传变异体。所述诱导变异体可以通过各种技术得到，如辐射或诱变剂等产生的随机突变，也可以通过如定点突变法或其他已知分子生物学的技术获得。所述“CTHRC1蛋白”还包括具有天然L型氨基酸的残基的类似物(如D型氨基酸)，以及具有非天然存在的或合成的氨基酸(如氨基酸、Y-氨基酸等)的类似物。
其中，CTHRC1核酸序列可以采用各种常规的制备方法制备。本发明的CTHRC1核酸序列通常可以用PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法，可根据本发明的核酸序列，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。获得了CTHRC1基因序列后，就可以将CTHRC1序列插入适当的表达载体，以获得重组表达质粒。由于核苷酸序列的特殊性，任何SEQ NO：1所示多核苷酸的变体，只要其与该多核苷酸具有70％以上同源性，均属于本发明保护范围之列。所述多核苷酸的变 体是指一种具有一个或多个核苷酸改变的多核苷酸序列。此多核苷酸的变体可以使生的等位变异体或非生的变异体，包括取代变异体、缺失变异体和插入变异体。如本领域所知的，等位变异体是一个多核苷酸的替换形式，它可能是一个多个核苷酸的取代、缺失或插入，但不会从实质上改变其编码的多肽的功能。
其中，CTHRC1的抗原表位(Epitope)，又称为抗原决定簇(Antigenicdeterminant)，是抗原分子上的一个免疫活性区，负责与抗体分子或免疫细胞表面的抗原受体结合。本发明中所述的CTHRC1的抗原表位的抗体，是指任何现有技术中已知的CTHRC1抗体，也包括任何通过本领域内技术人员已知的各种技术进行制备得到的抗体。
CTHRC1抗体可用于免疫组织化学技术中，检测活检标本中的CTHRC1蛋白，还可以作为用于预防肝纤维化或肝硬化的特异性治疗剂。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。
CTHRC1核酸探针是指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；亦或者该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。
本发明有以下特点：
本发明在肝硬化/纤维化领域首次提出利用CTHRC1基因及其产物的检测来达到早期检测以及治疗的效果。CTHRC1具有成为良好的生物标记的潜应用前景。
附图说明
本发明将参考附图进行进一步的解释，其中：
图1a-b为免疫组化染色图以及CTHRC1在组织中的表达水平的统计图；
图2为CCl4和TAA化学诱导模型小鼠中，小鼠肝纤维化程度的组织染色图；
图3为分别经CTHRC1中和抗体处理和IgG处理的CCl4和TAA化学诱导模型小鼠中，小鼠肝纤维化程度的组织染色图；
图4为CCl4和TAA化学诱导模型小鼠中，小鼠肝损伤程度示意图；
图5为分别经CTHRC1中和抗体处理和IgG处理的CCl4和TAA化学诱导模型小鼠中，小鼠肝损伤程度示意图；
图6为分别经CTHRC1中和抗体处理和IgG或mAb处理的肝星状细胞胶收缩实验示意图；
图7分别经CTHRC1中和抗体处理和IgG或mAb处理的肝星状细胞迁移实验示意图。
下述实施例中，如无特殊说明，均为常规方法。下述实施例中所用的实验材料或生物制剂，如无特殊说明，均为可从市场上购得的常规试剂。以下实施例中的定量实验，均设置三次重复实验，结果取平均值。
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，通常按照常规条件和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。
具体实施方式
以下实施例主要包括了如下内容：
检测了CTHRC1在人肝硬化组织芯片中的表达情况，发现CTHRC1在肝硬化组织中显著上调。
采用CCl4和TAA化学诱导模型小鼠，发现野生型小鼠肝纤维化和肝损伤程度均重于CTHRC1敲除小鼠。这些实验证实了CTHRC1具有加剧肝纤维化进展，加速肝功能损伤的作用。
构建了CTHRC1的表达质粒，并转染到293T细胞中，纯化出重组CTHRC1蛋白。通过胶收缩和Transwell实验，分别观测到重组CTHRC1蛋白可以促进肝星状细胞收缩和迁移，使用CTHRC1中和抗体可逆转此作用。
在野生型小鼠中CTHRC1中和抗体可减轻CCl4和TAA所诱导的肝纤维化和肝损伤。
以上实验证实了使用CTHRC1中和抗体可以很大程度上减缓肝纤维化病变的进展，以下将详细描述各个实验：
实施例1 CTHRC1在人肝硬化组织芯片中的免疫染色
1.1主要试剂
CTHRC1一抗购自Abcam公司，HRP二抗购自Abmart公司；DAB显色液购自Thermo公司；中性树胶购自上海生工生物技术有限公司；肝硬化组织芯片购自西安艾丽娜生物科技有限公司，产品编号：LV805。组织芯片包含30例正常肝组织、10例肝炎组织、40例肝硬化组织。
1.2对上述组织芯片进行免疫组化染色并统计CTHRC1在组织中的表达水平：
①脱蜡至水化：二甲苯20分钟 1/2二甲苯10分钟 无水乙醇10分钟 95％乙醇10分钟 85％乙醇10分钟 75％乙醇10分钟 PBS(磷酸盐缓冲液)洗至水化；
②抗原热修复：沸水加热0.01M柠檬酸钠抗原修复液(pH6.0)至95℃左右，然后小心放入组织芯片或组织切片水浴加热15分钟，取出后可自然冷却至室温；α-SMA抗体染色不需抗原热修复步骤；
③消除内源性过氧化物酶活性：使用0.3％过氧化氢在37℃孵育30分钟，然后用PBS冲洗；
④抗原封闭：用10％牛血清(BSA)室温封闭1小时；在湿盒内进行；
⑤一抗孵育：向玻片的组织上滴加一抗150-200ul，4℃孵育过夜，PBS冲洗3次；置湿盒进行反应；
⑥二抗孵育：滴加二抗(山羊抗兔-HRP标记二抗1∶500稀释，兔抗小鼠-HRP标记二抗1∶200稀释)150-200ul，在室温孵育1小时，置湿盒内进行，孵育完毕后用PBS洗3次；
⑦发色：使用DAB显色液(现用现配)，DAB显色一般持续1-5分钟， 显微镜下控制发色程度，用自来水终止反应，然后PBS冲洗；
⑧使用苏木精复染细胞核1-2分钟，自来水冲洗5-10分钟；
⑨脱水，75％乙醇5分钟 85％乙醇5分钟 95％乙醇5分钟 无水乙醇5分钟 1/2二甲苯10分钟 二甲苯20分钟，自然晾干。
⑩用中性树胶封片、镜检。
染色结果如图1a所示，统计结果如图1b所示，可发现CTHRC1在人肝硬化组织中显著上调。
实施例2.CTHRC1蛋白的构建和纯化，制得CTHRC1蛋白以备后续实验所用
2.1 CTHRC1基因和表达载体
CTHRC1基因和表达载体均为本实验室保存，CTHRC1基因序列如SEQ ID NO：1所示，表达载体为V152，V162质粒。
2.2重组CTHRC1蛋白亲和层析纯化
收集CTHRC1-V152转染的293细胞培养液上清，进行蛋白亲和层析纯化获取重组CTHRC1-StrepII标签融合蛋白。具体步骤如下。
2.2.1组装层析柱子。层析柱由层析管、垫片、出口塞、顶盖以及管内琼脂糖磁珠组成。CTHRC1-StrepII tagged融合蛋白所用的亲和Beads是由德国iba公司(Goettingen，Germany)生产的Strep-Tactin Sepharose磁珠。Strep Tag II是仅含8个氨基酸的新型融合蛋白标签，V152载体是基于这个标签序列开发出的完备的融合蛋白真核生物表达系统。Strep Tag纯化技术的原理是基于biotin(生物素)/streptavidin(抗生物素蛋白链菌素)特异性结合的特点。Strep Tag II可以结合到Streptavidin的生物素口袋上，Strep-Tactin是改造过的Streptavidin，Strep-Tactin与Strep Tag II的亲和力远远超出普通Streptavidin约100倍，是非常高效的亲和层析分子。实验开始时向层析柱中加入2ml Strep-Tactin Sepharose。
2.2.2先加入5ml Buffer W洗涤层析柱1次。
2.2.3再分次加入收集的细胞培养液上清，至总上样量约50ml左右。加 入培养液上清后，让液体由重力控制缓慢流过层析柱，全程在4℃条件下操作。细胞培养液在上柱前进行离心，14,000rpm，5min，4℃，去除杂质。
2.2.4洗柱。用Buffer W洗柱5次，每次用Buffer W约2ml。
2.2.5用2.5mM Buffer E溶解洗脱蛋白。每次加入Buffer E 1ml，分6次加入共6ml Buffer E，分次接纳Buffer E洗脱下来的蛋白溶液，依序标记，每管留出20μl蛋白溶液用于SDS-PAGE检测。
2.2.6柱子再生。用Buffer R洗柱子3次使柱子再生。每次用Buffer R10ml，直至层析柱颜色由黄变红。
2.2.7洗柱。用Buffer W洗柱子2次，每次用Buffer W 8ml洗掉Buffer R。
2.2.8洗柱及再生后，将层析柱上灌注约2ml Buffer W(PH8.0)，并盖好顶盖，压好出口塞，保存在4℃冰箱，再生的柱子可以使用3-5次。
2.2.9收集到的蛋白溶液用NanoDrop2000进行蛋白浓度检测并记录，加入甘油后分装冻存于-80℃冰箱。每管留出的20μl蛋白溶液用于Western Blotting检测(检测CTHRC1抗体、StrepII抗体)，考马斯亮蓝检测蛋白条带单一性和准确性。
实施例3.CCl4和TAA化学诱导小鼠模型
3.1化学诱导肝纤维化动物模型实验
SPF级CTHRC1基因敲除C57小鼠(CTHRC1-/-C57BL/6mouse)从Hiroshi Sasaki(Lab for Embryonic Induction，Kobe，Japan)购买。CCl4诱导的肝纤维化模型：选择6周龄的同笼雄性C57野生型和CTHRC1-/-小鼠腹腔注射CCl4，每周两次，注射的CCl4需要按照1∶3的比例用橄榄油稀释，按照每只小鼠0.5μL/g(体重)的剂量注射。总共需要注射16次，最后一次注射后过48h处死全部小鼠，留取肝脏及血清，进行实验。
TAA(硫代乙酰胺)化学诱导肝纤维化实验：选择6周龄的同笼雄性C57野生型和CTHRC1-/-小鼠腹腔注射TAA(超纯水溶解，避光保存)，每周三次，按照每只小鼠0.2mg/g(体重)的剂量注射。总共需要注射8周， 最后一次注射后过48h处死全部小鼠，留取肝脏及血清，进行实验。
CTHRC1中和抗体抗纤维化治疗的动物实验：化学诱导实验包括CCl4和TAA诱导模型同上，实验动物选择6周龄雄性C57野生型小鼠，分为CTHRC1中和抗体处理组和IgG处理组。CTHRC1处理组以及IgG处理组分别给予中和抗体(在杭州华安公司订制)或IgG 1mg/只/次，腹腔注射，每周三次，与化学药物注射时间隔日进行，共处理8周。最后一次注射后过48h处死全部小鼠，留取肝脏及血清，进行实验。
3.2肝脏组织切片天狼星红染色
对实验3.1中获得的肝脏组织进行染色，苦味酸天狼星红染色可以使胶原持久染色，在偏振光显微镜下观察能增强胶原纤维的双折光性，从而可以特异性地显示胶原组织。在偏光显微镜下，I型胶原呈现黄或红色，III型胶原呈现绿色，II性胶原呈现蓝绿或灰蓝色。0.1％苦味酸天狼星红溶液配制方法：天狼星红0.1g，饱和苦味酸溶液100ml，苦味酸饱和液(1.22％)。组织切片需要常规脱蜡至水化，然后浸入0.1％苦味酸天狼星红染色至少1小时，然后用自来水流水冲洗4min；苏木素复染3min(非必须)，梯度乙醇脱水，脱水时间应短，晾干，中性树胶封片。实验结果如图2及图3所示。
结果表明CCl4和TAA化学诱导模型小鼠中，野生型(WT)小鼠肝纤维化程度重于CTHRC1敲除小鼠(图2)。而使用CTHRC1中和抗体可减轻CCl4和TAA所诱导的肝纤维化(图3)。
3.3动物血清肝功指标ALT、AST检测
检测实验3.1中所获得的小鼠血清，ALT、AST的检测试剂盒购自上海申索佑福医学诊断用品有限公司。包括丙氨酸氨基转移酶(ALT)测定试剂盒，门冬氨酸氨基转移酶(AST)测定试剂盒。检测的计算公式是：ALT(U/L)＝(ΔABS/min×Vt×1000)/(6.22×Vs×d)；检测方法如下；设定分光光度计测量程序为：主波长340nm，副波长600nm；血清每孔上样7.5μl，R-1试剂加150μl，R-2试剂加50μl，样品校准方式：K因子(K＝4448)，反应方向：下降；测定温度：37℃；血清样品与R-1混匀后反应5min，加入R-2混合 后延迟53秒测光密度值，延迟247秒测光密度值；具体流程如下：血清样品7.5μl+R-1 150μl，5min后加入R-2 50μl，过53秒后测光密度值(A340nm)，过247秒后测第二次光密度值(A340nm)，ΔABS/min是指每分钟平均吸光度值变化，即两次A340nm差值/6min；Vt＝总反应体积(0.2075ml)，Vs＝样品体积(0.0075ml)，6.22＝NADH的毫摩尔吸光系数，d＝比色杯光径(1cm)。AST检测方法跟ALT相同。实验结果如图4及图5所示。
结果表明CCl4和TAA化学诱导模型小鼠中，野生型小鼠肝损伤程度重于CTHRC1敲除小鼠(图4)。而使用CTHRC1中和抗体可减轻CCl4和TAA所诱导的肝损伤(图5)。
实施例4.肝星状细胞胶收缩实验
4.1肝星状细胞株
肝星状LX-2细胞株为本室保存，购自Millipore公司(实施例5同)。
4.2胶原晶格收缩实验
4.2.1 LX-2细胞先用无血清DMEM培养液饥饿培养过夜；用胰酶消化细胞，离心，计数；然后配制胶原晶格混合液，全程在冰上进行。按需要的总体积制成混合液体。然后向每个3.5cm皿中加入混合液2mL，摇匀，将培养皿置于37℃细胞培养箱培养；
4.2.2“摇松”，细胞培养45min至1小时凝胶凝固后，打开培养皿盖，小心加入0.5mL DMEM培养液，小心地上下、左右晃动使凝胶漂浮于液体中；间隔每4-6小时观察凝胶面积大小，去培养皿盖进行拍照，拍照时培养皿旁边放直尺；拍照者需戴口罩和帽子，不讲话。
4.2.3使用IPP软件或PhotoShop软件计算凝胶面积或凝胶占培养皿的面积比，用EXEL自带的TTEST法统计学分析。
4.2.4重组CTHRC1蛋白处理的胶原晶格收缩实验步骤和方法
配制胶原蛋白晶格混合液，分为至少7个处理组，分别加入实施例2中所获得的重组CTHRC1蛋白，0nM，20nM，50nM，以及加入mAb蛋白20nM，50nM，和IgG蛋白20nM，50nM，每组内各三皿复孔，每皿需加混合液2ml， 计算总量。具体操作全部在冰上进行。
细胞准备：将2个10cm皿长满的LX-2细胞消化，离心，加入11ml含10％血清的细胞培养液重悬细胞，取出10ml细胞悬液加入到配好的胶原蛋白混合液中，用电动移液器反复吹打混匀，然后以7ml为一组将细胞/胶原蛋白混合液吸出分别加到4个50ml灭菌离心管中，在7个离心管中分别加入重组CTHRC1蛋白使蛋白浓度达到0nM、20nM、50nM，同时加入mAb蛋白达到20nM，50nM，和IgG蛋白达到20nM，50nM。将每个离心管的混合液用电动移液器充分混匀，以2ml/皿-的量将混合液分别加到各个3.5cm培养皿中，2ml可分两次加。培养箱培养。1小时后进行“摇松”操作，每隔4小时拍照1次，注意拍照应关掉闪关灯，避免液体表面反光。对所获得的照片，使用IPR软件计算凝胶占培养皿的面积比，并用EXEL自带的TTEST法统计学分析，获得图6所示结果。
实验结果表明重组CTHRC1蛋白可以促进肝星状细胞收缩，而CTHRC1中和抗体可逆转此作用(图6)。
实施例5.肝星状细胞迁移实验
5.1选择对数生长期的LX-2细胞，饥饿培养过夜，然后用0.25％胰酶消化并计数，根据计数结果将细胞悬液稀释成20×104/ml的密度。
5.2将用不含血清DMEM培养基重悬的LX-2细胞以4×104/200μl的密度种到每个Transwell小室(Millicell)上层，将小室置于24孔板内，小室下层加入800μl含10％FBS的DMEM培养基，注意避免产生气泡。37℃培养箱培养17小时使细胞穿过小室的滤膜。
蛋白处理对LX-2细胞迁移能力影响的实验需注意，在小室下层的培养液内加入实施例2中所获得的重组CTHRC1蛋白达到蛋白浓度：0nM，20nM，50nM，以及加入mAb蛋白20nM，50nM和IgG蛋白20nM，50nM。下室用低血清培养液(含1％-2％血清的DMEM培养液)，小室内用不含血清的DMEM重悬细胞。
5.3取出Transwell小室，用1×PBS洗涤小室1次，用干净的小棉花团 或棉签擦净小室内膜表面附着的细胞，将小室浸入2％戊二醛溶液或者4％多聚甲醛溶液中进行固定15min。
5.4固定结束后，取出Transwell小室，用PBS洗涤小室2次，稍晾干小室，然后将小室置于0.1％结晶紫染色液中染色1小时，注意小室外膜与结晶紫染液间避免产生气泡。
5.5染色结束后，取出Transwel小室，用1×PBS洗涤小室内、外膜表面的结晶紫3次，直至洗涤的PBS不呈紫色为止。
5.6将染色洗涤后的小室倒置晾干，完全干燥后用正置显微镜观察、拍照、计数、统计。结果如图7所示。
实验结果表明重组CTHRC1蛋白可以促进肝星状细胞迁移，而CTHRC1中和抗体可逆转此作用(图7)。
综上所述，CTHRC1参与调节肝纤维化的进展。CTHRC在动物体内可以加剧肝纤维化进展，加速肝功能损伤。在体外CTHRC1可以加速肝星状细胞的活化，促进星状细胞的运动和收缩。使用CTHRC1中和抗体针对CTHRC1进行阻断后，可逆转CTHRC1的作用。而在动物体内使用CTHRC1中和抗体后，可以很大程度上减缓肝纤维化病变的进展。以上结果说明该基因或其探针可以成为肝硬化诊断和治疗的最新靶标。
应当注意的是，本发明的实施例有较佳的实施性，且并非对本发明作任何形式的限制，任何熟悉该领域的技术人员可能利用上述揭示的技术内容变更或修饰为等同的有效实施例，但凡未脱离本发明技术方案的内容，依据本发明的技术实质对以上实施例所作的任何修改或等同变化及修饰，均仍属于本发明技术方案的范围内。
[参考文献]
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Claims (10)

  1. CTHRC1核酸序列或蛋白作为在样品中检测肝硬化或肝纤维化的试剂中的应用，其中所述CTHRC1的核酸序列如SEQ ID NO：1所示，所述CTHRC1蛋白如SEQ ID NO：2所示。
  2. 如权利要求1所述的应用，其中所述试剂是用于识别CTHRC1抗原表位的抗体。
  3. 如权利要求1所述的应用，其中所述试剂是用于识别CTHRC1核酸序列的核酸探针。
  4. CTHRC1核酸序列或蛋白作为在制备预防或治疗肝硬化或肝纤维化的药物中的应用，其中所述CTHRC1的核酸序列如SEQ ID NO：1所示，所述CTHRC1蛋白如SEQ ID NO：2所示。
  5. 如权利要求4所述的应用，其中所述药物包括用于识别CTHRC1抗原表位的抗体。
  6. 如权利要求4所述的应用，其中所述药物包括CTHRC1核酸序列的反义核酸。
  7. CTHRC1核酸序列或蛋白作为体外筛选和制备肝硬化或肝纤维化药物的药靶的应用，其中所述CTHRC1的核酸序列如SEQ ID NO：1所示，所述CTHRC1蛋白如SEQ ID NO：2所示。
  8. 一种肝硬化或肝纤维化的试剂盒，所述试剂盒中包括一容器，所述容器中含有CTHRC1抗体。
  9. 如权利要求8所述的试剂盒，所述容器中含有CTHRC1抗体作为一抗，以及HRP标记的山羊抗兔抗体作为二抗。
  10. 一种肝硬化或肝纤维化的试剂盒，所述试剂盒中包括一容器，所述容器中含有CTHRC1特异性探针。
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