WO2013104102A1 - 乳腺癌诊断和预示的标志物 - Google Patents

乳腺癌诊断和预示的标志物 Download PDF

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WO2013104102A1
WO2013104102A1 PCT/CN2012/070147 CN2012070147W WO2013104102A1 WO 2013104102 A1 WO2013104102 A1 WO 2013104102A1 CN 2012070147 W CN2012070147 W CN 2012070147W WO 2013104102 A1 WO2013104102 A1 WO 2013104102A1
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seq
breast cancer
cst1
cystatin
cleavage
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PCT/CN2012/070147
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English (en)
French (fr)
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王弢
渠香云
陈菲
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苏州工业园区为真生物医药科技有限公司
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Priority to GB1414085.9A priority Critical patent/GB2513275B/en
Priority to JP2014550606A priority patent/JP6138154B2/ja
Priority to US14/371,083 priority patent/US20150292024A1/en
Priority to CN201280066466.0A priority patent/CN104039962B/zh
Priority to PCT/CN2012/070147 priority patent/WO2013104102A1/zh
Publication of WO2013104102A1 publication Critical patent/WO2013104102A1/zh

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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
    • G01N33/57411—Specifically defined cancers of cervix
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/8139—Cysteine protease (E.C. 3.4.22) inhibitors, e.g. cystatin
    • 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
    • G01N33/57415—Specifically defined cancers of breast
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/81—Protease inhibitors
    • G01N2333/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • G01N2333/8139—Cysteine protease (E.C. 3.4.22) inhibitors, e.g. cystatin

Definitions

  • the present invention relates to the field of biomedical technology, and relates to a molecular marker useful for tumor diagnosis and prediction, and reagents, kits and chips for detecting the molecular marker. Background technique
  • Cystatin family as a cathepsin is mainly an endogenous inhibitor of cysteine protease, which plays an important role in the occurrence, development, infiltration and metastasis of tumors. The role.
  • the results of the study showed that several members of the Cystatin family have increased expression levels in different tumors. For example, Cystatin C has different levels of expression in ovarian cancer and head and neck cancer, and stefm A is in non-small cell lung cancer.
  • Cystatin F increased significantly in a variety of tumors, probably due to the need for the participation of cathepsins in tumorigenesis and development, first to induce an increase in the expression level, and then activate the body itself.
  • the stress mechanism leads to an increase in the expression level of Cystatin to inhibit excessive cathepsin activity.
  • the expression level of Cystatin is not positively correlated with the development of tumor.
  • the low expression of Cystatin C means the late stage of the disease, the patient's survival is shorter, and it is easy to relapse. This may be due to the tumor. In the later stages, there are other mechanisms that regulate the level of Cystatin to promote further tumor progression.
  • Cystatin SN is a member of the human Cystatin family. It is encoded by the CST1 gene and contains 141 amino acids. It has two disulfide bonds in its molecule and has a molecular weight of 16.4Kda. It is a typical secreted protein distributed in various body fluids and secretions, such as tears. , saliva, serum, plasma, etc. It is reported in the literature that the expression level of CST1 in gastric cancer tissues is higher than that in normal gastric mucosa tissues. The expression sites in gastric cancer cell lines are consistent with those in gastric cancer tissues, and the expression rate decreases with the decrease of cell line differentiation.
  • CST1 Clinical data analysis shows that The expression of CST1 was associated with depth of invasion, distant metastasis, and TNM stage; survival analysis showed that the 5-year survival rate of CST1 positive group was significantly higher than that of non-expression group; Cox regression analysis showed that CST1 was an independent prognostic factor; thus suggesting CST1 It may play a similar role as a tumor suppressor in the development and progression of gastric cancer. But so far, the relationship between CST1 and breast cancer has not been reported in the literature.
  • the object of the present invention is to study the expression of CST1 in breast cancer, and to analyze its relationship with clinical pathological parameters and biological behavior of breast cancer, and then discover that CST1 is involved in the occurrence, development and metastasis of breast cancer. The role played.
  • the present invention compared the expression of CST1 in different tissues of human body, and found that CST1 is lowly expressed in normal human tissues other than salivary glands, which is extremely advantageous for the detection of CST1 under pathological conditions.
  • the present invention compares the expression differences of CST124, CST1, CST2, CST4 in breast cancer and adjacent tissues, and found that CST1 has the greatest difference in breast cancer and adjacent tissues. Since CST1 mRNA (SEQ ID No. 44) selectively cleaves to form two cleavage, the first cleavage (SEQ ID No. 48) contains CST1 exon 1 (SEQ ID No. 45) exon 2 (SEQ ID No. 46) and exon 3 (SEQ ID No.
  • the second cleavage contains only CST1 exon 1 (SEQ ID No. 45) and Receptor 2 (SEQ ID No. 46)
  • the present invention compares the expression differences between the two CSTl cleavers in surgical specimens of breast cancer and paracancerous tissues, and finds that the first cleavage is in cancer and adjacent tissues. The degree of expression difference is better than the second type of shear.
  • the present invention compares the first cut of CST1 in breast cancer, paracancerous and normal tissue surgical specimens, breast cancer, mastitis and normal tissue puncture specimens, breast cancer metastasis-positive lymph nodes and negative lymph nodes, breast cancer, mastitis
  • the difference in expression between the patient and normal human cell-free RNA found that the first CST1 cleavage is abnormally highly expressed in breast cancer and breast cancer metastasis tissues.
  • breast cancer and cancer can be detected.
  • Side and normal tissues, breast cancer metastasis positive tissue and negative tissue are distinguished, high sensitivity and good specificity.
  • the present invention compares the CST1 encoding protein Cystatin SN (amino acid sequence as shown in SEQ ID No. 52, encoding gene sequence as shown in SEQ ID No. 53) in breast cancer cell line culture supernatant and normal humans.
  • the expression of serum and breast cancer patients was significantly different from that of normal human serum.
  • Cystatin SN was found to be abnormally high in the serum of breast cancer cell culture supernatant and breast cancer patients.
  • breast cancer cell lines can be cultured. Clear and normal human serum, breast cancer patients serum and normal human serum, high sensitivity, specificity.
  • CST1 first cleavage and Cystatin SN can be used as molecular markers for breast cancer diagnosis and prediction.
  • the epitope peptide of Cystatin SN can also be used as a molecular marker for diagnosis and prediction of breast cancer.
  • the amino acid sequence of Cystatin SN epitope peptide can be as shown in SEQ ID No. 54 (ie, removing Cystatin SN).
  • the diagnosis and prediction of breast cancer can be performed, including differential diagnosis and/or susceptibility analysis of breast cancer or breast cancer tissue metastasis, breast cancer or breast cancer tissue metastasis treatment drugs, treatment methods, Assessment of treatment efficacy and prognosis, assessment of the risk of breast cancer or breast cancer tissue metastasis in the relevant population.
  • the method One: determining the content or expression level of the first type of CST1 or Cystatin SN in the sample to be tested, comparing the detection result with a threshold value, and judging as a positive value if the threshold value is exceeded; the threshold value can be compared with a normal person and a breast
  • the content or expression level of CST1 first cleavage or Cystatin SN in body fluids or tissues of cancer patients was statistically obtained.
  • the present invention further investigates the above-mentioned molecular marker detection reagents, kits and detection chips for use in breast cancer diagnosis and prediction.
  • PCR polymerase chain reaction
  • NASBA quantitative real-time PCR
  • TMA Transcription-median amplification
  • LCR Ligase chain reaction
  • tSDA thermophilic strand displacement amplification
  • a breast cancer diagnostic and predictive reagent application can be made as long as a primer or probe that specifically recognizes the first splice of CST1 or its cDNA (collectively referred to as the capture agent of the first scissor of CST1).
  • the present invention preferably prefers a primer or probe that specifically recognizes CST1 exon 1 or a primer or probe that specifically recognizes CST1 exons 2 and 3, and more preferably a primer or probe that specifically recognizes CST1 exon 1.
  • the primer is preferably at least one of nucleotide sequences as shown in SEQ ID Nos. 1 to 2, 4 to 21, 34 and 39 to 42, more preferably SEQ ID No. 1 and SEQ ID No. 2
  • the probe is preferably at least one of the nucleotide sequences as shown in SEQ ID No. 3, 35-38 and 43, more preferably as shown in SEQ ID No. 3.
  • Cystatin SN capture agents can be used in breast cancer diagnostic and predictive reagent applications.
  • the antibody may be a polyclonal antibody or a monoclonal antibody, and may be subjected to various types of enzymes such as alkaline phosphatase, luciferase, peroxidase, ⁇ -galactosidase, and various fluorescent compounds such as fluorescein.
  • the label which can also be labeled with biotin, amplifies the reaction signal using the avidin-enzyme complex.
  • labeled as an enzyme it can be quantified by adding an enzyme substrate to change the light absorption.
  • enzyme substrate an enzyme substrate to change the light absorption.
  • fluorescent compound it can be quantified by exciting it with ultraviolet light to emit fluorescence.
  • the above detection reagents can be combined in various combinations or combined with other auxiliary detection reagents to prepare a breast cancer diagnosis and predictive kit.
  • Commercially available or documented kits containing the above detection reagents can also be directly used in breast cancer diagnostic and predictive kit applications.
  • the CST1 first cleavage detection kit based on TaqMan hydrolysis probe real-time PCR comprises at least one pair of primers and one probe for specifically identifying the cDNA of the first cleavage of the CST1 gene.
  • the nucleotide sequence of the primer is shown in SEQ ID No. 1 and SEQ ID No. 2, and the nucleotide sequence of the probe is SEQ.
  • ID No. 3 the 5' end of the probe is labeled with a fluorophore and the 3' end is labeled with a quencher group.
  • the CST1 first cleavage detection kit based on dye real-time PCR comprises at least one pair of primers which specifically recognize the cDNA of the first cleavage of the CST1 gene, and the CST1 gene is cleaved
  • the capture agent is a primer that specifically recognizes the cDNA of the CST1 gene cleavage, and the nucleotide sequence of the primer is shown in SEQ ID No. 1 to 2, or as shown in SEQ ID Nos. 4 to 5, or As shown in SEQ ID Nos. 6 to 7, or as shown in SEQ ID Nos. 8 to 9, or as shown in SEQ ID 10 os. 10 to 11, or as shown in SEQ ID ⁇ ⁇ . 12 to 13, or as SEQ.
  • the IDs are shown in SEQ ID Nos. 14 to 15, or as shown in SEQ ID ⁇ ⁇ . 16 to 17, or as shown in SEQ ID 18 ⁇ . 18 to 19, or as shown in SEQ ID Nos. 20 to 21.
  • the NASBA or TMA-based CST1 first cleavage detection kit comprises at least one pair of primers and one probe that specifically recognize the cDNA of the first cleavage of the CST1 gene,
  • the nucleotide sequence of the primer is shown in SEQ ID No. 34 and SEQ ID No. 2
  • the nucleotide sequence of the probe is shown in SEQ ID No. 3, and the 5' end of the probe is labeled with a fluorophore.
  • the 3' end is labeled with a quenching group.
  • the LCR-based CST1 first cleavage detection kit comprises at least four probes that specifically recognize the first cleavage of the CST1 gene, and the nucleotide sequence of the probe is as As shown in SEQ ID Nos. 35 to 40, the 5' end of each probe was labeled with a hapten.
  • the tSDA-based CST1 first cleavage detection kit comprises at least two pairs of primers and one probe that specifically recognize the cDNA of the first cleavage of the CST1 gene, and the primers
  • the nucleotide sequence is shown in SEQ ID Nos. 39 to 44, the nucleotide sequence of the probe is shown in SEQ ID No. 43, and the 5' end of the probe is labeled with a radioisotope.
  • one or more auxiliary detection reagents may be added as appropriate, including but not limited to: 1 visualizing the amplification of the corresponding amplicon of the primer a reagent, for example, a reagent for visualizing an amplicon by agarose gel electrophoresis, enzyme-linked gel method, chemiluminescence method, in situ hybridization method, fluorescence detection method, etc.; 2 RA extraction reagent; 3 reverse transcription reagent; 4 cDNA Amplification reagents, such as PCR, real-time quantitative PCR, NASBA, TMA, LCR, tSDA related reagents; 5 standards for preparing standard curves, such as recombinant plasmids containing CST1 first cleavage amplicon; 6 positive control Products, such as human breast cancer cell lines HCC1937, SK-BR-3, MCF-7; 7 negative control, such as human normal breast cell Hs578Bst.
  • the competitive ELISA-based Cystatin SN detection kit comprises at least a Cystatin SN antigen, an anti-Cystatin SN monoclonal antibody, an enzyme-labeled secondary antibody, and an enzyme substrate.
  • the ELISA plate was coated with Cystatin SN antigen, and the test sample was sequentially incubated with anti-Cystatin SN monoclonal antibody on the plate, and the anti-Cystatin SN bound to the plate was detected by the reporter component (the enzyme-labeled secondary antibody and the enzyme substrate).
  • the amount of monoclonal antibody according to this Determine the amount of Cystatin SN in the sample.
  • the Cystatin SN detection kit based on double-anti-sandwich ELISA comprises at least anti-Cystatin SN monoclonal antibody, biotin-labeled anti-Cystatin SN polyclonal antibody, avidin-enzyme complex and enzyme bottom Things.
  • the plate was coated with Cystatin SN monoclonal antibody, and the test sample and biotin-labeled anti-Cystatin SN polyclonal antibody were sequentially incubated on the plate and detected by the reporter component (Avidin-enzyme complex and enzyme substrate).
  • the amount of Cystatin SN in the sample was determined based on the amount of anti-Cystatin SN monoclonal antibody bound to the plate.
  • one or more auxiliary detection reagents may be added, including but not limited to: 1 blocking solution; 2 antibody dilution solution; 3 washing buffer; 4 coloring stop solution; 5 Prepare the Cystatin SN standard used in the standard curve.
  • the probe that specifically recognizes the first cleavage of CST1 or its cDNA can also be immobilized on the surface of the solid phase carrier to make a breast cancer diagnosis and prediction chip.
  • Commercially available or documented microarrays with these probes can also be directly fabricated into breast cancer diagnostic and predictive chip applications.
  • the detection reagent, kit or chip of the invention can be used to determine whether breast cancer is susceptible or formed, perform breast cancer pTNM staging, evaluate breast cancer progression or treatment effect, and determine whether metastasis or recurrence.
  • the test sample may include surgical tissue, puncture tissue, lymph node tissue, bone marrow, serum sample, plasma sample, whole blood sample, blood fraction sample, urine sample, and the like.
  • the subject may be a person who is treated for breast discomfort, a person with a history of familial breast cancer, or a patient with breast cancer.
  • the beneficial effects of the present invention are as follows:
  • the present invention discloses a CST1 gene cleavage represented by SEQ ID No. 48 and a CST1 gene-encoding protein Cystatin SN represented by SEQ ID No. 52 as a molecular marker for diagnosis and prognosis of breast cancer.
  • the present invention also discloses a capture agent of the CST1 gene cleavage agent and a capture agent of Cystatin SN Application in preparing breast cancer diagnosis and predictive reagents, kits and chips, and disclosing a breast cancer diagnosis and predictive kit containing the above-mentioned capture agent, and a description of the chip
  • Figure 1 is a map of the recombinant plasmid (pMD18-T-CSTl) containing the first splicing amplicon of CST1.
  • Figure 2 shows the CST1 gene in normal human tissues (tonils, posterior pituitary, thyroid, salivary gland, skeletal muscle, bone marrow, peripheral blood, lung, stomach, liver, heart, kidney, adrenal gland, intestine, colon, pancreas Expression of visceral, spleen, bladder, prostate, breast, ovary, uterus, placenta and testis), human breast cancer cell lines (HCC1937, SK-BR-3, and MCF-7) and human normal breast cell line Hs578Bst.
  • Figure 3 shows the differential expression of CST124, CST1, CST2, and CST4 in breast cancer and adjacent tissues by dye-based real-time quantitative PCR.
  • Figure 4 shows the expression of ACTB in breast cancer and paracancerous tissue samples by real-time quantitative PCR assay.
  • N1-N20 and T1 ⁇ T20 represent 20 pairs of breast cancer and paracancerous tissue paired samples, respectively.
  • Figure 5 shows the differential expression of the first splicel and the second splicing (splice2) of CST1 in breast cancer and paracancerous tissue samples by real-time quantitative PCR assay.
  • Figure 6 shows the differential expression of CST1 first shear in the surgical specimens of breast cancer, paracancerous and normal tissues by real-time PCR absolute quantification.
  • Figure 7 is a real-time PCR absolute quantitative method for the detection of CST1 first cleavage in breast cancer and mastitis puncture samples.
  • Figure 8 is a real-time PCR absolute quantitative method for detecting CST1 first cleavage in breast cancer metastasis-positive lymph nodes and negative Differences in expression in lymph nodes.
  • Figure 9 shows the differential expression of CST1 first cleavage in breast cancer patients, mastitis patients, and normal human cell-free R A by real-time PCR absolute quantification.
  • FIG 10 shows the receiver operator characteristic curve (ROC curve) for real-time PCR absolute quantification.
  • Figure 11 shows the difference in the expression of CST1 first cleavage in plasma circulating R A of breast cancer patients, mastitis patients and normal subjects by Ligase chain reaction (LCR).
  • LCR Ligase chain reaction
  • Figure 12 shows the detection of thermophilic strand displacement amplification (tSDA)
  • CST1 Differential expression of CST1 first splice in plasma circulating R A in breast cancer patients, mastitis patients, and normal subjects.
  • Figure 13 shows the differential expression of CST1 first cleavage in the urine circulation of breast cancer patients, mastitis patients and normal subjects by Nucleic Acid based Amplificatin (NASBA).
  • NASBA Nucleic Acid based Amplificatin
  • Figure 14 shows the comparison of the expression level of the first cleavage of CST1 in peripheral blood of patients with breast cancer and cytology.
  • Figure 15 shows the comparison of the expression level of the first cleavage of CST1 in breast cancer patients with cytology.
  • Figure 16 shows the differential expression of CST1 first cleavage in plasma p-NM of different pTNM stages in breast cancer by Transcription-mediated amplification (TMA).
  • TMA Transcription-mediated amplification
  • Figure 17 shows the difference in expression of Cystatin SN in serum of breast cancer cell lines and normal human serum.
  • Figure 18 shows the difference in expression of Cystatin SN in serum of normal human serum and breast cancer patients.
  • Figure 19 shows the difference in expression of Cystatin SN in serum between normal and breast cancer patients by competitive ELISA.
  • Figure 20 shows the differential expression of Cystatin SN in serum of normal and breast cancer patients by double-antibody sandwich ELISA.
  • Figure 21 shows the sensitivity and specificity of ELISA for the determination of serum Cystatin SN and carcinoembryonic antigen (CEA) in breast cancer patients.
  • Figure 22 shows the median array of Cystatin SN protein expression and the disease-free survival curve below the median array after treatment in patients with breast cancer.
  • the specimens used in the preferred embodiments were obtained by hospital-scheduled means after signing the informed consent form with the patient.
  • the pathological site specimen obtained by the needle biopsy was compared with the non-pathological site sample.
  • Samples such as lymph nodes obtained during surgery were immediately extracted or stored in liquid nitrogen or R Alater (Ambion); peripheral blood, bone marrow or urine samples were centrifuged at 4 ° C, 4000 rpm for 20 minutes, and the supernatant was taken. After centrifugation at 4 ° C, 13,000 rpm for 10 minutes, the supernatant and the precipitate were separated, and RA was immediately extracted or stored at -20 to -80 ° C.
  • the human tissue specimens used in this example were obtained from the cooperative hospitals except for the normal breast tissue specimens, and other tissue specimens were obtained from the formal commercial channels.
  • Affymetrix nucleotide chip HG-U95Av was used to detect the relative expression of CST1 mRNA in different tissues of human body. The specific operation was based on the nucleotide chip specification, and the relative expression value was normalized by the housekeeping gene ⁇ -actin fluorescence value. The normalized signal value after processing. The results are shown in Figure 2.
  • CST1 is not expressed in other tissues except for its high expression in salivary glands, indicating that the background value of CST1 expression in normal tissues is very low, which is used for the detection of CST1 under pathological conditions. Extremely beneficial.
  • CST1 was overexpressed in human breast cancer cell lines HCC1937, SK-BR-3 and B MCF-7, and was not expressed in human normal breast cell Hs578Bst, suggesting that CST1 is highly likely to be a marker for molecular diagnosis of breast cancer.
  • Example 2 Differences in expression of CST124, CST1, CST2, and CST4 in surgical specimens of breast cancer and paracancerous tissues were detected by dye real-time quantitative PCR to detect CST124, CST1, CST2, and CST4 mRNA in 20 breast cancer and paracancerous tissues. Relative expression level.
  • the total RA of the sample was extracted using Trizol reagent.
  • the RA was reverse transcribed into cDNA using a commercial reverse transcription kit and operated according to the kit instructions.
  • the PCR amplification primer sequences of the target gene are shown in Table 1.
  • the fluorescent dyes are SYBR Green, Eve Green, LC Green and the like.
  • the test results are shown in Figure 3.
  • Example 3 Difference in expression of CST1 first cleavage and second cleavage in surgical specimens of breast cancer and paracancerous tissues
  • Example 4 Differential expression of CST1 first splicing in breast cancer, paracancerous and normal tissue surgical samples.
  • Real-time PCR absolute quantification was used to detect CST1.
  • the first cleavage was performed in 100 cases of breast cancer, paracancerous and normal tissue.
  • the difference in expression, the primer sequence is shown in SEQ ID No. 1 and SEQ ID No. 2, and the amplicon sequence is shown in SEQ ID No. 50.
  • the results are shown in Figure 6.
  • the first type of CST1 is highly expressed in malignant pathological conditions of the breast, but is low in adjacent tissues and normal tissues.
  • the median copy of the cancer specimen is the median copy of normal tissue specimens. 15.6 times; from the 223.20 copy, the cancer tissue can be distinguished from normal tissue. Therefore, 223.20 copies can be used as a reference for breast cancer diagnosis of surgically sampled tissue samples.
  • Example 5 Difference in expression of CST1 first splice in breast cancer and mastitis puncture samples
  • Puncture sampling and surgical sampling of the sample are quite different, mainly in the sample of the puncture sample, the proportion of breast cancer cells varies greatly, and sometimes may only account for a small part of the whole sample, sometimes not even. Therefore, this example uses real-time PCR absolute quantitative method to detect the difference in the expression of CST1 first cleavage in 40 specimens of breast cancer and mastitis patients.
  • the primer sequences are SEQ ID No. 1 and SEQ ID No. 2. Shown.
  • the test results are shown in Figure , CST1
  • the first type of splicing was highly expressed in breast cancer specimens, but was lowly expressed in mastitis specimens.
  • the median copy of cancer specimens was 11.0 times the median copy of inflammatory specimens; from 120.66 copies, Cancer can be distinguished from inflammation, so 120.66 copies can be used as a reference for breast cancer diagnosis of puncture samples.
  • Example 6 Differential expression of CST1 first splice in breast cancer metastasis-positive lymph nodes and negative lymph nodes 30 lymph nodes with pathologically proven breast cancer metastasis were obtained by surgery, and the metastases were of different sizes. Pathologically negative lymph nodes 30 were mainly obtained from patients with early breast cancer, and the lymph nodes with negative pathological diagnosis but actually micrometastasis were minimized to avoid experimental errors. Real-time PCR absolute quantification was used to detect the difference in expression of CST1 first cleavage in breast cancer metastasis-positive lymph nodes and negative lymph nodes.
  • the primer sequences are shown in SEQ ID No. 1 and SEQ ID No. 2, and the amplicon sequences are as follows. SEQ ID No. 50 is shown.
  • the results are shown in Figure 8.
  • the first type of CST1 is highly expressed in metastatic lymph nodes and low in negative lymph nodes.
  • the median copy of metastatic lymph node specimens is 6.3 times the median copy of negative lymph node specimens.
  • From the 82.45 copy two of the negative lymph nodes were found to be weakly positive for the first cut of CST1. After careful observation by serial sections, the two negative lymph nodes had micrometastases. Therefore, 82.45 copies were not only 100. % distinguishes the results of cytology, and can detect lymph nodes with micrometastases that cannot be detected by cytology.
  • the first cleavage copy number detection of CST1 is more sensitive than cytological detection.
  • Example 7 PCR detection of differential expression of CST1 first splice in plasma circulating RNA of breast cancer, inflammatory patients and normal humans
  • the difference in expression of the primers is shown in SEQ ID No. 1 and SEQ ID No. 2, and the amplicon sequence is shown in SEQ ID No. 50.
  • the median number of copies of the first type of CST1 in cancer was 8.8 times that of inflammation and 32 times that of normal, respectively; from the line of 65.23, it was possible to distinguish cancer, inflammation, and normal. Come.
  • the ROC curve in Figure 10 shows that the method for diagnosing breast cancer based on CST1 first cleavage expression level has high sensitivity and specificity, and the area under the curve is 0.987. Therefore, the CST1 first cleavage can be used as non-invasive plasma. A specific marker for the diagnosis of breast cancer in the sample.
  • Example 8 LCR detection of differential expression of CST1 first splice in plasma circulating RNA of breast cancer, inflammatory patients and normal subjects
  • the circulating RA in plasma was extracted by commercial kit.
  • the expression of CST1 first cleavage in plasma circulating RA was detected by LCR in 50 patients with breast cancer, 30 patients with mastitis and 30 normal controls.
  • the primer sequence was SEQ.
  • the amplicon sequence is shown in SEQ ID No. 50.
  • the median number of relative light units (RLU) of the first type of CST1 in the cancer was 12.38 times that of inflammation and 40.12 times that of normal; the line was drawn from 18.51 RLU. It can distinguish cancer, inflammation and normal.
  • Example 9 tSDA detects differential expression of CST1 first cleavage in plasma circulating RNA of breast cancer, inflammatory patients and normal subjects
  • the circulating RA in plasma was extracted by commercial kit, tSDA was used to detect the difference of CST1 first cleavage in 50 patients with breast cancer, 30 patients with mastitis and 30 normal controls.
  • the primer sequence was SEQ. As shown in ID No. 1 and SEQ ID No. 2, the amplicon sequence is shown in SEQ ID No. 50.
  • the median number of RLU of the first type of CST1 in cancer was 41.3 times that of inflammation and 42.86 times that of normal; from the line of 24.81 RLU, cancer, inflammation and normal were distinguished. .
  • NASBA detects differences in expression of CST1 first splice in urine circulating RNA in breast cancer, inflammatory patients and normal subjects
  • NASBA detected differential expression of CST1 first cleavage in 20 patients with breast cancer, 10 patients with mastitis, and 10 normal subjects with urinary circulation RA.
  • Primer sequences As shown in SEQ ID No. 1 and SEQ ID No. 2, the amplicon sequence is shown as SEQ ID No. 50.
  • the median number of RLU in the first type of CST1 in cancer was 18 times that of inflammation and 18.87 times that of normal; from 22.93 RLU, it was possible to distinguish cancer, inflammation and normal. That is, 22.93 RLU can be used as a reference value for breast cancer diagnosis in non-invasive urine samples.
  • Example 11 Detection of CST1 first cleavage expression level in peripheral blood of breast cancer patients compared with cytological detection RA was extracted from peripheral blood nucleated cells of breast cancer patients excluding red blood cells and platelets, and CST1 was detected by real-time PCR absolute quantitative method.
  • the first cleavage expression level of CST1 can confirm the positive result of cytological identification by 100%, and can detect the case of partial metastasis in the case of negative cytological identification, indicating that CST1 is the first type.
  • Shear expression level detection is more sensitive than cytology and can detect the presence of micrometastases that are not detectable by cytology.
  • Example 12 Comparison of the expression level of the first cleavage of CST1 in breast cancer patients compared with cytology.
  • the bone marrow of breast cancer patients obtained by biopsy sampling was detected by real-time PCR absolute quantitative method to detect the expression level of CST1 first cleavage.
  • the primer sequence is as shown in SEQ ID No. 1 and B SEQ ID No. 2
  • the amplicon sequence is as shown in SEQ ID No. 50
  • whether the first type of cleavage of CST1 is determined by comparison with the expression level of normal bone marrow High expression, thereby confirming the presence or absence of metastasis and micrometastasis in the bone marrow, and comparing it with the results of cytology.
  • the results are shown in Figure 15.
  • the first cleavage expression level of CST1 can confirm the positive result of cytological identification by 95%, and the positive rate is higher than that of cytology. This indicates that the sensitivity of CST1 first spit expression level detection is higher than that of cytology.
  • Example 13 CST1 first cleavage expression level for breast cancer pTNM staging
  • the circulating RA in plasma was extracted by commercial kit, and the expression of CST1 first splice in plasma circulating RA was detected by TMA method in different pTNM stages of 80 cases of breast cancer (I+II 30 cases, III+IV 50 cases).
  • the primer sequence is shown in SEQ ID No. 1 and SEQ ID No. 2, and the amplicon sequence is shown in SEQ ID No. 50.
  • the results are shown in Figure 16.
  • the median RLU of the first cleavage of CST1 in breast cancer ⁇ +IV is 15 times that of ⁇ + ⁇ , indicating that the expression of the first cleavage of CST1 can be used for breast cancer pTNM staging. .
  • Example 14 CST1 first cleavage expression level for dynamic monitoring during breast cancer treatment
  • the expression level of the first cleavage of CST1 in the blood of the patient is detected by real-time quantitative PCR, the primer sequence is shown in SEQ ID No. 1 and SEQ ID No. 2, and the amplicon sequence is shown in SEQ ID No. 50.
  • Real-time monitoring of the treatment effect of breast cancer patients (6 patients with chemotherapy and 4 patients with radiotherapy). The results are shown in Table 2.
  • the expression level of the first type of CST1 in the blood of the therapeutically effective patients gradually decreased with the increase of the course of treatment.
  • the imaging findings showed that the breast cancer mass gradually decreased; while the CST1 in the blood of patients with treatment failure
  • the expression level of a kind of cleavage gradually increased with the increase of the course of treatment, and the imaging observation showed that the breast cancer lumps gradually became larger. Therefore, the expression level of the first type of CST1 can be used as an evaluation index for the treatment of breast cancer patients, and it can be used for dynamic monitoring during breast cancer treatment.
  • Example 15 CST1 first cleavage expression level for prognosis of breast cancer patients
  • the expression level of the first cleavage of CST1 in the blood of the patient is detected by real-time quantitative PCR, the primer sequence is shown in SEQ ID No. 1 and SEQ ID No. 2, and the amplicon sequence is shown in SEQ ID No. 50.
  • the prognostic effects of 5 breast cancer patients were examined at 1 month, 3 months, and 1 year. The results are shown in Table 3.
  • the expression of the first type of CST1 in the blood of two patients who relapsed after one year gradually increased with time, and was confirmed by imaging findings when it reached about 1000 copies;
  • the expression of CST1 first cleavage in the blood of three patients who did not relapse during the year did not change significantly with time, and no abnormalities were found in imaging. Therefore, the expression level of the first type of CST1 can be used as an indicator of prognosis in breast cancer patients.
  • the first cleavage detection kit includes at least the following components: 1 Primer for CST1 first cleavage: Upstream primer: 5'- tctcaccctcctctcctg -3 ' ( SEQ ID No. 1 ); downstream primer: 5'-ttatcctatcctcctcttgg-3, (SEQ ID No. 2); 2 probe for CST1 first cleavage: 5*-ctccagctttgtgctctgcctct-3* (SEQ ID No.3), the 5' end is labeled with FAM and the 3' end is labeled with TAMRA.
  • RNA extraction reagent reverse transcription reagent
  • deoxyribonucleotide dNTP
  • buffer MgCl 2
  • DNA polymerase containing the first cut of CST1 Recombinant plasmid standard for the amplicon (sequence shown in SEQ ID No. 51) [its map is shown in Figure 1; primer for CST1 first cleavage: upstream primer: 5 '-gggctccctgcctcgggctctcac-3 ' ( SEQ ID No. 22); downstream primer: 5'-acggtctgttgcctggct-cttagt-3' (SEQ ID No. 23)] positive control (breast cancer cell line HCC1937), negative control (human normal breast cell line Hs578Bst).
  • dNTP deoxyribonucleotide
  • buffer MgCl 2
  • DNA polymerase DNA polymerase
  • the experimental group, the positive control group, the negative control group and the recombinant plasmid standard were subjected to real-time PCR amplification, and the concentration gradient of the recombinant plasmid standard and the corresponding CP value after amplification were used as a standard curve, and then according to the standard.
  • the curves give the copy number of the experimental group, the positive control group, and the negative control group.
  • Example 17 CST1 first shear detection kit based on dye method real-time PCR
  • Primer for the first cleavage of CST1 1 upstream primer: 5'-tctcaccct-ccctctcctg-3' (SEQ ID No. 1); downstream primer: 5 '-ttatcctatcctcctcttgg-3 ' (SEQ ID No. 2); 2 upstream reference 5'-ccctgggagaacagaaggtcc-3, (SEQ ID No. 4); downstream primer: 5'-ggtggtggctggtgcgaat-3, (SEQ ID No.
  • 3 upstream primer 5'-cattcgcaccagccac-3, (SEQ ID No .6); downstream primer: 5'-agaagcaa-gaaggaaggagggag-3' (SEQ ID No. 7); 4 upstream bow I: 5 '-cagcgtgcccttcacttcg-3 ' (SEQ ID No. 8); downstream primer: 5 ' -cggtctgttgcctggctctta-3 ' (SEQ ID No. 9); 5 upstream primer: 5'-cattcgcaccagcca-ccac-3' (SEQ ID No.
  • downstream primer 5 '-cagggctatagaagcaagaaggaa-3 ' (SEQ ID No. ll 6 upstream primer: 5 '-ggtacagcgtgcccttcacttc-3 ' (SEQ ID No. 12); downstream primer: 5 '-cggtctgttgcctggctctta-3 '
  • upstream primer 5 '-gagaacagaaggtccctggtgaa-3 ' (SEQ ID No. 14); downstream primer: 5'-ggtggtggctggtgcgaat-3, (SEQ ID No. 15); 8 upstream primer: 5 '-tgggtacagcgtgcccttca-3,
  • Primers for the internal reference gene ⁇ -actin upstream primer: 5'- aagatcattgctcctcctg-3' (SEQ ID No. 32); downstream primer: 5'-cgtcatactcctgcttgc- 3' (SEQ ID No. 33); RNA extraction reagent, reverse transcription reagent, fluorescent dye (such as SYBR Green) dNTP, buffer, MgCl 2 , DNA polymerase.
  • Primer for CST1 first cleavage Upstream primer: 5'-aattctaatacgactca- ctatagggtctcaccctcctctcctg-3 ' (SEQ ID No. 34); Downstream primer: 5 '-ttatcctatcctcctcttgg-3 ' (SEQ ID No. 2); 2 probe for the first cleavage of CST1: 5'-ctccagctttgtgctctgcctct-3' (SEQ ID No. 3), the 5' end is labeled with FAM and the 3' end is labeled with DABSYL.
  • RA extraction reagent may also include any one or more of the following components: RA extraction reagent, reverse transcription reagent, RA fluorescent dye (such as Ribo-Green fluorescent dye), T7 RNA polymerase, RNase H, avian leukosis virus (AMV) Reverse transcriptase, ribonucleotide (NTP), dNTP.
  • RA fluorescent dye such as Ribo-Green fluorescent dye
  • T7 RNA polymerase such as Ribo-Green fluorescent dye
  • RNase H avian leukosis virus (AMV) Reverse transcriptase
  • NTP ribonucleotide
  • Primer for CST1 first cleavage Upstream primer: 5'-aattctaatacgactc-actatagggtctcaccctcctctcctg-3 ' (SEQ ID No. 34); Downstream primer: 5 '-ttatcctatcctcctcttgg-3 ' ( SEQ ID No. 2); 2 probe for the first cleavage of CST1: 5'-ctccagctttgtgctctgcctct-3' (SEQ ID No. 3), the 5' end is labeled with FAM and the 3' end is labeled with DABSYL.
  • RA extraction reagent may also include any one or more of the following components: RA extraction reagent, reverse transcription reagent, RA fluorescent dye (eg Ribo-Green fluorescent dye), T7 RA polymerase, RNase H, avian myeloid leukemia virus (AMV) reverse transcriptase, NTP, dNTP.
  • RA fluorescent dye eg Ribo-Green fluorescent dye
  • T7 RA polymerase eg Ribo-Green fluorescent dye
  • RNase H avian myeloid leukemia virus (AMV) reverse transcriptase
  • AMV avian myeloid leukemia virus
  • Example 20 LCR-based CST1 first shear quantitative assay kit
  • At least the following four probes are included: 5'-agtatctgagtaccctgctgctcctgc-3, (SEQ ID No. 35); 5'-accctagct- gtggccctggcctggag-3 ' (SEQ ID No. 36); 5 '-catagactcatgggacgacg-3 ' (SEQ ID No. 37 ); 5'- acaccgggaccggacctc-3' (SEQ ID No. 38); The 5' end of each probe is labeled with a hapten.
  • R A extraction reagent reverse transcription reagent
  • T4 DNA ligase dNTP
  • CST1 B1 bow I 5'-tgggtacagc- gtgcccttcactt-3' (SEQ ID No. 39);
  • Material 5 '- Ccgctcgagtacagcgtgcccttcacttcgc-3 ' (SEQ ID No. 40);
  • CST1 B21 5 '-caacggtctgttgcctggctctta-3 ' (SEQ ID No.
  • CST1 S2 Primer 5 '-gacctcgaggttgcctggctcttagtacccg-3 ' (SEQ ID No. 42 2)
  • the probe for the first cleavage of CST1 5 '-gtgctcgagtcagcgagtataacaaggccaccaaagatgactac-3 ' (SEQ ID No. 43), and the 5' end was labeled with 32 P.
  • Cystatin SN recombinant protein used in the preferred embodiment rabbit anti-Cystatin SN polyclonal antibody was purchased from NOVUS Biologicals, mouse anti-human Cystatin SN monoclonal antibody (specific recognition sequence such as Cystatin SN epitope shown in SEQ ID No. 54) Purchased from R&D, TMB Peroxidase Substrate (containing TMB solution A and Peroxidase Solution B) is hooked from Kirkegaard and Perry Laboratories Inc.
  • the serum sample is prepared by placing the whole blood sample at room temperature for 2 hours or 4 ° C overnight, centrifuging at 1000 g for 20 minutes, taking the supernatant for detection, or storing at -20 ° C or -80 ° C to avoid repeated freezing and thawing. .
  • the plasma sample is prepared by using EDTA or heparin as an anticoagulant. The specimen is centrifuged at 2 to 8 ° C and 1000 g for 15 minutes within 30 minutes after collection. The supernatant can be detected, or set at - 20 ° C or -80 ° C. Save, avoid repeated freezing and thawing.
  • the serum or plasma samples were diluted 10 times with PBS pH 7.0 to 7.2 and 0.1 M, and then measured.
  • Example 22 Differential expression of Cystatin SN in culture supernatant of human breast cancer cell lines and normal human serum
  • the protein was electrotransferred onto a nitrocellulose membrane containing 5% skim milk powder and 0.1% Tween- 20 PBS was blocked at room temperature for 2 hours, added anti-Cystatin SN polyclonal antibody, incubated overnight at 4 ° C, washed 3 times with PBS containing 0.1% Tween-20, and then labeled with horseradish peroxidase (HRP).
  • HRP horseradish peroxidase
  • the goat anti-rabbit IgG was incubated at 37 °C for 1 hour, washed 4 times with PBS containing 0.1% Tween-20, and once with PBS, and then detected by TMB peroxidase substrate, with ⁇ - Actin is an internal reference protein.
  • ⁇ - Actin is an internal reference protein.
  • a strong band of about 16 Kda was detected in lanes 1-4, and only a weak band of about 16 Kda was detected in lanes 5-8, indicating that the CST1-encoded Cystatin SN was abnormally high in human breast cancer cells. expression.
  • Example 23 Differential expression of Cystatin SN in serum of breast cancer patients and normal subjects
  • Serum samples from normal subjects (lanes 1-2) and serum samples from breast cancer patients (lanes 3-6) were subjected to SDS-PAGE, protein electroporation and immunoblotting as described in Example 22.
  • SDS-PAGE Serum samples from normal subjects
  • serum samples from breast cancer patients (lanes 3-6) were subjected to SDS-PAGE, protein electroporation and immunoblotting as described in Example 22.
  • Fig. 18 As a result, as shown in Fig. 18, only weak bands were detected in lanes 1-2, and strong bands were detected in lanes 3-6, indicating that Cystatin SN was abnormally highly expressed in the serum of breast cancer patients.
  • Example 24 Competition ELISA for differential expression of Cystatin SN in breast cancer patients and normal humans ELISA plates were coated with 5 ug/ml Cystatin SN, and plates were blocked with 3% BSA, serum samples and anti-Cystatin SN monoclonal antibodies (1) :2000 ) Incubate for 1 hour at 37 ° C, wash with TBS (pH 7.5 containing 154 mM NaCl, 10 mM Tris-HCl), and add 0.08 ug/ml HRP-labeled goat anti-rabbit IgG at 37 ° C.
  • TBS pH 7.5 containing 154 mM NaCl, 10 mM Tris-HCl
  • Example 25 Double-anti-sandwich ELISA assay for differential expression of Cystatin SN in serum of breast cancer patients and normal humans ELISA plates were coated with 5 ug/ml mouse anti-human Cystatin SN monoclonal antibody, and plates were blocked with 3% BSA, serum samples were added. Incubate for 1 hour at 37 ° C, wash with TBS, add biotinylated rabbit anti-Cystatin SN polyclonal antibody (1:1000) for 1 hour at 37 ° C, wash with TBS, then add avidin - The peroxidase complex was incubated at 37 ° C for 1 hour, washed with TBS, and TMB peroxidase substrate was added to each well and quantified by enzyme-linked instrument.
  • Example 26 Comparison of Sensitivity and Specificity of Cystatin SN and Carcinoembryonic Antigen (CEA) in Breast Cancer Patients by ELISA
  • Cystatin SN serum levels were determined as described in Example 25.
  • CEA assays were performed using a commercial CEA ELISA kit and operated according to the kit instructions. A total of 30 normal human serum and 30 breast cancer patients were measured. Clear. The results are shown in Figure 21.
  • the area under the Cystatin SN curve is 0.994, and the area under the CEA curve is 0.833, indicating that Cystatin SN is superior to CEA in sensitivity and specificity.
  • coated antigen (Cystatin SN); primary antibody (murine anti-human Cystatin SN monoclonal antibody); enzyme-labeled secondary antibody (HRP-labeled goat anti-rabbit IgG); enzyme substrate (o-phenylenediamine) ).
  • BSA blocking solution
  • TBS washing buffer
  • the ELISA plate was coated with 5 mg/ml Cystatin SN, and the plate was blocked with 3% BSA.
  • the serum sample was incubated with anti-Cystatin SN monoclonal antibody (1:2000) for 1 hour at 37 ° C, washed with TBS, and then added with 0.08 ug. /ml HRP-labeled goat anti-rabbit IgG was incubated at 37 ° C for 1 hour, washed with TBS, and then added with 0.4 mg / ml o-phenylenediamine (dissolved in pH 5, 500 mM phosphate-citrate buffer). Quantitatively determined by enzyme-linked instrument.
  • Example 28 Cystatin SN quantitative detection kit based on double-antibody sandwich ELISA
  • Cystatin SN standard prepared or dissolved in PBST containing 1% BSA before use to prepare a series of gradient solutions, such as concentrations of 10, 5, 2.5, 1, 0.5, 0.25 ng / ml
  • Coated antibody mouse anti-human Cystatin SN monoclonal antibody
  • biotinylated secondary antibody biotinylated rabbit anti-Cystatin SN polyclonal antibody
  • avidin-peroxidase complex peroxidase bottom (TMB peroxidase substrate).
  • coated antibody dilution pH 9, 0.05M NaHC0 3
  • blocking solution PBST containing 3% BSA
  • secondary antibody dilution containing 1% BSA
  • wash buffer PBST, ie PBS containing 0.05% Tween-20
  • stop solution 2N H 2 S0 4
  • the coated antibody was diluted to a protein content of 5 g/ml with a coating antibody buffer, added to an ELISA plate, 0.1 ml per well, incubated overnight at 4 ° C, the liquid in the well was discarded, and washed 3 times with washing buffer. , every 3 minutes. 200 ul of blocking solution was added to each well, incubated at 37 ° C for 1 hour or overnight at 4 ° C, and the liquid in the well was discarded and washed 3 times with washing buffer for 3 minutes each time.
  • avidin-peroxidase complex 100 ⁇ l was added to each well, and the mixture was incubated at 37 ° C for 1 hour, and the liquid in the well was discarded, and washed 5 times with a washing buffer for 1 to 2 minutes each time.
  • the reaction was stopped by adding 50 ⁇ l of the stop solution to each well (blue to yellow).
  • the optical density (OD value) of each well was measured by an enzyme-coupled instrument at a wavelength of 405 nm.
  • a scatter curve is drawn based on the standard concentration and the corresponding OD value.
  • the R 2 and curve equations are calculated. For example, R 2 0.95, the OD value of the sample is substituted into the curve equation to calculate the concentration of Cystatin SN in the serum or plasma sample.
  • Example 29 Cystatin SN expression levels for breast cancer pTNM staging
  • Example 28 Using the kit described in Example 28 and its method of use, the expression levels of Cystatin SN in serum of 20 patients with pathologically confirmed breast cancer T, 30 patients with N phase, and 30 patients with M phase were detected. The results are shown in Table 4. It can be seen that as the disease progresses, the expression level of Cystatin SN also increases, suggesting that the expression level of Cystatin SN can be used for breast cancer pTNM staging.
  • Example 30 Cystatin SN expression levels were used to assess whether breast cancer metastasized
  • Example 28 The kit of the method described in Example 28 and the method of using the same were used to detect the expression level of Cystatin SN in 20 patients with pathologically confirmed breast cancer non-metastatic patients and 30 patients with metastatic patients. The results are shown in Table 5, and the breast cancer metastasis patients can be seen. Cystatin SN expression levels were higher than those without metastasis, suggesting that Cystatin SN expression levels can be used to assess whether breast cancer metastasizes.
  • Example 31 Cystatin SN expression levels are used to assess the efficacy of breast cancer chemotherapy combined with endocrine therapy
  • the disease-free survival rate was higher in patients with a median expression above Cystatin SN of 20%, and the disease-free survival rate was lower in patients with a median expression of Cystatin SN of 60%, suggesting Cystatin SN expression.
  • Levels can be used to assess the efficacy of breast cancer chemotherapy combined with endocrine therapy.

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Abstract

提供SEQ ID NO.48所示的CST1基因剪切子和SEQ ID NO.52所示的CST1基因编码蛋白Cystatin SN作为乳腺癌诊断和预示标志物的应用。通过测定这些标志物的表达量，可以进行乳腺癌或乳腺癌组织转移的鉴别诊断和/或易感性分析，进行乳腺癌或乳腺癌组织转移治疗药物、治疗方法、治疗疗效及预后的评估，进行相关人群乳腺癌或乳腺癌组织转移患病风险的评估等，具有灵敏度高、特异性好、结果准确可靠的优点。还提供了所述CST1基因剪切子的捕获剂和Cystatin SN的捕获剂在制备乳腺癌诊断和预示试剂、试剂盒及芯片中的应用，以及提供含有CST1基因剪切子的捕获剂和Cystatin SN的捕获剂的乳腺癌诊断和预示试剂盒和芯片。

Description

乳腺癌诊断和预示的标志物 技术领域 本发明属于生物医学技术领域， 涉及一种可用于肿瘤诊断和预示的分子标志物， 以及用 于检测该分子标志物的试剂、 试剂盒和芯片。 背景技术
据世界卫生组织 （WHO) 统计， 全世界每年约有 120万妇女罹患乳腺癌， 约有 54万乳 腺癌患者死亡， 且乳腺癌的发病率及致死率还在逐年上升。 因此， 乳腺癌的防治是目前亟待 解决的问题， 包括如何更早发现、 及时干预， 如何实现疗效评估和监测， 以及如何对术后患 者进行实时准确的复发监控等。 其中， 灵敏度高、 特异性强的乳腺癌诊断试剂的研制是提高 乳腺癌早期检出率、 改善患者预后的关键之一。
近年来， 随着对肿瘤发病机理研究的不断深入， 发现 Cystatin家族作为组织蛋白酶主要 是半胱氨酸蛋白酶的内源性抑制剂， 在肿瘤的发生、 发展、 浸润和转移过程中起着非常重要 的作用。 研究结果显示， Cystatin家族的几个成员在不同肿瘤中的表达水平有所上升， 例如 Cystatin C在卵巢癌和头颈部癌症中的表达有不同水平的上升， stefm A在非小细胞肺癌中的 表达水平有所增加， Cystatin F 在多种肿瘤中的表达水平都有显著增加， 很可能是由于在肿 瘤发生、 发展过程中需要组织蛋白酶的参与， 先诱导其表达量增加， 再激活机体本身的应激 机制， 导致 Cystatin的表达量上升以抑制过盛的组织蛋白酶活性。 但 Cystatin的表达量并不 与肿瘤的发展成正相关关系， 例如在胶质瘤中， Cystatin C 的低表达意味着疾病的晚期， 患 者的生存期较短， 且易于复发， 这可能是到了肿瘤的后期阶段， 又会有一些其他的机制对 Cystatin的水平进行调控， 以促进肿瘤的进一步恶化。
Cystatin SN是人类 Cystatin家族成员， 由 CST1基因编码， 含 141个氨基酸， 分子中有 两个二硫键， 分子量为 16.4Kda， 为典型的分泌蛋白， 分布于多种体液和分泌物中， 如眼 泪、 唾液、 血清、 血浆等。 文献报道， CST1 在胃癌组织中的表达量高于正常胃粘膜组织， 在胃癌细胞系中的表达部位与胃癌组织较一致， 且表达率随细胞系的分化程度降低而降低； 临床资料分析表明， CST1 的表达与浸润深度、 远处转移以及 TNM 分期相关； 生存分析表 明， CST1表达阳性组的 5年生存率显著高于无表达组； Cox回归分析表明， CST1是一个独 立预后因子； 从而提示 CST1 可能在胃癌的发生、 发展过程中发挥类似肿瘤抑制因子的作 用。 但迄今为止， CST1与乳腺癌的关系尚未见文献报道。 发明内容 有鉴于此， 本发明的目的在于研究 CST1在乳腺癌中的表达情况， 并分析其与乳腺癌临 床病理参数和生物学行为的关系， 进而发现 CST1在乳腺癌发生、 发展、 转移过程中所起的 作用。
首先， 从 mRNA水平， 本发明比较了 CST1在人体不同组织中的表达， 发现 CST1在除 唾液腺外的人体正常组织中均低表达， 这对于 CST1用于病理条件下检测极为有利。 接着， 本发明比较了 CST124、 CST1、 CST2、 CST4在乳腺癌与癌旁组织手术标本中的表达差异， 发现 CST1在乳腺癌与癌旁组织中的表达差异最大。 由于 CSTl mRNA ( SEQ ID No.44) 选 择性剪切形成 2种剪切子， 第一种剪切子 （SEQ ID No.48 ) 包含 CSTl外显子 1 ( SEQ ID No.45 ) 外显子 2 ( SEQ ID No.46) 和外显子 3 ( SEQ ID No.47), 第二种剪切子 ( SEQ ID No.49) 仅包含 CSTl 外显子 1 ( SEQ ID No.45 ) 和外显子 2 ( SEQ ID No.46), 本发明接着 比较了 CSTl 两种剪切子在乳腺癌与癌旁组织手术标本中的表达差异， 发现第一种剪切子在 癌与癌旁组织中的表达差异程度优于第二种剪切子。 接着， 本发明分别比较了 CST1第一种 剪切子在乳腺癌、 癌旁及正常组织手术标本， 乳腺癌、 乳腺炎与正常组织穿刺标本， 乳腺癌 转移阳性淋巴结与阴性淋巴结， 乳腺癌、 乳腺炎患者与正常人血浆循环 （cell-free) RNA 中 的表达差异， 发现 CST1第一种剪切子在乳腺癌及乳腺癌转移组织中异常高表达， 通过测定 其表达量， 可以将乳腺癌、 癌旁与正常组织， 乳腺癌转移阳性组织与阴性组织区分开来， 灵 敏度高， 特异性好。
再次， 从蛋白水平， 本发明比较了 CST1编码蛋白 Cystatin SN (氨基酸序列如 SEQ ID No.52所示， 编码基因序列如 SEQ ID No.53所示） 在乳腺癌细胞株培养上清与正常人血清、 乳腺癌患者血清与正常人血清中的表达差异， 发现 Cystatin SN在乳腺癌细胞株培养上清与 乳腺癌患者血清中异常高表达， 通过测定其表达量， 可以将乳腺癌细胞株培养上清与正常人 血清、 乳腺癌患者血清与正常人血清区分开来， 灵敏度高， 特异性好。
综合上述研究结果， CST1第一种剪切子和 Cystatin SN可以作为乳腺癌诊断和预示的分 子标志物应用。 根据本领域的公知常识， Cystatin SN的表位肽也同样可以作为乳腺癌诊断和 预示的分子标志物应用， Cystatin SN表位肽的氨基酸序列可以如 SEQ ID No.54所示 （即去 除 Cystatin SN前端的分泌信号肽序列）。
通过测定上述分子标志物的表达量， 可以进行乳腺癌的诊断和预示， 包括乳腺癌或乳腺 癌组织转移的鉴别诊断和 /或易感性分析， 乳腺癌或乳腺癌组织转移治疗药物、 治疗方法、 治疗疗效及预后的评估， 相关人群乳腺癌或乳腺癌组织转移患病风险的评估等。 例如， 方法 之一： 测定待测样品中 CST1 第一种剪切子或 Cystatin SN的含量或表达水平， 将检测结果 与阈值进行比较， 如果超过阈值则判断为阳性； 所述阈值可通过对比正常人与乳腺癌患者体 液或组织中 CST1第一种剪切子或 Cystatin SN的含量或表达水平统计得到。
测定上述分子标志物的表达量需要使用检测试剂、 试剂盒或检测芯片等。 因此， 本发明 进一步研究了上述分子标志物的检测试剂、 试剂盒和检测芯片， 以用于乳腺癌诊断和预示。
检测 mR A表达的方法有很多种， 包括但不限于多聚酶链式反应 （polymerase chain reaction, PCR)、 实时定量 PCR (quantitative real-time PCR) 基于核酸序列扩增法 （Nucleic Acid based Amplificatin, NASBA )、 转录介导的扩增 ( Transcription-median amplification, TMA)、 连接酶链反应 (Ligase chain reaction, LCR)、 适温链置换反应 (thermophilic strand displacement amplification, tSDA) 等。 根据上述检测方法， 只要特异性识别 CST1第一种剪 切子或其 cDNA的引物或探针 （统称为 CST1第一种剪切子的捕获剂） 都可以制成乳腺癌诊 断和预示试剂应用。 本发明优选特异性识别 CST1 外显子 1 的引物或探针或特异性识别 CST1 外显子 2和 3 的引物或探针， 更优选特异性识别 CST1 外显子 1 的引物或探针。 例 如， 所述引物优选为核苷酸序列如 SEQ ID No.l〜2、 4〜21、 34和 39〜42所示的至少一种， 更 优选如 SEQ ID No.l和 SEQ ID No.2所示； 所述探针优选为核苷酸序列如 SEQ ID No.3、 35-38和 43所示的至少一种， 更优选如 SEQ ID No.3所示。
检测蛋白表达的方法有很多种， 包括但不限于酶联免疫吸附测定 （ELISA) 例如竞争性 ELISA和双抗夹心 ELISA、 免疫印迹、 ELISA和免疫印迹的组合等。 根据上述检测方法， 抗 Cystatin SN的抗体 （统称为 Cystatin SN的捕获剂） 都可以制成乳腺癌诊断和预示试剂应 用。 所述抗体可以是多克隆抗体或单克隆抗体， 可以被各种类型的酶如碱性磷酸酶、 荧光素 酶、 过氧化物酶、 β-半乳糖苷酶以及各种荧光化合物如荧光素等标记， 也可以被生物素标 记， 再利用亲合素 -酶复合物放大反应信号。 当标记为酶时， 可通过添加酶作用底物使光吸 收发生变化而进行定量。 当标记为荧光化合物时， 可通过用紫外光激发使其发射荧光而进行 定量。
为了检测更方便、 快速， 上述检测试剂可以多种组合或与其它辅助检测试剂组合制成乳 腺癌诊断和预示试剂盒。 商业化的或文献报道的含有上述检测试剂的试剂盒也可以直接制成 乳腺癌诊断和预示试剂盒应用。
作为一种优选的技术方案， 基于 TaqMan水解探针法实时 PCR的 CST1第一种剪切子检 测试剂盒至少包含特异性识别 CST1基因第一种剪切子的 cDNA的 1对引物和 1条探针， 所 述引物的核苷酸序列如 SEQ ID No.l和 SEQ ID No.2所示， 所述探针的核苷酸序列如 SEQ ID No.3所示， 探针的 5'端用荧光基团标记， 3'端用淬灭基团标记。
作为一种优选的技术方案， 基于染料法实时 PCR 的 CST1 第一种剪切子检测试剂盒至 少包含特异性识别 CST1基因第一种剪切子的 cDNA的 1对引物， 所述 CST1基因剪切子的 捕获剂为特异性识别 CST1基因剪切子的 cDNA的引物， 所述引物的核苷酸序列如 SEQ ID No.l〜2所示， 或者如 SEQ ID No.4〜5所示， 或者如 SEQ ID No.6〜7所示， 或者如 SEQ ID No.8〜9所示， 或者如 SEQ ID Νο.10〜11所示， 或者如 SEQ ID Νο.12〜13所示， 或者如 SEQ ID Νο.14〜15所示， 或者如 SEQ ID Νο.16〜17所示， 或者如 SEQ ID Νο.18〜19所示， 或者如 SEQ ID No.20~21所示。
作为一种优选的技术方案， 基于 NASBA或 TMA的 CST1第一种剪切子检测试剂盒至 少包含特异性识别 CST1基因第一种剪切子的 cDNA的 1对引物和 1条探针， 所述引物的核 苷酸序列如 SEQ ID No.34和 SEQ ID No.2所示， 所述探针的核苷酸序列如 SEQ ID No.3所 示， 探针的 5'端用荧光基团标记， 3'端用淬灭基团标记。
作为一种优选的技术方案， 基于 LCR 的 CST1 第一种剪切子检测试剂盒至少包含特异 性识别 CST1基因第一种剪切子的 4条探针， 所述探针的核苷酸序列如 SEQ ID No.35〜40所 示， 每条探针的 5'端用半抗原标记。
作为一种优选的技术方案， 基于 tSDA的 CST1第一种剪切子检测试剂盒至少包含特异 性识别 CST1基因第一种剪切子的 cDNA的 2对引物和 1条探针， 所述引物的核苷酸序列如 SEQ ID No.39〜44所示， 所述探针的核苷酸序列如 SEQ ID No.43所示， 探针的 5'端用放射 性同位素标记。
在上述 CST1 第一种剪切子检测试剂盒中， 还可以视情况加入一种或多种辅助检测试 剂， 所述辅助试剂包括但不限于： ①使所述引物对应的扩增子可视化的反应试剂， 例如通过 琼脂糖凝胶电泳法、 酶联凝胶法、 化学发光法、 原位杂交法、 荧光检测法等使扩增子可视化 的试剂； ② R A 提取试剂； ③逆转录试剂； ④ cDNA 扩增试剂， 例如 PCR、 实时定量 PCR、 NASBA、 TMA、 LCR、 tSDA的相关试剂； ⑤制备标准曲线所用的标准品， 例如包含 CST1 第一种剪切子扩增子的重组质粒； ⑥阳性对照品， 例如人乳腺癌细胞株 HCC1937、 SK-BR-3、 MCF-7; ⑦阴性对照品， 例如人正常乳腺细胞 Hs578Bst。
作为一种优选的技术方案， 基于竞争性 ELISA 的 Cystatin SN 检测试剂盒至少包含 Cystatin SN抗原、 抗 Cystatin SN单克隆抗体、 酶标二抗和酶作用底物。 用 Cystatin SN抗原 包被 ELISA平板， 待测样品与抗 Cystatin SN单克隆抗体依次加于平板进行孵育， 通过报告 组分 （酶标二抗和酶作用底物） 检测结合在平板上的抗 Cystatin SN 单克隆抗体的量， 据此 确定样品中 Cystatin SN的量。
作为一种优选的技术方案， 基于双抗夹心 ELISA的 Cystatin SN检测试剂盒至少包含抗 Cystatin SN单克隆抗体、 生物素标记的抗 Cystatin SN多克隆抗体、 亲合素-酶复合物和酶作 用底物。 用 Cystatin SN单克隆抗体包被平板， 待测样品和生物素标记的抗 Cystatin SN多克 隆抗体依次加于平板进行孵育， 通过报告组分 （亲合素 -酶复合物和酶作用底物） 检测结合 在平板上的抗 Cystatin SN单克隆抗体的量， 据此确定样品中 Cystatin SN的量。
在上述 Cystatin SN检测试剂盒中， 还可以加入一种或多种辅助检测试剂， 所述辅助试 剂包括但不限于： ①封闭液； ②抗体稀释液； ③洗涤缓冲液； ④显色终止液； ⑤制备标准曲 线所用的 Cystatin SN标准品。
为了检测更方便、 快速， 特异性识别 CST1第一种剪切子或其 cDNA的探针还可以固定 在固相载体表面制成乳腺癌诊断和预示芯片。 商业化的或文献报道的固定有上述探针的芯片 也可以直接制成乳腺癌诊断和预示芯片应用。
本发明的检测试剂、 试剂盒或芯片可用于判断乳腺癌是否易感或形成， 进行乳腺癌 pTNM 分期， 评估乳腺癌进展或治疗效果， 判断是否转移、 复发。 受检样品可包括手术组 织、 穿刺组织、 淋巴结组织、 骨髓、 血清样品、 血浆样品、 全血样品、 血液级分样品、 尿样 等。 受试者可以是因乳房不适就医者、 有家族性乳腺癌病史的人群或乳腺癌患者等。
本发明的有益效果在于： 本发明公开了 SEQ ID No.48所示的 CST1基因剪切子和 SEQ ID No.52所示的 CST1基因编码蛋白 Cystatin SN作为乳腺癌诊断和预示的分子标志物的应 用， 通过测定所述分子标志物的表达量， 可以进行乳腺癌或乳腺癌组织转移的鉴别诊断和 / 或易感性分析， 乳腺癌或乳腺癌组织转移治疗药物、 治疗方法、 治疗疗效及预后的评估， 相 关人群乳腺癌或乳腺癌组织转移患病风险的评估等， 灵敏度高， 特异性好， 结果准确可靠； 本发明还公开了所述 CST1 基因剪切子的捕获剂和 Cystatin SN的捕获剂在制备乳腺癌诊断 和预示试剂、 试剂盒及芯片中的应用， 并公开了包含上述捕获剂的乳腺癌诊断和预示试剂 盒、 芯片 附图说明
图 1为包含 CST1第一种剪切子扩增子的重组质粒 （pMD18-T-CSTl ) 图谱。
图 2为 CST1基因在人体正常组织 （扁桃体、 垂体后叶、 甲状腺、 唾液腺、 骨骼肌、 骨 髓、 除去红细胞和血小板的外周血、 肺、 胃、 肝脏、 心脏、 肾、 肾上腺、 肠、 结肠、 胰脏、 脾脏、 膀胱、 前列腺、 乳腺、 卵巢、 子宫、 胎盘和睾丸）、 人乳腺癌细胞株 （HCC1937、 SK-BR-3、 和 MCF-7) 及人正常乳腺细胞株 Hs578Bst中的表达情况。 图 3为染料法实时定量 PCR检测 CST124、 CST1、 CST2、 CST4在乳腺癌及癌旁组织 手术样本中的表达差异。
图 4 为染料法实时定量 PCR检测 ACTB 在乳腺癌及癌旁组织手术样本中的表达， N1-N20和 T1〜T20分别表示 20对乳腺癌、 癌旁组织配对样本。
图 5 为染料法实时定量 PCR 检测 CST1 第一种剪切子 （splicel )、 第二种剪切子 (splice2) 在乳腺癌及癌旁组织手术样本中的表达差异。
图 6为实时 PCR绝对定量法检测 CST1第一种剪切子在乳腺癌、 癌旁及正常组织手术 样本中的表达差异。
图 7为实时 PCR绝对定量法检测 CST1第一种剪切子在乳腺癌及乳腺炎穿刺样本中的 图 8为实时 PCR绝对定量法检测 CST1第一种剪切子在乳腺癌转移阳性淋巴结和阴性 淋巴结中的表达差异。
图 9为实时 PCR绝对定量法检测 CST1第一种剪切子在乳腺癌患者、 乳腺炎患者和正 常人血浆循环 （cell-free) R A中的表达差异。
图 10 为实时 PCR 绝对定量法检测的受试者工作特征曲线 （receiver operator characteristic curve, ROC曲线)。
图 11为连接酶链反应 （Ligase chain reaction, LCR) 检测 CST1第一种剪切子在乳腺癌 患者、 乳腺炎患者和正常人血浆循环 R A中的表达差异。
图 12 为适温链置换反应 (thermophilic strand displacement amplification, tSDA) 检测
CST1第一种剪切子在乳腺癌患者、 乳腺炎患者和正常人血浆循环 R A中的表达差异。
图 13为核酸序列扩增法 （Nucleic Acid based Amplificatin, NASBA) 检测 CST1第一种 剪切子在乳腺癌患者、 乳腺炎患者和正常人尿液循环 R A中的表达差异。
图 14为乳腺癌患者外周血 CST1第一种剪切子表达水平检测与细胞学检测对比。
图 15为乳腺癌患者骨髓 CST1第一种剪切子表达水平检测与细胞学检测对比。
图 16为转录介导的扩增 （Transcription-mediated amplification, TMA) 检测 CST1第一 种剪切子在乳腺癌不同 pTNM分期血浆循环 R A中的表达差异。
图 17为 Cystatin SN在乳腺癌细胞株培养上清和正常人血清中的表达差异。
图 18为 Cystatin SN在正常人血清和乳腺癌患者血清中的表达差异。
图 19为竞争 ELISA法测定正常人和乳腺癌患者血清中 Cystatin SN的表达差异。
图 20为双抗夹心 ELISA法测定正常人和乳腺癌患者血清中 Cystatin SN的表达差异。 图 21 为 ELISA测定乳腺癌患者血清 Cystatin SN和癌胚抗原 （CEA) 的灵敏度和特异 性比较。
图 22为乳腺癌患者经治疗后高于 Cystatin SN蛋白表达中位数组及低于该中位数组的无 病生存曲线。 具体实施方式 为了使本发明的目的、 技术方案和优点更加清楚， 下面将结合附图对本发明的优选实施 例进行详细的描述。 优选实施例中未注明具体条件的实验方法， 通常按照常规条件， 例如分 子克隆实验指南 （第三版， J.萨姆布鲁克等著， 黄培堂等译， 科学出版社， 2002 年） 中所述 的条件， 或按照制造厂商所建议的条件。 除非另有说明， 优选实施例中所述百分比均为质量 百分比。
(一） mRNA 7j平
优选实施例中所用标本均是在与病人签署知情同意书后按医院规定途径获得。 穿刺活检 获取的病理部位标本与非病理部位样本作为对比。 手术中获取的淋巴结等样本立即提取 R A或置于液氮或 R Alater (Ambion公司） 中保存； 外周血、 骨髓或尿液样本先在 4°C、 4000rpm条件下离心 20分钟， 取上清， 再在 4°C、 13000rpm条件下离心 10分钟， 分离上清 和沉淀， 立即提取 R A或置 -20〜- 80°C保存。
实施例 1.人体不同组织中的 CST1表达差异
本实施例中使用的人体组织标本除正常乳腺组织标本为发明人从合作医院采集外， 其它 各组织标本均从正规商业渠道获得。 采用 Affymetrix的核苷酸芯片 HG-U95Av检测人体不同 组织标本中 CSTl mRNA的相对表达量， 具体操作参照核苷酸芯片说明书， 相对表达量的值 为通过管家基因 β -actin荧光值进行归一化处理后的标准化信号值。 检测结果如图 2所示， CST1 除在唾液腺中的表达量较高外， 在其它组织中均不表达， 说明 CST1 表达在正常组织 中的背景值很低， 这对 CST1 用于病理条件下检测极为有利。 此外， CST1 在人乳腺癌细胞 株 HCC1937、 SK-BR-3禾 B MCF-7 中过表达， 在人正常乳腺细胞 Hs578Bst中不表达， 说明 CST1极有可能作为乳腺癌分子诊断的标志物。
实施例 2.乳腺癌及癌旁组织手术样本中 CST124、 CST1、 CST2、 CST4的表达差异 采用染料法实时定量 PCR 分别检测 20 对乳腺癌、 癌旁组织手术样本中 CST124、 CST1、 CST2、 CST4 mRNA的相对表达量。 样本总 R A的提取采用 Trizol试剂。 R A逆 转录为 cDNA采用商业化的逆转录试剂盒并按照试剂盒说明书进行操作。 目的基因的 PCR 扩增引物序列见表 1。 荧光染料为 SYBR Green, Eve Green, LC Green等。 检测结果如图 3 CST124、 CST1、 CST2、 CST4的 PCR扩增引物序列
Figure imgf000010_0001
实施例 3.乳腺癌及癌旁组织手术样本中 CST1第一种剪切子、 第二种剪切子的表达差 异
首先， 采用染料法实时定量 PCR检测 20 对乳腺癌、 癌旁组织手术样本中管家基因 ACTB的表达量。 检测结果如图 4所示， 各样本中 ACTB的 CP ( cross point) 值基本一致。 然后， 采用染料法实时定量 PCR检测 CST1第一种剪切子、 第二种剪切子在 20对乳腺癌、 癌旁组织手术样本中癌旁与对应癌组织的 CP值之差的中位数， 具体方法和引物序列参照实 施例 2。 检测结果如图 5 所示， CST1 第一种剪切子在乳腺癌及癌旁组织中的表达量差异明 显优于第二种剪切子， 提示 CST1第一种剪切子在判别是否为癌时更具有优越性。
实施例 4.乳腺癌、 癌旁及正常组织手术样本中 CST1第一种剪切子的表达差异 采用实时 PCR绝对定量法检测 CST1 第一种剪切子在 100例乳腺癌、 癌旁及正常组织 手术样本中的表达差异， 引物序列如 SEQ ID No.l和 SEQ ID No.2所示， 扩增子序列如 SEQ ID No.50所示。 检测结果如图 6所示， CST1第一种剪切子在乳腺恶性病理条件下特异性高 表达， 而在癌旁及正常组织低表达， 癌标本拷贝的中位数是正常组织标本拷贝中位数的 15.6 倍； 从 223.20 拷贝处划线， 可将癌组织与正常组织区分开来， 因此， 223.20 拷贝可作为手 术取样组织样本乳腺癌诊断的一个参考值。
实施例 5.乳腺癌及乳腺炎穿刺样本中 CST1第一种剪切子的表达差异
穿刺取样与手术取样的样本具有较大差别， 主要表现在穿刺取样的样本中乳腺癌细胞的 比例变动较大， 有时可能仅占到整块样本的很小一部分， 有时甚至没有。 因此， 本实施例采 用实时 PCR绝对定量法检测了 CST1第一种剪切子在 40例乳腺癌、 乳腺炎患者穿刺样本中 的表达差异， 引物序列如 SEQ ID No.l和 SEQ ID No.2所示。 检测结果如图 Ί所示， CST1 第一种剪切子在乳腺癌标本中特异性高表达， 而在乳腺炎标本中低表达， 癌标本拷贝的中位 数是炎标本拷贝中位数的 11.0 倍； 从 120.66 拷贝处划线， 可将癌与炎区分开来， 因此， 120.66拷贝可作为穿刺取样样本乳腺癌诊断的一个参考值。
实施例 6.乳腺癌转移阳性淋巴结和阴性淋巴结中 CST1第一种剪切子的表达差异 手术获得经病理证明为乳腺癌转移阳性的淋巴结 30 枚， 且转移灶大小不等。 病理阴性 淋巴结 30 枚主要取自早期乳腺癌患者， 尽可能减少病理诊断阴性但实际有微转移存在的淋 巴结， 以避免实验误差。 采用实时 PCR绝对定量法检测 CST1 第一种剪切子在乳腺癌转移 阳性淋巴结和阴性淋巴结中的表达差异， 引物序列如 SEQ ID No.l和 SEQ ID No.2所示， 扩 增子序列如 SEQ ID No.50所示。 检测结果如图 8所示， CST1第一种剪切子在转移阳性淋巴 结中高表达， 而在阴性淋巴结中低表达， 转移阳性淋巴结标本拷贝的中位数是阴性淋巴结标 本拷贝中位数的 6.3倍； 从 82.45拷贝处划线， 阴性淋巴结中有 2例检出 CST1第一种剪切 子弱阳性， 经连续切片细致观察发现， 这 2例阴性淋巴结有微转移的存在， 因此， 82.45 拷 贝不仅 100%区分了细胞学检测结果， 而且能检测出细胞学不能检出的有微转移存在的淋巴 结， CST1第一种剪切子拷贝数检测相比细胞学检测具有更高的灵敏度。
实施例 7. PCR检测乳腺癌、 炎患者及正常人血浆循环 RNA中 CST1第一种剪切子的 表达差异
采用商品化试剂盒抽提血浆中的循环 （Cell-free) RNA, 实时 PCR绝对定量法检测 50 例乳腺癌患者、 30例乳腺炎患者与 30例正常人血浆循环 R A中 CST1第一种剪切子的表 达差异， 引物序列如 SEQ ID No.l和 SEQ ID No.2所示， 扩增子序列如 SEQ ID No.50所 示。 结果如图 9所示， CST1第一种剪切子在癌中拷贝的中位数分别是炎的 8.8倍和正常的 32 倍； 从 65.23 拷贝处划线， 可将癌、 炎和正常区分开来。 图 10 的 ROC 曲线显示基于 CST1 第一种剪切子表达水平诊断乳腺癌的方法具有较高的灵敏度和特异性， 曲线下面积为 0.987， 因此， CST1第一剪切子可作为非侵入性血浆样本乳腺癌诊断的特异性标志物。
实施例 8. LCR检测乳腺癌、 炎患者及正常人血浆循环 RNA中 CST1第一种剪切子的 表达差异
采用商品化试剂盒抽提血浆中的循环 R A， LCR检测 50例乳腺癌患者、 30例乳腺炎患 者与 30例正常人血浆循环 R A 中 CST1 第一种剪切子的表达差异， 引物序列如 SEQ ID No.l和 SEQ ID No.2所示， 扩增子序列如 SEQ ID No.50所示。 结果如图 11所示， CST1第 一种剪切子在癌中相对荧光强度 （relative light units, RLU) 的中位数分别是炎的 12.38倍和 正常的 40.12倍； 从 18.51RLU处划线， 可将癌、 炎和正常区分开来。 实施例 9. tSDA检测乳腺癌、 炎患者及正常人血浆循环 RNA中 CST1第一种剪切子的 表达差异
采用商品化试剂盒抽提血浆中的循环 R A， tSDA检测 50例乳腺癌患者、 30例乳腺炎 患者与 30例正常人血浆循环 R A中 CST1第一种剪切子的表达差异， 引物序列如 SEQ ID No.l和 SEQ ID No.2所示， 扩增子序列如 SEQ ID No.50所示。 结果如图 12所示， CST1第 一种剪切子在癌中 RLU的中位数是炎的 41.3倍和正常的 42.86倍； 从 24.81RLU处划线， 可将癌、 炎和正常区分开来。
实施例 10. NASBA检测乳腺癌、 炎患者及正常人尿液循环 RNA中 CST1第一种剪切子 的表达差异
采用商品化试剂盒抽提尿液中的循环 R A， NASBA检测 20例乳腺癌患者、 10例乳腺 炎患者与 10例正常人尿液循环 R A中 CST1第一种剪切子的表达差异， 引物序列如 SEQ ID No.l和 SEQ ID No.2所示， 扩增子序列如 SEQ ID No.50所示。 结果如图 13所示， CST1 第一种剪切子在癌中 RLU的中位数是炎的 18倍和正常的 18.87倍； 从 22.93RLU处划线， 可将癌、 炎和正常区分开来， 即 22.93RLU可作为非侵入性尿液样本乳腺癌诊断的一个参考 值。
实施例 11.乳腺癌患者外周血 CST1第一种剪切子表达水平检测与细胞学检测对比 将乳腺癌患者除去红细胞和血小板的外周血有核细胞提取 R A， 采用实时 PCR绝对定 量法检测 CST1第一种剪切子的表达水平， 引物序列如 SEQ ID No.l和 SEQ ID No.2所示， 扩增子序列如 SEQ ID No.50所示， 通过与乳腺炎患者及正常人的表达量比较， 判断 CST1 第一种剪切子是否高表达， 从而确认乳腺癌患者的外周血中是否有游离乳腺癌细胞存在， 并 与细胞学检测结果进行比对。 结果如图 14所示， CST1第一种剪切子表达水平检测可 100% 确认细胞学鉴定的阳性结果， 同时可检测出细胞学鉴定阴性的病例中存在部分转移的病例， 说明 CST1第一种剪切子表达水平检测比细胞学检测具有更高的灵敏度， 能检测到细胞学无 法检测的微转移的存在。
实施例 12.乳腺癌患者骨髓 CST1第一种剪切子表达水平检测与细胞学检测对比 将穿刺活检取样获得的乳腺癌患者骨髓采用实时 PCR绝对定量法检测 CST1 第一种剪 切子的表达水平， 引物序列如 SEQ ID No.l禾 B SEQ ID No.2所示， 扩增子序列如 SEQ ID No.50所示， 通过与正常骨髓的表达量比较， 判断 CST1 第一种剪切子是否高表达， 从而确 认骨髓是否存在转移和微转移， 并与细胞学检测结果进行比对。 结果如图 15所示， CST1第 一种剪切子表达水平检测可 95%确认细胞学鉴定的阳性结果， 同时阳性率高于细胞学检测， 表明 CST1第一种剪切子表达水平检测的灵敏度高于细胞学检测。
实施例 13. CST1第一种剪切子表达量用于乳腺癌 pTNM分期
采用商品化试剂盒抽提血浆中的循环 R A， TMA法检测 80例乳腺癌不同 pTNM分期 (I+II 30例、 III+IV 50例） 血浆循环 R A中 CST1第一种剪切子的表达差异， 引物序列如 SEQ ID No.l和 SEQ ID No.2所示， 扩增子序列如 SEQ ID No.50所示。 结果如图 16所示， 乳腺癌 ΠΙ+IV中 CST1第一种剪切子的 RLU中位数是 Ι+Π的 15倍， 说明 CST1第一种剪切 子的表达量可用于乳腺癌 pTNM分期。
实施例 14. CST1第一种剪切子表达量用于乳腺癌治疗过程中的动态监测
通过实时定量 PCR法检测患者血液中 CST1第一种剪切子的表达量， 引物序列如 SEQ ID No.l和 SEQ ID No.2所示， 扩增子序列如 SEQ ID No.50所示， 实时监测乳腺癌患者 （化 疗 6人， 放疗 4人） 的治疗效果。 结果如表 2所示， 治疗有效患者血液中 CST1第一种剪切 子的表达量随着疗程增加而逐渐降低， 同时影像学观察发现乳腺癌肿块逐渐减小； 而治疗无 效患者血液中 CST1第一种剪切子的表达量随着疗程的增加而逐渐上升， 同时影像学观察发 现乳腺癌肿块逐渐变大。 因此， CST1 第一种剪切子的表达量可作为乳腺癌患者治疗效果的 评价指标， 用于乳腺癌治疗过程中的动态监测。
表 2 实时定量 PCR检测放化疗过程中乳腺癌患者血液中 CST1第一种剪切子的表达量
Figure imgf000013_0001
实施例 15. CST1第一种剪切子表达量用于乳腺癌患者的预后判断
通过实时定量 PCR法检测患者血液中 CST1第一种剪切子的表达量， 引物序列如 SEQ ID No.l和 SEQ ID No.2所示， 扩增子序列如 SEQ ID No.50所示， 分别在术后或放化疗后的 1个月、 3个月、 1年考察 5个乳腺癌患者的预后效果。 结果如表 3所示， 一年后复发的 2 个患者血液中 CST1 第一种剪切子的表达量随着时间的延长而逐渐增高， 当达到约 1000拷 贝时被影像学发现确诊； 而 1年内没有复发的 3个患者血液中 CST1第一种剪切子的表达量 随着时间的延长并无明显变化， 影像学也无发现异常。 因此， CST1 第一种剪切子的表达量 可作为乳腺癌患者预后判断的指标。
表 3 实时定量 PCR检测术后或放化疗后乳腺癌患者血液中 CST1第一种剪切子的表达量
Figure imgf000014_0001
实施例 16.基于 TaqMan水解探针法实时 PCR的 CST1第一种剪切子检测试剂盒 至少包括以下组分： ①针对 CST1第一个剪切子的引物： 上游引物： 5'- tctcaccctcctctcctg -3' ( SEQ ID No.l ); 下游引物： 5'-ttatcctatcctcctccttgg-3， ( SEQ ID No.2); ②针对 CST1第一 个剪切子的探针： 5*-ctccagctttgtgctctgcctct-3* ( SEQ ID No.3 ) , 5'端用 FAM 标记， 3'端用 TAMRA标记。
还可以包括以下组分中的任一种或多种： RNA提取试剂、 逆转录试剂、 脱氧核糖核苷 酸 （dNTP)、 缓冲液、 MgCl2、 DNA聚合酶、 包含 CST1第一种剪切子扩增子 （序列如 SEQ ID No.51所示） 的重组质粒标准品 [其图谱如图 1所示； 针对 CST1第一种剪切子的引物： 上游引物： 5 '-gggctccctgcctcgggctctcac-3 ' ( SEQ ID No.22); 下游引物： 5'-acggtctgttgcctggct- cttagt-3' ( SEQ ID No.23 )] 阳性对照 （乳腺癌细胞株 HCC1937)、 阴性对照 （人正常乳腺细 胞株 Hs578Bst)。
检测时， 分别将实验组、 阳性对照组、 阴性对照组与重组质粒标准品进行实时 PCR扩 增， 根据重组质粒标准品的浓度梯度和扩增后对应的 CP值作标准曲线， 再依据该标准曲线 给出实验组、 阳性对照组和阴性对照组的拷贝数。
实施例 17.基于染料法实时 PCR的 CST1第一种剪切子检测试剂盒
至少包括以下任一对引物： 针对 CST1 第一个剪切子的引物： ①上游引物： 5'-tctcaccct- cctctcctg-3' ( SEQ ID No.l ); 下游引物： 5 ' -ttatcctatcctcctccttgg-3 ' ( SEQ ID No.2); ②上游引 物： 5'-ccctgggagaacagaaggtcc-3， (SEQ ID No.4); 下游引物： 5'-ggtggtggctggtgcgaat-3， (SEQ ID No.5); ③上游引物： 5'-cattcgcaccagccaccac-3， (SEQ ID No.6); 下游引物： 5'-agaagcaa- gaaggaaggagggag-3' (SEQ ID No.7); ④上游弓 I物： 5 ' -cagcgtgcccttcacttcg-3 ' (SEQ ID No. 8)； 下游引物： 5 ' -cggtctgttgcctggctctta-3 ' (SEQ ID No.9); ⑤上游引物： 5'-cattcgcaccagcca- ccac-3' (SEQ ID No.10); 下游引物： 5 ' -cagggctatagaagcaagaaggaa-3 ' (SEQ ID No.ll)； ⑥上 游引物： 5 '-ggtacagcgtgcccttcacttc-3 ' (SEQ ID No.12); 下游引物： 5 '-cggtctgttgcctggctctta-3 '
(SEQ ID No.13); ⑦上游引物： 5 ' -gagaacagaaggtccctggtgaa-3 ' (SEQ ID No.14); 下游引 物： 5'-ggtggtggctggtgcgaat-3， ( SEQ ID No.15 )； ⑧上游引物： 5'-tgggtacagcgtgcccttca-3，
(SEQ ID No.16); 下游引物： 5 '-cggtctgttgcctggctctta-3 ' (SEQ ID No.17); ⑨上游引物： 5'- ccctgggagaacagaaggtcc-3 ' (SEQ ID No.18); 下游引物： 5 '-tggtggctggtgcgaatgg-3 ' (SEQ ID No. 19)； ⑩上游引物： 5'-ttccctgggag- aacagaaggtcc-3' (SEQ ID No.20); 下游引物： 5 ' -tggtggctggtgcgaatgg-3 ' (SEQ ID No.21)o
还可以包括以下组分中的任一种或多种： 针对内参基因 β-actin 的引物： 上游引物： 5'- aagatcattgctcctcctg-3' (SEQ ID No.32); 下游引物： 5'-cgtcatactcctgcttgc-3' (SEQ ID No.33); RNA提取试剂、 逆转录试剂、 荧光染料 （如 SYBR Green ) dNTP、 缓冲液、 MgCl2、 DNA 聚合酶。
实施例 18.基于 NASBA的 CST1第一种剪切子检测试剂盒
至少包括以下组分： ①针对 CST1第一个剪切子的引物： 上游引物： 5'-aattctaatacgactca- ctatagggtctcaccctcctctcctg-3 ' (SEQ ID No.34); 下游引物： 5 '-ttatcctatcctcctccttgg-3 ' (SEQ ID No.2); ②针对 CST1第一个剪切子的探针： 5'-ctccagctttgtgctctgcctct-3' (SEQ ID No.3)， 5'端 用 FAM标记， 3'端用 DABSYL标记。
还可以包括以下组分中的任一种或多种： R A提取试剂、 逆转录试剂、 R A荧光染料 (如 Ribo-Green fluorescent dye)、 T7 RNA聚合酶、 RNase H、 禽骨髓白血病病毒 （AMV) 逆转录酶、 核糖核苷酸 （NTP)、 dNTP。
实施例 19.基于 TMA的 CST1第一种剪切子定量检测试剂盒
至少包括以下组分： ①针对 CST1 第一个剪切子的引物： 上游引物： 5'-aattctaatacgactc- actatagggtctcaccctcctctcctg-3 ' (SEQ ID No.34); 下游引物： 5 ' -ttatcctatcctcctccttgg-3 ' ( SEQ ID No.2); ②针对 CST1第一个剪切子的探针： 5'-ctccagctttgtgctctgcctct-3' (SEQ ID No.3)， 5'端 用 FAM标记， 3'端用 DABSYL标记。
还可以包括以下组分中的任一种或多种： R A提取试剂、 逆转录试剂、 R A荧光染料 (如 Ribo-Green fluorescent dye)、 T7 R A聚合酶、 RNase H、 禽骨髓白血病病毒 （AMV) 逆转录酶、 NTP、 dNTP。
实施例 20.基于 LCR的 CST1第一种剪切子定量检测试剂盒
至少包括以下 4条探针： 5'-agtatctgagtaccctgctgctcctgc-3， ( SEQ ID No.35 ); 5'-accctagct- gtggccctggcctggag-3 ' ( SEQ ID No.36 )； 5 '-catagactcatgggacgacg-3 ' ( SEQ ID No.37 )； 5'- acaccgggaccggacctc-3' ( SEQ ID No.38 ); 每条探针的 5'端用半抗原标记。
还可以包括以下组分中的任一种或多种： R A提取试剂、 逆转录试剂、 T4 DNA连接 酶、 dNTP。
实施例 21.基于 tSDA的 CST1第一种剪切子检测试剂盒
至少包括以下组分： ①针对 CST1第一个剪切子的引物： CST1 B1弓 I物： 5'-tgggtacagc- gtgcccttcactt-3' ( SEQ ID No.39); CST1 SI弓 |物： 5 '-ccgctcgagtacagcgtgcccttcacttcgc-3 ' ( SEQ ID No.40); CST1 B2弓 I物: 5 '-caacggtctgttgcctggctctta-3 ' ( SEQ ID No.41 ); CST1 S2引物: 5 '-gacctcgaggttgcctggctcttagtacccg-3 ' ( SEQ ID No.42); ②针对 CST1第一个剪切子的探针： 5 ' -gtgctcgagtcagcgagtataacaaggccaccaaagatgactac-3 ' ( SEQ ID No.43 ), 5 '端用 32P标记。
还可以包括以下组分中的任一种或多种： R A 提取试剂、 逆转录试剂、 dCTPctS、 dATP、 dGTP、 dTTP、 Bsobl酶、 exo-Bca酶。
(二） 蛋白水平
优选实施例中使用的 Cystatin SN重组蛋白， 兔抗 Cystatin SN多克隆抗体购自 NOVUS Biologicals, 鼠抗人 Cystatin SN 单克隆抗体 （特异性识别序列如 SEQ ID No.54 所示的 Cystatin SN表位） 购自 R&D， TMB过氧化物酶底物 （TMB Peroxidase Substrate, 包含 TMB solution A禾口 Peroxidase Solution B ) 贝勾自 Kirkegaard and Perry Laboratories Inc.。
血清样品的制备是将全血标本于室温放置 2小时或 4°C过夜后， 1000g离心 20分钟， 取 上清即可检测， 或置 -20°C或 -80°C保存， 避免反复冻融。 血浆样品的制备是用 EDTA或肝素 作为抗凝剂， 标本采集后 30分钟内于 2〜8°C、 1000g离心 15分钟， 取上清即可检测， 或置- 20°C或 -80°C保存， 避免反复冻融。 血清或血浆样品用 pH7.0〜7.2、 0.1M 的 PBS稀释 10倍 后进行测定。
实施例 22.人乳腺癌细胞株培养上清与正常人血清中 Cystatin SN的表达差异
取 CST1 mR A高表达的人乳腺癌细胞株 HCC1937 (泳道 1-2)、 MCF-7 (泳道 3-4 ) 的 培养上清以及正常人的血清样本 S1 (泳道 5-6 ) 和 S2 (泳道 7-8 )， 进行 15%的 SDS- PAGE。 电泳完毕后， 将蛋白电转移至硝酸纤维素膜上， 用含有 5%脱脂奶粉和 0.1% Tween- 20的 PBS于室温下封闭 2小时， 加入抗 Cystatin SN多克隆抗体于 4°C温育过夜， 用含有 0.1% Tween-20的 PBS洗涤 3次， 再加入辣根过氧化物酶 （HRP) 标记的山羊抗兔 IgG于 37 °C温育 1小时， 用含有 0.1% Tween-20的 PBS洗涤 4次， 再用 PBS洗涤 1次， 之后用 TMB 过氧化物酶底物显色检测， 以 β-actin为内参蛋白。 结果如图 17所示， 泳道 1-4中检测到约 16Kda的强条带， 而泳道 5-8仅检测到约 16Kda的弱条带， 说明 CST1 编码的 Cystatin SN 在人乳腺癌细胞中异常高表达。
实施例 23.乳腺癌患者与正常人血清中 Cystatin SN的表达差异
取正常人的血清样本 （泳道 1-2) 和乳腺癌患者的血清样本 （泳道 3-6)， 按照实施例 22 所述方法进行 SDS-PAGE、 蛋白电转和免疫印迹。 结果如图 18所示， 泳道 1-2仅检测到弱 条带， 而泳道 3-6检测到强条带， 说明 Cystatin SN在乳腺癌患者血清中异常高表达。
实施例 24.竞争 ELISA测定乳腺癌患者与正常人血清中 Cystatin SN的表达差异 用 5ug/ml Cystatin SN 包被 ELISA 平板， 再用 3% BSA 封闭平板， 血清样品与抗 Cystatin SN 单克隆抗体 （1 :2000 ) 于 37°C温育 1 小时， 用 TBS (含有 154mM NaCl 的 pH7.5、 10mM Tris-HCl) 洗涤， 再加入 0.08ug/ml HRP标记的山羊抗兔 IgG于 37°C温育 1小 时， 用 TBS洗涤， 再加入 0.4mg/ml邻苯二胺 （溶于 pH5、 500mM磷酸盐-柠檬酸盐缓冲液 中） 反应， 用酶联仪定量。 共测定了 20 例正常人血清和 30 例乳腺癌患者血清。 结果如图 19所示， 正常人血清中 Cystatin SN浓度中位数为 1.7ng/ml， 乳腺癌患者血清中 Cystatin SN 浓度中位数为 4.1ng/ml， 从 3.25ng/ml处划线， 基本可以将癌和正常区分开来。
实施例 25.双抗夹心 ELISA测定乳腺癌患者与正常人血清中 Cystatin SN的表达差异 用 5ug/ml鼠抗人 Cystatin SN单克隆抗体包被 ELISA平板， 再用 3%BSA封闭平板， 加 入血清样品于 37°C温育 1小时， 用 TBS洗涤， 再加入生物素标记的兔抗 Cystatin SN多克隆 抗体 （1 :1000 ) 于 37°C温育 1小时， 用 TBS洗涤， 再加入亲合素-过氧化物酶复合物于 37°C 温育 1 小时， 用 TBS洗涤， 再每孔加入 TMB过氧化物酶底物， 用酶联仪定量。 共测定了 30例正常人血清和 50例乳腺癌患者血清。 结果如图 20所示， 正常人血清中 Cystatin SN浓 度中位数为 0.525ng/ml， 乳腺癌患者血清中 Cystatin SN 浓度中位数为 2.99ng/ml， 从 1.48ng/ml处划线， 基本可以将癌和正常区分开来。
实施例 26. ELISA测定乳腺癌患者血清 Cystatin SN和癌胚抗原 （CEA) 的灵敏度和特 异性比较
按照实施例 25所述方法测定 Cystatin SN血清水平。 采用商品化的 CEA ELISA试剂盒 进行 CEA检测， 按照试剂盒说明书操作。 共测定了 30例正常人血清和 30例乳腺癌患者血 清。 结果如图 21 所示， Cystatin SN 曲线下面积为 0.994， CEA 曲线下面积为 0.833， 说明 ELISA测定 Cystatin SN比测定 CEA在灵敏度和特异性方面更具有优越性。
实施例 27.基于竞争 ELISA法的 Cystatin SN定量检测试剂盒
1、 试剂盒的组成
至少包括以下组分： 包被抗原 （Cystatin SN ) ; 一抗 （鼠抗人 Cystatin SN 单克隆抗 体）； 酶标二抗 （HRP标记的山羊抗兔 IgG); 酶作用底物 （邻苯二胺）。
还可以包括以下组分中的任一种或多种： 封闭液 （3%BSA)、 洗涤缓冲液 （TBS , 即含 有 154mM NaCl的 pH7.5、 10mM Tris-HCl)。
2、 试剂盒的使用方法
用 5mg/ml Cystatin SN 包被 ELISA 平板， 再用 3%BSA 封闭平板， 血清样品与抗 Cystatin SN单克隆抗体 （1 :2000) 于 37°C温育 1小时， 用 TBS洗涤， 再加入 0.08ug/ml HRP 标记的山羊抗兔 IgG于 37°C温育 1 小时， 用 TBS洗涤， 再加入 0.4mg/ml邻苯二胺 （溶于 pH5、 500mM磷酸盐-柠檬酸盐缓冲液中） 反应， 用酶联仪定量。
实施例 28.基于双抗夹心 ELISA法的 Cystatin SN定量检测试剂盒
1、 试剂盒的组成
至少包括以下组分： Cystatin SN标准品， 临用前用含有 1%BSA的 PBST溶解或稀释制 成一系列梯度溶液， 如浓度分别为 10、 5、 2.5、 1、 0.5、 0.25ng/ml ; 包被抗体 （鼠抗人 Cystatin SN单克隆抗体）； 生物素标记二抗 （生物素标记的兔抗 Cystatin SN多克隆抗体）； 亲合素-过氧化物酶复合物； 过氧化物酶作用底物 （TMB过氧化物酶底物）。
还可以包括以下组分中的任一种或多种： 包被抗体稀释液 （pH9、 0.05M的 NaHC03)、 封闭液 （含有 3%BSA 的 PBST )、 二抗稀释液 （含有 1%BSA 的 PBST )、 洗涤缓冲液 (PBST, 即含有 0.05% Tween-20的 PBS)、 终止液 （2N H2S04)。
2、 试剂盒的使用方法
用包被抗体缓冲液将包被抗体稀释至蛋白含量为 5 g/ml， 加至 ELISA平板中， 每孔 0.1ml, 4°C孵育过夜， 弃去孔内液体， 用洗涤缓冲液洗涤 3 次， 每次 3 分钟。 再每孔加入 200ul封闭液， 37°C温育 1 小时或 4°C孵育过夜， 弃去孔内液体， 用洗涤缓冲液洗涤 3次， 每次 3 分钟。 分别设空白孔、 标准品孔、 样品孔， 空白孔每孔加样品稀释溶剂即 pH7.0〜7.2、 0.1M 的 PBS ΙΟΟμΙ, 标准品孔每孔加 Cystatin SN标准品溶液 100μ1， 样品孔每 孔加血清或血浆样品 100μ1， 37°C温育 2小时， 弃去孔内液体， 甩干。 再每孔加入生物素标 记二抗 ΙΟΟμΙ (使用前用二抗稀释液进行 1 :200稀释）， 37°C温育 1小时， 弃去孔内液体， 用 洗涤缓冲液洗涤 3次， 每次 1〜2分钟。 再每孔加入亲合素 -过氧化物酶复合物 100μ1， 37°C温 育 1小时， 弃去孔内液体， 用洗涤缓冲液洗涤 5次， 每次 1〜2分钟。 再每孔加入过氧化物酶 作用底物 50μ1， 37°C避光显色 （15分钟内， 待肉眼可见标准品前 4-5孔有明显的梯度兰色、 后 4-5孔梯度不明显时即可终止）。 再每孔加入终止液 50μ1终止反应 （蓝色立转黄色）。 用酶 联仪于 405nm波长处测量各孔光密度 （OD值）。 根据标准品浓度和对应的 OD值绘制散点 曲线， 计算 R2和曲线方程式， 如 R2 0.95， 则将样品 OD值代入曲线方程式中， 即可算出 血清或血浆样品中的 Cystatin SN浓度。
实施例 29. Cystatin SN表达水平用于乳腺癌 pTNM分期
采用实施例 28所述试剂盒及其使用方法检测经病理确诊的乳腺癌 T期患者 20人、 N期 患者 30人和 M期患者 30人血清中的 Cystatin SN表达水平， 结果如表 4所示， 可见随着病 程的进展， Cystatin SN 表达水平亦随之提高， 提示 Cystatin SN 表达水平可用于乳腺癌 pTNM分期。
表 4 不同病理分期的乳腺癌患者 Cystatin SN表达水平
Figure imgf000019_0001
实施例 30. Cystatin SN表达水平用于评估乳腺癌是否转移
采用实施例 28所述试剂盒及其使用方法检测经病理确诊的乳腺癌未转移患者 20人和转 移患者 30 人血清中的 Cystatin SN 表达水平， 结果如表 5 所示， 可见乳腺癌转移患者的 Cystatin SN 表达水平高于未转移者， 提示 Cystatin SN 表达水平可用于评估乳腺癌是否转 移。
表 5 转移与未转移乳腺癌患者 Cystatin SN表达水平
Figure imgf000019_0002
实施例 31. Cystatin SN表达水平用于评估乳腺癌化疗联合内分泌治疗的效果
2885例 N0-1期乳腺癌患者， 行 4个周期的 AC (多柔比星 +环磷酰胺） 或 AT (多柔比 星 +紫杉醇） 方案化疗， 再根据激素受体 （HR) 状况选择是否行辅助内分泌治疗， 采用实施 例 28所述试剂盒及其使用方法检测治疗周期结束后患者血清中的 Cystatin SN表达水平， 有 效数据 776例， Cystatin SN表达中位数为 4.06ng/ml， 之后随访 76个月， 记录患者的无病生 存情况。 结果如图 22所示， 高于上述 Cystatin SN表达中位数的患者无病生存率为 20%， 低于上述 Cystatin SN表达中位数的患者的无病生存率为 60%， 提示 Cystatin SN表达水平可 用于评估乳腺癌化疗联合内分泌治疗的效果。
虽然通过上述具体实施例对本发明进行了比较详细的描述， 但可以理解的是， 本发明为 了清楚而在各个实施例中描述的技术特征也可按需要重新组合应用， 所有技术特征的等同替 换、 修饰和改变对本领域技术人员来说都是显而易见的， 因此， 这些等同替换、 修饰和改变 也包括在本发明范围之内。

Claims

权利要求书
1. CST1基因剪切子、 CST1基因编码蛋白 Cystatin SN及其表位肽作为乳腺癌诊断和预示 标志物的应用， 所述 CST1 基因剪切子的核苷酸序列如 SEQ ID No.48 所示， 所述 Cystatin SN的氨基酸序列如 SEQ ID No.52所示。
2. 根据权利要求 1 所述的应用， 其特征在于， 所述 Cystatin SN 表位肽的氨基酸序列如 SEQ ID No.54所示。
3. 根据权利要求 1或 2所述的应用， 其特征在于， 所述乳腺癌诊断和预示包括乳腺癌或乳 腺癌组织转移的鉴别诊断和 /或易感性分析， 乳腺癌或乳腺癌组织转移治疗药物、 治疗方 法、 治疗疗效及预后的评估， 以及相关人群乳腺癌或乳腺癌组织转移患病风险的评估。
4. CST1基因剪切子的捕获剂和 CST1基因编码蛋白 Cystatin SN的捕获剂在制备乳腺癌诊 断和预示试剂、 试剂盒及芯片中的应用， 所述 CST1 基因剪切子的捕获剂为特异性识别 CST1基因剪切子或其 cDNA的引物和 /或探针， 所述 Cystatin SN的捕获剂为抗 Cystatin SN的抗体， 所述 CST1基因剪切子的核苷酸序列如 SEQ ID No.48所示， 所述 Cystatin SN的氨基酸序列如 SEQ ID No.52所示。
5. 根据权利要求 4所述的应用， 其特征在于， 所述引物为核苷酸序列如 SEQ ID No.l~2、 4~21、 34和 39~42所示的至少一种， 所述探针为核苷酸序列如 SEQ ID No.3、 35~38和 43所示的至少一种。
6. 根据权利要求 5所述的应用， 其特征在于， 所述引物的核苷酸序列如 SEQ ID N0.l~2所 示， 所述探针的核苷酸序列如 SEQ ID No.3所示。
7. 包含 CST1基因剪切子的捕获剂或 CST1基因编码蛋白 Cystatin SN的捕获剂的试剂盒在 制备乳腺癌诊断和预示试剂盒中的应用， 所述 CST1 基因剪切子的捕获剂为特异性识别 CST1基因剪切子或其 cDNA的引物和 /或探针， 所述 Cystatin SN的捕获剂为抗 Cystatin SN的抗体， 所述 CST1基因剪切子的核苷酸序列如 SEQ ID No.48所示， 所述 Cystatin SN的氨基酸序列如 SEQ ID No.52所示。
8. 根据权利要求 7所述的应用， 其特征在于， 所述引物为核苷酸序列如 SEQ ID No.l~2、 4~21、 34和 39~42所示的至少一种， 所述探针为核苷酸序列如 SEQ ID No.3、 35~38和 43所示的至少一种。
9. 根据权利要求 8所述的应用， 其特征在于， 所述引物的核苷酸序列如 SEQ ID N0.l~2所 示， 所述探针的核苷酸序列如 SEQ ID No.3所示。
10. 表面固定有 CST1 基因剪切子的捕获剂的芯片在制备乳腺癌诊断和预示芯片中的应用， 所述 CST1基因剪切子的捕获剂为特异性识别 CST1基因剪切子或其 cDNA的探针， 所 述 CST1基因剪切子的核苷酸序列如 SEQ ID No.48所示。
11. 根据权利要求 10 所述的应用， 其特征在于， 所述探针为核苷酸序列如 SEQ ID No.3、 35-38和 43所示的至少一种。
12. 根据权利要求 11所述的应用， 其特征在于， 所述探针的核苷酸序列如 SEQ ID No.3所 示。
13. 包含 CST1 基因剪切子的捕获剂的乳腺癌诊断和预示试剂盒， 其特征在于， 所述 CST1 基因剪切子的捕获剂为特异性识别 CST1基因剪切子的 cDNA的引物和探针， 所述引物 的核苷酸序列如 SEQ ID No.l~2所示， 所述探针的核苷酸序列如 SEQ ID No.3所示， 探 针的 5'端用荧光基团标记， 3'端用淬灭基团标记。
14. 根据权利要求 13 所述的乳腺癌诊断和预示试剂盒， 其特征在于， 所述试剂盒中还包括 含有 CST1 第一种剪切子扩增子的重组质粒标准品， 所述 CST1第一种剪切子扩增子的 核苷酸序列如 SEQ ID No.51所示。
15. 根据权利要求 14 所述的乳腺癌诊断和预示试剂盒， 其特征在于， 所述试剂盒中还包含 阳性对照乳腺癌细胞株 HCC1937和阴性对照人正常乳腺细胞株 Hs578Bst。
16. 包含 CST1 基因剪切子的捕获剂的乳腺癌诊断和预示试剂盒， 其特征在于， 所述 CST1 基因剪切子的捕获剂为特异性识别 CST1基因剪切子的 cDNA的引物和探针， 所述引物 的核苷酸序列如 SEQ ID No.34和 SEQ ID No.2所示， 所述探针的核苷酸序列如 SEQ ID No.3所示， 探针的 5'端用荧光基团标记， 3'端用淬灭基团标记。
17. 包含 CST1 基因剪切子的捕获剂的乳腺癌诊断和预示试剂盒， 其特征在于， 所述 CST1 基因剪切子的捕获剂为特异性识别 CST1基因剪切子的 cDNA的引物和探针， 所述引物 的核苷酸序列如 SEQ ID No.39-44所示， 所述探针的核苷酸序列如 SEQ ID No.43所示， 探针的 5'端用放射性同位素标记。
18. 包含 CST1 基因剪切子的捕获剂的乳腺癌诊断和预示试剂盒， 其特征在于， 所述 CST1 基因剪切子的捕获剂为特异性识别 CST1基因剪切子的 cDNA的引物， 所述引物的核苷 酸序列如 SEQ ID No.l~2所示， 或者如 SEQ ID No.4~5所示， 或者如 SEQ ID No.6~7所 示， 或者如 SEQ ID No.8~9 所示， 或者如 SEQ ID Νο.10~11 所示， 或者如 SEQ ID Νο.12~13所示， 或者如 SEQ ID Νο.14~15所示， 或者如 SEQ ID Νο.16~17所示， 或者如 SEQ ID No.18-19所示， 或者如 SEQ ID No.20~21所示。
19. 包含 CST1 基因剪切子的捕获剂的乳腺癌诊断和预示试剂盒， 其特征在于， 所述 CST1 基因剪切子的捕获剂为特异性识别 CST1基因剪切子的 cDNA的探针， 所述探针的核苷 酸序列如 SEQ ID No. 35-38所示， 每条探针的 5'端用半抗原标记。
20. 包含 CST1 基因编码蛋白 Cystatin SN 的捕获剂的乳腺癌诊断和预示试剂盒， 其特征在 于， 所述 CST1基因编码蛋白 Cystatin SN的捕获剂为抗 Cystatin SN单克隆抗体， 所述 试剂盒中还包含 Cystatin SN抗原、 酶标第二抗体和酶作用底物。
21. 包含 CST1 基因编码蛋白 Cystatin SN 的捕获剂的乳腺癌诊断和预示试剂盒， 其特征在 于， 所述 CST1基因编码蛋白 Cystatin SN的捕获剂为抗 Cystatin SN单克隆抗体和生物 素标记的抗 Cystatin SN多克隆抗体， 所述试剂盒中还包含 Cystatin SN标准品、 亲合素- 酶复合物和酶作用底物。
22. 运用权利要求 13~21任一项所述的乳腺癌诊断和预示试剂盒诊断或预示乳腺癌的方法， 其特征在于， 用所述试剂盒检测待测样品中 CST1 基因剪切子或 CST1 基因编码蛋白 Cystatin SN的含量或表达水平， 将检测结果与阈值进行比较， 如果超过阈值则判定为阳 性； 所述阈值是通过对比正常人与乳腺癌患者体液或组织中 CST1 基因剪切子或 CST1 基因编码蛋白 Cystatin SN的含量或表达水平统计得到； 所述待测样品为手术组织、 穿刺 组织、 淋巴结组织、 骨髓、 血清样品、 血浆样品、 全血样品、 血液级分样品或尿样。
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