CN116083581B - Kit for detecting early digestive tract tumor - Google Patents
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Abstract
The application discloses a kit for detecting early-stage digestive tract tumors, and belongs to the technical field of biological detection. The kit comprises a protein marker and a DNA methylation biomarker, and the double-marker composition is related to digestive tract tumorigenesis. The detection data output after the combination of the two markers is suitable for assisting doctors in judging early digestive tract tumors. The NEBNext Enzymatic Methyl-seq kit is used for converting cytosine into uracil, so that cfDNA is not broken, and the truest methylation state in blood can be preserved; the detection data output after the combination of the two markers can obviously improve the detection rate and the accuracy, can detect and effectively distinguish liver cancer, gastric cancer and colorectal cancer simultaneously, is suitable for large-scale popularization and application in view of the noninvasive property of the detection of the in-vitro blood sample.
Description
Technical Field
The application belongs to the technical field of biological detection, and particularly relates to a kit for detecting early-stage digestive tract tumors.
Background
Digestive tract tumors are the type of the current high-incidence tumor, and according to the statistics of the current national tumor annual report, the incidence rates of liver cancer, gastric cancer and colorectal cancer are respectively located at 3, 4 and 5 sites of the high-incidence tumor, and the death rate is located at the front of the death rate of the cancer. Such high morbidity and mortality means that most digestive tract tumors are found only in the middle and late stages. In clinical practice, early diagnosis of digestive tract tumors remains a great challenge, and the tumor diagnosis has only a 20-30% excision rate, and the 5-year survival rate after operation is 30-50%.
The current means for early screening of tumors include imaging examination, serological molecular marker examination, pathological examination and the like. The imaging examination can detect adenoma with the diameter more than or equal to 1cm, but the detection sensitivity is lower for earlier lesions, and certain radiation exists, so that cancerous lesions can be induced. Pathological examination causes trauma to the mind and body of a liver cancer patient, is greatly influenced by physical and psychological quality of the patient, and cannot be performed for multiple times in a short time. Serological molecular markers are currently the conventional means available for detecting and monitoring digestive tract tumors, but their clinical utility is limited by low sensitivity.
Currently, most of the examination means are directed only to single cancers, such as alpha fetoprotein combined with ultrasound examination for liver cancer screening; helicobacter pylori detection is used for gastric cancer screening and enteroscopy is used to screen colorectal cancer patients. The detection sensitivity of the methods for early digestive tract cancers is generally low, and the methods can be used simultaneously to screen liver cancer, stomach cancer and colorectal cancer, so that the method is not beneficial to popularization of the screening of digestive tract tumors.
Disclosure of Invention
The first objective of the present application is to provide a kit for detecting early stage digestive tract tumor, which solves the technical problems of complicated detection process, limitation of detection performance caused by singly using any one of the markers, inaccurate result or more false positive/negative in the prior art by combining detection by using a dual-marker composition. The kit can detect and distinguish the tumors with the first three morbidity in the digestive tract simultaneously through one-time detection, is simple to operate and is beneficial to large-scale popularization.
The second purpose of the application is to provide a kit for detecting early digestive tract tumors, which uses NEBNext Enzymatic Methyl-seq kit to convert cytosine into uracil, and solves the technical problems that the traditional bisulfite conversion method is easy to cause the breakage of cfDNA, thereby causing the change of methylation state and inaccurate detection result.
The application is realized by the following technical scheme:
a kit for detecting early stage gut tumor, the kit comprising a dual marker composition;
the dual tag composition includes a protein tag and a DNA methylation biomarker;
the protein markers include a combination of one or more of alpha fetoprotein, gastrin and glycoprotein antigen 24-2;
the DNA methylation biomarker comprises a composition of one or more of chr7:45018849-45018850 in the hg19 human genome.
DNA methylation is an apparent regulatory modification that participates in regulating the expression of proteins without changing the base sequence. There is a change in the methylation state of DNA in tumor cells, and methylation modification plays a critical role in early tumor development. While blood cfDNA methylation markers proved to be able to detect early stage tumor patients more sensitively than other blood tumor markers. In addition, as the blood alpha fetoprotein level, the gastrin protein level and the glycoprotein antigen 24-2 can be specifically increased or decreased in liver cancer, gastric cancer and colorectal cancer patients respectively, the detection means of blood cfDNA methylation combined with serum protein markers can improve the detection rate of early cancers, and can simply and effectively trace digestive tract tumors, thereby being a simple, convenient, quick, economical and feasible early digestive tract tumor detection method.
Preferably, the kit further comprises a probe of a dual label composition and a protein quantification antibody.
Preferably, the probe is a hybridization capture DNA sequence fragment containing the dual-marker composition.
Preferably, the protein quantification antibody comprises alpha fetoprotein antibody, gastrin protein antibody and glycoprotein antigen 24-2 antibody.
Preferably, the DNA methylation component in the kit adopts the following detection platform: reagents used in PCR amplification, digital PCR, fluorescent quantitative PCR, methylation chip method, liquid phase chip method, bisulfite sequencing, first generation sequencing, second generation sequencing, third generation sequencing, or a combination thereof.
The above-described preference is given to using a digital PCR platform.
Preferably, the detection of the protein component in the kit employs immunofluorescence quantification.
Compared with the prior art, the application has at least the following technical effects:
the application provides a kit for detecting early stage digestive tract tumor, which comprises a protein marker and a DNA methylation biomarker, wherein the double-marker composition is related to digestive tract tumor generation. The detection data output after the combination of the two markers is suitable for assisting doctors in judging early digestive tract tumors.
(II) the kit uses NEBNext Enzymatic Methyl-seq kit for cytosine to uracil conversion. Compared with the traditional bisulfite conversion, the reagent kit adopts a method instruction which can not cause the breakage of cfDNA and can preserve the truest state of methylation in blood.
And thirdly, as cfDNA methylation modification is singly used or the protein markers have certain limitation on the detection performance of digestive tract tumor patients in different periods, the detection data output after the two markers are combined is provided, the detection rate and the accuracy can be remarkably improved, and the method is suitable for large-scale popularization and application in view of the noninvasive property of in-vitro blood sample detection.
Drawings
FIG. 1 is a schematic diagram of the screening and detection technique of cfDNA methylation markers and protein markers of the isolated blood sample of example 1;
FIG. 2 is a schematic representation of the ROAUC of the cfDNA methylation biomarkers of example 2 in independent detection of gut tumor samples;
FIG. 3 is a box plot of methylation levels of cfDNA methylation biomarkers in TCGA gut tumor tissue and paracancestral tissue in example 2;
FIG. 4 is a ROAUC diagram of the isolated liver cancer blood sample from the isolated colorectal cancer and gastric cancer blood sample using alpha fetoprotein concentration as a marker in example 3;
FIG. 5 is a box plot of alpha fetoprotein concentration in an ex vivo blood sample of a patient with a digestive tract tumor in example 3;
FIG. 6 is a ROAUC diagram of the isolated blood sample of colorectal cancer and the isolated blood sample of liver cancer and stomach cancer using the concentration of glycoprotein antigen 24-2 as a marker in example 3;
FIG. 7 is a box plot of the concentration of glycoprotein antigen 24-2 in an ex vivo blood sample of a patient with a digestive tract tumor in example 3;
FIG. 8 is a ROAUC graph of the isolated blood sample of gastric cancer versus the isolated blood sample of liver cancer and colorectal cancer using the gastrin protein concentration as a marker in example 3;
FIG. 9 is a box plot of gastrin protein concentration in an ex vivo blood sample of a patient with a digestive tract tumor in example 3.
Detailed Description
Embodiments of the present application will be described in detail below with reference to the following examples, which are to be construed as merely illustrative and not limitative of the scope of the application, but are not intended to limit the scope of the application to the specific conditions set forth in the examples, either as conventional or manufacturer-suggested, nor are reagents or apparatus employed to identify manufacturers as conventional products available for commercial purchase.
Example 1:
this example discloses methylation markers of blood cfDNA for detection of early stage gut tumors. Specifically comprises the following gene fragments with obvious differential methylation in an in vitro blood sample of a patient with digestive tract tumor: chr7, 45018849-45018850. The methylation marker is obtained by screening an isolated blood sample of a TCGA and GEO digestive tract tumor methylation public database and a clinical digestive tract tumor patient by combining a statistical method with a machine learning model, and is proved to be capable of accurately detecting the isolated blood sample of a pan digestive tract tumor patient, including liver cancer, gastric cancer and colorectal cancer patients.
In order to solve the problems of low cfDNA content in plasma and DNA damage by bisulfite, specific embodiments of the present application use NEBNext Enzymatic Methyl-seq kit for cytosine to uracil conversion.
FIG. 1 is a schematic diagram showing the detection flow of an isolated sample of digestive tract tumor blood combined with cfDNA methylation markers and protein markers.
The experiment uses 100 cases of healthy physical examination control blood samples, 30 cases of liver cancer patient ex-vivo blood samples, 30 cases of stomach cancer patient ex-vivo blood samples and 30 cases of colorectal cancer patient ex-vivo blood samples. The specific sample statistics are shown in the following table:
TABLE 1 sample gender statistics
| Liver cancer | Stomach cancer | Colorectal cancer | Normal state | |
| Man's body | 22 | 21 | 16 | 39 |
| Female | 8 | 9 | 14 | 61 |
Table 2 sample age statistics
| Liver cancer | Stomach cancer | Colorectal cancer | Normal state | |
| <Age 40 | 1 | 5 | 3 | 22 |
| Age of 40-50 years | 9 | 4 | 5 | 49 |
| Age 50-60 years | 10 | 6 | 12 | 19 |
| >Age 60 | 10 | 15 | 10 | 10 |
Example 2: methylation marker detection based on ddPCR platform
The present example is based on 30 liver cancer ex vivo blood samples, 30 colorectal cancer ex vivo blood samples, 30 gastric cancer ex vivo blood samples, and 100 healthy physical ex vivo blood samples, and the effect of the cfDNA methylation markers obtained by the above screening on predicting pan-digestive tract tumor samples was verified.
And taking an in-vitro blood sample of a liver cancer patient, a colorectal cancer patient and a gastric cancer patient as a tumor group, and taking an in-vitro blood sample of a healthy physical examination volunteer as a control group.
The application platform of the experiment is a ddPCR platform, and the specific experimental steps are as follows:
step 1: extracting cfDNA of blood plasma: specific procedures for plasma cfDNA extraction the cassette "free DNA extraction kit (suction filtration method) #c02-1" was used for the extraction of free DNA from the organisms uze, guangzhou.
Step 2: cfDNA mass QC: and (5) carrying out subsequent Qubit detection after gently shaking and uniformly mixing the collected DNA sample.
mu.L was taken for the Qubit 3.0 assay and the concentration of the sample was measured. Total amount standard is extracted: and the total cfDNA is equal to or greater than 20 ng.
Step 3: DNA methylation transformation: cfDNA conversion and PCR amplification were performed using the guangzhou euzem liver cancer methylation Gene detection kit (digital PCR method) #c01-1) reference method.
Step 4: ddPCR methylation assay: this example uses a Bio-rad digital PCR instrument (QX 200Droplet Digital PCR (ddPCR) TM ) And 3) detecting the methylation rate of the cfDNA amplified by the conversion in the step 3, and calculating the methylation rate of the detected tumor marker.
Methylation of the sites was analyzed according to the methylation rate of the methylation markers calculated on the ddPCR platform. AUC analysis was performed on cfDNA methylation sites and AUC values were calculated for cfDNA methylation markers that independently distinguish blood from healthy physical examination volunteers from blood from patients with digestive tract tumors.
As shown in fig. 2, the ROAUC profile of cfDNA methylation biomarkers detected independently in gut tumor samples.
The AUC value for distinguishing healthy volunteer blood from patients with digestive tract tumors using the methylation marker chr7:45018849-45018850 was 0.919.
To verify the effect of the methylation marker in tumor tissue data, the site methylation was verified using TCGA gut tumor tissue data (liver cancer sample 50 pair, colorectal cancer sample 38 pair, gastric cancer sample 3 pair) download.
As shown in FIG. 3, the methylation level of chr7:45018849-45018850 in the tumor tissue of the digestive tract was significantly higher than that of the control tissue beside the cancer, and the difference test was performed on the two groups of data by using the paired t test, and the p-val was 1.43E-07.
The analysis results show that: the blood methylation marker can better distinguish an isolated blood sample of a patient with digestive tract tumor from an isolated blood sample of a healthy physical examination volunteer.
Example 3: use of tumor marker proteins for tracing digestive tract tumor types
Serum protein carbohydrate antigen protein 24-2 (CA 24-2), alpha Fetoprotein (AFP) and gastrin protein (G17) were detected in serum using 30 cases of colorectal cancer ex vivo blood samples, 30 cases of liver cancer ex vivo blood samples and 30 cases of gastric cancer ex vivo blood samples collected (hereinafter, abbreviated as protein).
The specific experimental procedure for protein marker detection is as follows:
step 1: sample processing: serum was separated 24 hours after venous blood sampling and the hemolyzed and lipidemic samples were not available for testing and were re-sampled.
Step 2: preparation before detection: the power of the instrument is firstly turned on to turn on light, the software is turned on to start initialization, and the instrument can be detected after being started for 30 minutes. Taking out the separated serum sample, thawing if the sample is frozen, balancing at room temperature for 30min, and balancing at room temperature for 30min after taking out the kit and the standard.
Step 3: scaling: and adding 500 mu L of calibrator diluent into each of calibrator 1 and calibrator 2 for reconstitution, and standing after dissolving the dry powder completely. And selecting a calibration product at an instrument operation interface, selecting a two-step method, and calibrating according to the selected protein marker. Taking out the reagent strips balanced in advance, adding 100 mu L of the redissolved standard substance for detection, and setting the double-hole detection for the standard substances 1 and 2. And after the calibration is finished, checking a standard curve, displaying that the calibration is successful, and detecting a sample.
Step 4: sample detection: when a new batch of reagent is used, a reagent batch number is required to be recorded, if the batch exists, the reagent batch number does not need to be recorded again, a corresponding number of reagent strips and samples are prepared, the reagent strips are loaded into the clamping grooves, 100 mu L of samples are simultaneously added into the corresponding reagent strips, the samples and enough gun heads are put into the instrument, the clicking is started, and the instrument starts to detect. And after the instrument detection is finished, cleaning the waste liquid box, cleaning the table top, and closing the instrument.
(1) Aiming at the liver cancer patient in-vitro blood sample, AFP protein is used for distinguishing the liver cancer patient in-vitro blood sample from other cancer in-vitro blood samples.
The AUC analysis and the concentration analysis were performed on AFP concentration data in the two groups of data using 30 ex-vivo blood samples of liver cancer patients as a tumor group, colorectal cancer patient ex-vivo blood samples (15 cases) and gastric cancer patient ex-vivo blood samples (15 cases) as a control group.
As shown in fig. 4, the AUC value of the blood sample for distinguishing liver cancer patients from colorectal cancer and gastric cancer patients using AFP was 0.752. The result shows that the blood of liver cancer patients can be better distinguished from the in vitro blood samples of other cancer patients by using AFP.
As shown in FIG. 5, AFP concentrations in ex vivo blood samples of different cancers were analyzed. The result shows that the concentration of AFP in the isolated blood sample of the liver cancer patient is far higher than that of the isolated blood sample of other cancer patients.
The results show that: the blood sample of the liver cancer patient can be better distinguished by the serum AFP protein concentration, and the blood sample of the other two (colorectal cancer and gastric cancer) digestive tract tumor patients can be better distinguished.
(2) For colorectal cancer ex vivo blood samples, CA24-2 was used to distinguish colorectal cancer patient ex vivo blood samples from other cancer patient ex vivo blood samples.
The AUC analysis and the concentration analysis were performed on the two sets of data using 30 cases of colorectal cancer patient ex-vivo blood samples as a tumor group, liver cancer patient ex-vivo blood samples (15 cases) and gastric cancer patient ex-vivo blood samples (15 cases) as a control group.
As shown in fig. 6, AUC value of the gastric cancer patient ex-vivo blood sample, which was used to distinguish colorectal cancer from liver cancer patient ex-vivo blood sample, was 0.79. The results show that the use of CA24-2 can better distinguish colorectal cancer in vitro blood samples from other two cancer patients.
As shown in FIG. 7, the concentration of CA24-2 in ex vivo blood samples of different cancers was simultaneously counted. The results show that the concentration of CA24-2 in the ex-vivo blood samples of colorectal cancer patients is far greater than that of other cancer patients.
The above results indicate that: the blood sample of the colorectal cancer patient isolated from the other two (liver cancer and stomach cancer) digestive tract tumor patients isolated from the blood sample can be better distinguished by detecting the concentration of the serum CA24-2 protein.
(3) Aiming at the gastric cancer patient in-vitro blood sample, G17 protein is used for distinguishing the gastric cancer patient in-vitro blood sample from other cancer in-vitro blood samples.
The AUC analysis and the concentration analysis were performed on the G17 concentration data in the two groups of data using 30 cases of the gastric cancer patient ex-vivo blood samples as the tumor group, the colorectal cancer patient ex-vivo blood samples (15 cases) and the liver cancer patient ex-vivo blood samples (15 cases) as the control group.
As shown in fig. 8, AUC analysis was performed on the gastric cancer patient's ex-vivo blood and the liver cancer patient's ex-vivo blood and colorectal cancer patient's ex-vivo blood samples using gastrin protein G17. Analysis showed that AUC reached 0.763. The G17 can better distinguish the in-vitro blood sample of the gastric cancer patient from the in-vitro blood sample of other cancer patients.
As shown in fig. 9, the G17 concentration of ex vivo blood samples from different cancer patients was analyzed. The concentration of G17 in the in vitro blood sample of the gastric cancer patient is obviously lower than that of the in vitro blood samples of other cancer patients.
The results show that: the blood sample isolated from gastric cancer patients and the blood sample isolated from other two (colorectal cancer and liver cancer) digestive tract tumors can be better distinguished through the serum G17 protein concentration.
In conclusion, the protein marker and the DNA methylation marker disclosed by the application are combined to assist in detecting early-stage digestive tract tumors, so that the protein marker and the DNA methylation marker have practical application value.
Finally, it should be noted that: the foregoing description is only of the preferred embodiments of the application and is not intended to limit the scope of the application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.
Claims (5)
1. A dual marker composition for detecting early stage gut tumor, wherein the dual marker composition is a protein marker and a DNA methylation biomarker;
the protein marker is alpha fetoprotein, gastrin and glycoprotein antigen 24-2;
the DNA methylation biomarker is chr7:45018849-45018850 in the hg19 human genome;
the serum alpha fetoprotein concentration can distinguish an in-vitro blood sample of a liver cancer patient from an in-vitro blood sample of a colorectal cancer and gastric cancer digestive tract tumor patient;
the blood sample of the colorectal cancer patient in vitro and the blood sample of the liver cancer and gastric cancer digestive tract tumor patient in vitro can be distinguished by detecting the concentration of the serum glycoprotein antigen 24-2 protein;
the serum gastrin protein concentration can be used for distinguishing an in-vitro blood sample of a gastric cancer patient from an in-vitro blood sample of colorectal cancer, liver cancer and digestive tract tumor.
2. Use of a reagent for detecting the dual marker composition of claim 1 in the manufacture of a kit for detecting early stage gut tumors, wherein the reagent comprises a probe for detecting the dual marker composition of claim 1 and a protein quantification antibody;
the probe is a hybridization capture DNA sequence fragment comprising the dual-marker composition of claim 1; the probe is used for detecting the methylation level of the DNA methylation biomarker;
the protein quantitative antibodies include alpha fetoprotein antibodies, gastrin protein antibodies and glycoprotein antigen 24-2 antibodies.
3. The use according to claim 2, wherein the DNA methylation component in the kit is detected using the following detection platform: reagents used in PCR amplification, methylation chip, liquid chip, bisulfite sequencing, first generation sequencing, second generation sequencing, third generation sequencing, or a combination thereof.
4. The use according to claim 3, wherein the PCR amplification method comprises digital PCR and fluorescent quantitative PCR.
5. The use according to claim 2, wherein the detection of the protein component in the kit is performed by immunofluorescence quantification.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110603329A (en) * | 2017-03-02 | 2019-12-20 | 优美佳肿瘤技术有限公司 | Methylation markers for diagnosis of hepatocellular carcinoma and lung cancer |
| CN110927392A (en) * | 2020-02-10 | 2020-03-27 | 首都医科大学附属北京友谊医院 | Marker for detecting digestive system cancer and application thereof |
| CN111560434A (en) * | 2020-05-18 | 2020-08-21 | 苏州唯善生物科技有限公司 | Primer probe set, kit, detection method and application for detecting digestive tract tumor marker |
| CN114317762A (en) * | 2022-03-16 | 2022-04-12 | 北京莱盟君泰国际医疗技术开发有限公司 | Three-marker composition for detecting early liver cancer and kit thereof |
| CN114574587A (en) * | 2022-04-06 | 2022-06-03 | 北京莱盟君泰国际医疗技术开发有限公司 | Marker composition for colorectal cancer detection and application thereof |
| WO2022121414A1 (en) * | 2020-12-08 | 2022-06-16 | 东莞市朋志生物科技有限公司 | Anti-ca24-2 antibody, reagent and kit for detecting ca24-2 |
| WO2022143226A1 (en) * | 2020-12-30 | 2022-07-07 | 苏州唯善生物科技有限公司 | Digestive track tumor marker combination, detection reagent kit, and uses thereof |
| WO2022226231A1 (en) * | 2021-04-21 | 2022-10-27 | Helio Health Inc. | Liver cancer methylation and protein markers and their uses |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20190300964A1 (en) * | 2016-07-06 | 2019-10-03 | YourHealth Biotech, Limited | Colon cancer methylation markers and uses thereof |
| US20190300965A1 (en) * | 2016-07-06 | 2019-10-03 | Youhealth Biotech, Limited | Liver cancer methylation markers and uses thereof |
| CN107847515B (en) * | 2016-07-06 | 2021-01-29 | 优美佳生物技术有限公司 | Solid tumor methylation marker and application thereof |
-
2022
- 2022-12-27 CN CN202211691474.6A patent/CN116083581B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110603329A (en) * | 2017-03-02 | 2019-12-20 | 优美佳肿瘤技术有限公司 | Methylation markers for diagnosis of hepatocellular carcinoma and lung cancer |
| CN110927392A (en) * | 2020-02-10 | 2020-03-27 | 首都医科大学附属北京友谊医院 | Marker for detecting digestive system cancer and application thereof |
| CN111560434A (en) * | 2020-05-18 | 2020-08-21 | 苏州唯善生物科技有限公司 | Primer probe set, kit, detection method and application for detecting digestive tract tumor marker |
| WO2022121414A1 (en) * | 2020-12-08 | 2022-06-16 | 东莞市朋志生物科技有限公司 | Anti-ca24-2 antibody, reagent and kit for detecting ca24-2 |
| WO2022143226A1 (en) * | 2020-12-30 | 2022-07-07 | 苏州唯善生物科技有限公司 | Digestive track tumor marker combination, detection reagent kit, and uses thereof |
| WO2022226231A1 (en) * | 2021-04-21 | 2022-10-27 | Helio Health Inc. | Liver cancer methylation and protein markers and their uses |
| CN114317762A (en) * | 2022-03-16 | 2022-04-12 | 北京莱盟君泰国际医疗技术开发有限公司 | Three-marker composition for detecting early liver cancer and kit thereof |
| CN114574587A (en) * | 2022-04-06 | 2022-06-03 | 北京莱盟君泰国际医疗技术开发有限公司 | Marker composition for colorectal cancer detection and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| Circulating tumor DNA methylation marker MYO1-G for diagnosis and monitoring of colorectal cancer;Wu‑Hao Lin 等;Clinical Epigenetics;第13卷(第1期);摘要 * |
| ctDNA在结直肠癌诊断中的应用进展;于恒响 等;四川生理科学杂志;第42卷(第3期);第364页左栏第2段 * |
| 刘丽娟 等.肿瘤标志物的研究进展和临床应用.云南科技出版社,2009,(第1版),第311-315页. * |
| 崔巍 等.医学检验科诊断常规.中国医药科技出版社,2020,(第2版),第71-82页. * |
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