CN113834941B - Marker for prognosis diagnosis of colon cancer based on B cell expression and application thereof - Google Patents
Marker for prognosis diagnosis of colon cancer based on B cell expression and application thereof Download PDFInfo
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Abstract
The invention discloses a marker for prognosis diagnosis of colon cancer based on B cell expression and application thereof, belongs to the field of molecular biology and clinical prognosis judgment, and particularly relates to application of a detection reagent for CD19 protein expression level in preparation of a diagnosis product for prognosis prediction of colon cancer, wherein colon cancer comprises colon cancer metastasis-free and colon cancer liver metastasis, the expression level of the CD19 protein is detected to reflect the expression level of B cells of a colon cancer patient and is used for diagnosis of prognosis prediction of the colon cancer patient, a diagnosis product or a diagnosis model for prognosis prediction of colon cancer is obtained, and the result of judging the prognosis condition of the colon cancer patient by the detection reagent is accurate, objective, convenient and rapid, thereby providing a new reference basis for clinical diagnosis and treatment of colon cancer.
Description
Technical Field
The invention relates to the field of molecular biology and clinical prognosis, in particular to a marker for prognosis diagnosis of colon cancer based on B cell expression and application thereof.
Background
Colon cancer is one of the most common malignant tumors worldwide, and due to the changes of life style and dietary structure, the incidence rate of colon cancer in China is gradually increased in recent years, the life health of people is seriously affected, wherein liver metastasis is one of the most common metastasis parts of colon cancer, the prognosis is poor, and the prognosis evaluation of colon cancer and colon cancer liver metastasis is still relatively lacking at present.
Previous studies have shown that immune cell infiltration of tumor tissue may be closely related to prognosis of tumor, and tumor immune microenvironment may play an important role in tumor metastasis and prognosis, such as regulatory T cells, cytotoxic T cells, etc. In fact, the composition of the colon tumor immune microenvironment is complex, the survival difference of patients can be large due to different microenvironment compositions, and the method for searching immune cells possibly playing an important role in the colon tumor immune microenvironment and analyzing and evaluating the prognosis of colon cancer and colon cancer liver metastasis patients according to the immune cells is needed to be solved; in addition, the research discovers that the B cells and the subpopulations thereof can inhibit liver metastasis of colon cancer and improve the immune microenvironment of the colon cancer, and therefore, the expression of the B cells is presumed to be possibly related to prognosis of the colon cancer.
Immunohistochemical staining is a relatively mature method for evaluating the expression of various molecules in tumor tissues, and the expression level of B cells can be reflected by detecting the expression of CD19, so that a method for evaluating prognosis of colon cancer patients is not reported at home and abroad.
Disclosure of Invention
In view of the shortcomings of colon cancer prognosis in the prior art, a first object of the present invention is to provide an application of a detection reagent for detecting the expression level of CD19 protein in preparing a diagnostic product for prognosis prediction of colon cancer, wherein the expression level of CD19 protein reflects the expression level of B cells of a colon cancer patient, and the diagnostic reagent is used for diagnosing prognosis prediction of colon cancer and colon cancer liver metastasis patients.
A second object of the present invention is to provide a diagnostic product for prognosis prediction of colon cancer.
A third object of the present invention is to provide a diagnostic model for prognosis prediction of colon cancer.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the invention provides application of a detection reagent for CD19 protein expression level in preparation of a diagnosis product for prognosis prediction of colon cancer, wherein the colon cancer comprises colon cancer metastasis-free and colon cancer liver metastasis.
As a preferred embodiment, the detection reagent for the expression level of CD19 protein includes an antibody that specifically binds to CD19 protein, or further includes a detection buffer.
Further, the antibody is a monoclonal antibody or a polyclonal antibody.
As a preferred technical scheme, the detection reagent for the expression level of the CD19 protein detects the CD19 protein in a tissue sample by an immunohistochemical method to determine the expression level of the CD19 protein, wherein the high expression level of the CD19 protein reflects the high expression level of B cells in the tissue sample, and represents that the prognosis of a colon cancer patient is good.
Further, the method for detecting the CD19 protein in the tissue sample by using the detection reagent for the expression level of the CD19 protein through an immunohistochemical method comprises the following steps:
(1) Taking an isolated colon cancer tissue sample, embedding paraffin wax and preparing a tissue section;
(2) And (3) HE staining colon cancer cells in the tissue sections, performing immunohistochemical expression scoring, namely H scoring, on the CD19 protein according to the percentage and the intensity of cell staining, and comparing the H scoring with an optimal threshold value of CD19 protein expression to judge the expression level of the CD19 protein in colon cancer tissues.
Further, the H-score ranges from 0 to 300, H-score = Σ (pi×i) = (weakly stained cell percentage×1) + (moderately stained cell percentage×2) + (strongly stained cell percentage×3), where:
PI represents the percentage of positive cell numbers to all cell numbers in the tissue section;
i represents the color intensity;
and calculating an optimal threshold value of CD19 protein expression by adopting X-tile software, judging that the expression level of the CD19 protein in colon cancer tissues is high when the H value is more than or equal to the optimal threshold value of CD19 protein expression, and judging that the expression level of the CD19 protein in colon cancer tissues is low when the H value is less than the optimal threshold value of CD19 protein expression.
Further, the optimal threshold for CD19 protein expression is 58.2.
The invention also provides a diagnosis product for prognosis prediction of colon cancer, which comprises a detection reagent for the expression level of CD19 protein and a product instruction; wherein the product specification includes: a step of detecting the expression level of CD19 protein by the detection reagent of the expression level of CD19 protein, and a judgment standard for prognosis of colon cancer patients, wherein the judgment standard is as follows: the high expression level of the CD19 protein reflects the high expression level of B cells in the tissue sample, and represents that the prognosis of a patient with colon cancer is good; wherein the colon cancer comprises colon cancer metastasis free and colon cancer liver metastasis.
As a preferred technical scheme, the detection reagent for the expression level of the CD19 protein comprises an antibody which specifically binds to the CD19 protein, or further comprises a detection buffer solution, and the expression level of the CD19 protein is determined by detecting the CD19 protein in a tissue sample through an immunohistochemical method.
Further, the diagnostic products include kits, chips, and test papers.
The invention also provides a diagnosis model for prognosis prediction of colon cancer, which comprises the diagnosis product for prognosis prediction of colon cancer.
The term "prognosis" as referred to herein is meant to provide a prediction of the likely course and outcome of colon cancer, both to determine the particular outcome of a disease (e.g., recovery, appearance or disappearance of certain symptoms, signs and complications, and other abnormalities, and death), and to provide a time cue, e.g., to predict the likelihood of a certain outcome occurring within a certain period of time, which may include the likelihood of complications, metastasis, spread of cancer, the likely outcome of cancer, likelihood of recovery, total survival, and/or total mortality. Preferably, prognosis is the probability of a patient recovering or having a recurrence/recurrence of cancer, e.g. "better prognosis" means that cancer is less prone to recurrence metastasis and "worse prognosis" means that cancer is more prone to recurrence metastasis, this information is useful not only to the patient, but also to the physician in determining the most effective course of treatment, determining the likelihood of recurrence or likelihood of metastasis of cancer helps the physician in determining whether a more conservative or more aggressive treatment should be taken. Prognosis may be used to select and classify patients predicted to benefit from a given treatment regimen, as well as to design appropriate therapies for treatment of colon cancer, for example by showing whether colon cancer is still in benign stages or whether colon cancer has progressed to stages requiring interventional therapy.
The term "high expression" or "low expression" as used herein refers to the expression level of CD19 protein in an isolated tissue sample from a test subject above or below a reference value which is a statistically significant average or median value obtained by one skilled in the art from a specific population, e.g., a population with a better prognosis of colon cancer, by conventional means.
The high and low expression of the CD19 protein is carried out according to the CD19 protein staining score, and then the evaluation is carried out by a professional, and the high and low expression are classified.
Compared with the prior art, the invention has the beneficial effects that:
the invention reflects the expression level of B cells of a colon cancer patient by detecting the expression level of the CD19 protein for the first time, and uses the expression level to diagnose the prognosis prediction of the colon cancer patient, thereby obtaining a diagnosis product or a diagnosis model for the prognosis prediction of the colon cancer patient, judging the prognosis situation of the colon cancer patient more accurately and objectively, being convenient and quick, and providing a new reference basis for clinically treating the colon cancer.
Drawings
FIG. 1 is a photograph of HE staining for immunohistochemical detection of CD19 protein expression in the example; wherein: (a) high expression of CD19 protein; (b) CD19 protein is expressed under.
FIG. 2 is a graph showing survival analysis of colon cancer patients in the examples; wherein: (a) CD19 protein low expression; (b) high expression of CD19 protein.
FIG. 3 is a survival analysis of colon cancer patients in the examples; wherein: (1) colon cancer non-metastatic patients; (2) a colon cancer liver metastasis patient; (3) Colon cancer in patients with liver metastasis only, (a) CD19 protein is under-expressed; (b) high expression of CD19 protein.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, which can be made by a person skilled in the art without creative efforts, based on the described embodiments of the present invention fall within the protection scope of the present invention.
The following examples provide methods for detecting CD19 protein in a tissue sample by immunohistochemical methods, comprising the steps of: (1) obtaining a fresh colon cancer tissue specimen and manufacturing a tissue chip; (2) embedding, dewaxing and hydrating tissue chips; (3) inactivating endogenous enzyme, soaking in antigen retrieval liquid, and sealing; (4) primary antibody to CD19 (ab 134114, abcam) was incubated overnight; (5) incubating the secondary antibody; (6) DBA color development; (7) hematoxylin staining; (8) sealing the tree glue and photographing; (9) immunohistochemical expression score for CD19 (H score); statistical analysis was performed using SPSS22.0 and GraphPad Prism 7.0, survival was plotted using Kaplan-Meier, and comparison of survival analysis between groups using Log-rank test to obtain P-value and HR-value, P-value <0.05 was considered to have significant statistical difference.
Example 1
1. Case selection
This example co-incorporates 401 colon cancer patients from 2008, 6 months to 2012, 11 months in general surgery in a middle mountain hospital affiliated with the university of double denier, 281 of which patients with no metastasis, 120 of which patients with liver metastasis of colon cancer (98 of which patients with liver metastasis) were treated, clinical information of the patients was recorded and followed up, and the follow up of all patients was stopped until 2018, 6 months. The experiment has been approved by the ethical committee of the secondary university affiliated zhongshan hospital, and all patients provided written informed consent.
2. Preparation of tissue chip
(1) Fresh colon cancer tissue specimens were taken, fixed by soaking in 10% formaldehyde for 24 to 48 hours, carefully trimmed with a surgical knife in a fume hood after removal, placed in an embedding box and numbered.
(2) The embedded boxes are placed in a dehydrator to dehydrate with ethanol, benzene and xylene with different concentrations, and are immersed in wax.
(3) Placing the tissue in an embedding machine, adding melted wax for embedding and marking, cooling and solidifying at-20 ℃, taking out the wax block after the solidification is completed, and trimming and slicing.
(4) And (3) placing the trimmed wax block into a slicing machine for slicing, setting the slicing thickness to be 4 microns, transferring the sliced sheet into a spreading machine after slicing, slightly picking up the flattened sliced sheet by using a glass slide, and storing the marked sliced sheet in a slicing box.
(5) And (3) performing HE staining on paraffin sections, observing and defining required tissues through a microscope, marking, designing an array, punching by using a tissue chip array manufacturing instrument, transferring the tissues marked in the donor wax blocks into acceptor wax block holes, taking the acceptor wax blocks after completion, placing the acceptor wax blocks into an incubator to fuse the tissues with the acceptor wax blocks, placing the fused tissues into a refrigerator at 4 ℃ for storage, and cutting tissue chips on a glass slide by using the method for subsequent experiments.
3. Immunohistochemical staining
(1) The slices were taken and placed in an oven at 60℃for 30 minutes.
(2) Taking out the baked slice, soaking in 3 times of xylene and 3 times of ethanol for dewaxing and hydrating.
(3) 3% Hydrogen peroxide at room temperature for 5-10 min to extinguish endogenous enzyme, ddH 2 O was washed 3 times.
(4) Soaking in antigen retrieval liquid, heating with high fire in a microwave oven for 5 minutes, powering off for 5-10 minutes, heating with medium and high fire for 5 minutes, taking out, and cooling at room temperature.
(5) After washing 2 times with PBS, BSA blocking solution was added dropwise, and incubated at room temperature for 20 minutes.
(6) The primary antibodies to CD19 were all according to 1:100, adding the primary antibody working solution, and incubating overnight at 4 ℃ in a wet box.
(7) After overnight the wet box was equilibrated at room temperature for 15 minutes, washed 2 times with PBS and goat anti-rabbit IgG concentrate 1:100 was diluted to working solution to completely cover the tissue on the slide and incubated at 37℃for 30 minutes.
(8) PBS is washed for 2 times, SABC-POD working solution (diluted 1:100) is added dropwise to the tissue after airing, the reaction is carried out for 30 minutes at 37 ℃, and the PBS is washed for several times.
(9) Airing, adding DBA working solution, observing whether color development is carried out or not under a microscope, and carrying out ddH after color development 2 The reaction was terminated by O-washing.
(10)ddH 2 O is washed 3 times, soaked in hematoxylin dye solution for dyeing for 1 minute, and washed by running water for 30 minutes.
(11) Immersing the dried glass slide in ethanol and dimethylbenzene for dehydration and transparency, naturally airing in a fume hood, sealing the glass slide with gum after airing, and drying overnight at 37 ℃.
(12) Observed under a microscope and photographed.
Table 1: experimental reagent
| Reagent name | Manufacturer(s) | Production area |
| Ethanol | Sinopharm Group Chemical Reagent Co., Ltd. | China |
| Xylene (P) | Sinopharm Group Chemical Reagent Co., Ltd. | China |
| Alcoholic benzene | Sinopharm Group Chemical Reagent Co., Ltd. | China |
| Formaldehyde | BIOSHARP Biotech Co.Ltd | China |
| Hydrogen peroxide | Sinopharm Group Chemical Reagent Co., Ltd. | China |
| PBS buffer | Biyun Tian Biotechnology Co.Ltd | China |
| SABC-POD kit | BOSTER Co Ltd | USA |
| Citrate antigen retrieval liquid | Biyun Tian Biotechnology Co.Ltd | China |
| Rabbit anti-human CD19 polyclonal Long Shan antibody | abcam Co Ltd | British UK |
| Anti-rabbit poly-HRP for Doxil sheep | YX Biotech Co Ltd | China |
| Anti-dilution liquid | Biyun Tian Biotechnology Co.Ltd | China |
| Secondary antibody diluent | Biyun Tian Biotechnology Co.Ltd | China |
| Bovine Serum Albumin (BSA) | Shanghai Ding national biotechnology Co.Ltd | China |
| Hematoxylin dyes | DAKO Co Ltd | Danish |
| Neutral gums | BIOSHARP Biotech Co.Ltd | China |
Table 2: experimental instrument and consumable
4. Statistical method
(1) Expression of immunohistochemistry: histologically scoring CD19 based on the percentage and intensity of positive cell staining (H-score) ranging from 0 to 300, H-score = Σ (pi×i) = (weak staining cell percentage×1) + (medium intensity staining cell percentage×2) + (strong staining cell percentage×3), where PI represents the percentage of positive cell number to all cell numbers in the section; i represents the color strength. The optimal threshold (high or low expression divided by this H value) was calculated using X-tile software and the subsequent analysis was performed based on this threshold (optimal threshold for CD19 was 58.2) (fig. 1).
(2) Statistical analysis was performed using SPSS22.0 and GraphPad Prism 7.0. The comparison between groups of survival analysis using Log-rank test to obtain P and HR values using Kaplan-Meier method, P <0.05 was considered to have significant statistical differences, where "×" in the statistical plot indicates P <0.05, "×" indicates P <0.01, "×" indicates P <0.001, "×" indicates P <0.0001.
5. Experimental results
As shown in fig. 2, survival analysis of all colon cancer patients in the group revealed that the prognosis of colon cancer patients with high expression of CD19 protein, i.e., B cells (p= 0.031,HR:1.734, 95%CI:1.127-2.667) was significantly better.
As shown in fig. 3, survival analysis was performed on colon cancer non-metastatic patients and colon cancer liver metastatic patients, respectively, and it was found that there was a better trend in prognosis of high-expression B cells in colon cancer non-metastatic patients, but there was no statistical difference (p= 0.270,HR:1.963, 95%CI:0.728-5.296) (fig. 3-1), whereas the prognosis of high-expression B cells in colon cancer liver metastatic patients was significantly better (p= 0.005,HR:2.156, 95%CI:1.370-3.391) (fig. 3-2), and the same result was found in patients with liver metastasis alone (p= 0.022,HR:2.019, 95%CI:1.210-3.369) (fig. 3-3), because colon cancer non-metastatic patients were better in general survival, suggesting that the expression level of B cells might be more suitable for survival assessment of colon cancer liver metastatic patients.
In conclusion, the invention is used for evaluating the prognosis of colon cancer and colon cancer liver metastasis based on the expression condition of the CD19 marker B cells, firstly, tissue sections of a primary colon cancer focus are prepared, the expression condition of the B cells is reflected by immune scoring based on the expression of the CD19 protein through immunohistochemical staining, and the B cells are grouped according to the immune scoring, so that the patients with the colon cancer and colon cancer liver metastasis with high expression of the B cells have better prognosis, the result is accurate, objective, convenient and quick, and a new reference basis is provided for clinical diagnosis and treatment of the colon cancer.
Claims (1)
1. Use of a detection reagent for the expression level of CD19 protein in the manufacture of a diagnostic product for prognosis prediction of colon cancer, including colon cancer metastasis-free and colon cancer liver metastasis;
the detection reagent for the expression level of the CD19 protein comprises an antibody which specifically binds to the CD19 protein, and also comprises a detection buffer solution;
the antibody is a monoclonal antibody or a polyclonal antibody;
the detection reagent for the expression level of the CD19 protein detects the CD19 protein in a tissue sample by an immunohistochemical method to determine the expression level of the CD19 protein, wherein the high expression level of the CD19 protein reflects the high expression level of B cells in the tissue sample, and represents that the prognosis of a colon cancer patient is good;
the method for detecting the CD19 protein in the tissue sample by using the detection reagent for the expression level of the CD19 protein through an immunohistochemical method comprises the following steps:
(1) Taking an isolated colon cancer tissue sample, embedding paraffin wax and preparing a tissue section;
(2) Performing HE staining on colon cancer cells in the tissue sections, performing immunohistochemical expression scoring on CD19 protein according to the percentage and intensity of cell staining, namely, H scoring, and comparing the H scoring with an optimal threshold value of CD19 protein expression to judge the expression level of the CD19 protein in colon cancer tissues;
the H-score ranges from 0 to 300, H-score = Σ (pi×i) = (weakly stained cell percentage×1) + (moderately stained cell percentage×2) + (strongly stained cell percentage×3), where:
PI represents the percentage of positive cell numbers to all cell numbers in the tissue section;
i represents the color intensity;
calculating an optimal threshold value of CD19 protein expression by adopting X-tile software, judging that the expression level of the CD19 protein in colon cancer tissues is high when the H value is more than or equal to the optimal threshold value of CD19 protein expression, and judging that the expression level of the CD19 protein in colon cancer tissues is low when the H value is less than the optimal threshold value of CD19 protein expression;
wherein the optimal threshold for CD19 protein expression is 58.2;
survival analysis is carried out on colon cancer non-metastatic patients and colon cancer liver metastatic patients respectively, and the prognosis of high-expression B cells of colon cancer non-metastatic patients is found to have better trend, but no statistical difference exists, P=0.270, HR:1.963 95% CI:0.728-5.296, whereas the prognosis of high expressing B cells in patients with colon cancer liver metastasis is significantly better, p=0.005, hr:2.156 95% CI:1.370-3.391, significantly better prognosis of high expressing B cells in patients with liver metastasis alone, p=0.022, hr:2.019 95% CI:1.210-3.369, since the general survival of patients without colon cancer metastasis is better, the expression level of B cells is suggested to be more suitable for survival assessment of patients with colon cancer liver metastasis.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2619340C1 (en) * | 2016-02-11 | 2017-05-15 | Федеральное государственное бюджетное учреждение "Ростовский научно-исследовательский онкологический институт" Министерства здравоохранения Российской Федерации | Method for local-distributed colon cancer metastasis |
| CN110763845A (en) * | 2018-07-27 | 2020-02-07 | 成都市第三人民医院 | Application of ligand for detecting protein marker in preparation of product for diagnosing colon cancer and kit |
| CN113281516A (en) * | 2021-06-29 | 2021-08-20 | 复旦大学附属中山医院 | Application of CUL9 as marker in colorectal cancer prognosis evaluation |
| CN114019163A (en) * | 2021-11-02 | 2022-02-08 | 复旦大学附属中山医院 | Marker for prognosis diagnosis of colon cancer based on expression of activated B cells and use thereof |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1833996B1 (en) * | 2005-01-06 | 2012-12-05 | Genentech, Inc. | Cancer prognostic methods |
| EP1777523A1 (en) * | 2005-10-19 | 2007-04-25 | INSERM (Institut National de la Santé et de la Recherche Médicale) | An in vitro method for the prognosis of progression of a cancer and of the outcome in a patient and means for performing said method |
| JP5970560B2 (en) * | 2012-01-20 | 2016-08-17 | アンスティチュ ナショナル ドゥ ラ サンテ エ ドゥ ラ ルシェルシュ メディカル | Method for predicting survival time of patients suffering from solid cancer based on B cell density |
| ES2707285T3 (en) * | 2012-01-20 | 2019-04-03 | Inst Nat Sante Rech Med | Method for the prognosis of the survival time of a patient suffering from a solid cancer |
| WO2018024608A2 (en) * | 2016-08-03 | 2018-02-08 | Cbmed Gmbh Center For Biomarker Research In Medicine | Antitumor compounds and tumor diagnosis |
| US20200095643A1 (en) * | 2017-02-07 | 2020-03-26 | Inserm (Institut National De La Sante Et De La Recherche Medicale) | Tim-3 for assessing the severity of cancer |
-
2021
- 2021-11-02 CN CN202111287602.6A patent/CN113834941B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2619340C1 (en) * | 2016-02-11 | 2017-05-15 | Федеральное государственное бюджетное учреждение "Ростовский научно-исследовательский онкологический институт" Министерства здравоохранения Российской Федерации | Method for local-distributed colon cancer metastasis |
| CN110763845A (en) * | 2018-07-27 | 2020-02-07 | 成都市第三人民医院 | Application of ligand for detecting protein marker in preparation of product for diagnosing colon cancer and kit |
| CN113281516A (en) * | 2021-06-29 | 2021-08-20 | 复旦大学附属中山医院 | Application of CUL9 as marker in colorectal cancer prognosis evaluation |
| CN114019163A (en) * | 2021-11-02 | 2022-02-08 | 复旦大学附属中山医院 | Marker for prognosis diagnosis of colon cancer based on expression of activated B cells and use thereof |
Non-Patent Citations (10)
| Title |
|---|
| Phenotype of peripheral blood leukocytes and survival of patients with metastatic colorectal cancer;P. Vesely´;《SHORT COMMUNICATION》;第20卷(第2期);第126-133页 * |
| Tumor-infiltrated activated B cells suppress liver metastasis of colorectal cancers;Yuqiu Xu;《Cell Reports 》;第1-25 * |
| 树突状细胞及其亚群在结肠直肠癌术后肝转移中的作用及其临床意义;秦建民;盛霞;杨林;撒忠秋;徐夏君;李琦;殷佩浩;张敏;陈腾;曹伟家;;外科理论与实践(第05期);全文 * |
| 结直肠癌患者化疗前后外周血T/B/NK淋巴细胞亚群水平研究;李世龙;张宝;宋杨;张海光;秦莉;吴丹;苗雅静;;标记免疫分析与临床(第11期);全文 * |
| 结直肠癌组织中BTG3蛋白表达及意义;布力布・吉力斯汉;唐勇;达热拜・热达提;赛福丁・柯尤木;;山东医药(第38期);全文 * |
| 结直肠癌肝转移与肝脏免疫功能;张森;大肠肛门病外科杂志(第02期);全文 * |
| 结直肠癌肝转移分子机制研究进展;王东亮;陈功;;中国实用外科杂志(第04期);全文 * |
| 结肠癌患者免疫状态研究进展;牛东生, 赵莉;国外医学.内科学分册(第07期);全文 * |
| 结肠癌术后行经导管肝动脉化疗栓塞联合区域灌注化疗对预后的影响;商斌;曹成海;;医学综述(第20期);全文 * |
| 肝动脉栓塞联合射频消融对结肠癌术后肝转移的疗效;蔡敏;殷涛;翁高龙;;中国病案(第07期);全文 * |
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