CN113721019A - Bile duct cancer postoperative survival prediction method, kit and application method of kit - Google Patents
Bile duct cancer postoperative survival prediction method, kit and application method of kit Download PDFInfo
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Abstract
The invention provides a bile duct cancer postoperative survival prediction method, a kit and an application method of the kit, relating to the technical field of bile duct cancer, wherein the method comprises the following steps: obtaining a bile duct cancer case specimen of a postoperative excision focus of a patient and a corresponding trefoil factor family gene expression level; wherein the trefoil factor family gene expression level is a prediction and prognosis index for the postoperative survival of the bile duct cancer; the TFF is used as an index for predicting the postoperative prognosis of the patient with the bile duct cancer, and the TFF gene expression level is input into the bile duct cancer prognosis prediction model constructed by the method based on the obtained TFF gene expression level to obtain the corresponding survival risk score, so that the survival condition of the patient with the bile duct cancer can be effectively predicted.
Description
Technical Field
The invention relates to the technical field of cholangiocarcinoma, in particular to a bile duct cancer postoperative survival prediction method, a kit and an application method of the kit.
Background
Cholangiocarcinoma (CCA) is a highly aggressive malignancy originating from biliary epithelia with a global incidence, especially on an increasing annual basis in asian countries with a 5-year survival rate of less than 5%. Bile duct cancer is hidden in early stage, has no obvious clinical manifestation, and is definitely diagnosed in the middle and late stages. Although clinical treatment means such as surgery, radiotherapy and chemotherapy exist at present, the prognosis of patients with cholangiocarcinoma is still poor. With intensive studies on the molecular level of CCA, several key regulators such as cancer suppressor genes p53, p16, BAP1, DPC4/SMAD4, others such as ARID1A, ARID1B, BAP1, PBRM1, TP53, STK11 and PTEN were found; the protooncogenes Ras family, C-erbB-2, C-ROS oncogene 1, IDH1/IDH2, and others such as KRAS, BRAF and PIK3CA may become therapeutic targets. In addition, the current commonly used targeting drugs in clinic mainly take EGFR, VEGFR, PDE-FR, BRAF, MEK, mTOR, LI-6, COX-2 and the like as action targets. Since the occurrence and development of CCA cancer involve mutation of multiple genes or interaction of multiple signal pathways, the genes can only be used as references for the occurrence and development of CCA, and the judgment of disease prognosis is very limited.
TFF (Trefoil factor family) has been studied to have characteristics of promoting epithelial cell migration and anti-apoptosis, and is considered to be highly related to tumor biological characteristics in various tumors, for example, TFF3 can be used as a serological detection index of lung cancer, TFF3 of non-small cell lung cancer tissue is highly expressed, and is related to pathological grading and lymph node metastasis; in the human papillary thyroid cancer cell line, TFF3 is possibly involved in the proliferation and apoptosis of TPC-1 cells by activating a PI 3K/Akt/NF-kB signal pathway, so that the occurrence and development of papillary thyroid cancer cells are influenced; for patients with colorectal cancer, the serum TFF3 level is remarkably increased, and the correlation with CEA, CA19-9 and TNM stage is remarkable; silencing TFF3 gene can reduce proliferation and invasion of ovarian cancer SKOV3 cells in vitro in ovarian cancer patients. Among related studies on CCA, there is no study on the correlation and prognosis prediction of the TFF family with CCA diseases. There remains a gap in the effective prediction of CCA prognosis.
In conclusion, the gene marker provided by the prior art is poor in the effect of judging the prognosis of a patient after CCA surgery, and the gene marker in the prior art is not ideal in the effect of selecting corresponding drugs for auxiliary treatment, so that the individual treatment effect on the CCA patient cannot be achieved; the TFF family is thought to be highly correlated with tumor biological properties in a variety of tumors, but has not been studied for prediction of CCA disease prognosis.
Disclosure of Invention
In view of the above, the invention provides a bile duct cancer postoperative survival prediction method, a kit and an application method of the kit, so as to solve the defect that the genetic marker in the prior art is poor in the effect of judging the prognosis of a patient after a CCA operation, and effectively predict the survival condition of the patient with bile duct cancer, thereby making the most favorable clinical decision for the patient.
Based on the above purpose, the invention provides a bile duct cancer postoperative survival prediction method, which comprises the following steps:
obtaining a bile duct cancer case specimen of a postoperative excision focus of a patient and a corresponding trefoil factor family gene expression level; wherein the trefoil factor family gene expression level is a prediction and prognosis index for the postoperative survival of the bile duct cancer;
inputting the trefoil factor family gene expression level into a bile duct cancer prognosis prediction model to obtain a survival risk score output by the bile duct cancer prognosis prediction model; the bile duct cancer prognosis prediction model is obtained by training based on the trefoil factor family gene expression level corresponding to the normal sample and the trefoil factor family gene expression level corresponding to the bile duct cancer sample.
Optionally, the bile duct cancer prognosis prediction model is obtained by training through the following steps:
obtaining the normal sample and the bile duct cancer sample;
obtaining trefoil factor family gene expression level corresponding to the normal sample and trefoil factor family gene expression level corresponding to the bile duct cancer sample;
and taking the trefoil factor family gene expression level as input data for training to obtain the bile duct cancer prognosis prediction model for generating the survival risk score of the bile duct cancer case specimen.
Optionally, the trefoil factor family gene expression level is detected by a kit, and the kit contains a trefoil factor family antibody and a trefoil factor family anti-antibody.
The invention also provides a kit for detecting the bile duct cancer determination marker, which comprises a kit liquid I and a kit liquid II, wherein the kit liquid I contains the trefoil factor family antibody, and the kit liquid II contains the trefoil factor family anti-antibody.
Optionally, the trefoil factor family includes trefoil factor 1, trefoil factor 2, and trefoil factor 3.
The invention also provides an application method of the kit, which comprises the following steps:
the biliary duct cancer case sample is separated in vitro, and the separated biliary duct cancer case sample is protected at low temperature;
paraffin-embedded tissues of isolated cholangiocarcinoma example samples;
slicing the tissue, and placing the glass slide containing the complete tissue in a water body at 40 ℃;
fishing out the glass slide and putting the glass slide into a 37 ℃ incubator for drying;
sequentially putting the dried glass slide into a reagent of dimethylbenzene-100% alcohol-95% alcohol-90% alcohol-80% alcohol-70% alcohol for dewaxing;
washing the dewaxed glass slide in clear water for a period of time, adding 3% H2O2Soaking in the solution for 10min, and then pouring off 3% H2O2Washing the solution twice in clear water, adding a citric acid buffer solution, cooking for 3min, cooling to room temperature just after boiling, cooking again, and cooling to room temperature to expose antigen sites;
pouring out the citric acid buffer solution, washing with clear water for 2 times and phosphate buffer salt solution for 2 times, wiping off the phosphate buffer salt solution around the tissue, adding serum immediately, and placing in a 37 ℃ incubator for 30 min;
taking out the glass slide, wiping the serum with absorbent paper, adding the first test kit liquid, and storing in a refrigerator at 4 ℃ overnight;
taking out the glass slide from the refrigerator, putting the glass slide into phosphate buffer saline solution for washing for 3 times, wiping the phosphate buffer saline solution around the tissue, adding the two liquid solutions of the kit, and then putting the two liquid solutions of the kit into a 37 ℃ incubator for 30 min;
taking out the glass slide from the incubator, washing the glass slide in phosphate buffer salt solution for 3 times, wiping the phosphate buffer salt solution around the tissue dry, adding a streptavidin-biotin compound, and then placing the glass slide in the incubator at 37 ℃ for 30 min;
taking out the tablet from the incubator, washing in phosphate buffer saline solution for 3 times, wiping off phosphate buffer saline solution around the tissue, and adding color developing agent.
Washing the developed slide glass with clear water for a period of time, and soaking the slide glass in hematoxylin for counterstaining;
washing the counterstained glass slide in clear water, sequentially putting the glass slide into 70% alcohol-80% alcohol-90% alcohol-95% alcohol-100% alcohol-xylene, finally soaking in xylene, and moving to a ventilation hood;
dropping neutral gum on the tissue side, covering with a cover glass, placing the sealed glass slide in a fume hood, and air drying.
Optionally, the thickness of the slice is 5 μm.
Optionally, in the dewaxing step, the dried glass slide is sequentially placed in xylene-100% alcohol-95% alcohol-90% alcohol-80% alcohol-70% alcohol reagents, and each reagent is placed for 10 min.
Optionally, the counterstained glass slide is washed with water, the glass slide is sequentially placed in 70% alcohol-80% alcohol-90% alcohol-95% alcohol-100% alcohol-xylene, and finally, the glass slide is soaked in xylene and moved to a ventilation hood, and each reagent is placed in the ventilation hood for 2 min.
Optionally, each time of washing with water in clean water and each time of washing with phosphate buffered saline solution is 5 min.
From the above, the bile duct cancer postoperative survival prediction method, the kit and the application method of the kit provided by the invention have the advantages that TFF is used as an index for predicting bile duct cancer patient postoperative prognosis, the TFF gene expression level is input into the bile duct cancer prognosis prediction model constructed by the method based on the obtained TFF gene expression level, the corresponding survival risk score is obtained, the survival condition of the bile duct cancer patient can be effectively predicted, and the most favorable clinical decision on the patient can be made. Furthermore, TFF markers show good performance in predicting survival and may be an independent prognostic indicator for survival prediction in patients with biliary tract cancer.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic flow chart of a bile duct cancer postoperative survival prediction method of the present invention;
FIG. 2 is a schematic flow chart of the method of using the kit of the present invention;
FIG. 3 is a TFF3 positive distribution chart of 121 CCA total samples tested by the kit of the invention;
FIG. 4 is a graph of overall survival for 121 total cases of CCA using the kit of the present invention;
FIG. 5 is a schematic diagram of immunohistochemistry for predicting high expression of TFF3 in Intrahepatic bile duct carcinoma (ICC) samples, which was the subject of the experiment using the kit of the present invention;
FIG. 6 is a schematic diagram of immunohistochemistry performed on an ICC sample with a low expression level of TFF3 in an experiment performed by using the kit of the present invention;
FIG. 7 is a schematic diagram of immunohistochemistry for high expression of TFF3 in Extrahepatic cholangiocarcinoma (ECC) samples, which was the subject of the experiment using the kit of the present invention;
FIG. 8 is a schematic diagram of immunohistochemistry performed on a sample of ECC on a TFF3 low-expression sample in an experiment performed by using the kit of the present invention;
FIG. 9 is a schematic diagram showing immunohistochemistry for high expression of TFF3 in a sample of Peripheral intrahepatic cholangiocarcinoma (PCC) tested using the kit of the present invention;
FIG. 10 is a schematic diagram of immunohistochemistry performed on samples of PCC with low expression of TFF3 according to the method of using the kit of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to specific embodiments and the accompanying drawings.
It is to be noted that technical terms or scientific terms used in the embodiments of the present invention should have the ordinary meanings as understood by those having ordinary skill in the art to which the present disclosure belongs, unless otherwise defined. The use of "first," "second," and similar terms in this disclosure is not intended to indicate any order, quantity, or importance, but rather is used to distinguish one element from another. The word "comprising" or "comprises", and the like, means that the element or item listed before the word covers the element or item listed after the word and its equivalents, but does not exclude other elements or items. The terms "connected" or "coupled" and the like are not restricted to physical or mechanical connections, but may include electrical connections, whether direct or indirect. "upper", "lower", "left", "right", and the like are used merely to indicate relative positional relationships, and when the absolute position of the object being described is changed, the relative positional relationships may also be changed accordingly.
As a preferred embodiment of the present invention, the present invention provides a method for predicting post-operation survival of cholangiocarcinoma, comprising the steps of:
obtaining a bile duct cancer case specimen of a postoperative excision focus of a patient and a corresponding trefoil factor family gene expression level; wherein the trefoil factor family gene expression level is a prediction and prognosis index for the postoperative survival of the bile duct cancer;
inputting the trefoil factor family gene expression level into a bile duct cancer prognosis prediction model to obtain a survival risk score output by the bile duct cancer prognosis prediction model; the bile duct cancer prognosis prediction model is obtained by training based on the trefoil factor family gene expression level corresponding to the normal sample and the trefoil factor family gene expression level corresponding to the bile duct cancer sample.
The invention also provides a kit for detecting the bile duct cancer determination marker, which comprises a kit liquid I and a kit liquid II, wherein the kit liquid I contains the trefoil factor family antibody, and the kit liquid II contains the trefoil factor family anti-antibody.
The invention also provides an application method of the kit, which comprises the following steps:
the biliary duct cancer case sample is separated in vitro, and the separated biliary duct cancer case sample is protected at low temperature;
paraffin-embedded tissues of isolated cholangiocarcinoma example samples;
slicing the tissue, and placing the glass slide containing the complete tissue in a water body at 40 ℃;
fishing out the glass slide and putting the glass slide into a 37 ℃ incubator for drying;
sequentially putting the dried glass slide into a reagent of dimethylbenzene-100% alcohol-95% alcohol-90% alcohol-80% alcohol-70% alcohol for dewaxing;
washing the dewaxed glass slide in clear water for a period of time, adding 3% H2O2Soaking in the solution for 10min, and then pouring off 3% H2O2Washing the solution twice in clear water, adding a citric acid buffer solution, cooking for 3min, cooling to room temperature just after boiling, cooking again, and cooling to room temperature to expose antigen sites;
pouring out the citric acid buffer solution, washing with clear water for 2 times and phosphate buffer salt solution for 2 times, wiping off the phosphate buffer salt solution around the tissue, adding serum immediately, and placing in a 37 ℃ incubator for 30 min;
taking out the glass slide, wiping the serum with absorbent paper, adding the first test kit liquid, and storing in a refrigerator at 4 ℃ overnight;
taking out the glass slide from the refrigerator, placing the glass slide into phosphate buffer salt solution for washing for 3 times, each time for 5min, wiping the phosphate buffer salt solution around the tissue, adding the two liquid solutions of the kit, and then placing the glass slide in a 37 ℃ incubator for 30 min;
taking out the glass slide from the incubator, washing the glass slide in phosphate buffer salt solution for 3 times, wiping the phosphate buffer salt solution around the tissue dry, adding a streptavidin-biotin compound, and then placing the glass slide in the incubator at 37 ℃ for 30 min;
taking out the tablet from the incubator, washing in phosphate buffer saline solution for 3 times, wiping off phosphate buffer saline solution around the tissue, and adding color developing agent.
Washing the developed slide glass with clear water for a period of time, and soaking the slide glass in hematoxylin for counterstaining;
washing the counterstained glass slide in clear water, sequentially putting the glass slide into 70% alcohol-80% alcohol-90% alcohol-95% alcohol-100% alcohol-xylene, finally soaking in xylene, and moving to a ventilation hood;
dropping neutral gum on the tissue side, covering with a cover glass, placing the sealed glass slide in a fume hood, and air drying.
By the bile duct cancer postoperative survival prediction method, the kit and the application method of the kit, TFF is used as an index for predicting bile duct cancer postoperative prognosis, and the TFF gene expression level is input into the bile duct cancer prognosis prediction model constructed by the method based on the obtained TFF gene expression level to obtain a corresponding survival risk score, so that the survival condition of a bile duct cancer patient can be effectively predicted, and the most favorable clinical decision on the patient can be made. Furthermore, TFF markers show good performance in predicting survival and may be an independent prognostic indicator for survival prediction in patients with biliary tract cancer.
The following describes preferred embodiments of the bile duct cancer postoperative survival prediction method, the kit and the application method of the kit according to the present invention with reference to the accompanying drawings.
Referring to fig. 1, the present invention provides a method for predicting survival after CCA operation, which includes the following steps:
s100, obtaining a CCA case sample of a focus of a patient after a surgery and a corresponding TFF gene expression level, wherein the TFF gene expression level in the method is a CCA postoperative survival prediction prognosis index, namely TFF in the method is used as a CCA prognosis determination marker.
S200, inputting the TFF gene expression level into a CCA prognosis prediction model to obtain a survival risk score output by the CCA prognosis prediction model. In the method, a CCA prognosis prediction model is obtained by training based on the TFF gene expression level corresponding to a normal sample and the TFF gene expression level corresponding to a CCA sample.
In addition, the method can also download gene expression profile data of a plurality of CCA samples and normal samples from a tumor genome map (TCGA) database, construct a TFF gene differential expression profile of the CCA in-vitro specimen, compare the TFF gene expression level corresponding to the CCA case specimen with the TFF gene differential expression profile of the CCA in-vitro specimen, and can also evaluate the survival risk of the patient to obtain the survival risk score.
In this method, the expression level of the TFF gene is detected by a kit containing a TFF antibody and a TFF anti-antibody.
Therefore, in the method, TFF is used as an index for predicting the postoperative prognosis of the CCA patient, the TFF gene expression level is input into the CCA prognosis prediction model constructed by the method based on the obtained TFF gene expression level, the corresponding survival risk score is obtained, the survival condition of the CCA patient can be effectively predicted, and the clinical decision which is most beneficial to the patient is made. Furthermore, TFF markers show good performance in predicting survival and may be an independent prognostic indicator for survival prediction in CCA patients.
It will be appreciated that the use of TFF as a prognostic assay marker for CCA may also find application in the molecular mechanisms of CCA development and progression. CCA has complex pathways and mechanisms in the occurrence and development process, more and more related genes are discovered through research, and cross network effect exists among the related genes. However, the research on the molecular mechanism of the TFF in CCA is temporarily lacked, so that the TFF as a CCA prognostic determination marker can provide help for the subsequent research on the molecular mechanism of CCA occurrence and progression.
It will be appreciated that the use of TFF as a prognostic CCA assay marker may also find application in tumour markers. The tumor marker is a substance which can reflect the occurrence and development of tumors and monitor the response of the tumors to treatment. The existing CCA markers for clinical detection have limited types and insufficient specificity, and can not be used for early diagnosis, prognosis judgment, treatment monitoring and the like of CCA diseases. Therefore, TFF as a CCA prognostic determination marker can provide a new tumor marker for clinic and is used for aspects such as prognosis judgment of CCA diseases and the like.
It will be appreciated that TFF as a prognostic CCA assay marker may also find application in drug targets. Biological targeted therapy is an interventional therapy strategy aimed at specific oncogenic sites (gene fragments, antigens/receptors or tumor microenvironment) to allow specific death of tumor cells by designing relevant therapeutic drugs into specific oncogenic sites without damaging normal tissues or cells. Currently, most of the CCA targeted therapies are still in experimental research stage and have less clinical applications. Therefore, TFF as a CCA prognostic determination marker can provide a new drug target for the targeted treatment of CCA, and provides a direction for subsequent research.
The CCA prognosis prediction model is obtained by training the following steps:
and A100, acquiring a normal sample and a CCA sample.
And A200, acquiring the TFF gene expression level corresponding to the normal sample and the TFF gene expression level corresponding to the CCA sample.
And A300, taking the TFF gene expression level as input data used for training to obtain a CCA prognosis prediction model for generating a survival risk score of a CCA case sample.
The invention also provides a kit comprising a marker for detecting CCA assay, i.e. for detecting TFF gene level, comprising a kit liquid and a kit liquid, wherein the kit liquid comprises a TFF antibody, i.e. a TFF primary antibody, and the kit liquid comprises a TFF anti-antibody, i.e. a TFF secondary antibody, and the TFF comprises TFF1, TFF2 and TFF 3.
Immunohistochemistry (IHC) is a technology for locating, characterizing and quantifying antigens in tissues by developing labeled antibodies through chemical reactions based on the principle of antigen-antibody specific binding. According to the principles of antigen-antibody reaction and chemo-coloration. Protein is extracted from tissues or cells to be detected to be used as antigen, the protein is firstly combined with primary antibody to form antigen-antibody reactant, then the antigen-antibody reactant is reacted with biotin or fluorescein labeled secondary antibody, the antigen-antibody reactant can be clearly observed under a microscope through chemical color development, and thus the distribution and the content of the antigen can be determined on tissue slices. The kit is based on the existing mature immunohistochemical technology, and specific antibodies, namely kit liquid I and kit liquid II, are added.
Referring to fig. 2, the present invention also provides an application method of the kit, which comprises the following steps:
a10, specimen in vitro: the CCA case specimens were isolated and the isolated CCA case specimens were cryoprotected.
A20, paraffin embedding: tissues of isolated CCA case samples were paraffin embedded.
A30, slicing: the tissue was sectioned and the slides containing intact tissue were placed in a40 ℃ water body. In this example, the thickness of the slices was 5 μm.
A40, drying: and taking out the glass slide and putting the glass slide into a 37 ℃ incubator for drying.
A50, dewaxing: and sequentially putting the dried glass slide into a dimethylbenzene-100% alcohol-95% alcohol-90% alcohol-80% alcohol-70% alcohol reagent for dewaxing. In step A50, each reagent was added for 10 min.
A60, antigen retrieval: washing the dewaxed glass slide in clear water for a period of time, adding 3% H2O2Soaking in the solution for 10min, and then pouring off 3% H2O2Washing the solution twice in clear water, adding citric acid buffer solutionAnd (3) cooking for 3min, cooling to room temperature just after boiling, cooking again, and cooling to room temperature to expose antigen sites.
A70, serum blocking: the citric acid buffer was decanted, washed 2 times in clear water and 2 times in PBS, the PBS surrounding the tissue was wiped dry, serum was immediately added, and the tissue was placed in a 37 ℃ incubator for 30 min.
A80, adding a primary antibody: the slides were removed, the serum was wiped dry with absorbent paper, the kit one was added and stored overnight in a4 ℃ freezer.
A90, plus secondary antibody: the slide was removed from the refrigerator, washed 3 times in Phosphate Buffered Saline (PBS), wiped to dry the PBS surrounding the tissue, added to the kit cocktail, and then placed in a 37 ℃ incubator for 30 min.
A100, adding SABC: the slide was removed from the incubator, washed 3 times in PBS, wiped to dry the PBS surrounding the tissue, followed by streptavidin-biotin complex (SABC), and then placed in an incubator at 37 ℃ for 30 min.
A110, adding a color developing agent: the slide was removed from the incubator, washed 3 times in PBS, wiped to dry the PBS surrounding the tissue, and then color developing agent was added.
A120, counterdyeing: and washing the developed glass slide with clear water for a period of time, and soaking the glass slide in hematoxylin for counterstaining.
A130, dehydration: washing the counterstained glass slide in clear water, sequentially placing the glass slide in 70% alcohol-80% alcohol-90% alcohol-95% alcohol-100% alcohol-xylene, finally soaking in xylene, and moving to a ventilation hood. In step A130, each reagent was added for 2 min.
A140, sealing piece: dropping neutral gum on the tissue side, covering with a cover glass, placing the sealed glass slide in a fume hood, and air drying. And obtaining the TFF gene expression level corresponding to the CCA case sample based on IHC.
In this example, each wash with water in clear water and each wash with PBS was 5 min.
Referring to fig. 3 to 10, a patient sample was collected 121 for routine hepatobiliary surgery and the pathology was diagnosed as "CCA" after the surgery. The immunohistochemical test of TFF3 was performed according to the method of using the kit provided by the present invention, and the positive distribution was shown in FIG. 3. From the results, a survival plot of CCA discriminatory expression TFF3 was plotted, as shown in fig. 4. Specifically, by comparing different position CCA conclusions, TFF3 is more meaningful for ICC disease prognosis.
Those of ordinary skill in the art will understand that: the discussion of any embodiment above is meant to be exemplary only, and is not intended to intimate that the scope of the disclosure, including the claims, is limited to these examples; within the idea of the invention, also features in the above embodiments or in different embodiments may be combined, steps may be implemented in any order, and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity.
The embodiments of the invention are intended to embrace all such alternatives, modifications and variances that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, substitutions, improvements and the like that may be made without departing from the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (10)
1. The bile duct cancer postoperative survival prediction method is characterized by comprising the following steps:
obtaining a bile duct cancer case specimen of a postoperative excision focus of a patient and a corresponding trefoil factor family gene expression level; wherein the trefoil factor family gene expression level is a prediction and prognosis index for the postoperative survival of the bile duct cancer;
inputting the trefoil factor family gene expression level into a bile duct cancer prognosis prediction model to obtain a survival risk score output by the bile duct cancer prognosis prediction model; the bile duct cancer prognosis prediction model is obtained by training based on the trefoil factor family gene expression level corresponding to the normal sample and the trefoil factor family gene expression level corresponding to the bile duct cancer sample.
2. The method for detecting an indicator of prognosis of postoperative survival of bile duct cancer according to claim 1, wherein the model for predicting prognosis of bile duct cancer is trained by the following steps:
obtaining the normal sample and the bile duct cancer sample;
obtaining trefoil factor family gene expression level corresponding to the normal sample and trefoil factor family gene expression level corresponding to the bile duct cancer sample;
and taking the trefoil factor family gene expression level as input data for training to obtain the bile duct cancer prognosis prediction model for generating the survival risk score of the bile duct cancer case specimen.
3. The method for detecting an index of prognosis of postoperative survival of bile duct cancer according to claim 1, wherein the trefoil factor family gene expression level is detected by a kit, and the kit comprises a trefoil factor family antibody and a trefoil factor family anti-antibody.
4. The kit is used for detecting the bile duct cancer determination marker and is characterized by comprising a kit liquid I and a kit liquid II, wherein the kit liquid I contains a trefoil factor family antibody, and the kit liquid II contains a trefoil factor family anti-antibody.
5. The kit of claim 4, wherein said trefoil factor family comprises trefoil factor 1, trefoil factor 2, and trefoil factor 3.
6. The method for using the kit according to claim 4 or 5, comprising the steps of:
the biliary duct cancer case sample is separated in vitro, and the separated biliary duct cancer case sample is protected at low temperature;
paraffin-embedded tissues of isolated cholangiocarcinoma example samples;
slicing the tissue, and placing the glass slide containing the complete tissue in a water body at 40 ℃;
taking out the glass slide and putting the glass slide into a 37 ℃ incubator for drying;
sequentially putting the dried glass slide into a reagent of dimethylbenzene-100% alcohol-95% alcohol-90% alcohol-80% alcohol-70% alcohol for dewaxing;
washing the dewaxed glass slide in clear water for a period of time, adding 3% H2O2Soaking in the solution for 10min, and then pouring off 3% H2O2Washing the solution twice in clear water, adding a citric acid buffer solution, cooking for 3min, cooling to room temperature just after boiling, cooking again, and cooling to room temperature to expose antigen sites;
pouring out the citric acid buffer solution, washing with clear water for 2 times and phosphate buffer salt solution for 2 times, wiping off the phosphate buffer salt solution around the tissue, adding serum immediately, and placing in a 37 deg.C incubator for 30 min;
taking out the glass slide, wiping the serum with absorbent paper, adding the first test kit liquid, and storing in a refrigerator at 4 ℃ overnight;
taking out the glass slide from the refrigerator, putting the glass slide into phosphate buffer saline solution for washing for 3 times, wiping the phosphate buffer saline solution around the tissue, adding the two liquid solutions of the kit, and then putting the two liquid solutions of the kit into an incubator at 37 ℃ for 30 min;
taking out the glass slide from the incubator, washing in phosphate buffer saline solution for 3 times, wiping off the phosphate buffer saline solution around the tissue, adding a streptavidin-biotin compound, and then placing in the incubator at 37 ℃ for 30 min;
taking out the tablet from the incubator, washing in phosphate buffer saline solution for 3 times, wiping off phosphate buffer saline solution around the tissue, and adding color developing agent.
Washing the developed slide glass with clear water for a period of time, and soaking the slide glass in hematoxylin for counterstaining; washing the counterstained glass slide in clear water, sequentially putting the glass slide into 70% alcohol-80% alcohol-90% alcohol-95% alcohol-100% alcohol-xylene, finally soaking in xylene, and moving to a ventilation hood; dropping neutral gum on the tissue side, covering with a cover glass, placing the sealed glass slide in a fume hood, and air drying.
7. The method for using the kit according to claim 6, wherein the thickness of the slice is 5 μm.
8. The method for applying the kit according to claim 6, wherein the dewaxing step is performed by sequentially placing the dried glass slide into xylene-100% alcohol-95% alcohol-90% alcohol-80% alcohol-70% alcohol reagents, each of which is placed for 10 min.
9. The application method of the kit according to claim 6, wherein the counterstained glass slide is washed with water, and then sequentially placed in 70% alcohol-80% alcohol-90% alcohol-95% alcohol-100% alcohol-xylene, and finally soaked in xylene, and then moved to a fume hood, and each reagent is placed in the fume hood for 2 min.
10. The method of using the kit according to claim 6, wherein each washing with clean water and each washing with phosphate buffered saline is 5 min.
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| US20090022708A1 (en) * | 2004-12-22 | 2009-01-22 | Lobie Peter E | Trefoil Factors and Methods of Treating Proliferation Disorders Using Same |
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| US20090022708A1 (en) * | 2004-12-22 | 2009-01-22 | Lobie Peter E | Trefoil Factors and Methods of Treating Proliferation Disorders Using Same |
| US20090304700A1 (en) * | 2008-06-06 | 2009-12-10 | Neuren Pharmaceuticals, Ltd. | Conformation specific antibodies that bind trefoil factors |
| CN111458508A (en) * | 2020-04-14 | 2020-07-28 | 中国人民解放军海军军医大学第三附属医院 | Molecular marker, kit and method for evaluating intrahepatic bile duct cancer prognosis |
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