CN113142135A - Construction method of digestive tract tumor PDX model and standardized model library - Google Patents
Construction method of digestive tract tumor PDX model and standardized model library Download PDFInfo
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Abstract
The invention provides a digestive tract tumor PDX model and a method for constructing a standardized model library, wherein the digestive tract tumor PDX model is sampled, the collected peripheral blood sample of the digestive tract tumor is processed to obtain tumor cells, the tumor cells are processed and cultured, the tumor cells are stably passaged to n generations to construct the PDX model of the digestive tract tumor, the model evaluation is carried out, the digestive tract tumor PDX standardized model library is used for establishing a human-derived digestive tract tumor transplantation animal model of Chinese population and a corresponding primary tumor cell line, an important in-vivo model is provided for the biological research of the tumor, the search of diagnostic markers and the drug screening, the medication randomness and blindness are avoided, the disturbance caused by factors such as original tissue samples and the like is avoided, the digestive tract tumor PDX model can be stably passaged to more than three generations, the construction of the digestive tract tumor PDX model standard sample library provides bioinformatics for exploring new targets and searching and analyzing the clinical characteristics of the digestive tract tumor, it can be used for assisting the research on digestive tract tumor lethal mechanism and assisting the tumor treatment of patients.
Description
Technical Field
The invention relates to the technical field of construction of biological and medical models and model libraries, in particular to a construction method of a digestive tract tumor PDX model and a standardized model library.
Background
According to statistics, digestive tract tumors in China account for 43.3 percent of the incidence rate of all cancers, almost account for the half-wall Jiangshan, and are the first few two maces in the tumor world. A human tissue xenograft model, also called a PDX model, is a tumor model constructed by transplanting tumor tissue of a tumor patient into a severe immunodeficiency mouse (NSG) and growing the tumor tissue in the mouse, and assists in tumor therapy. The PDX model reserves the microenvironment and the basic characteristics of cells of the primary tumor to form a first generation of transplanted tumor, and the transplanted tumor tissue is taken to be aseptically operated and transplanted into a new batch of mice when the transplanted tumor grows to a certain size to form a second generation of transplanted tumor, and the third generation, the fourth generation and the like of the transplanted tumor are formed by repeated operation. The model reserves the microenvironment for the growth of the parent tumor as much as possible, such as lymphocytes infiltrated around the tumor cells, extracellular matrix, microvasculature and the like, is favorable for better expressing the parent tumor characters, and maintain the heterogeneity of the tumor, the common PDX model transplantation methods are subcutaneous transplantation, kidney under-envelope xenotransplantation and orthotopic transplantation, compared with a CDX model, the PDX model can keep the molecular, genetic and pathological characteristics of the primary tumor, but the current research situation is less in the research of the PDX model of the digestive tract tumor, because the PDX model has differentiation characteristics without a uniform standard, the PDX model constructed by transplanting and passaging an immune deficient mouse (NSG) is easily disturbed by the factors of a primary tissue sample, the flow normalization is poor, the success rate of the model construction is influenced, and a PDX model standard sample library of the digestive tract tumor is not constructed, so that bioinformatics cannot be provided for exploring a new target and searching and analyzing clinical characteristics of the digestive tract tumor.
Disclosure of Invention
The invention aims to provide a digestive tract tumor PDX model and a construction method of a standardized model library, which are beneficial to auxiliary treatment of digestive tract tumors of patients, provide an important in-vivo model for biological research of tumors, search of diagnostic markers and drug screening, avoid the randomness and blindness of medication, avoid the disturbance of factors such as original tissue samples and the like, stably passage the digestive tract tumor PDX model to more than three generations, enlarge the scale of clinical samples for multiple detection, standardize the flow of the digestive tract tumor PDX model, improve the success rate of PDX model construction, provide bioinformatics for exploring new targets and searching and analyzing clinical characteristics of the digestive tract tumors by constructing the standard sample library of the digestive tract tumor PDX model, the clinical evaluation of the anti-tumor effect of the medicine on the digestive tract is carried out, the reaction of a patient to a therapeutic medicine is predicted, and the research on the fatal mechanism of the digestive tract tumor and the treatment of the tumor of the patient are assisted.
The technical scheme adopted by the invention is as follows: a PDX model of a gut tumor, comprising: the method comprises the following steps:
(1) sampling: clinically screening and taking a peripheral blood sample and a tissue sample from a digestive tract tumor patient and placing the peripheral blood sample and the tissue sample into a centrifugal tube containing tissue fluid; and preparing peripheral blood samples and tissue samples of the digestive tract tumor patients according to the specifications by combining a clinical pathology database.
(2) Processing the peripheral blood sample of the digestive tract tumor collected in the step (1) to obtain tumor cells which grow vigorously and have no ulcer and necrosis;
(3) processing the tissue samples collected in the step (1), and comparing on a methodology level;
(4) culturing the tumor cells obtained by separation in the step (2) in a culture dish containing RPMI-1640 (a culture dish containing 10% fetal calf serum and 10% dimethyl sulfoxide), storing in liquid nitrogen, sterilizing the cultured specimen by 75% alcohol, then putting into a puncture needle, slowly pushing the tumor cells into the middle position of the oxter of a mouse at the puncture needle inserting position, pulling out after the puncture needle is stopped for 2-4s, suturing and sterilizing the puncture needle inserting position, growing by virtue of a biological microenvironment provided by the mouse to obtain a P1 generation mouse, stably passaging the tumor cells to n generations, taking out the tumor, shearing, transplanting into a new mouse to obtain a Pn generation mouse, and constructing a PDX model of the digestive tract tumor by virtue of the tumor in the Pn generation mouse, wherein n is an integer not less than 3.
(5) Evaluating the digestive tract tumor PDX model constructed in the step (3), and screening out the digestive tract tumor PDX model sensitive to the drugs and/or the chemoradiotherapy according to the drugs and the drug administration scheme.
Preferably, the tumor cells in step (4) are passaged to the n passage with a volume of 200-1000mm3。
Preferably, the PDX model evaluation of the digestive tract tumor is specifically:
s1: recording and judging a tumor growth boundary in the constructed digestive tract tumor PDX model, wherein the tumor growth boundary is a tumor cell volume critical value when the transplanted tumor grows in a mouse and is necrotic or liquefied;
s2: performing pathological and immunohistochemical staining evaluation on tumor cells in the constructed PDX model of the digestive tract tumor;
s3: and (3) carrying out mutation detection on key genes of tumor cells in the constructed digestive tract tumor PDX model, and screening out the digestive tract tumor PDX model sensitive to the drugs/chemoradiotherapy according to mutation conditions.
Preferably, the gene mutation types are: L858R, L861Q, G719C or Exon19 were deleted.
A method for constructing a PDX standardized model library of digestive tract tumors comprises the steps of carrying out clinical pathological typing and staging on the digestive tract tumors of Chinese population and establishing a large-scale standardized humanized digestive tract tumor transplantation animal model of the Chinese population and a corresponding primary tumor cell line, determining the typing of the digestive tract tumor cell diseases of the Chinese population, and developing a personalized research and development system and a treatment scheme.
As a preferable scheme of the construction method of the PDX standardized model library of the digestive tract tumor, the primary tumor cell line is formed by covering the PDX model of the digestive tract tumor on the digestive tract tumor in a large scale.
As a preferable scheme of the construction method of the PDX standardized model library of the digestive tract tumor, the human digestive tract tumor transplantation animal model of Chinese population divides the peripheral blood-derived tumor cells of the Chinese digestive tract tumor patients into three parts: the first part is used for DNA/RNA extraction; the second part is used for pathological examination, fixed in formalin and embedded in paraffin; the third part is used for being implanted into an immunodeficiency mouse or being put into a freezing medium and being preserved in liquid nitrogen, growth factors are used for assisting the growth of digestive tract tumor cells in the immunodeficiency mouse, and pathological section detection and genetic detection and identification are carried out.
As a preferable scheme of the construction method of the PDX standardized model library of the digestive tract tumors, the human digestive tract tumor transplantation model of the Chinese population comprises clinical information, pathotype classification, histopathology, gene mutation and gene transcription expression of patients with complete digestive tract tumors.
As a preferable scheme of the construction method of the PDX standardized model library of the digestive tract tumor, the digestive tract tumor transplantation model of Chinese people transplants peripheral blood-derived tumor cells or fresh tumor tissues of Chinese digestive tract tumor patients onto immunodeficient mice.
As a preferable scheme of the construction method of the digestive tract tumor PDX standardized model library, the digestive tract tumor PDX standardized model library establishes a drug in-vitro rapid screening and clinical close in-vivo evaluation method system and a drug effect combined application or toxic and side effect mechanism.
Drawings
In order to more clearly illustrate the embodiments of the present application or technical solutions in the prior art, the drawings required to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present invention.
FIG. 1 is an enlarged view under a microscope of a control group A and an experimental group B in example 2 of the present invention;
FIG. 2 is an enlarged view under a microscope of test group C and test group D in example 2 of the present invention.
Has the advantages that: the invention relates to a digestive tract tumor PDX model and a construction method of a standardized model library, the digestive tract tumor PDX model keeps the molecular, genetic and pathological characteristics of primary tumors, is beneficial to auxiliary treatment of the digestive tract tumors of patients, the differentiation degree, morphological characteristics, structural characteristics, molecular characteristics and the like of tumor cells from peripheral blood are more similar to the tumor characteristics of the patients, an important in-vivo model is provided for biological research of tumors, search of diagnosis markers and drug screening, the model is applied to personalized medication guidance of targeted treatment of tumor cells, medication randomness and blindness are avoided, the construction process of the digestive tract tumor PDX model is optimized, the original microenvironment of the digestive tract tumor is simulated, the survival rate and the tumor formation rate of mice are high, the model is prevented from being disturbed by factors such as original tissue samples and the like, the digestive tract tumor PDX model can be stably passaged to more than three generations, the method has the advantages of expanding clinical sample scale for multiple detection, standardizing the process of a digestive tract tumor PDX model, improving the success rate of PDX model construction, providing bioinformatics for exploring a new target and searching and analyzing the clinical characteristics of the digestive tract tumor by constructing a digestive tract tumor PDX model standard sample library, clinically evaluating the anti-digestive tract tumor effect of a medicament on the cell and animal level, predicting the response of a patient to the treatment medicament, including curative effect, toxic and side effects, absorption degree and the like, researching the targeted treatment necessity and clinical approach of the digestive tract tumor, and assisting the research of the vital mechanism of the digestive tract tumor and the treatment of the patient tumor.
Detailed Description
The specific sorting, identifying and detecting method of CTC in serum of biliary tract tumor of the present invention is further described in detail below.
Example 1
A PDX model of a gut tumor, comprising: the method comprises the following steps:
(1) sampling: clinically screening and taking a peripheral blood sample and a tissue sample from a digestive tract tumor patient and placing the peripheral blood sample and the tissue sample into a centrifugal tube containing interstitial fluid, and preparing the peripheral blood sample and the tissue sample of the digestive tract tumor patient according to a standard by combining with a clinical pathology database; examining the tumor patients, making a definite diagnosis, and making a detailed data record;
(2) processing the peripheral blood sample of the digestive tract tumor collected in the step (1) to obtain tumor cells which grow vigorously and have no ulcer and necrosis;
the tumor cells from peripheral blood exist in the tumor cells which can escape from host immune killing and survive in the peripheral blood circulation of tumor patients, compared with tumor cell lines from other sources, the differentiation degree, morphological characteristics, structural characteristics and molecular characteristics of the tumor cells from peripheral blood are more consistent with the tumor characteristics of digestive tract tumor patients, so that an important in vivo model is provided for the biological research of tumors, the search of diagnostic markers and the drug screening;
(3) processing the tissue samples collected in the step (1), and comparing on a methodology level;
(4) culturing the tumor cells obtained by separation in the step (2) in a culture dish containing RPMI-1640, sterilizing the cultured specimen by 75% alcohol, then loading the sterilized specimen into a puncture needle, slowly pushing the tumor cells into the puncture needle at the position of the middle part of the mouse axilla, pulling out the tumor cells after the puncture needle stays for 4s, suturing the puncture needle, sterilizing, growing by depending on a biological microenvironment provided by the mouse, taking out the tumor for the P1 generation mouse, stably passaging the tumor cells to 3 generations, taking out the tumor, cutting into pieces, transplanting the tumor cells into a new mouse body, taking out the P3 generation mouse, and constructing a PDX model of the digestive tract tumor by using the tumor in the P3 generation mouse.
(5) Evaluating the digestive tract tumor PDX model constructed in the step (3), and screening out a digestive tract tumor PDX model sensitive to drugs and/or chemoradiotherapy according to drugs and drug administration schemes, wherein the digestive tract tumor PDX model keeps the molecular, genetic and pathological characteristics of primary tumors, is beneficial to auxiliary treatment of the digestive tract tumors of patients, and the differentiation degree, morphological characteristics, structural characteristics, molecular characteristics and the like of tumor cells from peripheral blood sources are more similar to the tumor characteristics of the patients, so that an important in-vivo model is provided for the biological research of tumors, the search of diagnostic markers and the drug screening, is applied to the personalized drug guidance of the targeted therapy of the tumor cells, and avoids the randomness and blindness of drug administration.
Preferably, the tumor cells in step (4) are passaged to 3 passages with a volume of 200mm3。
Preferably, the PDX model evaluation of the digestive tract tumor is specifically:
s1: recording and judging a tumor growth boundary in the constructed digestive tract tumor PDX model, wherein the tumor growth boundary is a tumor cell volume critical value when the transplanted tumor grows in a mouse and is necrotic or liquefied;
s2: performing pathological and immunohistochemical staining evaluation on tumor cells in the constructed PDX model of the digestive tract tumor;
s3: and (3) carrying out mutation detection on key genes of tumor cells in the constructed digestive tract tumor PDX model, and screening out the digestive tract tumor PDX model sensitive to the drugs/chemoradiotherapy according to mutation conditions.
Preferably, the gene mutation types are: L858R, L861Q.
In order to verify the scientificity of a theoretical basis and evaluate the actual effect of the digestive tract tumor PDX model, 20 mice are divided into a control group A, an experimental group B, an experimental group C and an experimental group D, 5 mice in each group, the experimental group B, the experimental group C and the experimental group D are used for constructing the digestive tract tumor PDX model by the method, 5 mice in the control group A are provided with wounds on the backs of the mice by a traditional method, tumor cells obtained by separation are transplanted to the affected parts and are stably passed to 3 generations, other experimental methods are the same as those in the experimental group B, C, D, the construction process of the digestive tract tumor PDX model is optimized, the original microenvironment of the digestive tract tumor is simulated, the survival rate and the tumor formation rate of the mice are high, and the mice are prevented from being disturbed by factors such as original tissue samples, and the passage effect is influenced. The digestive tract tumor PDX model can be stably passed to more than three generations, the scale of clinical samples is enlarged to carry out multiple detection, the process of the digestive tract tumor PDX model is standardized, and the success rate of constructing the PDX model is improved.
The mice of the control group and the experimental group are respectively continuously observed in an animal room cage for 2 months, the mice are sent to a PET-CT for inspection in the second week for observation, the tumor formation and metastasis conditions are observed, the control group A is used as a contrast test of the non-injected medicament, the mice are killed and tumor is taken out, the tumor bodies are evaluated to find that the tumor formation profile is poor, one part is frozen, the other part is subjected to pathological detection and gene sequencing, and the excessive proliferation and abnormal shape of tumor cells are observed under a visible microscope; the experimental group B is used as a contrast test of non-injected medicaments, but the method of the application is adopted to construct a digestive tract tumor PDX model, mice are sacrificed to take tumors, tumor bodies are evaluated to find that the tumor formation profile is better, one part of the tumor bodies is frozen, the other part of the tumor bodies is subjected to pathological detection and gene sequencing, the normal proliferation of tumor cells, complete shape and no obvious cell death are observed under a visible microscope, the experimental group mice adopting the original microenvironment for simulating the digestive tract tumor can simulate the real digestive tract tumor environment better than the control group A and simulate the proliferation of the digestive tract tumor cells, the observation and the recording of the control group A and the experimental group B have no influence on diet and drinking water, the individuals have no obvious difference, the behavior of the mice has no obvious abnormality, the activity capacity is normal, the hair color has no change, no adverse reactions such as abnormal function and infection are caused, and the defecation is normal, experiment group C adopts epirubicin drug to carry out intraperitoneal injection on mice, the dosage concentration of each drug is 20ug/ml, epirubicin is the drug acting on digestive tract tumor, one part is frozen and preserved, the other part is carried out pathological detection and gene sequencing, as shown in a microscope, most cells of tumor cells are cracked and killed, the epirubicin drug inhibition effect is obvious, an experimental group D adopts etoposide drug to carry out intraperitoneal injection on mice, the dose concentration of each drug is 20ug/ml, the etoposide is drug acting on digestive tract tumors, one part is frozen and the other part is subjected to pathological detection and gene sequencing, the lysis and apoptosis of most tumor cells can be observed under a visible microscope, and the inhibition effect of etoposide on the tumor cells in the digestive tract is more obvious than that of epirubicin, which is shown in table 1 in detail.
TABLE 1 comparative protocol for PDX model of digestive tract tumors
The digestive tract tumor PDX model keeps the molecular, genetic and pathological characteristics of primary tumor, is beneficial to auxiliary treatment of the digestive tract tumor of a patient, has the tumor cell differentiation degree, morphological characteristics, structural characteristics, molecular characteristics and the like of peripheral blood source which are more similar to the tumor characteristics of the patient, provides an important in vivo model for biological research of tumor, search of diagnosis markers and drug screening, is applied to personalized medication guidance of targeted treatment of tumor cells, avoids medication randomness and blindness, optimizes the construction process of the digestive tract tumor PDX model, simulates the original microenvironment of the digestive tract tumor, has high survival rate and tumor formation rate of mice, avoids disturbance of factors such as primary tissue samples and the like, can be stably passed to more than three generations, enlarges clinical sample scale for multiple detection, standardizes the process of the digestive tract tumor PDX model, and the success rate of PDX model construction is improved.
Example 2
A PDX model of a gut tumor, comprising: the method comprises the following steps:
(1) sampling: clinically screening and taking a peripheral blood sample and a tissue sample from a digestive tract tumor patient and placing the peripheral blood sample and the tissue sample into a centrifugal tube containing tissue fluid; examining the tumor patients, making a definite diagnosis, and making a detailed data record;
(2) processing the peripheral blood sample of the digestive tract tumor collected in the step (1) to obtain tumor cells which grow vigorously and have no ulcer and necrosis;
the tumor cells from peripheral blood exist in the tumor cells which can escape from host immune killing and survive in the peripheral blood circulation of tumor patients, compared with tumor cell lines from other sources, the differentiation degree, morphological characteristics, structural characteristics and molecular characteristics of the tumor cells from peripheral blood are more consistent with the tumor characteristics of digestive tract tumor patients, so that an important in vivo model is provided for the biological research of tumors, the search of diagnostic markers and the drug screening;
(3) processing the tissue samples collected in the step (1), and comparing on a methodology level;
(4) culturing the tumor cells obtained by separation in the step (2) in a culture dish containing RPMI-1640, sterilizing the cultured specimen with 75% alcohol, then loading the sterilized specimen into a puncture needle, slowly pushing the tumor cells into the position of the middle part of the mouse axilla at the puncture position of the puncture needle with the depth of 1.8cm, staying for 2s, then pulling out the specimen, suturing the puncture position of the puncture needle, sterilizing, growing by virtue of a biological microenvironment provided by the mouse, taking out the tumor, cutting into pieces, transplanting the tumor cells into a new mouse body to serve as a Pn generation mouse, constructing a PDX model of the digestive tract tumor by using the tumor in the Pn generation mouse, the construction process of the PDX model of the digestive tract tumor is optimized, the original microenvironment of the digestive tract tumor is simulated, the survival rate and the tumor formation rate of the mouse are high, and the disturbance of factors such as an original tissue sample and the like is avoided, so that the passage effect is influenced. The digestive tract tumor PDX model can be stably passed to more than three generations, the scale of clinical samples is enlarged to carry out multiple detection, the process of the digestive tract tumor PDX model is standardized, and the success rate of constructing the PDX model is improved.
(5) Evaluating the digestive tract tumor PDX model constructed in the step (3), and screening out a digestive tract tumor PDX model sensitive to drugs and/or chemoradiotherapy according to drugs and drug administration schemes, wherein the digestive tract tumor PDX model keeps the molecular, genetic and pathological characteristics of primary tumors, is beneficial to auxiliary treatment of the digestive tract tumors of patients, and the differentiation degree, morphological characteristics, structural characteristics, molecular characteristics and the like of tumor cells from peripheral blood sources are more similar to the tumor characteristics of the patients, so that an important in-vivo model is provided for the biological research of tumors, the search of diagnostic markers and the drug screening, is applied to the personalized drug guidance of the targeted therapy of the tumor cells, and avoids the randomness and blindness of drug administration.
Preferably, the tumor cells in step (4) are passaged to P4 passage and have a volume of 500mm3。
Preferably, the tumor cells in step (4) are passaged to P5 passage with a volume of 1000mm3。
Preferably, the PDX model evaluation of the digestive tract tumor is specifically:
s1: recording and judging a tumor growth boundary in the constructed digestive tract tumor PDX model, wherein the tumor growth boundary is a tumor cell volume critical value when the transplanted tumor grows in a mouse and is necrotic or liquefied;
s2: performing pathological and immunohistochemical staining evaluation on tumor cells in the constructed PDX model of the digestive tract tumor;
s3: and (3) carrying out mutation detection on key genes of tumor cells in the constructed digestive tract tumor PDX model, and screening out the digestive tract tumor PDX model sensitive to the drugs/chemoradiotherapy according to mutation conditions.
Preferably, the gene mutation types are: L858R, L861Q and G719C.
In order to verify the scientificity of a theoretical basis and evaluate the actual effect of the digestive tract tumor PDX model, 20 mice are divided into a control group A, an experimental group B, an experimental group C and an experimental group D, 5 mice in each group, the experimental group B, the experimental group C and the experimental group D are used for constructing the digestive tract tumor PDX model by the method, 5 mice in the control group A are provided with wounds on the backs of the mice by a traditional method, tumor cells obtained by separation are transplanted to the affected parts and are stably passed to more than 4 generations, other experimental methods are the same as the experimental group B, C, D, the construction process of the digestive tract tumor PDX model is optimized, the original microenvironment of the digestive tract tumor is simulated, the survival rate and the tumor formation rate of the mice are high, and the mice are prevented from being disturbed by factors such as original tissue samples, and the like, so that the passage effect is influenced. The digestive tract tumor PDX model can be stably passed to more than three generations, the scale of clinical samples is enlarged to carry out multiple detection, the process of the digestive tract tumor PDX model is standardized, and the success rate of constructing the PDX model is improved.
Referring to fig. 1-2, mice in a control group and a test group are respectively continuously observed in an animal room cage for 2 months, the mice are subjected to PET-CT inspection for observation of the second week, the tumor formation and metastasis conditions are observed, the control group A is used as a contrast test of non-injected drugs, the mice are killed and tumor is taken out, tumor bodies are evaluated to find that the tumor formation profile is poor, one part is frozen, the other part is subjected to pathological detection and gene sequencing, and the excessive proliferation and morphological abnormality of tumor cells are observed under a visible microscope; the experimental group B is used as a contrast test of non-injected medicaments, but the method of the application is adopted to construct a digestive tract tumor PDX model, mice are sacrificed to take tumors, tumor bodies are evaluated to find that the tumor formation profile is better, one part of the tumor bodies is frozen, the other part of the tumor bodies is subjected to pathological detection and gene sequencing, the normal proliferation of tumor cells, complete shape and no obvious cell death are observed under a visible microscope, the experimental group mice adopting the original microenvironment for simulating the digestive tract tumor can simulate the real digestive tract tumor environment better than the control group A and simulate the proliferation of the digestive tract tumor cells, the observation and the recording of the control group A and the experimental group B have no influence on diet and drinking water, the individuals have no obvious difference, the behavior of the mice has no obvious abnormality, the activity capacity is normal, the hair color has no change, no adverse reactions such as abnormal function and infection are caused, and the defecation is normal, experiment group C adopts epirubicin drug to carry out intraperitoneal injection on mice, the dosage concentration of each administration is 40-100ug/ml, epirubicin is drug acting on digestive tract tumor, one part is frozen, the other part is carried out pathological detection and gene sequencing, most cells of tumor cells are observed to be cracked and dead under a visible microscope, the inhibition effect of epirubicin drug is more obvious, experiment group D adopts etoposide drug to carry out intraperitoneal injection on mice, the dosage concentration of each administration is 40-100ug/ml, etoposide is drug acting on digestive tract tumor, one part is frozen, the other part is carried out pathological detection and gene sequencing, the majority of cells of tumor cells are observed to be cracked and apoptotic under the visible microscope, the inhibition effect of etoposide on digestive tract tumor cells is more obvious than that of epirubicin, see table 2 for details.
TABLE 2 comparative protocol for PDX model of digestive tract tumors
The digestive tract tumor PDX model keeps the molecular, genetic and pathological characteristics of primary tumor, is beneficial to auxiliary treatment of the digestive tract tumor of a patient, has the tumor cell differentiation degree, morphological characteristics, structural characteristics, molecular characteristics and the like of peripheral blood source which are more similar to the tumor characteristics of the patient, provides an important in vivo model for biological research of tumor, search of diagnosis markers and drug screening, is applied to personalized medication guidance of targeted treatment of tumor cells, avoids medication randomness and blindness, optimizes the construction process of the digestive tract tumor PDX model, simulates the original microenvironment of the digestive tract tumor, has high survival rate and tumor formation rate of mice, avoids disturbance of factors such as primary tissue samples and the like, can be stably passed to more than three generations, enlarges clinical sample scale for multiple detection, standardizes the process of the digestive tract tumor PDX model, and the success rate of PDX model construction is improved.
Example 3
A method for constructing a PDX standardized model library of digestive tract tumors comprises the steps of carrying out clinical pathological classification and staging on the digestive tract tumors of Chinese population, establishing a large-scale standardized humanized digestive tract tumor transplantation animal model of the Chinese population and a corresponding primary tumor cell line, and establishing a clinical phenotype database of the digestive tract tumors.
As a preferable scheme of the construction method of the PDX standardized model library of the digestive tract tumor, the primary tumor cell line is formed by covering the PDX model of the digestive tract tumor on the digestive tract tumor in a large scale.
As a preferable scheme of the construction method of the PDX standardized model library of the digestive tract tumor, the human digestive tract tumor transplantation animal model of Chinese population divides the peripheral blood-derived tumor cells of the Chinese digestive tract tumor patients into three parts: the first part is used for DNA/RNA extraction; the second part is used for pathological examination; the third part is used for being implanted into an immunodeficiency mouse or being put into a freezing medium and being preserved in liquid nitrogen, growth factors are used for assisting the growth of digestive tract tumor cells in the immunodeficiency mouse, and pathological section detection and genetic detection and identification are carried out.
As a preferred scheme of the construction method of the PDX standardized model library of the digestive tract tumors, the human-derived digestive tract tumor transplantation model of Chinese population comprises clinical information, pathotyping classification, histopathology, gene mutation and gene transcription expression of patients with complete digestive tract tumors, and simultaneously constructs a primary tumor cell line of the patients corresponding to a CDX model of the digestive tract tumors, so that the near-clinical comprehensive evaluation of the anti-digestive tract tumor effect of the medicine can be carried out on the cell and animal level.
As a preferable scheme of the construction method of the digestive tract tumor PDX standardized model library, the Chinese population human digestive tract tumor transplantation model transplants peripheral blood-derived tumor cells or fresh tumor tissues of Chinese digestive tract tumor patients onto immunodeficient mice to establish the digestive tract tumor PDX standardized model library.
As a preferable scheme of a construction method of a digestive tract tumor PDX standardized model library, the digestive tract tumor PDX standardized model library establishes a drug in-vitro rapid screening and clinical close in-vivo evaluation method system and drug efficacy combined application or toxic and side effect mechanism, carries out drug sensitivity detection by applying a digestive tract tumor PDX test, provides medication guidance for patients, evaluates clinical treatment effect through imaging detection, and establishes a unified drug efficacy database.
At present, the construction method of the PDX model standard sample library of the digestive tract tumor is not unified, the unified standard is lacked, the construction of the PDX model standard sample library of the digestive tract tumor provides bioinformatics for exploring a new target and searching and analyzing clinical characteristics of the digestive tract tumor, clinical evaluation is carried out on the anti-digestive tract tumor effect of a medicament on the level of cells and animals, the reaction of a patient to the treatment medicament is predicted, including curative effect, toxic and side effect, absorption degree and the like, the targeted treatment necessity and clinical approach of the digestive tract tumor are researched, and the research on the lethal mechanism of the digestive tract tumor and the treatment of the tumor of the patient are assisted.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. A PDX model of a gut tumor, comprising: the method comprises the following steps:
(1) sampling: clinically screening and taking a peripheral blood sample and a tissue sample from a digestive tract tumor patient and placing the peripheral blood sample and the tissue sample into a centrifugal tube containing tissue fluid;
(2) processing the peripheral blood sample of the digestive tract tumor collected in the step (1) to obtain tumor cells which grow vigorously and have no ulcer and necrosis;
(3) processing the tissue samples collected in the step (1), and comparing on a methodology level;
(4) putting the tumor cells obtained by separation in the step (2) into a culture dish containing RPMI-1640 for culture, sterilizing the cultured specimen by 75% alcohol, then putting the sterilized specimen into a puncture needle, slowly pushing the tumor cells into the puncture needle at the position of the middle part of the mouse axilla, pulling out the specimen after the specimen stays for 2-4s, sewing the puncture needle, sterilizing, growing by depending on a biological microenvironment provided by the mouse, stably passaging the tumor cells to n generations, taking out the tumor, cutting into pieces, transplanting the tumor cells into a new mouse body, constructing a PDX model of the digestive tract tumor for the Pn generation mouse by using the tumor in the Pn generation mouse, wherein n is an integer not less than 3;
(5) evaluating the digestive tract tumor PDX model constructed in the step (3), and screening out the digestive tract tumor PDX model sensitive to the drugs and/or the chemoradiotherapy according to the drugs and the drug administration scheme.
2. The PDX model of gut tumor according to claim 1, wherein: the volume of the tumor cells after passage to the n generation in the step (4) is 200-3。
3. The PDX model of gut tumor according to claim 1, wherein:
the PDX model evaluation of the digestive tract tumor specifically comprises the following steps:
s1: recording and judging a tumor growth boundary in the constructed digestive tract tumor PDX model, wherein the tumor growth boundary is a tumor cell volume critical value when the transplanted tumor grows in a mouse and is necrotic or liquefied;
s2: performing pathological and immunohistochemical staining evaluation on tumor cells in the constructed PDX model of the digestive tract tumor;
s3: and (3) carrying out mutation detection on key genes of tumor cells in the constructed digestive tract tumor PDX model, and screening out the digestive tract tumor PDX model sensitive to the drugs/chemoradiotherapy according to mutation conditions.
4. The PDX model of claim 3, wherein:
the gene mutation types are as follows: L858R, L861Q, G719C or Exon19 were deleted.
5. A method for constructing a PDX standardized model library of digestive tract tumors is characterized by comprising the following steps: the PDX standardized model library for the digestive tract tumors comprises clinical pathological classification and staging of the digestive tract tumors of Chinese population, digestive tract tumor characteristic indexes, establishment of large-scale and standardized humanized digestive tract tumor transplantation animal models of the Chinese population and corresponding primary tumor cell lines.
6. The method for constructing the PDX standardized model library of the digestive tract tumor according to claim 5, wherein the PDX standardized model library of the digestive tract tumor comprises the following steps: the primary tumor cell line is used for covering the digestive tract tumor PDX model on a large scale.
7. The method for constructing the PDX standardized model library of the digestive tract tumor according to claim 6, wherein the PDX standardized model library of the digestive tract tumor is prepared by the following steps:
the animal model for transplanting the human-derived digestive tract tumor of the Chinese population divides the peripheral blood-derived tumor cells of the Chinese digestive tract tumor patient into three parts: the first part is used for DNA/RNA extraction; the second part is used for pathological examination; the third part is used for being implanted into an immunodeficiency mouse or being put into a freezing medium and being preserved in liquid nitrogen, growth factors are used for assisting the growth of digestive tract tumor cells in the immunodeficiency mouse, and pathological section detection and genetic detection and identification are carried out.
8. The method for constructing the PDX standardized model library of the digestive tract tumor according to claim 7, wherein the PDX standardized model library of the digestive tract tumor is prepared by the following steps: the human-derived digestive tract tumor transplantation model of Chinese population comprises clinical information, pathological typing classification, histopathology, gene mutation and gene transcription expression of patients with complete digestive tract tumor.
9. The method for constructing the PDX standardized model library of the digestive tract tumor according to claim 8, wherein the PDX standardized model library of the digestive tract tumor comprises the following steps: the Chinese population human digestive tract tumor transplantation model transplants peripheral blood-derived tumor cells or fresh tumor tissues of Chinese digestive tract tumor patients onto immunodeficient mice.
10. The method for constructing the PDX standardized model library of the digestive tract tumor according to claim 5, wherein the PDX standardized model library of the digestive tract tumor comprises the following steps: the PDX standardized model library for the digestive tract tumor establishes an in-vivo evaluation method system for rapid in-vitro screening and clinical application of a medicament and a medicament effect combined application or toxic and side effect mechanism.
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