CN115251008A - Construction method and application of nasopharyngeal carcinoma PDX model - Google Patents
Construction method and application of nasopharyngeal carcinoma PDX model Download PDFInfo
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Abstract
The invention discloses a construction method and application of a nasopharyngeal carcinoma PDX model, wherein the construction method comprises the following steps: collecting tumor tissue, and inoculating the tumor tissue to a mouse; constructing a P0 generation PDX model, observing the construction condition of the nasopharyngeal carcinoma PDX model, measuring the size of a tumor body, calculating the volume of the tumor, taking materials and fixing tumor tissues, and detecting morphological indexes. And can be further passaged to obtain a passage model mouse. The method does not involve the steps of sample pretreatment, cell digestion and the like, and the success rate of primary nasopharyngeal carcinoma PDX is 58 percent after the primary nasopharyngeal carcinoma PDX is successfully established; compared with the prior art, the method shortens the planting time, simplifies the planting steps, and builds up the individual PDX platform for nasopharyngeal carcinoma tumors of Chinese population, thereby making up for the huge vacancy in the transformation research of the nasopharyngeal carcinoma PDX model at home and abroad and meeting the requirements of the individual drug evaluation system for nasopharyngeal carcinoma tumors with Chinese population characteristics in the clinical transformation research.
Description
Technical Field
The invention belongs to the technical field of animal models, and particularly relates to a construction method and application of a nasopharyngeal carcinoma PDX model.
Background
Nasopharyngeal carcinoma refers to a malignant tumor that occurs in the top and side walls of the nasopharyngeal cavity. The method aims to build a Chinese population nasopharyngeal carcinoma tumor individualized PDX platform, make up for the huge vacancy in the domestic and foreign transformation research of a nasopharyngeal carcinoma PDX model, and meet the requirements of a nasopharyngeal carcinoma tumor individualized drug evaluation system with Chinese population characteristics in the clinical transformation research, but the prior art can not achieve a satisfactory expected effect on better PDX construction. Clinically, nasopharyngeal carcinoma is not high in malignancy degree and is regional and obvious. In addition, the tumor volume is small, and the success rate in the PDX construction process is extremely low, so an efficient construction method of the nasopharyngeal carcinoma PDX model is urgently needed. Therefore, on the basis of the construction of other cancer PDX, the existing scheme is referred to, and the adjustment is carried out so as to improve the success rate of the construction of the nasopharyngeal carcinoma PDX model.
Disclosure of Invention
The invention aims at providing a method for constructing a nasopharyngeal carcinoma PDX model.
The second aspect of the present invention is to provide the application of the above-mentioned method for constructing a nasopharyngeal carcinoma PDX model in constructing a nasopharyngeal carcinoma PDX model.
The third aspect of the present invention is to provide an application of the nasopharyngeal carcinoma PDX model constructed by the above-mentioned method for constructing a nasopharyngeal carcinoma PDX model in the research of the molecular mechanism of occurrence and development of nasopharyngeal carcinoma.
The fourth aspect of the present invention is to provide an application of the nasopharyngeal carcinoma PDX model constructed by the above-mentioned method for constructing a nasopharyngeal carcinoma PDX model in screening or evaluating a drug capable of preventing, alleviating or treating nasopharyngeal carcinoma.
The technical scheme adopted by the invention is as follows:
the invention provides a method for constructing a nasopharyngeal carcinoma PDX model, which comprises the following steps: the tumor tissue was inoculated to mice to construct a P0 generation PDX model.
In some embodiments of the present invention, the construction method further comprises observing the construction condition of the nasopharyngeal carcinoma PDX model, measuring the tumor size and calculating the tumor volume, and performing morphological index detection after sampling and fixing the tumor tissue.
In some embodiments of the invention, the method for collecting tumor tissue comprises: collecting a fresh tumor tissue specimen; the tumor tissue is put into a serum-free culture solution containing streptomycin and penicillin, rinsed for 2-5 times, each time for 2-3min, and fat is removed.
In some embodiments of the invention, the culture medium is a cell culture medium commonly used in the art, wherein streptomycin is 0.05-0.2 mg/ml, penicillin is 0.3-0.7 mg/ml.
In some embodiments of the invention, the inoculating the tumor tissue into the mouse is: tumor tissue was inoculated into the dorsal axilla of the hind limb of mice.
In some embodiments of the present invention, the specific steps of inoculating the tumor tissue into the mouse are: the method comprises the steps of cutting a wound of about 0.5cm under the skin of the back of a near hind limb of a mouse, punching a notch with the length of 1-1.5cm between the skin and the muscle in the direction of the forelimb by using an ophthalmological forceps, inoculating the thinned tissue along the notch under the armpit of the forelimb by using the ophthalmological forceps, and suturing and marking the wound by using a wound clip.
In some embodiments of the invention, the morphological index detection comprises HE staining, immunohistochemical staining, in situ hybridization.
In some embodiments of the invention, the strain of mouse is NOD-SCID, BALC/c-nu.
In some embodiments of the invention, tumors are isolated from the P0 generation PDX model and inoculated into new immunodeficient mice for passaging.
In some embodiments of the invention, the tumor tissue is divided into (0.8-1.2) mm x (0.8-1.2) mm sized tissue pieces when inoculated into new immunodeficient mice for passaging.
In a second aspect of the present invention, there is provided a use of the method of the first aspect of the present invention in constructing a nasopharyngeal carcinoma PDX model.
In a third aspect of the present invention, an application of the nasopharyngeal carcinoma PDX model constructed by the construction method of the first aspect of the present invention in the research of the molecular mechanism of occurrence and development of nasopharyngeal carcinoma is provided.
In a fourth aspect of the present invention, an application of the nasopharyngeal carcinoma PDX model constructed by the construction method of the first aspect of the present invention in screening or evaluating a drug capable of preventing, alleviating or treating nasopharyngeal carcinoma is provided.
The beneficial effects of the invention are:
the invention directly inoculates the tumor tissue specimen of the patient to the mouse, does not involve the steps such as sample pretreatment and cell digestion, etc. in the middle, the success rate of transmitting the PDX of the primary nasopharyngeal carcinoma to the first generation mouse is 58 percent after the PDX of the primary nasopharyngeal carcinoma is successfully established, and the other models can not successfully grow new tumor when transmitting the PDX to the first generation mouse; compared with the existing invention, the method shortens the planting time, simplifies the planting steps, and builds the individual PDX platform of the nasopharyngeal carcinoma tumor of Chinese population, thereby making up for the huge gap in the transformation research of the nasopharyngeal carcinoma PDX model at home and abroad and meeting the requirement of the individual drug evaluation system of the nasopharyngeal carcinoma tumor with the characteristics of Chinese population in the clinical transformation research.
Drawings
FIG. 1 shows the morphological examination of the first and second generation tumor tissues of PDX-NPC 01C.
FIG. 2 shows the first and second generation tumor weights and volumes for PDX-NPC 01C.
FIG. 3 shows the morphological examination of PDX-NPC01H first and second generation tumor tissues.
FIG. 4 shows the first and second generation tumor weights and volumes for PDX-NPC 01H.
Detailed Description
The idea of the invention and the resulting technical effects will be clearly and completely described below in connection with the embodiments, so that the objects, features and effects of the invention can be fully understood. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention.
Human-derived tissue xenograft (PDX) model: the tumor tissue of a tumor patient is transplanted into an immune deficient mouse, so that the tumor tissue grows in the mouse to form a first generation transplanted tumor, which is also called a primary PDX model.
Passage of PDX mice: tumors were removed from the N generation of PDX mice and necrotic tissue was removed before tumor tissue was inoculated into another new mouse, referred to as the N +1 generation of PDX mice.
RPMI 1640: medical cell culture solution.
NOD-SCID: the mouse has the characteristics of T and B lymphocyte combined immunodeficiency, low NK cell activity, no circulating complement, macrophage and antigen presenting cell function damage and the like, and has become the best research model of human tumor transplantation tumor in recent years.
BALC NU nude mice/NU-NU nude mice: mutant mice that are deficient in congenital thymus.
Immunohistochemical staining: that is, the principle of specific binding between an antigen and an antibody, and a chemical reaction is used to develop a color-developing agent (fluorescein, enzyme, metal ion, isotope) for labeling the antibody, thereby determining the antigen (polypeptide and protein) in the tissue cell, and performing the research of localization, characterization and relative quantification of the antigen and the protein.
In situ hybridization: in situ hybridization refers to the process of hybridizing specifically labeled nucleic acids of known sequence as probes to nucleic acids in cells or tissue sections to precisely quantify the specific nucleic acid sequence. In situ hybridization can be performed on a cell or tissue specimen.
HE staining: hematoxylin-eosin staining (HE staining) is one of the staining methods commonly used in paraffin section technology. The hematoxylin staining solution is alkaline, and mainly makes the chromatin in the cell nucleus and the nucleic acid in the cytoplasm bluish; eosin is an acid dye that primarily reddens components in the cytoplasm and extracellular matrix. The HE staining method is the most basic and widely used technical method in histology, embryology, pathology teaching and scientific research.
Example 1
1. Purchasing 4-5 weeks old NOD-SCID or BALC NU nude mice;
2. the quarantine of the mouse is carried out for 2 to 5 days after the mouse arrives, and the tissue can be inoculated after 2 to 5 days of observation;
3. serum-free 1640 samples were collected and 0.1mg/ml streptomycin and 0.5mg/ml penicillin were added. Specimen processing must be performed under sterile conditions: placing the specimen into 1640 containing streptomycin and penicillin without serum, rinsing for 3 times (2-3 min each time), and removing fat.
And (3) inoculation step:
1. after the specimen is processed according to the specimen processing method, the tissue is cut to be as thin as possible but not broken by an ophthalmic scissors; usually tumor tissue from one patient with nasopharyngeal carcinoma is inoculated into one mouse. In the case of cervical lymph node or other large tissue specimen with metastasis, the tumor specimen is divided into several 1mmX1mm size portions, which are inoculated to several mice.
2. The skin of the back of the near hind limb of the mouse is cut to form a wound of about 0.5cm, a 1-1.5cm long notch is formed between the skin and the muscle in the direction of the fore limb by using an ophthalmic forceps, the cut tissue is inoculated to the axilla of the fore limb along the notch by using the ophthalmic forceps, the tissue is not taken out as much as possible, and the wound is sutured by using a wound clamp and marked.
Mice inoculated with nasopharyngeal carcinoma tissues need to be observed every other day for the state and the tumorigenesis condition, mice which are not tumorigenic for more than four months are euthanized, and P0 generation tumorigenic mice (namely primary nasopharyngeal carcinoma PDX) are counted, as shown in Table 1.
TABLE 1
When the tumor growth of the 0 th generation (P0 generation) tumor-forming mice reaches the "plateau stage" (about 50-70d days), namely the tumor growth trend is mild or the tumor surface is ulcerated, the mice are killed by cervical dislocation, the tumors are carefully dissected out, envelope and necrotic tissues are removed, the tumors are separated from the outer layer and divided into tissue blocks with the size of 1mm multiplied by 1mm, and the tissue blocks are inoculated to new immunodeficient mice to construct first generation nasopharyngeal carcinoma tumor mice with the success rate of about 58% (Table 1); counting the weight and volume of the first generation tumor forming mice, and carrying out morphological detection on the first generation tumor forming mice, wherein the identification of nasopharyngeal carcinoma pathological histology is mainly carried out after paraffin embedding, and the identification comprises HE staining, immunohistochemical staining and in-situ hybridization:
HE staining: hematoxylin-eosin staining (HE staining) is one of the staining methods commonly used in paraffin section technology. The hematoxylin staining solution is alkaline, and mainly makes the chromatin in the cell nucleus and the nucleic acid in the cytoplasm bluish; eosin is an acid dye that primarily reddens components in the cytoplasm and extracellular matrix. The HE staining method is the most basic and widely used technical method in histology, embryology, pathology teaching and scientific research.
Immunohistochemical staining: the principle of antigen-antibody specific binding is utilized to locate a special auxiliary chemical staining mode of a certain antigen in tissues and cells, so as to achieve the combination of morphological function and metabolic change. The method is widely applied to clinical pathological work at present and is an indispensable diagnostic technique. The results of immunohistochemical staining must be closely correlated with tissue morphological features and clinical manifestations. Because nasopharyngeal carcinoma is a poorly differentiated and undifferentiated carcinoma mainly derived from nasopharyngeal mucosal epithelial cells, epithelial markers such as CK are usually selected for identification in immunohistochemical staining. However, nasopharyngeal carcinoma is similar in morphology to lymphoma, so that in the same model, lymphoma markers such as LCA and CD19 are also needed for differentiation.
In situ hybridization: the complementary base sequence between the single strands of nucleic acid molecules is utilized to complementarily pair the radioactive or nonradioactive exogenous nucleic acid (i.e. probe) with the DNA or RNA to be detected on the tissue, cell or chromosome to form a specific nucleic acid hybrid molecule, and the position of the nucleic acid to be detected on the tissue, cell or chromosome is displayed by a certain detection means. The increase of the EB virus capsid antigen-IgA antibody (VCA-IgA antibody) in the serum of a nasopharyngeal carcinoma patient is most obvious, so that whether the EB virus is positive in a model is detected by taking EBERs as a probe in situ hybridization.
The morphological detection of the PDX-NPC01C first-generation tumor tissue is shown in FIG. 1, the weight and volume of the PDX-NPC01C first-generation tumor are shown in FIG. 2; the morphological examination of the first generation tumor tissue of PDX-NPC01H is shown in FIG. 3, and the weight and volume of the first generation tumor of PDX-NPC01H is shown in FIG. 4. The characteristic of nasopharyngeal carcinoma can be obviously seen in HE staining, the index expression of epithelial cells can be clearly seen in immunohistochemical staining, and the EBERs positive expression can be clearly seen in situ hybridization, which indicates that the nasopharyngeal carcinoma is a tumor.
The first generation of tumor-forming mice are further selected and passaged to construct the second generation of tumor-forming mice. Performing tumor weight, tumor volume statistics and tumor tissue morphology detection, wherein the morphology detection of the second generation tumor tissue of PDX-NPC01C is shown in figure 1, and the weight and volume of the second generation tumor of PDX-NPC01C is shown in figure 2; the morphological examination of the PDX-NPC01H second generation tumor tissue is shown in FIG. 3, and the weight and volume of the PDX-NPC01H second generation tumor is shown in FIG. 4. It can be seen that the constructed PDX tumor model of nasopharyngeal carcinoma can be stably passed.
The present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.
Claims (10)
1. A construction method of a nasopharyngeal carcinoma PDX model is characterized by comprising the following steps: inoculating the tumor tissue to the mice; and constructing a P0 generation PDX model.
2. The construction method according to claim 1, further comprising observing construction conditions of the nasopharyngeal carcinoma PDX model, measuring tumor size and calculating tumor volume, taking materials and fixing tumor tissues, and detecting morphological indexes.
3. The construction method according to claim 1, wherein the collection method of tumor tissue comprises: collecting a fresh tumor tissue specimen; the tumor tissue is put into a serum-free culture solution containing streptomycin and penicillin, rinsed for 2-5 times, each time for 2-3min, and fat is removed.
4. The construction method according to claim 1, wherein the specific steps of inoculating the tumor tissue to the mouse are as follows: inoculated into the dorsal axilla of the mouse hind limb.
5. The method of construction of claim 1, further comprising isolating a tumor from the P0 generation PDX model and inoculating to a new immunodeficient mouse for passage.
6. The method of claim 5, wherein the tumor tissue is divided into (0.8-1.2) mm x (0.8-1.2) mm-sized tissue pieces when inoculated into new immunodeficient mice for passaging.
7. The method of claim 1, wherein the strain of mouse is NOD-SCID, BALC/c-nu.
8. Use of the method of any one of claims 1 to 7 for constructing a PDX model of nasopharyngeal carcinoma.
9. Use of the PDX model for nasopharyngeal carcinoma constructed by the construction method according to any one of claims 1 to 7 for studying the molecular mechanism of occurrence and development of nasopharyngeal carcinoma.
10. Use of the PDX model for nasopharyngeal carcinoma constructed by the construction method according to any one of claims 1 to 7 in screening or evaluating a drug capable of preventing, alleviating or treating nasopharyngeal carcinoma.
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