CN112425567A - Construction method of human tumor xenograft model - Google Patents
Construction method of human tumor xenograft model Download PDFInfo
- Publication number
- CN112425567A CN112425567A CN202011451604.XA CN202011451604A CN112425567A CN 112425567 A CN112425567 A CN 112425567A CN 202011451604 A CN202011451604 A CN 202011451604A CN 112425567 A CN112425567 A CN 112425567A
- Authority
- CN
- China
- Prior art keywords
- tissue
- tumor
- xenograft model
- tumor tissue
- target animal
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000010276 construction Methods 0.000 title claims abstract description 28
- 238000013415 human tumor xenograft model Methods 0.000 title claims abstract description 18
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 89
- 239000012634 fragment Substances 0.000 claims abstract description 46
- 241001465754 Metazoa Species 0.000 claims abstract description 40
- 238000002347 injection Methods 0.000 claims abstract description 34
- 239000007924 injection Substances 0.000 claims abstract description 34
- 239000000243 solution Substances 0.000 claims description 69
- 230000002669 organ and tissue protective effect Effects 0.000 claims description 31
- 238000000034 method Methods 0.000 claims description 28
- 108010082117 matrigel Proteins 0.000 claims description 15
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 11
- 239000003242 anti bacterial agent Substances 0.000 claims description 9
- 230000003115 biocidal effect Effects 0.000 claims description 9
- 229940088710 antibiotic agent Drugs 0.000 claims description 8
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 claims description 8
- 238000010254 subcutaneous injection Methods 0.000 claims description 6
- 239000007929 subcutaneous injection Substances 0.000 claims description 6
- 208000010507 Adenocarcinoma of Lung Diseases 0.000 claims description 4
- 201000008424 adenosquamous lung carcinoma Diseases 0.000 claims description 4
- 201000005249 lung adenocarcinoma Diseases 0.000 claims description 4
- 229960005322 streptomycin Drugs 0.000 claims description 4
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 3
- 210000004072 lung Anatomy 0.000 claims description 3
- 201000005202 lung cancer Diseases 0.000 claims description 3
- 208000020816 lung neoplasm Diseases 0.000 claims description 3
- 239000002504 physiological saline solution Substances 0.000 claims description 3
- 206010041823 squamous cell carcinoma Diseases 0.000 claims description 3
- 230000004614 tumor growth Effects 0.000 abstract description 5
- 238000002054 transplantation Methods 0.000 abstract description 4
- 238000013459 approach Methods 0.000 abstract description 3
- 230000002068 genetic effect Effects 0.000 abstract description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 26
- 230000000052 comparative effect Effects 0.000 description 7
- 238000007920 subcutaneous administration Methods 0.000 description 7
- 206010061598 Immunodeficiency Diseases 0.000 description 6
- 208000029462 Immunodeficiency disease Diseases 0.000 description 6
- 230000029087 digestion Effects 0.000 description 6
- 230000007813 immunodeficiency Effects 0.000 description 6
- 230000001954 sterilising effect Effects 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 230000005740 tumor formation Effects 0.000 description 6
- 229930182555 Penicillin Natural products 0.000 description 5
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 5
- 230000001745 anti-biotin effect Effects 0.000 description 5
- 239000011159 matrix material Substances 0.000 description 5
- 229940049954 penicillin Drugs 0.000 description 5
- 238000003860 storage Methods 0.000 description 5
- 241000699670 Mus sp. Species 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 238000011282 treatment Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000013414 tumor xenograft model Methods 0.000 description 3
- 206010002091 Anaesthesia Diseases 0.000 description 2
- 230000037005 anaesthesia Effects 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000002689 xenotransplantation Methods 0.000 description 2
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 230000003444 anaesthetic effect Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 201000005243 lung squamous cell carcinoma Diseases 0.000 description 1
- 210000004088 microvessel Anatomy 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000011369 optimal treatment Methods 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/12—Animals modified by administration of exogenous cells
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0331—Animal model for proliferative diseases
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Cell Biology (AREA)
- Animal Behavior & Ethology (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a construction method of a human tumor xenograft model, which relates to the technical field of xenograft tumor transplantation, and comprises the following steps: an injection containing a fragment of tumor tissue from a donor patient is injected subcutaneously into the target animal. The construction method combines the concept of primary tumor growth microenvironment, creates a more effective and rapid construction approach, the model constructed by the construction method can keep high genetic consistency with donor patients, and compared with the traditional construction method, the construction period and the success rate of the model are obviously improved.
Description
Technical Field
The invention relates to the technical field of xenotransplantation of tumors, in particular to a construction method of a human tumor xenotransplantation model.
Background
The treatment of malignant tumors is a worldwide problem. Currently, clinical tumor chemotherapy can be deployed in conjunction with genomic sequencing, but the actual utility of targeted drugs remains uncertain. Therefore, in the process of clinical treatment, the selection of the optimal target drug by using animal model tests and the test of the actual curative effect of the drug are the best approaches to solve the problem.
The human-Derived tumor Xenograft model (PDX) established by directly transplanting tumor tissues of a Patient to an immunodeficient mouse retains the characteristics of most primary tumors on the histopathology, molecular biology and gene level, can simulate a tumor growth microenvironment, and is one of the most effective tools for drug screening.
However, the conventional PDX model has a long manufacturing period and a low success rate, and is not favorable for the reference basis of treatment of patients with malignant tumors.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a construction method of a human tumor xenograft model.
The invention is realized by the following steps:
the embodiment of the invention provides a method for constructing a human tumor xenograft model, which comprises the following steps: an injection containing a fragment of tumor tissue from a donor patient is injected subcutaneously into the target animal.
Preferably, the volume of each fragment of the tumor tissue fragment is less than or equal to 1mm3。
Preferably, the target animal is injected subcutaneouslyThe volume of the tumor tissue fragments contained in the liquid is 30-70 mm3。
Preferably, the volume of the tumor tissue fragments contained in the subcutaneous injection of the target animal is 40-60 mm3。
Preferably, the injection also comprises a tissue protection solution, and 30-70 mm of the tissue protection solution is added into every 100 mu L of the tissue protection solution3The tumor tissue fragment of (a).
Preferably, the tissue protection solution further comprises: at least one of matrigel and physiological saline.
Preferably, when the tissue protection solution comprises matrigel and normal saline, the volume ratio of the matrigel to the normal saline is 1 (1-3).
Preferably, the tissue protection solution further comprises antibiotics;
preferably, the antibiotics are added in the following amount: adding 50-150 mu L of antibiotics into every 1mL of tissue protection solution;
preferably, the antibiotic is a streptomycin diabody.
Preferably, the tumor tissue is lung cancer tissue;
preferably, the tumor tissue is: at least one of lung adenocarcinoma, lung squamous carcinoma and lung adenosquamous carcinoma tissue.
Preferably, the target animal is a mouse;
preferably, the target animal is an NCG mouse.
The invention has the following beneficial effects:
the embodiment of the invention provides a method for constructing a human tumor xenograft model, which comprises the following steps: an injection containing a fragment of tumor tissue from a donor patient is injected subcutaneously into the target animal. The construction method combines the concept of primary tumor growth microenvironment, creates a more effective and rapid construction approach, the model constructed by the construction method can keep high genetic consistency with donor patients, and compared with the traditional construction method, the construction period and the success rate of the model are obviously improved.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a graph comparing PDX results of examples and comparative examples in test example 1;
FIG. 2 is a graph showing the growth of tumor tissue in mice in test example 1.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The embodiment of the invention provides a method for constructing a human tumor xenograft model (PDX), which comprises the following steps: an injection containing a fragment of tumor tissue from a donor patient is injected subcutaneously into the target animal.
The inventor creates the construction method by paying a series of creative works and combining the concept of primary tumor growth microenvironment, the model constructed by the construction method can keep high genetic consistency with donor patients, and compared with the traditional PDX model, the construction method greatly improves the manufacturing efficiency and the success rate.
Preferably, the volume of each fragment of the tumor tissue fragment is less than or equal to 1mm3。
Under the volume setting range, the tumor tissue can enter the body of a target animal in an injection mode without anesthesia and surgical operation on a mouse, so that the exposure time of the tumor tissue in vitro is effectively shortened, and the wound on the mouse is smaller.
Preferably, the volume of the tumor tissue fragment can be 0.01-1 mm3。
Specifically, it may be 0.05mm3、0.10mm3、0.15mm3、0.20mm3、0.25mm3、0.30mm3、0.35mm3、0.40mm3、0.45mm3、0.50mm3、0.55mm3、0.60mm3、0.65mm3、0.70mm3、0.75mm3、0.80mm3、0.85mm3、0.90mm3、0.95mm3And 1mm3Any volume of (a).
Preferably, the volume of the tumor tissue fragments contained in the subcutaneous injection of the target animal is 30-70 mm3。
The volume of the tumor tissue fragment contained in the subcutaneous injection liquid for the target animal may be 30mm3、32mm3、34mm3、36mm3、38mm3、40mm3、42mm3、44mm3、46mm3、48mm3、50mm3、52mm3、54mm3、56mm3、58mm3、60mm3、62mm3、64mm3、66mm3、68mm3、70mm3Any volume of (a).
Preferably, the volume of the tumor tissue fragments contained in the subcutaneous injection of the target animal is 40-60 mm3。
Injecting the mixture into a target animal subcutaneously for 40-60 mm3Can ensure a high tumor formation rate, and if the tumor formation rate is lower than the range, the tumor formation rate can be obviously reduced.
Preferably, the injection also comprises a tissue protection solution, and 30-70 mm of the tissue protection solution is added into every 100 mu L of the tissue protection solution3The tumor tissue fragment of (a). The tissue protection solution can protect tumor tissue fragments on one hand, and can enable the tumor tissue fragments to enter a target animal body in an injection mode on the other hand, so that a model is constructed.
Preferably, the tissue protection solution further comprises: at least one of matrigel and physiological saline.
Preferably, when the tissue protection solution comprises matrigel and normal saline, the volume ratio of the matrigel to the normal saline is 1 (1-3). The specific tissue protection solution can improve the success rate of the construction method, effectively protect tumor tissues and ensure that the injection process is smoother.
Preferably, the tissue protection solution further comprises antibiotics;
preferably, the antibiotics are added in the following amount: adding 50-150 mu L of antibiotics into every 1mL of tissue protection solution;
preferably, the antibiotic is a streptomycin diabody.
Preferably, the tumor tissue is lung cancer tissue;
preferably, the tumor tissue is: at least one of lung adenocarcinoma, lung squamous carcinoma and lung adenosquamous carcinoma tissue.
Preferably, the target animal is a mouse;
preferably, the target animal is an NCG mouse.
Preferably, the construction method further comprises obtaining a tumor tissue sample, and shearing the tumor tissue sample until the volume of each fragment in the tumor tissue fragments is less than or equal to 1mm3The state of (1).
The present invention is not limited to a specific injection method, and can be carried out by a conventional injection method. Preferably. A1 mL syringe is selected, and a 20G needle can be used as a syringe needle.
The embodiment of the invention provides a construction method for supporting the tumor tissue of a donor patient by using a special matrigel component on the basis of the preparation of an original human-derived tumor xenograft model, so that the tumor tissue can grow better after being inoculated to the subcutaneous part of a mouse, and simultaneously absorb nutrient components more quickly to promote the formation of micro-vessels around the tumor, improve the success rate of model preparation to 90 percent and effectively shorten the preparation period.
The invention originally discloses an inoculation method for injecting a graft by using an injector, which does not need anesthesia and operation on a mouse, is more convenient and faster in transplantation operation, shortens the exposure time of tumor tissues in vitro, and simultaneously causes less wound on an immunodeficiency mouse. The obtained model mouse can quickly screen clinical treatment drugs and verify the curative effect, so that patients can quickly obtain an optimal treatment scheme and the prognosis is improved.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The embodiment provides a method for constructing a human tumor xenograft model, which comprises the following steps.
(1) Obtaining tumor tissue:
tumor tissue from donor patients was harvested (after collection, may be stored in a tissue protective solution, stored under cryogenic storage conditions and transported to the laboratory).
The tissue protection solution is prepared by the following method: mixing Matrigel matrix and sterilized normal saline in a volume ratio of 1: 2.
Then 1% (volume percentage: 100. mu.L of antibiotic per 1mL of tissue protective solution) of the penicillin anti-biotin was added to the tissue protective solution.
(2) And (3) breaking tumor tissues:
in a biological safety cabinet, fresh tumor tissue is cut into pieces with the volume of each piece smaller than 1mm by using a physical digestion mode3Thereafter, until no obvious fragments are visible to the naked eye.
(3) And (3) injection:
selecting target animals for local physical unhairing, and sterilizing after subcutaneous full exposure; in this example, the target animal is a triple severe immunodeficiency NCG mouse.
Mixing the obtained tumor tissue fragments with tissue protective solution, adding 50mm of tissue protective solution per 100 μ L3Obtaining an injection solution from the tumor tissue fragment.
Then, 100. mu.L of the injection solution containing the tumor tissue fragment was injected subcutaneously into the target animal using a 1mL syringe (needle size: 20G). The injection volume of 100. mu.L is the volume of the liquid in the injection solution, and the tumor tissue contained in the injection solutionThe pieces are solid and contain 50mm per 100. mu.L in this example3Thus, injection of 100. mu.L means injection of 50mm subcutaneously into the target animal3The tumor tissue fragment of (a), and the like in other embodiments.
Example 2
The embodiment provides a method for constructing a human tumor xenograft model, which specifically comprises the following steps.
(1) Obtaining tumor tissue:
tumor tissue from donor patients was harvested (after collection, may be stored in a tissue protective solution, stored under cryogenic storage conditions and transported to the laboratory).
The tissue protection solution is prepared by the following method: mixing Matrigel matrix and sterilized normal saline in a volume ratio of 1: 1.
Then 1% (volume percentage: 100. mu.L of antibiotic per 1mL of tissue protective solution) of the penicillin anti-biotin was added to the tissue protective solution.
(2) And (3) breaking tumor tissues:
in the biological safety cabinet, fresh tumor tissue is cut into pieces with volume less than 1mm by physical digestion3Thereafter, until no obvious fragments are visible to the naked eye.
(3) And (3) injection:
selecting target animals for local physical unhairing, and sterilizing after subcutaneous full exposure; in this example, the target animal is a triple severe immunodeficiency NCG mouse.
Mixing the obtained tumor tissue fragments with tissue protective solution, adding 50mm of tissue protective solution per 100 μ L3Obtaining an injection solution from the tumor tissue fragment.
Then, 100. mu.L of the injection solution containing the tumor tissue fragment was injected subcutaneously into the target animal using a 1mL syringe (needle size: 20G).
Example 3
The embodiment provides a method for constructing a human tumor xenograft model, which specifically comprises the following steps.
(1) Obtaining tumor tissue:
tumor tissue from donor patients was harvested (after collection, may be stored in a tissue protective solution, stored under cryogenic storage conditions and transported to the laboratory).
The tissue protection solution is prepared by the following method: mixing Matrigel matrix and sterilized normal saline in a volume ratio of 1: 3.
Then 1% (volume percentage: 100. mu.L of antibiotic per 1mL of tissue protective solution) of the penicillin anti-biotin was added to the tissue protective solution.
(2) And (3) breaking tumor tissues:
in the biological safety cabinet, fresh tumor tissue is cut into pieces with volume less than 1mm by physical digestion3Thereafter, until no obvious fragments are visible to the naked eye.
(3) And (3) injection:
selecting target animals for local physical unhairing, and sterilizing after subcutaneous full exposure; in this example, the target animal is a triple severe immunodeficiency NSG mouse.
Mixing the obtained tumor tissue fragments with tissue protective solution, adding 50mm of tissue protective solution per 100 μ L3Obtaining an injection solution from the tumor tissue fragment.
Then, 100. mu.L of the injection solution containing the tumor tissue fragment was injected subcutaneously into the target animal using a 1mL syringe (needle size: 20G).
Example 4
The embodiment provides a method for constructing a human tumor xenograft model, which specifically comprises the following steps.
(1) Obtaining tumor tissue:
tumor tissue from donor patients was harvested (after collection, may be stored in a tissue protective solution, stored under cryogenic storage conditions and transported to the laboratory).
The tissue protection solution is prepared by the following method: mixing Matrigel matrix and sterilized normal saline in a volume ratio of 1: 2.
Then 1% (volume percentage: 100. mu.L of antibiotic per 1mL of tissue protective solution) of the penicillin anti-biotin was added to the tissue protective solution.
(2) And (3) breaking tumor tissues:
in the biological safety cabinet, fresh tumor tissue is cut into pieces with volume less than 1mm by physical digestion3Thereafter, until no obvious fragments are visible to the naked eye.
(3) And (3) injection:
selecting target animals for local physical unhairing, and sterilizing after subcutaneous full exposure; in this example, the target animal is a triple severe immunodeficiency NCG mouse.
Mixing the obtained tumor tissue fragments with tissue protective solution, adding 30mm of tissue protective solution per 100 μ L3Obtaining an injection solution from the tumor tissue fragment.
Then, 100. mu.L of the injection solution containing the tumor tissue fragment was injected subcutaneously into the target animal using a 1mL syringe (needle size: 20G).
Example 5
The embodiment provides a method for constructing a human tumor xenograft model, which specifically comprises the following steps.
(1) Obtaining tumor tissue:
tumor tissue from donor patients was harvested (after collection, may be stored in a tissue protective solution, stored under cryogenic storage conditions and transported to the laboratory).
The tissue protection solution is prepared by the following method: mixing Matrigel matrix and sterilized normal saline in a volume ratio of 1: 2.
Then 1% (volume percentage: 100. mu.L of antibiotic per 1mL of tissue protective solution) of the penicillin anti-biotin was added to the tissue protective solution.
(2) And (3) breaking tumor tissues:
in the biological safety cabinet, fresh tumor tissue is cut into pieces with volume less than 1mm by physical digestion3Thereafter, until no obvious fragments are visible to the naked eyeUntil now.
(3) And (3) injection:
selecting target animals for local physical unhairing, and sterilizing after subcutaneous full exposure; in this example, the target animal is a triple severe immunodeficiency NCG mouse.
Mixing the obtained tumor tissue fragments with tissue protective solution, adding 70mm per 100 μ L tissue protective solution3Obtaining an injection solution from the tumor tissue fragment.
Then, 100. mu.L of the injection solution containing the tumor tissue fragment was injected subcutaneously into the target animal using a 1mL syringe (needle size: 20G).
Comparative example 1
The comparative example provides a method for constructing a human-derived tumor xenograft model, which is mainly different from example 1 in that tumor tissues are directly surgically placed in a target animal instead of being injected, and specifically comprises the following steps.
(1) Fresh human lung adenosquamous carcinoma tissue from a patient source is collected, placed in a tissue protective solution and transported to a laboratory under a low temperature condition, and 100 mu L of streptomycin double antibody is added into 10mL of the tissue protective solution.
(2) Cutting fresh tumor tissue into pieces of 1mm by physical digestion in biological safety cabinet3Hereinafter, no obvious fragments were visible to the naked eye.
(3) Selecting a 6-week-old female NCG mouse, sterilizing with 75% alcohol after physical unhairing, injecting anesthetic to anesthetize the mouse, performing tumor tissue transplantation at a subcutaneous incision, and suturing a wound after transplantation.
(4) The mice were returned to their cages after they were resuscitated. Mouse tumorigenesis to 500mm3The average time is 55 days, and the tumor formation rate is about 60%.
Test example 1
The construction was performed by the construction method provided in example 1, and in the experimental example, the tumor tissue was obtained from fresh human lung adenocarcinoma tissue collected from the patient, and the target animal was obtained from 6-week-old female NCG mouse.
Is small after injectionThe mouse is returned to the mouse cage, and the detection shows that the mouse has the tumor of 500mm3The average time is 32 days, and the tumor formation rate is over 90 percent.
The results of example 1(Optimized PDX) were compared with those of comparative example 1 (comparative PDX), and the results of size comparison of the models constructed in example 1 and comparative example 1 were shown in FIG. 1, and the growth graphs of the mouse tumors in example 1 and comparative example 1 were shown in FIG. 2. The result shows that the construction method of the embodiment 1 of the present application has a shorter model construction period and a higher success rate.
Test example 2
The construction was carried out by the construction method provided in example 1, and in the experimental example, the tumor tissue was fresh human squamous cell lung carcinoma tissue collected from the patient, and the target animal was a 6-week-old female NCG mouse.
After injection, the mice are returned to the mouse cage, and the detection shows that the tumors of the mice are formed to 500mm3The average time is 36 days, and the tumor formation rate is about 85 percent.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A construction method of a human tumor xenograft model is characterized by comprising the following steps: an injection containing a fragment of tumor tissue from a donor patient is injected subcutaneously into the target animal.
2. The method of claim 1, wherein the volume of each of the fragments of tumor tissue is less than or equal to 1mm3。
3. The method of claim 2, wherein the target animal is a subcutaneous injection solution containing the human tumor xenograft modelThe volume of the broken blocks of the tumor tissue is 30-70 mm3。
4. The method for constructing the human tumor xenograft model according to claim 3, wherein the volume of the tumor tissue fragment contained in the subcutaneous injection of the target animal is 40 to 60mm3。
5. The method for constructing the human tumor xenograft model according to any one of claims 1 to 4, wherein the injection further comprises a tissue protective solution, and 30 to 70mm is added to each 100 μ L of the tissue protective solution3The tumor tissue fragment of (a).
6. The method of claim 5, wherein the tissue protection solution further comprises: at least one of matrigel and physiological saline.
7. The method for constructing the human tumor xenograft model according to claim 6, wherein when the tissue protective solution comprises matrigel and normal saline, the volume ratio of the matrigel to the normal saline is 1 (1-3).
8. The method of claim 5, wherein the tissue protective solution further comprises an antibiotic;
preferably, the antibiotics are added in the following amount: adding 50-150 mu L of antibiotics into every 1mL of tissue protection solution;
preferably, the antibiotic is a streptomycin diabody.
9. The method for constructing the human tumor xenograft model according to any one of claims 1 to 4, wherein the tumor tissue is a lung cancer tissue;
preferably, the tumor tissue is: at least one of lung adenocarcinoma, lung squamous carcinoma and lung adenosquamous carcinoma tissue.
10. The method for constructing the human tumor xenograft model according to any one of claims 1 to 4, wherein the target animal is a mouse;
preferably, the target animal is an NCG mouse.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011451604.XA CN112425567A (en) | 2020-12-10 | 2020-12-10 | Construction method of human tumor xenograft model |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011451604.XA CN112425567A (en) | 2020-12-10 | 2020-12-10 | Construction method of human tumor xenograft model |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112425567A true CN112425567A (en) | 2021-03-02 |
Family
ID=74691121
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011451604.XA Pending CN112425567A (en) | 2020-12-10 | 2020-12-10 | Construction method of human tumor xenograft model |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112425567A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110623770A (en) * | 2019-09-23 | 2019-12-31 | 中南大学湘雅二医院 | Method for constructing malignant schwannoma xenograft mouse model |
| CN111110385A (en) * | 2019-12-31 | 2020-05-08 | 南京普恩瑞生物科技有限公司 | Construction method of human tumor xenograft model |
| CN111500541A (en) * | 2020-04-12 | 2020-08-07 | 江苏安泰康健康科技有限公司 | Method for shortening period of human tumor xenograft PDX model |
-
2020
- 2020-12-10 CN CN202011451604.XA patent/CN112425567A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110623770A (en) * | 2019-09-23 | 2019-12-31 | 中南大学湘雅二医院 | Method for constructing malignant schwannoma xenograft mouse model |
| CN111110385A (en) * | 2019-12-31 | 2020-05-08 | 南京普恩瑞生物科技有限公司 | Construction method of human tumor xenograft model |
| CN111500541A (en) * | 2020-04-12 | 2020-08-07 | 江苏安泰康健康科技有限公司 | Method for shortening period of human tumor xenograft PDX model |
Non-Patent Citations (2)
| Title |
|---|
| 周勇: "人源肿瘤异种移植（PDTX）模型建立及药效学检测初步探讨", 《中国胸心血管外科临床杂质》 * |
| 尹东涛: "人源肺癌异种移植模型的建立及其在纳米药物临床前研究中的应用", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111500541A (en) | Method for shortening period of human tumor xenograft PDX model | |
| CN113142135A (en) | Construction method of digestive tract tumor PDX model and standardized model library | |
| CN111903603B (en) | Transplant for constructing bile duct cancer xenograft model and preparation method and application thereof | |
| Bashford et al. | General results of propagation of malignant new growths | |
| CN105873639A (en) | Autologous tumor vaccines and methods | |
| CN102277434B (en) | The purposes of people ZFX gene and related drugs thereof | |
| CN112425567A (en) | Construction method of human tumor xenograft model | |
| CN111227981B (en) | Construction method of neuroblastoma adrenal gland in-situ transplantation tumor animal model | |
| CN112210538A (en) | Human esophageal squamous carcinoma cell line NCCE1, and establishment method and application thereof | |
| CN112481215B (en) | Stable and efficient PDX tumor model modeling method | |
| CN105580774B (en) | A kind of method for building up of rabbit tongue cancer animal model | |
| CN104726408B (en) | The rapid extracting method of vascular endothelial cell | |
| CN107446024A (en) | It is a kind of can antagonism DDX3 protein rna binding activity polypeptide DIP 13 and its application | |
| CN114574442A (en) | Human brain glioma cell line and method and application for constructing in-situ transplantation model thereof | |
| CN114561360B (en) | Mouse lung cancer brain transfer cell LLC-BMT3 and construction method and application thereof | |
| Castro et al. | Maintenance of human tumours and tissues in immunosuppressed mice | |
| CN106636444A (en) | Application of FAM78A gene | |
| CN113951212A (en) | HER2 positive gastric cancer drug-resistant PDX model and construction method and application thereof | |
| CN106491630A (en) | The detection and application of the miRNA of one new liver cancer targeting stem cell | |
| CN107460170A (en) | The foundation and its application of Pituitary adenoma cell system | |
| CN104114111A (en) | Methods and apparatuses for harvesting, modifying and reimplantation of dermal micro -organs | |
| CN114657129B (en) | Bile duct cancer lymph node metastasis animal model and construction method thereof | |
| CN115029316B (en) | Primary cervical cancer cell line with radiotherapy sensitivity and radiotherapy tolerance characteristics, and construction method and application thereof | |
| CN112522262B (en) | Pancreatic cancer-related tRF and application thereof | |
| KR102040499B1 (en) | An animal model for real-time monitoring of cancer cells metastasized to bone marrow and a method of producing thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210302 |