CN110692594A - Human hemangioma animal model and high-success-rate construction method thereof - Google Patents
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Abstract
The invention discloses a human hemangioma animal model and a high-success-rate construction method thereof, wherein the construction method comprises the following steps: s1, taking isolated human hemangioma cells for later use; s2, resuspending the human hemangioma cells uniformly by PBS; s3, inoculating the resuspended human hemangioma cells to the subcutaneous part of the immunodeficient animal; s4, injecting estradiol into the abdominal cavity to obtain mother tumor; s5, picking the mother tumor, peeling off and washing; s6, dividing the washed parent tumor into 30-70mm3The tumor tissue of (a); s7, inoculating the tumor tissue to the subcutaneous tissue of the immunodeficient animal, and feeding to obtain the final product. The invention has the advantages that: the hemangioma tumor forming rate is extremely high and can reach 100%; the cell composition of the formed hemangioma is completely humanized, and the hemangioma is very suitable for researching pathogenesis, new drug evaluation and treatment method of human hemangioma.
Description
Technical Field
The invention relates to an animal model and a construction method thereof, in particular to a human hemangioma animal model constructed by human hemangioma mother tumor constructed by human hemangioma endothelial cells and a high-success-rate construction method thereof.
Background
Hemangioma is one of the most frequently occurring angiogenesis diseases of infants, is better to be developed on the body surfaces of the head, the face, the limbs and the like, and accounts for 2.5-12% of all newborns. Generally, tumor bodies begin to regress within one year after birth, and can naturally and slowly regress and return to normal within 10 years. However, some superficial tumors may affect the aesthetic and physiological function of the patient, and some may cause ulceration, bleeding, infection, high flow congestive heart failure, and even be life threatening.
At present, the formation mechanism of hemangioma is still unknown, and a simple and feasible human hemangioma animal model construction method does not exist, so that the problem limits the deep research on the hemangioma pathogenesis and the treatment method to a certain extent. The difficulty of establishing animal models is further increased because human hemangioma cannot occur in other species. At present, the construction methods of a plurality of reported human hemangioma animal models mainly comprise a hemangioma tissue transplantation method and a cell suspension inoculation method. However, the sources of hemangioma tissues are more limited, and the tissues are difficult to obtain; the inoculation method of the cell suspension is simple, but the tumor formation rate is low, and multiple reseeding is needed. Neither method can simply and rapidly construct a human hemangioma model.
Chinese patent application No. 201110118832.X discloses a human hemangioma animal model and a construction method thereof, and the human hemangioma animal model is established by applying a human hemangioma stem cell and human umbilical vein endothelial cell co-transplantation method. However, the method is complicated and tedious to operate and has low tumor formation rate. Therefore, it is necessary to establish an experimental animal model of hemangioma, which can overcome the disadvantages of the existing animal model, establish the characteristics completely similar to human hemangioma, and be used for researching the generation mechanism and treatment method of hemangioma.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a human hemangioma animal model and a high-success-rate construction method thereof.
The purpose of the invention is realized by the following technical scheme:
in a first aspect, the present invention provides a method for constructing a human hemangioma animal model, comprising the following steps:
s1, taking isolated human hemangioma cells for later use;
s2, resuspending the human hemangioma cells uniformly by PBS;
s3, inoculating the resuspended human hemangioma cells to the subcutaneous part of the immunodeficient animal;
s4, injecting estradiol into the abdominal cavity to obtain mother tumor;
s5, picking the mother tumor, peeling off and washing;
s6, dividing the washed parent tumor into 30-70mm3The tumor tissue of (a);
s7, inoculating the tumor tissue to the subcutaneous part of an animal with immunodeficiency, and feeding to obtain a human hemangioma animal model;
the human hemangioma cells comprise one or a mixture of human hemangioma endothelial cells and human hemangioma stem cells.
Preferably, in step S2, the human hemangioma cells are cultured, proliferated, digested, and then the number of human hemangioma cells is 1 × 106～1×108The digests were collected, centrifuged, the supernatant removed and resuspended in PBS.
Preferably, the culture is performed in DMEM complete medium and the digestion is performed with 0.25% pancreatin.
Preferably, the number of the human hemangioma cells is 1 × 106～5×107The volume of the mother tumor tissue is 40-70 mm3。
Preferably, the number of the human hemangioma cells is 5 × 106～1×107The volume of the mother tumor tissue is 50-60 mm3。
More preferably, the number of the human hemangioma cells is 1 × 107The volume of the parent tumor tissue is 60mm3。
The cell number can affect the tumor formation rate of the parent tumor, the tissue volume of the parent tumor can affect the tumor formation rate and the growth speed of the hemangioma, and the cell number range and the tissue volume range of the parent tumor can be obtained through earlier experiments by the inventor.
Preferably, the immunodeficient animal is a T cell immunodeficient nude mouse or a SCID mouse with severe combined immunodeficiency of T cells and B cells.
Preferably, the immunodeficient animal is a 6-8 week old animal.
Preferably, the injection amount of the estradiol is 0.1-1.0 mg, and more preferably 0.5 mg. Estradiol is an important hormone related to the growth of hemangioma, and is injected for the purpose of promoting the growth of hemangioma and preventing regression during the growth process.
Preferably, in step S5, the step of extracting the parent tumor includes: immunodeficient animals containing the parent tumor were sacrificed and the parent tumor was dissected.
Preferably, in step S6, the inoculation specifically comprises the following steps: tumor tissues are inoculated subcutaneously into 6-8 weeks old immunodeficiency animals by implanting through an implantation trocar.
The invention also provides a human hemangioma animal model constructed according to the method. The human hemangioma animal model can be applied to research on the occurrence mechanism and treatment method of human hemangioma.
Compared with the prior art, the invention has the following beneficial effects:
1. the success rate of hemangioma (i.e. parent tumor) obtained by the step S4 is very low, less than 50%, so that the parent tumor forming animal model cannot be directly prepared by adopting the step S4; the success rate of hemangioma of an animal model formed by the parent tumor and the subsequent steps can reach 100 percent, the tumor formation rate is extremely high, and the operation is simple and convenient.
2. The hemangioma animal model formed by the invention has the pathological characteristics of human hemangioma completely, and basic experiments of histopathology prove that a large number of new capillaries exist in hemangioma tissues and cells proliferate vigorously, and the pathological characteristics are very similar to those of human hemangioma tissues.
3. The animal model of the invention is very suitable for the research of the generation mechanism and the treatment method of the human hemangioma, and the evaluation of the curative effect of various drugs for treating hemangioma and the screening of the drugs for treating hemangioma.
Drawings
Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:
FIG. 1 is a tumor growth curve;
FIG. 2 is a picture of a tumor; A. tumor pictures of nude mice; B. picture of tumor in vitro;
FIG. 3 is tumor section staining and identification; A. HE staining of tumor sections; B. tumor section CD31 staining; C. tumor section CD34 staining; D. tumor sections were Ki-67 stained.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that it would be obvious to those skilled in the art that various changes and modifications can be made without departing from the spirit of the invention. All falling within the scope of the present invention.
Example 1
The embodiment provides a method for constructing a human hemangioma animal model, which comprises the following specific steps:
① culture of hemangioma endothelial cells (HemECs)
a. Purchasing or separating and extracting to obtain human hemangioma endothelial cells;
b. cells were cultured in DMEM complete medium (10% FBS + 1% diabody + 89% DMEM basal medium), proliferated, and counted on a hemocytometer.
② hemangioma endothelial cells (HemECs) construction of hemangioma animal model parent tumors
a. Digesting the cultured hemangioma endothelial cells with 0.25% pancreatin;
b. after counting, take 1X 106Centrifuging the cell digestive juice in a 15mL centrifuge tube at 1200rpm for 5 minutes, and removing the supernatant;
c. adding PBS to resuspend the cells, centrifuging and discarding the supernatant, and adding 100 mu LPBS to resuspend the cells.
d. Taking a 1mL syringe, sucking 100 mu L of cell suspension, and injecting the cell suspension into the subcutaneous part of a BALB/c-nu nude mouse with the age of 6-8 weeks;
e. immunodeficient animals were injected intraperitoneally with 0.5mg estradiol.
③ hemangioma endothelial cell mother tumor construction hemangioma animal model
a. Killing the female tumor mice (i.e. BALB/c-nu nude mice with formed female tumors), and stripping the female tumors;
b. removing redundant tissues on the surfaces of the parent tumors, and washing the parent tumors with PBS;
c. placing parent tumor into cell culture dish, adding a little PBS for infiltration, and cutting tumor block into 60mm with ophthalmologic operation scissors3Tumor tissues with fixed sizes were washed clean with PBS;
d. immunodeficient animals were anesthetized in advance, and prepared tumor tissues were inoculated subcutaneously into 6-8 week old BALB/c-nu nude mice using an implantation trocar.
Referring to FIG. 1, FIG. 1 is a graph showing the growth of subcutaneous tumors in immunodeficient mice. The figure shows that the growth speed of hemangioma is fast, and the volume of hemangioma can reach 5000mm in four weeks3。
Referring to fig. 2, fig. 2 is a tumor picture. A. The tumor picture of the immunodeficient mouse shows that the hemangioma has uniform shape and is similar to a sphere, and abundant blood vessels on the surface of the tumor can be seen through subcutaneous tissue. B. The picture of the isolated tumor, 6 tumors in the picture are parallel samples obtained by the method of the embodiment 1, the surface of the tumor tissue is compact and compact, the blood vessels are rich, and the shape is similar to spherical and grows uniformly.
The tissue structure of the hemangioma animal model established in the embodiment is similar to human hemangioma, the hemangioma rate is high, the growth speed is high, and obvious tumor bulge can be observed after one week.
Referring to FIG. 3, FIG. 3 shows the staining and identification of tumor sections. A. HE staining of tumor sections. B. The tumor section is subjected to immunohistochemical staining by CD31, and positive cells are mostly distributed around blood vessels, so that the blood vessels in the tumor are rich in distribution, and the growth activity of the tumor is high. C. The tumor section is subjected to CD34 immunofluorescence staining, and positive cells are mostly distributed around blood vessels, so that the blood vessels in the tumor are rich in distribution, and the growth activity of the tumor is high. D. The tumor section Ki-67 is subjected to immunohistochemical staining, and the Ki-67 is a nuclear antigen which is specifically related to cell proliferation, and the abundant expression of the Ki-67 in the tumor indicates that the hemangioma cell proliferation activity is high. Tissue staining results show that the hemangioma animal model established by the invention has characteristics equivalent to those of human hemangioma. The animal model is very suitable for researching pathogenesis of human hemangioma, developing drugs or methods for treating human hemangioma, evaluating various drugs for treating hemangioma and screening drugs for treating hemangioma.
The hemangioma animal model established by the invention combines the tissue transplantation method and the cell suspension inoculation method, the final tumor formation rate is 100%, the problems of difficult acquisition of human hemangioma tissues and low tumor formation rate of cell suspension inoculation are avoided, and a simple and efficient hemangioma animal model construction method is provided.
Since the inoculated cells are humanized cells, the hemangioma model formed completely has the pathological characteristics of human hemangioma, and tissue section staining also proves that: a large number of new capillaries exist in hemangioma tissues, the cell proliferation activity is high, and the tumor growth speed is high. These pathological features are very similar to human hemangioma tissue.
Example 2
The embodiment provides a method for constructing a human hemangioma animal model, which comprises the following specific steps:
① culture of hemangioma stem cells
a. Purchasing or separating and extracting to obtain human hemangioma stem cells;
b. cells were cultured in DMEM complete medium (10% FBS + 1% diabody + 89% DMEM basal medium), proliferated, and counted on a hemocytometer.
② hemangioma stem cell construction of hemangioma animal model parent tumor
a. Digesting the cultured hemangioma stem cells with 0.25% pancreatin;
b. after counting, take 1X 106Centrifuging the cell digestive juice in a 15mL centrifuge tube at 1200rpm for 5 minutes, and removing the supernatant;
c. adding PBS to resuspend the cells, centrifuging and discarding the supernatant, and adding 100 mu LPBS to resuspend the cells.
d. Taking a 1mL syringe, sucking 100 mu L of cell suspension, and injecting the cell suspension into the subcutaneous part of a BALB/c-nu nude mouse with the age of 6-8 weeks;
e. immunodeficient animals were injected intraperitoneally with 0.5mg estradiol.
③ hemangioma stem cell mother tumor construction hemangioma animal model
a. Killing the female tumor mice (i.e. BALB/c-nu nude mice with formed female tumors), and stripping the female tumors;
b. removing redundant tissues on the surfaces of the parent tumors, and washing the parent tumors with PBS;
c. placing parent tumor into cell culture dish, adding a little PBS for infiltration, and cutting tumor block into 60mm with ophthalmologic operation scissors3Tumor tissues with fixed sizes were washed clean with PBS;
d. immunodeficient animals were anesthetized in advance, and prepared tumor tissues were inoculated subcutaneously into 6-8 week old BALB/c-nu nude mice using an implantation trocar.
Example 3
The embodiment provides a method for constructing a human hemangioma animal model, which has the following specific steps basically the same as those in embodiment 1:
① human hemangioma endothelial cells were cultured and expanded.
② mixing the raw materials together by 1 × 106The hemangioma endothelial cells were resuspended evenly with 100 μ L PBS;
③ inoculating the human hemangioma endothelial cells to the subcutaneous part of an immunodeficient BALB/c-nu nude mouse by a syringe;
④ intraperitoneal injecting estradiol 0.5mg to obtain mother tumor;
⑤ taking mother tumor, peeling off, and washing with PBS;
⑥ divide mother tumor into 30mm3Small pieces (tumor tissue);
⑦ tumor tissues were inoculated subcutaneously into immunodeficient BALB/c-nu nude mice using an implantation trocar.
The animal model morphology and the tissue structure of the hemangioma established by the invention are very similar to those of human hemangioma, and the success rate of the hemangioma is not high. The growth speed is moderate after planting, and subcutaneous tumor bulge can be obviously observed after two weeks. A large number of capillaries can be seen by section staining, and simultaneously, the cell proliferation activity is high and the tumor growth speed is high.
Example 4
The embodiment provides a method for constructing a human hemangioma animal model, which has the following specific steps basically the same as those in embodiment 1:
① human hemangioma endothelial cells were cultured and expanded.
② mixing 5X 106The hemangioma endothelial cells were resuspended evenly with 100 μ L PBS;
③ inoculating the human hemangioma endothelial cells to the subcutaneous part of an immunodeficient BALB/c-nu nude mouse by a syringe;
④ intraperitoneal injecting estradiol 0.1mg to obtain mother tumor;
⑤ taking mother tumor, peeling off, and washing with PBS;
⑥ divide mother tumor into 40mm3Small pieces (tumor tissue);
⑦ tumor tissues were inoculated subcutaneously into immunodeficient BALB/c-nu nude mice using an implantation trocar.
The animal model morphology and the tissue structure of the hemangioma established by the invention are very similar to those of human hemangioma, and the success rate of the hemangioma is not high. The growth speed is fast after planting, and subcutaneous tumor bulge can be obviously observed after half a week. A large number of capillaries can be seen by section staining, and simultaneously, the cell proliferation activity is high and the tumor growth speed is high.
Example 5
The embodiment provides a method for constructing a human hemangioma animal model, which has the following specific steps basically the same as those in embodiment 1:
① human hemangioma endothelial cells were cultured and expanded.
② mixing the raw materials together by 1 × 107The hemangioma endothelial cells were resuspended evenly with 100 μ L PBS;
③ inoculating the human hemangioma endothelial cells to the subcutaneous part of an immunodeficient BALB/c-nu nude mouse by a syringe;
④ intraperitoneal injecting estradiol 1.0mg to obtain mother tumor;
⑤ taking mother tumor, peeling off, and washing with PBS;
⑥ divide mother tumor into 60mm3Small pieces (tumor tissue);
⑦ tumor tissues were inoculated subcutaneously into immunodeficient BALB/c-nu nude mice using an implantation trocar.
The animal model morphology and the tissue structure of the hemangioma established by the invention are very similar to those of human hemangioma, and the success rate of the hemangioma is high. After planting, the growth speed is high, and subcutaneous tumor bulge can be obviously observed after one week. A large number of capillaries can be seen by section staining, and simultaneously, the cell proliferation activity is high and the tumor growth speed is high.
Example 6
The embodiment provides a method for constructing a human hemangioma animal model, which has the following specific steps basically the same as those in embodiment 1:
① human hemangioma endothelial cells were cultured and expanded.
② mixing 5X 107The hemangioma endothelial cells were resuspended evenly with 100 μ L PBS;
③ inoculating the human hemangioma endothelial cells to the subcutaneous part of an immunodeficient BALB/c-nu nude mouse by a syringe;
④ intraperitoneal injecting estradiol 0.5mg to obtain mother tumor;
⑤ taking mother tumor, peeling off, and washing with PBS;
⑥ divide mother tumor into 50mm3Small pieces (tumor tissue);
⑦ tumor tissues were inoculated subcutaneously into immunodeficient BALB/c-nu nude mice using an implantation trocar.
The animal model morphology and the tissue structure of the hemangioma established by the invention are very similar to those of human hemangioma, and the success rate of the hemangioma is high. After planting, the growth speed is high, and subcutaneous tumor bulge can be obviously observed after one week. A large number of capillaries can be seen by section staining, and simultaneously, the cell proliferation activity is high and the tumor growth speed is high.
Example 7
The embodiment provides a method for constructing a human hemangioma animal model, which has the following specific steps basically the same as those in embodiment 1:
① human hemangioma endothelial cells were cultured and expanded.
② mixing the raw materials together by 1 × 108The hemangioma endothelial cells were resuspended evenly with 100 μ L PBS;
③ inoculating the human hemangioma endothelial cells to the subcutaneous part of an immunodeficient BALB/c-nu nude mouse by a syringe;
④ intraperitoneal injecting estradiol 0.5mg to obtain mother tumor;
⑤ taking mother tumor, peeling off, and washing with PBS;
⑥ divide mother tumor into 70mm3Small pieces (tumor tissue);
⑦ tumor tissues were inoculated subcutaneously into immunodeficient BALB/c-nu nude mice using an implantation trocar.
The animal model morphology and the tissue structure of the hemangioma established by the invention are very similar to those of human hemangioma, and the success rate of the hemangioma is high. The growth speed is very fast after planting, and subcutaneous tumor bulge can be obviously observed after one week. A large number of capillaries can be seen by section staining, simultaneously, the cell proliferation activity is high, the tumor growth speed is high, and the skin necrosis on the surface of the tumor can be observed.
The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes or modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention. The embodiments and features of the embodiments of the present application may be combined with each other arbitrarily without conflict.
Claims (10)
1. A construction method of a human hemangioma animal model is characterized by comprising the following steps:
s1, taking isolated human hemangioma cells for later use;
s2, resuspending the human hemangioma cells uniformly by PBS;
s3, inoculating the resuspended human hemangioma cells to the subcutaneous part of the immunodeficient animal;
s4, injecting estradiol into the abdominal cavity to obtain mother tumor;
s5, picking the mother tumor, peeling off and washing;
s6, dividing the washed parent tumor into 30-70mm3The tumor tissue of (a);
s7, inoculating the tumor tissue to the subcutaneous part of an animal with immunodeficiency, and feeding to obtain a human hemangioma animal model;
the human hemangioma cells comprise one or a mixture of human hemangioma endothelial cells and human hemangioma stem cells.
2. The method for constructing a human hemangioma animal model according to claim 1, wherein in step S2, the human hemangioma cells are cultured, proliferated, digested, and then the number of human hemangioma cells is 1 x 106～1×108The digests were collected, centrifuged, the supernatant removed and resuspended in PBS.
3. The method for constructing an animal model of human hemangioma according to claim 2, wherein said culturing is performed in a DMEM complete medium and said digestion is performed in 0.25% pancreatin.
4. The method for constructing an animal model of human hemangioma according to claim 2, wherein the number of human hemangioma cells is 1 x 106～5×107The volume of the mother tumor tissue is 40-70 mm3。
5. The method for constructing an animal model of human hemangioma according to claim 4, wherein the number of human hemangioma cells is 5 x 106～1×107The volume of the mother tumor tissue is 50-60 mm3。
6. The method of claim 1, wherein the immunodeficient animal model is a T cell immunodeficient nude mouse or a T cell and B cell severe combined immunodeficient SCID mouse at step S3.
7. The method for constructing an animal model of human hemangioma according to claim 1, wherein in step S4, the injection amount of estradiol is 0.1-1.0 mg.
8. The method for constructing an animal model with human hemangioma according to claim 1, wherein in step S5, the step of extracting the parent tumor comprises: immunodeficient animals containing the parent tumor were sacrificed and the parent tumor was dissected.
9. The method for constructing an animal model of human hemangioma according to claim 1, wherein in step S6, the inoculation comprises the following steps: tumor tissues are inoculated subcutaneously into 6-8 weeks old immunodeficiency animals by implanting through an implantation trocar.
10. An animal model of human hemangioma constructed according to the method of any one of claims 1 to 9.
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