CN114795570A - Novel breast cancer bone metastasis model modeling mode - Google Patents
Novel breast cancer bone metastasis model modeling mode Download PDFInfo
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- CN114795570A CN114795570A CN202210465426.9A CN202210465426A CN114795570A CN 114795570 A CN114795570 A CN 114795570A CN 202210465426 A CN202210465426 A CN 202210465426A CN 114795570 A CN114795570 A CN 114795570A
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- A—HUMAN NECESSITIES
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Abstract
The invention discloses a novel breast cancer bone metastasis model modeling mode, wherein the first step is based on body position inclination as an operation basis; secondly, exposing the iliac artery and vein based on a stereoscopic dissection microscope; thirdly, separating arteriovenous based on a stereoscopic dissection microscope; the fourth step is preparation before iliac artery injection; the fifth step is to use the insulin needle to inject the tumor cells; the sixth step is to suture the wound; and seventhly, managing the temperature after the operation. Compared with the efficiency and the selective modeling of internal iliac artery injection microscopic bone metastasis disclosed by JoVE, the instant success rate of 60 percent and the survival rate of 20 percent in 3 days, the time for completing iliac artery injection of tumor cells for individual BALB/c-nu nude mice by the breast cancer bone metastasis model modeling method is 15-20min, the instant success rate of breast cancer bone metastasis model modeling reaches 100 percent, the survival rate of 3 days reaches 80 percent, the success rate of tumor cell injection is improved, bleeding of BALB/c-nu nude mice is reduced, and the survival rate of modeling is increased.
Description
Technical Field
The invention relates to the technical field of bone tumor pain models, in particular to a novel breast cancer bone metastasis model making mode.
Background
Breast cancer metastasis is one of the major causes of eventual death, and can metastasize to almost any part of the human body, with several common metastatic sites being lymph nodes, bone, liver, lung and brain. Among them, bone is the most common site of metastasis of breast cancer, bone metastasis is associated with poor prognosis, and patients with bone metastasis have poor quality of life at the end of cancer due to severe pain, mobility difficulties, hypercalcemia, osteolysis and fracture, and spinal cord compression and bone marrow dysplasia.
The establishment of a mouse breast cancer model is a common method for researching breast cancer, and has far-reaching significance for researching the etiology and the occurrence mechanism of the breast cancer, searching a drug target and guiding clinical treatment. Due to the particularity of bone tissues, the establishment of the mouse bone metastasis model at present has three main ways: tail vein injection (tumor cells), left atrium injection, iliac artery injection. However, the method of tail vein injection of tumor cells is easier to form lung and liver metastasis, and the probability of successfully establishing a mouse breast cancer bone metastasis model is not high; the left atrial injection causes too much trauma to mice and has low modeling survival rate, so that the iliac artery is generally adopted to inject tumor cells to establish a mouse breast cancer bone metastasis model. In view of the above, the JoVE website discloses efficiency and selective modeling of internal iliac artery injection microscleral bone metastasis, reveals that the iliac artery is used as a target of injected cancer cells, and requires iliac veins and arteries to be separated from surrounding tissues, and D-fluorescein labeling is carried out while breast cancer cells are labeled by GFP, so that observation of location of posterior iliac artery tumor cells is realized.
Disclosure of Invention
The invention aims to provide a novel breast cancer bone metastasis model modeling mode, which is characterized in that in the process of separating the iliac artery of a nude mouse model, the blood flow of the iliac artery is increased by using the methods of an inclined position, distal end suspension of an artery and a vein, temperature management and the like to achieve the purpose of increasing the diameter of the iliac artery, so that tumor cells can be further injected, the success rate of injection is improved, the bleeding of a BALB/c-nu nude mouse is reduced, and the survival rate of modeling is increased, so that the problems in the background technology are solved.
In order to achieve the purpose, the invention provides the following technical scheme:
a novel breast cancer bone metastasis model modeling method is characterized in that:
the first step is based on body position inclination;
secondly, exposing the iliac artery and vein based on a stereoscopic dissection microscope;
thirdly, separating arteriovenous based on a stereoscopic dissection microscope;
the fourth step is preparation before iliac artery injection;
the fifth step is to use the insulin needle to inject the tumor cells;
the sixth step is to suture the wound;
seventhly, managing the temperature after operation;
the instant success rate of the breast cancer bone metastasis model molding reaches 100%, and the 3-day survival rate reaches 80%.
Preferably, the breast cancer bone metastasis model is modeled by the following specific modeling method:
s1, inclining the posture, fixing the four limbs of the anesthetized BALB/c-nu nude mouse on a panel of an adjusting device, and adjusting the angle of the adjusting device;
s2, exposing iliac artery and veins, sterilizing the 4 th and 5 th nipple areas of the right lower abdomen of the BALB/c-nu nude mouse by iodophor, cutting an incision on the skin of the sterilized area by using sterile scissors, wherein the incision length is 1.0-1.2 cm, and shielding the position of the BALB/c-nu nude mouse except the incision by using sterile surgical cloth; fixing two sides of the incision by using a draw hook, and shearing subcutaneous fascia by using another sterile scissors, wherein the iliac artery, vein and nerve can be seen to be mixed together; using vertical precise forceps to break through the connective tissue between blood vessels and nerves under a stereoscopic dissection microscope, and separating iliac artery and vein from the connective tissue;
s3, separating the artery and the vein, passing the 4-0 non-absorbable surgical suture through the bottom of the iliac artery and the vein by using a bent precision forceps, lifting the 4-0 non-absorbable surgical suture at the proximal end to increase the tension of the gap between the iliac artery and the vein, and gently puncturing the connective tissue at the gap in a direction parallel to the blood vessel by using a vertical precision forceps, so that the complete separation of the iliac artery and the vein is realized; after the iliac artery and vein are completely separated, the 4-0 non-absorbable surgical suture is laid down and is not drawn out for subsequent use;
s4, preparing before iliac artery injection, pressing iliac artery by using a cotton swab heated by water bath to increase the temperature of iliac artery, moving 4-0 non-absorbable surgical suture at the bottom of blood vessel to the far end, and suspending 4-0 non-absorbable surgical suture, wherein iliac artery is obviously thickened;
s5, injecting tumor cells, namely, using a U40 (32G) sterile insulin syringe with a needle head diameter of 0.25mm to absorb 0.2ml of cell suspension, placing the needle head in parallel with the direction of a blood vessel, slowly pushing the needle head into the blood vessel, after the needle head is pricked into the blood vessel under a microscope, observing the syringe during injection, pressing a sterile cotton swab head above the needle head immediately after injection, and immediately pressing the cotton swab on the blood vessel after needle withdrawal so as to achieve the purpose of compression hemostasis and reduce bleeding amount;
withdrawing the suspended 4-0 non-absorbable surgical suture;
s6, suturing the wound, and after the BALB/c-nu nude mouse is completely stopped, suturing the subcutaneous fascia layer by layer and suturing the skin layer by using a needle head of a 4-0 absorbable surgical suture;
s7, managing temperature after operation, wherein the BALB/c-nu nude mouse is in an anesthesia state, the temperature adjusting function is weak, a heat blanket is arranged on a panel of an adjusting device to prevent the BALB/c-nu nude mouse from dying due to too low temperature, and the heat blanket can be powered off until the BALB/c-nu nude mouse completely revives from the anesthesia;
s8, putting the completely recovered BALB/c-nu nude mice into a cage box for breeding.
Preferably, the time for completing iliac artery injection of tumor cells to individual BALB/c-nu nude mice by the breast cancer bone metastasis model modeling method is 15-20 min.
Compared with the prior art, the invention has the beneficial effects that:
compared with the efficiency and the selective modeling of internal iliac artery injection microscopic bone metastasis disclosed by JoVE, the instant success rate of 60 percent and the survival rate of 20 percent in 3 days, the time for completing iliac artery injection of tumor cells for an individual BALB/c-nu nude mouse by a single time of the breast cancer bone metastasis model modeling method is 15-20min, the instant success rate of the breast cancer bone metastasis model modeling reaches 100 percent, and the survival rate of 3 days reaches 80 percent.
In the process of separating the iliac artery of a nude mouse model, the blood flow of the iliac artery is increased by using methods of oblique position, distal end suspension of arteriovenous, temperature management and the like to achieve the purpose of increasing the diameter of the iliac artery, thereby facilitating further injection of tumor cells, improving the success rate of tumor cell injection, reducing bleeding of BALB/c-nu nude mice and increasing the survival rate of molding.
Drawings
FIG. 1 is a comparison of arteriovenous separation of a breast cancer bone metastasis model and a JOVE bone metastasis model based on a stereotactic dissecting microscope in accordance with the present invention;
FIG. 2 is a comparison graph of bone metastasis of an intracardiac injection animal observed by in vivo imaging of a breast cancer bone metastasis model and a JOVE bone metastasis model according to the present invention;
FIG. 3 is a comparison of iliac artery injection of tumor cells for breast cancer bone metastasis models of the present invention versus JOVE bone metastasis models;
FIG. 4 is a comparison of breast cancer bone metastasis modeling survival rate of the present invention and JOVE bone metastasis modeling survival rate.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to fig. 1-4, the present invention provides a technical solution, a novel breast cancer bone metastasis model modeling method, which comprises a first step of operating on the basis of posture inclination; secondly, exposing the iliac artery and vein based on a stereoscopic dissection microscope; thirdly, separating arteriovenous based on a stereoscopic dissection microscope; the fourth step is preparation before iliac artery injection; the fifth step is to use the insulin needle to inject the tumor cells; the sixth step is to suture the wound; and seventhly, managing the temperature after the operation.
The invention relates to a breast cancer bone metastasis model modeling method, which comprises the following specific modeling methods:
s1, inclining the posture, fixing the four limbs of the anesthetized BALB/c-nu nude mouse on a panel of an adjusting device, and adjusting the angle of the adjusting device;
s2, exposing iliac artery and veins, sterilizing the 4 th and 5 th nipple areas of the right lower abdomen of the BALB/c-nu nude mouse by iodophor, cutting an incision on the skin of the sterilized area by using sterile scissors, wherein the incision length is 1.0-1.2 cm, and shielding the position of the BALB/c-nu nude mouse except the incision by using sterile surgical cloth; fixing two sides of the incision by using a draw hook, and shearing subcutaneous fascia by using another sterile scissors, wherein the iliac artery, vein and nerve can be seen to be mixed together;
breaking through connective tissue between blood vessels and nerves by using vertical precision forceps under a stereoscopic dissection microscope, or slightly rubbing iliac artery and vein by using a cotton swab to separate the iliac artery and vein from the peripheral nerves and connective tissue;
s3, separating the artery and the vein, using a bending precision forceps to pass the 4-0 non-absorbable surgical suture through the bottom of the iliac artery and the vein, and then lifting the 4-0 non-absorbable surgical suture at the proximal end, so that the tension of the gap between the iliac artery and the vein is increased, and the separation is facilitated; at the gap of the iliac artery and vein with increased tension, the connective tissue at the gap is punctured softly by using a vertical precise forceps in the direction parallel to the blood vessel, so as to realize the complete separation of the iliac artery and vein; after the iliac artery and vein are completely separated, the 4-0 non-absorbable surgical suture is laid down and is not drawn out for subsequent use;
s4, preparing before iliac artery injection, pressing the iliac artery by using a cotton swab heated by a water bath to increase the temperature of the iliac artery, so as to increase the iliac artery blood flow, moving a 4-0 non-absorbable surgical suture at the bottom of a blood vessel to the far end of the heart, and suspending the 4-0 non-absorbable surgical suture, wherein the iliac artery is obviously thickened;
s5, injecting tumor cells, sucking 0.2ml of cell suspension by using a U40 (32G) sterile insulin syringe with the needle diameter of 0.25mm, placing the needle direction and the blood vessel direction in parallel, and slowly pushing the cell suspension into the blood vessel; during the injection, an operator pricks the needle into a blood vessel under a microscope, and enables an assistant to observe the injector at any time during the injection, the operator stops the injection after the injection is finished, meanwhile, the assistant presses the sterile cotton swab head above the needle, and after the operator pulls the needle, the assistant presses the cotton swab on the blood vessel immediately, so that the purpose of compression hemostasis is achieved, and the blood flow of a BALB/c-nu nude mouse is reduced; withdrawing the suspended 4-0 non-absorbable surgical suture;
s6, suturing the wound, and after the BALB/c-nu nude mouse is completely stopped, suturing the subcutaneous fascia layer by layer and suturing the skin layer by using a needle head of a 4-0 absorbable surgical suture;
s7, managing temperature after operation, wherein the BALB/c-nu nude mouse is in an anesthesia state, the temperature adjusting function is weak, a heat blanket is arranged on a panel of an adjusting device to prevent the BALB/c-nu nude mouse from dying due to too low temperature, and the heat blanket can be powered off until the BALB/c-nu nude mouse completely revives from the anesthesia;
s8, putting the completely recovered BALB/c-nu nude mice into a cage box for breeding.
The molding method of the breast cancer bone metastasis model has the time for completing iliac artery injection of tumor cells to individual BALB/c-nu nude mice for 15-20 min; the instant success rate of the breast cancer bone metastasis model molding reaches 100%, and the 3-day survival rate reaches 80%.
It is worth noting that the tumor cells injected by the method of the invention carry fluorescent dye, after the tumor cells are injected for 2-3 days, the fluorescent color developing agent is injected into the abdominal cavity of the nude mouse, then the nude mouse living body fluorescence imaging is carried out, the density of the tumor cells at the ilium part is considerable, which shows that the breast cancer bone metastasis model modeling method of the invention effectively injects the tumor cells into the ilium artery.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes in the embodiments and/or modifications of the invention can be made, and equivalents and modifications of some features of the invention can be made without departing from the spirit and scope of the invention.
Claims (3)
1. A novel breast cancer bone metastasis model modeling method is characterized in that:
the first step is based on body position inclination;
secondly, exposing the iliac artery and vein based on a stereoscopic dissection microscope;
thirdly, separating arteriovenous based on a stereoscopic dissection microscope;
the fourth step is preparation before iliac artery injection;
the fifth step is to use the insulin needle to inject the tumor cells;
the sixth step is to suture the wound;
seventhly, managing the temperature after operation;
the instant success rate of the breast cancer bone metastasis model molding reaches 100%, and the 3-day survival rate reaches 80%.
2. The modeling method of the new breast cancer bone metastasis model according to claim 1, characterized in that: the specific molding method is as follows:
s1, inclining the posture, fixing the four limbs of the anesthetized BALB/c-nu nude mouse on a panel of an adjusting device, and adjusting the angle of the adjusting device;
s2, exposing iliac artery and veins, sterilizing the 4 th and 5 th nipple areas of the right lower abdomen of the BALB/c-nu nude mouse by iodophor, cutting an incision on the skin of the sterilized area by using sterile scissors, wherein the incision length is 1.0-1.2 cm, and shielding the position of the BALB/c-nu nude mouse except the incision by using sterile surgical cloth; fixing two sides of the incision by using a draw hook, and shearing subcutaneous fascia by using another pair of sterile scissors;
using vertical precise forceps to break through the connective tissue between blood vessels and nerves under a stereoscopic dissection microscope, and separating iliac artery and vein from the connective tissue;
s3, separating the artery and the vein, passing the 4-0 non-absorbable surgical suture through the bottom of the iliac artery and the vein by using a bent precision forceps, lifting the 4-0 non-absorbable surgical suture at the proximal end to increase the tension of the gap between the iliac artery and the vein, and gently puncturing the connective tissue at the gap in a direction parallel to the blood vessel by using a vertical precision forceps, so that the complete separation of the iliac artery and the vein is realized; after the iliac artery and vein are completely separated, the 4-0 non-absorbable surgical suture is laid down and is not drawn out for subsequent use;
s4, preparing before iliac artery injection, pressing iliac artery by using a cotton swab heated by water bath to increase the temperature of iliac artery, moving 4-0 non-absorbable surgical suture at the bottom of blood vessel to the far end, and suspending 4-0 non-absorbable surgical suture, wherein iliac artery is obviously thickened;
s5, injecting tumor cells, namely, using a U40 (32G) sterile insulin syringe with a needle head diameter of 0.25mm to absorb 0.2ml of cell suspension, placing the needle head in parallel with the direction of a blood vessel, slowly pushing the needle head into the blood vessel, after the needle head is pricked into the blood vessel under a microscope, observing the syringe during injection, pressing a sterile cotton swab head above the needle head immediately after injection, and immediately pressing the cotton swab on the blood vessel after needle withdrawal so as to achieve the purpose of compression hemostasis and reduce bleeding amount;
withdrawing the suspended 4-0 non-absorbable surgical suture;
s6, suturing the wound, and after the BALB/c-nu nude mouse is completely stopped, suturing the subcutaneous fascia layer by layer and suturing the skin layer by using a needle head of a 4-0 absorbable surgical suture;
s7, managing temperature after operation, wherein the BALB/c-nu nude mouse is in an anesthesia state, the temperature adjusting function is weak, a heat blanket is arranged on a panel of an adjusting device to prevent the BALB/c-nu nude mouse from dying due to too low temperature, and the heat blanket can be powered off until the BALB/c-nu nude mouse completely revives from the anesthesia;
s8, putting the completely recovered BALB/c-nu nude mice into a cage box for breeding.
3. The modeling method of the new breast cancer bone metastasis model according to claim 2, characterized in that: the molding method of the breast cancer bone metastasis model completes the iliac artery injection of tumor cells for 15-20min for individual BALB/c-nu nude mice once.
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Application publication date: 20220729 |