Nude mice are immunodeficient animals because they have no thymus glands, so they lack immune response. In normal mice, the thymus is located behind the manubrium, close to the trachea, and on the ventral surface of the heart. The thymus gland is closely related to the body's immune system. It is now known that the thymus gland can secrete several biologically active polypeptide substances, collectively called thymosin, which can stimulate lymphoid tissue to produce lymphocytes; The blast cells mature into T lymphocytes with cellular immune activity, thereby exerting immune function. Nude mice are congenitally athymic, and there is a different debate between IgM and antigen responses when T cells are suppressed, but there is no IgG and IgA responses at all in the absence of helper T cells.
Nude mice are an indispensable animal model in the current field of cancer research. It has special value in safety assessment and activity screening experiments of oncology, immunology, pharmacology and biological agents. Nude mice have great potential in scientific research because of the unique genetic characteristics of the nu gene. At present, the nu gene has been introduced into different inbred animals, and it has become a series of animal models. There is only one mouse model. Nude mice are a new breed of mutant mice due to the congenital thymus defect, due to the homozygosity of the Nu/Nu gene in the 11th pair of chromosomes. Nude mice appear hairless (although a fine-haired follicle is visible histologically) and lack a normal thymus. Its characteristics include T cell deficiency, so it can accept foreign transplanted cells, and it is less likely to have immune rejection. It is suitable for xenograft (Xenograft Model), and the most common is to take subcutaneous injection (Subcutaneous, SC) in the buttocks. Cancer cells produce tumors , but must be reared under sterile conditions.
The discovery of nude mice was made by Dr. Grist of Glasgow Hospital, UK, in 1962, who accidentally discovered individual hairless mice among non-inbred mice. After 1966, Dr. Flanagan of the Edinburgh Institute of Zoology confirmed that this hairless mouse was caused by a mutation in the nu gene on the chromosome. The skin histology is different from the previous hairless mice. It was found that there is a hairless gene due to chromosomal degeneration, so it is considered to be a new spontaneous mutant, and named "Nude" (Nude) mice, with "nu" Represented as gene symbols. Before 1968, the study of cellular immunology usually used drugs to inhibit the production and function of T cells in mice, or surgical removal of the thymus. However, these methods often failed due to incomplete inhibition or suboptimal surgery. In 1968, Dr. Pantelouris discovered that nude mice had no thymus, which aroused great interest among medical biologists. Due to the poor physical resistance of the nude mouse stock (probably lack of T cells), fertility is low and life span is shorter. Therefore, after breeding and transformation, they were bred with pure line BALB/c, C3H, C57BL/6 mice, and continuously selected and bred, and three new breeds of athymic nude mice were obtained, but their shortcomings were still not overcome. Finally, BALB/c nude mice were mated with NIH Swiss mice to obtain outbred athymic nude mice, which overcome the shortcomings of poor resistance, low fertility and short lifespan, so they can be bred and promoted in large numbers. The successful breeding of nude mice provides an ideal animal model for the research and development of thymus function, immunological mechanism and pharmacological activity.
Because nude mice must be in a specific pathogen-free environment, they can survive for 22 months. Nude mice can receive xenografted tissue, including normal skin, as well as xenografted cells, including cancer cells and normal vascular cells. Therefore, it can provide the best research model for skin transplantation, cancer and angiogenesis. It is worth noting that nude mice still have normal B cells, natural killer cells (NK cells), macrophages (macrophages) and granulocytes (granulocytes), so not all cancer cells can be transplanted successfully to induce cancer cells.
Nude rats are currently mainly used in human cancer transplantation research. Because nude rats are athymic and lack T cells, they can successfully transplant xenogeneic skin and xenogeneic tumors, including mouse tumors and human tumors.
Medicilon Pharmacology Service provides you with experiments on tumor formation in nude mice and subcutaneous tumor formation in nude mice. For details, please contact us. Email: marketing@medicilon.com
1. Operation plan design
2. Method development
3. Cell Preparation
4. Tumor formation in nude mice
Grouping: SW620 cell group, SW620 transfected irrelevant siRNA group, SW620 transfected target gene (6 cells in each group).
Cell preparation and injection in nude mice: Go to the animal room to prepare for the experiment.
Take out the untreated mice and the mouse cages for the treated mice, and place them in the clean table in order. Mice with larger body size were selected and weighed, and the mice were anesthetized by intraperitoneal injection of 10% chloral hydrate at a dose of 3 μl/g. After about 5 minutes, the mice began to enter a quiet state and were immobilized in their supine position. Do not overdose on anesthetics.
(1) Spread a white cloth on the mouse board, fix the four paws of the mouse in the supine position, and disinfect the ventral side with iodophor cotton balls up to the neck, down to the groin, and back to the posterior axillary line. Sterilize twice. Lay a surgical drape on the mouse, make a notch (4*4cm) on its left side, expose its left abdomen, and sterilize it with iodophor once. Cut down 2cm of skin at 1cm below the left costal arch to expose the abdominal wall muscles. The abdominal wall muscles are pulled with forceps, avoiding the spleen in the abdominal cavity, and the abdominal wall muscles are cut open to expose the internal organs. Dip a cotton swab in PBS and pull out the lower pole of the spleen. Do not pull it to avoid damage to the spleen and pancreas.
(2) At the same time of finding the spleen, mix 25 microliters of Matrigel into the cell tube, suspend the cell pellet, and make the cells evenly dispersed into a single cell suspension. Use a BD needle to aspirate the single-cell suspension in the EP tube, withdraw the needle core a little, blow out air bubbles, insert the needle at the inner side of the lower pole (concave surface) of the spleen and start injecting cells about 2 mm toward the hilum of the spleen, and stay for about 30 seconds after the injection. Withdraw the needle slowly to prevent cell exudation. At the same time, use a cotton ball dipped in PBS to compress the surrounding bleeding point to stop bleeding.
(3) About 400 microliters of gentamicin injection (diluted with normal saline 250:1) was sucked with a syringe, injected into the abdominal cavity, and the antibiotics exuded from the skin margin were dipped in a cotton ball. Begin suturing the abdominal wall muscles (4 stitches in a row). Draw about 400 microliters of gentamicin injection with a syringe again to flush the suture, and dipped a cotton ball to dry. Suture the skin (4 stitches with interrupted sutures). Sterilize the suture with iodophor cotton balls. The fixation was removed, and the mice were placed in a natural position and placed in a rat cage.
(4) After the injection of all cells, cut the ear marks, and record the cut ear marks and groupings.
Day5-7: Pay attention to the status of the mice, and find out in time whether the mice have died due to surgical stimulation, pancreatic damage, infection, etc.
Day 42: Cervical vertebrae were severed, the mice were sacrificed, and the cadavers were dissected to observe the growth of spleen tumors, and whether there were metastases in the liver, lungs and other organs. All suspicious tissues were marked and photographed. If there were nodules on the surface, they were counted and frozen in liquid nitrogen. Depending on the results, the samples were fixed with 10% formaldehyde solution overnight and then pathologically performed.
1. The cell state is the key to the tumorigenic experiment, so the cell growth state must be good. Take the cells in the logarithmic growth phase, and the density of the cells is about 80-90%. The fresh medium is replaced the night before the cells are collected.
2. After trypsinizing the cells, wash twice with pre-cooled PBS to remove serum from the cells.
3. Use PBS or serum-free medium to pipet the cells to a suitable concentration. Generally, the amount of cells inoculated for subcutaneous tumors is 1-5×10^6 cells/branch, and the inoculation volume is 0.1ml. Therefore, the concentration of the cell suspension is 1-5×10^7 cells/ml.
4. The cells should be inoculated into nude mice subcutaneously as soon as possible after digestion, generally within half an hour, and the cell suspension should be placed on ice on the way to reduce cell metabolism.
5. The selected nude mice are generally 5-8 weeks old and weigh about 18-20g. The implantation site is selected in areas with rich blood supply, such as the middle and rear of the armpit and the middle and upper part of the groin.
6. Before inoculation, use a gun to blow off the cell suspension sufficiently to prevent cells from agglomerating and reduce the cell survival rate.
7. When inoculating, the needle should be inserted a little deeper under the skin, about 1cm deep, to reduce the overflow of cell suspension from the needle eye after injection.
8. How to hold the nude mouse? Pinch the skin of the back of the neck of the nude mouse with the thumb and index finger of the left hand, and hold the tail of the nude mouse with the little finger.
9. About a week after inoculation, you can see lumps under the skin.
10. According to the characteristics of tumor cells and the amount of inoculated cells, the period of subcutaneous tumor formation is generally 1-2 months. Tumors should not grow too large, generally not more than 1000mm3. Too large tumor burden is often made difficult by the magazine for violating animal ethics (because this issue was tragically rejected by plos one).
11. The tumor size is generally measured twice a week, and the longest and shortest parts of the tumor are measured by vernier calipers. V=1/2*a*b2 (a is the long axis, b is the short axis).
12. After the subcutaneous tumor was removed, a part of frozen liquid nitrogen was used for protein and RNA extraction later, and a part was fixed in formalin for immunohistochemistry and immunofluorescence.
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