RU2388064C1 - Method for simulation of malignant process in experiment - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention concerns experimental medicine, namely experimental oncology and can be used for creating a malignant tumour growing in a pulmonary tissue. Therefore, for 14-21th day following transplantation of sarcomas C-45 in outbread male albino rats, a tumour of one animal is removed with a sterile appliance; connective tissue capsule is separated, a piece of a viable greyish-pink tumour tissue is selected and transferred to a sterile Petri dish. Then it is washed with a physiologic saline and transferred in a sterile homogeniser; the tumour is crushed. The prepared tumour pulp is dissolved in sterile physiologic saline in the volume ratio 1:5 and filled in a sterile syringe. The outbread male albino rats are fixed laying on back to introduce the tumour slurry into the subclavian vein in a dose 0.5 ml with observing the aseptic procedure.

EFFECT: method provides 100 % reproducibility, ensuring engrafting of tumour transplants in all cases, and also possibility of mass experiments for relatively short time and in a greater amount with simplicity of implementation.

Description

The invention relates to experimental medicine, namely to experimental oncology, and can be used to obtain a malignant tumor growing in lung tissue.

The development of tumor transplantation techniques marked the beginning of experimental oncology, since the information obtained in the study of transplanted tumors played an extremely important role in the development of modern ideas about the nature of malignant growth and methods of antitumor therapy. In 1921, Fleten, changing the traditional grafting under the skin, transplanted experimental tumors into the rat brain, which served as the beginning of a new direction in the study of the clinic, the pathomorphological picture and the effect of antitumor effects on developing neoplasms.

It is known that transplantable tumors, despite their greater remoteness from human tumors than spontaneous or induced, have several advantages. This is, firstly, the possibility of staging mass experiments in a relatively short time, since they can quickly be obtained in large quantities. Another advantage of transplantable tumor strains is the relative constancy of their structure and biological properties. These advantages make them an indispensable tool in the field of experimental oncology, especially during the initial selection of anticancer drugs (Konoplev VP. Tumors to be transplanted. // in the book “Models and methods of experimental oncology (practical guide).” Ed. A.D. Timofeevsky, M., 1960 .-- S. 144-162). One of the convenient models for biochemical, immunological and pharmacological studies is considered to be S-45 sarcoma. This sarcoma is transplanted to outbred rats in almost 100% of cases. Spontaneous resorption of tumors reaching 1-1.5 cm in diameter is noted in less than 1% of cases. The tumor has a moderate growth rate. On days 5-8, its diameter reaches 8-15 mm, and on days 21-25, 40-60 mm. Animals die, as a rule, on days 25-30 with an average tumor weight of 37-38 g. The sarcoma grows evenly, seasonal fluctuations in transplantability and growth rate are small.

However, this strain of the tumor is still transplanted and grows in the subcutaneous tissue, and all these positive points have not been used to attempt to reproduce C-45 in lung tissue.

There is a method of transplanting a tumor into the lung tissue of a mouse by introducing a suspension of tumor tissue into the nose ("Reproduction of diseases in animals for experimental and therapeutic research." Edited by N. Lazarev, "Medgiz", Leningrad Branch, 1954. - P.90 -92), we have chosen as a prototype. The technique of transplantation is as follows. Mice undergo light ether anesthesia, for which they are placed in a glass jar closed with a lid with a cotton ball placed on the bottom with ether. As soon as the mouse is in a prone position, it is removed and a thin elongated Pasteur pipette connected to a soft rubber tube with a mouthpiece or a tuberculin syringe is applied to the tip of the mouse’s nose, the ascitic fluid obtained from Ehrlich carcinoma of the mice, so that both nostrils are closed . Usually 10 drops are applied one after the other (only about 0.05 ml), as they are drawn into the nose of the mouse. With proper anesthesia, the percentage of successful inoculations is quite high - more than 90%. Macroscopically noticeable tumor nodules are detected on days 12-15, and significant tumors on days 20-25. Soon, all the mice die.

However, as the authors themselves indicate, the success of the inoculation depends on the proper anesthesia of the mice. With insufficient anesthesia, the mice quickly wake up, stop drawing the applied liquid into the airways and begin to sneeze, which removes the applied suspension from the external airways. If the anesthesia is too deep, the mice poorly absorb the fluid and, often, a significant part of the ascitic fluid enters the esophagus. In addition, with too deep anesthesia, many mice die soon after inoculation. The second point that makes this method of reproducing a transplantable lung tumor practically unacceptable at present is the use of anesthetized medical air, which is included in the list of drugs that are specially registered. And finally, the only animal that can reproduce malignant lung tumors that are adequate to those in humans is a rat. This is due to the fact that lungs of rats are more similar to human lungs than in mice. In rats, in particular as in humans, there are mucous glands in the bronchi that are absent in mice.

The aim of the invention is to simplify the method of obtaining an inoculant tumor growing in rat lung tissue.

This goal is achieved in that on days 14-21 from the moment of traditional transplantation of S-45 sarcoma to males of white outbred rats, one animal is killed, a tumor is removed with boxing tools, the connective tissue capsule is removed, a piece of viable tissue with a grayish-pink color is selected, and transferred to a sterile Petri dish. A piece is rinsed two to three times with saline and transferred to a sterile homogenizer. The tumor is ground, the resulting tumor slurry is diluted with sterile saline in a ratio of 1: 5 by volume and collected in a sterile syringe. Suspension tumors are administered to male white outbred rats fixed on the back, following the rules of asepsis in the subclavian vein at a dose of 0.5 ml.

The invention "A method for reproducing a malignant process in an experiment" is new, since it is unknown from the level of medicine, namely in experimental oncology, when a malignant tumor grows in the lung tissue.

Analysis of known methods for reproducing an experimental malignant process in the lungs gives reason to talk about the novelty of the proposed method, which is represented by morphologically confirmed sarcoma and 100% reproducibility.

The novelty of the invention lies in the fact that, in contrast to the prototype, the proposed “Method for reproducing a malignant process in an experiment” is carried out without prior anesthesia of animals, is performed using white outbred rats and obeys the requirements for experimental tumors, one of which postulates that tumor transplants should interbreed in 100% of cases, grow and lead animals to death.

The developed method has an inventive step, as it obviously does not follow for a specialist from the known level of development of experimental oncology and the reproduction of transplantable malignant tumors in rat lungs. In well-known sources of information in Russia, the CIS countries and abroad, a similar method was not found.

The invention is industrially applicable, since it can be repeatedly reproduced in research institutions of oncological profile with vivariums.

The method is as follows.

Tailoring is made in boxing. Tools, dishes, hands are sanitized in the usual way. The assistant slaughter an animal with S-45 sarcoma, which has traditionally grown by decapitation, fixes it, lubricates the skin over the tumor and the adjacent area with 70% ethanol with a small amount of iodine tincture.

The experimenter using sterile instruments, 1-2 cm away from the tumor, performs an incision almost the entire length of the animal, and separates the skin. The edge of the skin flap must be turned out so that the hair, which is poorly disinfected, does not fall on the tumor. To do this, the edge of the skin flap is captured with 2-3 Kocher clamps. A tumor is incised in the longitudinal direction, from its peripheral part facing the muscles of the abdominal wall (and not to the skin), a piece of viable tissue with a grayish-pink color is cut out and transferred to a sterile Petri dish. The connective tissue capsule is separated with surgical tweezers in situ. A piece of the tumor is rinsed with a sterile surgical solution and transferred to another Petri dish. This procedure is repeated 2-3 times to wash away the remnants of blood and microscopic lesions of necrosis. The tumor is transferred to a sterile homogenizer and crushed, the resulting tumor slurry is diluted with sterile saline in a ratio of 1: 5 by volume and put into a sterile syringe, homogenized, twice passed through a press and nylon, diluted with sterile saline in a ratio of 1: 5, and cell viability is checked. with trypan blue.

The rat being grafted is fixed in the supine position, the supraclavicular region is lubricated with 70% alcohol with a small iodine solution, then under sterile conditions a suspension of tumor cells is injected into the subclavian vein in a volume of 0.5 ml of suspension containing 2 million tumor cells. The puncture site is tightly clamped with a cotton swab with alcohol and held for 1 minute.

The procedure was performed on 350 animals that died within 35-42 days from the moment of inoculation.

After opening the animals, in all cases, multifocal tumor growth was detected in the lungs.

A microscopic examination conducted weekly until the death of the animals revealed the following. In 1-2 weeks: the blood vessels in the lungs significantly increase blood pressure, mechanical stretching of the airways, especially the alveoli. The development of interstitial and intraolveolar edema, the development of dystrophy of alveolocytes and endotheliocytes of blood capillaries is observed. Those. reduction of blood flow and hemorrhage into the lung tissue can also be considered one of the triggers for the development of a malignant tumor in this organ. The second important point is the development of small and large distelectases and peribronchial formation of lymphocytic infiltrates. By the end of the second week, in some places, proliferation of the alveolar epithelium can be seen. At the same time, alol-like and glandular structures, false bronchi, strands of epithelial cells appear in the connective tissue, which do not differentiate into new alveoli and do not provide their functions, but remain in the connective tissue, which takes the form of an “aerated scar”. Against such a structural and metabolic background, starting from 3 weeks from the moment of inoculation, the development of a true tumor process is observed in the form of formation of tumor foci along the periphery and in the center of the lungs. The cells have a clear fusiform shape. Such active seizure of the lung territory is observed at this time more often in the lower, less often in the upper lobes of the lungs. Tumor invasion is found not only in the interalveolar septum, but also in the main respiratory structures, including bronchioles. In some areas, hyperplasia of the connective tissue, which forms a multinuclear layer of fibroblasts and histiocytes, is noted; this is probably the process of building tumor cells of their own stroma due to the material of the host organ. At the terminal stage (5-6 weeks), a total tumor of the lungs is observed. In the central part of the lung, diffuse growth with foci of necrosis. In addition to the traditional fusiform shape, the tumor cells are round and oval in shape with various figures of pathological mitoses.

The technical and economic efficiency of the “Method for reproducing a malignant process in an experiment” lies in the fact that tumor grafts are transplanted in 100% of cases, provide 100% reproducibility, grow and lead animals to death. A simplified method for producing a transplantable tumor growing in rat lung tissue. The use of white outbred rats without prior anesthesia has several advantages: the possibility of staging mass experiments in a relatively short time and in large numbers, the similarity of rat lungs to human lungs, the relative constancy of the structure and biological properties of tumors.

Claims (1)

A method of reproducing a malignant process in the lungs in an experiment, including transplanting a tumor, characterized in that on the 14-21 day from the moment of transplantation of S-45 sarcoma to male white outbred rats, one animal’s tumor is removed with sterile instruments, the connective tissue capsule is separated, a piece of viable tumor tissue is selected grayish-pink in color and transferred to a sterile Petri dish, rinsed with saline and transferred to a sterile homogenizer, the tumor is crushed, the resulting tumor porridge Itza is diluted with sterile saline in a ratio of 1: 5 by volume and is taken into a sterile syringe, male white outbred rats are fixed on the back and tumor suspension is introduced in compliance with aseptic rules in the subclavian vein at a dose of 0.5 ml.