RU2658427C1 - Method of intravital determination of the cytoproliferative factor (aging factor) in animals - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to biochemistry. Group of inventions is described, including a method of intravital determining the level of a cytoproliferative factor (aging factor) in a mammal, excluding a person, the method of intravital determination of the level of a cytoproliferative factor in a dog, and a method of intravital determining the level of a cytoproliferative factor in a cat. In one embodiment, the method comprises the steps of: obtaining blood serum by centrifugation, dilution and heating in a water bath for 30 minutes at 60 °C, followed by repeated centrifugation; incubation of serum for 96 hours at 37 °C unspecialized syngeneic cells; addition of a mixture of versene with chymopsin; cell counting.

EFFECT: invention expands the range of methods for predicting the lifespan of mammals, in particular dogs and cats, by determining in the sera of the cytoproliferative factor using non-specialized syngeneic cells.

8 cl, 4 dwg, 6 tbl, 1 ex

Description

The invention relates to biotechnology and veterinary medicine. The invention is based on the use of non-specialized mammalian cells to determine the cytoproliferative factor (aging factor). The invention can be used to predict the life expectancy of mammals, in particular dogs and cats, by determining the serum cytoproliferative factor (aging factor - FS) using non-specialized syngeneic cells. In this case, FS in dogs is determined using MDCK cells (cells derived from a dog’s kidney), dog bone marrow stem cells, glial cells or dog neurons (primarily trypsinized or transplanted). In cats, FS is determined on cat's embryonic fibroblasts or on cells of glial origin or cat neurons (primarily trypsinized or transplanted). Cells are seeded in 24-well plates at a concentration of 50,000 cells per 1 ml in a volume of 900 μl per well and 100 μl of warmed-up serum are added to the same wells in 3 replicates. The plates are incubated for 96 hours at 37 ° C. After incubation, a supernatant was taken from each well and 200 μl of a mixture of versene with chymopsin was added to the well. After the cells creep in, add a supernatant and medium up to 1000 μl. Cell counting is performed using a Scepter ™ Millipore ™ manual automated cell counter (Germany). The aging factor is calculated by the proliferation index, i.e. by the ratio: the number of cells in the experiment / the number of cells in the control. The method allows to determine the presence of a cytoproliferative factor in the body of a mammal, in particular dogs and cats, during their lifetime.

Several years ago, a factor sharply stimulating the proliferation of glial cells in culture was found in the tissue of the brain of aging mice [1]. Such a cytoproliferative factor has been found in the serum of aging mice and aging people. It accumulates, starting from the end of the first third of the average life expectancy of animals and humans, followed by a progressive increase in its activity. As a result of the active proliferation of glial cells, two cardinal signs of the aging process of the mammalian brain are formed - the death of neurons and gliosis. The causative role of the factor in the aging process has been proved by experiments on accelerated artificial aging of young mice after the introduction of brain extracts or blood serum of old animals [3]. Under these conditions, by the 5th month of their life, mice recorded a rapid and increased accumulation of the indicated factor in the blood and the development of clinical signs of aging - lethargy, sluggish reaction to food and graying (C57Black / 6 mice). Moreover, morphometric analysis of the brain of such 5-month-old mice allowed them to detect cardinal morphological signs of aging - severe gliosis and pronounced death of neurons [4]; [5].

Therefore, the detected cytoproliferative factor was designated as an “aging factor”. It is distinguished by species specificity of action, low molecular weight (about 10 kD), resistance to elevated temperature, trypsin, ultrasound, ultraviolet and high sensitivity to protease K. A similar factor is found in the blood serum of people, starting from their 25 years of age with a subsequent increase its concentration with age [4], [5].

The wide distribution of pets among the population is accompanied by the natural desire of owners to increase the life expectancy of their pets, using various feeding schemes or commercially available products. For an objective assessment of the effectiveness of measures taken, the method of the present invention can be used.

The prior art method for intravital determination of the cytoproliferative factor in the body of aging mammals and humans, characterized in that the serum is intravitally taken, heated for 30 minutes at 90 ° C, centrifuged to remove coagulated proteins, syngeneic cells are added to the supernatant as primary or transplanted cultures and incubated for 72-96 h at 37 ° C in an atmosphere of CO ₂ followed by cell counting in the Goryaev chamber [2].

The specified method is the closest analogue of the claimed invention.

As studies have shown to determine the cytoproliferative factor (aging factor, PS), in the blood serum of dogs and cats using different cell lines, the effectiveness of determining PS in dogs and cats depends on the origin of cell cultures. Unexpectedly, it was found that PS is most effectively determined using non-specialized syngeneic cells in dogs, for example, using MDCK cells (cells derived from a dog’s kidney) or dog bone marrow stem cells, and in cats, for example, on cat’s embryonic fibroblasts , in particular on SS-81 cells.

The technical result is to increase the efficiency - the accuracy of the diagnosis of cytoproliferative factor in the body of mammals, in particular dogs and cats, during life. That is, accurately diagnose whether the longevity of the animals used for this feeding scheme or drugs increases the life expectancy of the pets.

The claimed method differs from the closest analogue using precisely not highly non-specialized cells, as well as lowering the temperature of serum heating from 90 to 60 ° C. Unexpectedly, it was found that these signs affect the accuracy of diagnosis of cytoproliferative factor.

Examples

Animals. 16 cats and 20 dogs of different ages were examined (Veterinary Clinic “Red Fox”).

Serum samples. Samples of coagulated blood of animals were delivered from the red fox veterinary clinic. Blood was centrifuged at 1500g for 15 minutes. The selected serum was diluted 10 times with medium 199 and heated in a water bath for 30 minutes at 60 ° C. After centrifugation at 1500 g for 15 minutes, the supernatant was stored at -20 ° C.

Cell culture. We used cell cultures: dogs MDCK (dog kidney) and Cf2Th (dog thymus), as well as cats CC81 (cat embryonic fibroblasts) and CRFK (cat kidney) obtained from the Museum of Cell Culture of the Institute of Virology named after DI. Ivanovo Federal State Budgetary Institution “National Research Center for Epidemiology and Microbiology named after N.F. Gamalei "Ministry of Health of the Russian Federation. In addition, we used dog stem cells obtained from the Department of Immunology, Federal State Budget Scientific Research Center named after N.F. Gamalei "from the doctor of medical sciences R.K. Chaylakhyana.

Nutrient media and solutions. Cells were cultured in a nutrient medium Needle MEM (manufactured by PIPVE named after MP Chumakov, Russia) with the addition of fetal calf serum (Gibco, South America). The same medium was used to dilute the serum. For the treatment of cells, a 0.02% versene solution (SSC VB Vektor, Russia) and a chymopsin solution at the rate of 0.1 mg / ml (Samson-Med LLC, Russia) were used.

Determination of the aging factor. Cells were scattered into 24-well plates at a concentration of 50,000 cells per 1 ml in a volume of 900 μl per well, and 100 μl of warmed-up serum was added to the same wells in 3 replicates. The plates were incubated 96 hours at 37 ° C. After incubation, a supernatant was taken from each well and 200 μl of a mixture of versene with chymopsin was added to the well. After the creeping of the cells, the supernatant and medium up to 1000 μl were added. Cell counting was performed using a Scepter ™ Millipore ™ manual automated cell counter (Germany).

The aging factor was calculated by the proliferation index, i.e. by the ratio: the number of cells in the experiment / the number of cells in the control [3].

RESULTS

In the first series, 20 sera of dogs of different ages and different breeds were used (Table 1).

The results of determining the aging factor of animals using dog kidney cell culture (MDCK) are separately presented in FIG. 1. As can be seen from the data of FIG. 1, in most animals under 6 years of age, it is not possible to detect the aging factor, while it is determined in older animals. However, in young animals, we observed 2 exceptions (Fig. 1), which may indicate either accelerated aging of these individuals, or, conversely, a particularly intensive accumulation of the aging factor in dogs of this breed, in which even at a young age the level of aging factor is somewhat elevated, while in old age it can be even higher. Of the 10 individuals of the older age group, 8 - the level of the aging factor was significantly increased, however, in two animals - 8 and 12 years old - the aging factor could not be detected (Fig. 1).

Tests of the sera of the same group of dogs on a culture of dog thymus cells (Cf2Th) were practically ineffective (Table 2).

In the third part of this section, blood serum of dogs was examined using a dog stem cell culture. For this purpose, 8 of the remaining set of sera were used (Table 3).

Five animals were older than 6 years old and 3 were younger than this age. It is characteristic that the indicators of the aging factor in 5 animals and when using a culture of MDCK cells, and when using a culture of stem cells almost coincided (table. 4).

At the same time, in the group of dogs older than 6 years old, indices lower than those in the control were also recorded (Fig. 2).

Also, 16 blood serums of cats of different ages and different breeds were tested. The results of determining the aging factor of animals using a culture of fibroblasts of a cat embryo (CC81) are presented in table. 5.

As can be seen from the data in table 5, in 8 cats of the breed "British" the aging factor was not determined regardless of their age. At the same time, in the remaining 8 cats of the breed Métis, Scottish fold, Sacred Burma and Bengal, the accumulation of the aging factor turned out to be characteristic of mammals depending on age, i.e. with a gradual increase in activity at an early age, followed by a decrease in activity when animals enter a more mature age (Fig. 3).

Somewhat similar results were obtained when examining feline sera using feline kidney cell cultures (CRFK) (Fig. 4, Table 6).

As can be seen from the data of FIG. 4 and tab. 6, in animals of the British breed, as well as when using a CC81 cell culture, the aging factor was not determined regardless of the age of the cats. At the same time, in cats of other breeds (“mixed breed”, “cattle fold”, “sacred burma” and “bengal”) aged 1.5 to 5 years, the aging factor was determined.

Analysis of the results of determining the aging factor in dogs with a high probability indicates a certain pattern associated with the appearance of the factor in the blood serum of animals after 6 years of age (Figs. 1 and 2). In this case, of course, one cannot fail to note isolated cases of determining increased values of the factor in animals at a young age, which was observed in 15% of animals using a MDCK cell culture and in 12.5% of animals using a stem cell culture.

Previously, the results of determining the aging factor in mice showed that cultures representing embryonic fibroblasts of the same animal are quite effective (Zuev et al., 2000 [1]). That is why in the presented results the most demonstrative dynamics of the accumulation of the aging factor in serum of cats of different ages is presented (although using an example of a small sample) when using a culture of CC81 cells - embryonic cat fibroblasts (Fig. 3). The use of cultures of highly specialized cells does not allow us to count on a positive result, as, for example, we observed when using a CRFK cell culture - a cat's kidney, or a Cf2Th cell culture - a dog thymus.

Thus, despite the fact that the breed of animals plays a role, however, the claimed method is applicable to most breeds for reliable in vivo determination of the level of cytoproliferative factor (aging factor) in the body.

Brief Description of the Drawings

FIG. 1. Determination of cytoproliferative factor in serum of dogs using a cell culture MDCK

FIG. 2. Determination of the cytoproliferative factor in serum of dogs using a culture of stem cells of bone marrow of dogs

FIG. 3. Determination of cytoproliferative factor in serum of cats using a cell culture CC-81

FIG. 4. Determination of cytoproliferative factor in serum of cats using CRFK cell culture

Note to FIG. 1, 2, 3, 4: the abscissa axis is the age of the animals (year), the ordinate axis is the cytoproliferative factor (U)

Bibliography

1. Zuev V.A., Viktorov I.V., Borodina N.P., Ignatova N.G., Bykovskaya S.I., Khalansky A.S. - The accumulation in an aging brain of mammals of a factor that dramatically stimulates the proliferative activity of glia // Bull. an expert. Biol and honey., 2000, t. 129, No. 3, p. 317-320.

2. Zuev V.A., Ignatova N.G. - A method for intravital determination of the cytoproliferative factor in the body of aging mammals and humans // Patent No. 2204135 of 05/10/2003.

3. Zuev V.A., Avtandilov G.G., Ignatova N.G., Bykovskaya S.I. - Artificial aging of mice // Bull. an expert. biol. and honey., 2003, t. 135, No. 9, p. 325-327.

4. Zuev V.A., Ignatova N.G., Avtandilov G.G. - Accumulation of the aging factor in mammals, including humans // Advances in gerontology - 2005, no. 17, p. 108-116.

5. Zuev V.A., Mezentseva M.V., Schaposchnikova G.M. - Apotential new biological marker of the biological age // Frontiers in Genetics, 2013, vol. 4, P. 1-3.05.

Claims (20)

1. The method of intravital determination of the level of cytoproliferative factor (aging factor) in a mammal, excluding humans, including: - obtaining blood serum, for which the blood is centrifuged, then the selected serum is diluted and heated in a water bath for 30 minutes at 60 ° C, then re-centrifuged; - the use of the obtained serum for incubation for 96 hours at 37 ° C non-specialized syngeneic cells; - adding a mixture of versene with chymopsin; - implementation after the sliding of cells of their counting, while the cytoproliferative factor (aging factor) is calculated by the proliferation index, i.e. according to the ratio of the number of cells in the experiment / the number of cells in the control, where an index of more than one indicates the presence of a cytoproliferative factor (aging factor), while the higher the index, the more cytoproliferative factor (aging factor) is present in the body. 2. A method for intravital determination of the level of cytoproliferative factor in dogs, including: - obtaining blood serum, for which the blood is centrifuged, then the selected serum is diluted and heated in a water bath for 30 minutes at 60 ° C, then re-centrifuged; - the use of the obtained serum for incubation for 24 hours to 8 days at 37 ° C of the MDCK cell culture (cells obtained from the dog’s kidney), dog bone marrow stem cells, glial cells or dog neurons (primarily trypsinized or transplanted); - adding a mixture of versene with chymopsin; - implementation after the sliding of the cells of their counting, while the cytoproliferative factor is calculated by the proliferation index, i.e. by the ratio of the number of cells in the experiment / the number of cells in the control, where an index of more than one indicates the presence of a cytoproliferative factor (aging factor), and the higher the index, the more cytoproliferative factor is present in the body. 3. A method for intravital determination of the level of cytoproliferative factor (aging factor) in cats, including: - obtaining blood serum, for which the blood is centrifuged, then the selected serum is diluted and heated in a water bath for 30 minutes at 60 ° C, then re-centrifuged; - the use of the obtained serum for incubation for 24 hours to 8 days at 37 ° C of a culture of cat embryonic fibroblast cells, glial cells or cat neurons (primarily trypsinized or transplanted); - adding a mixture of versene with chymopsin; - implementation after the sliding of the cells of their counting, while the cytoproliferative factor (aging factor) is calculated by the proliferation index, i.e. according to the ratio of the number of cells in the experiment / the number of cells in the control, where an index of more than one indicates the presence of a cytoproliferative factor, and the higher the index, the more cytoproliferative factor (aging factor) is present in the body. 4. The method according to PP. 1-3, where the serum is diluted preferably 10 times with medium 199. 5. The method according to PP. 1-3, where the serum is obtained by centrifugation at 1500g for 15 minutes clotted blood. 6. The method according to PP. 1-3, where re-centrifugation is carried out at 1500g for 15 minutes. 7. The method according to PP. 1-3, where a 0.02% versene solution is used. 8. The method according to PP. 1-3, where the cells are seeded into 24-well plates at a concentration of 50,000 cells per 1 ml in a volume of 900 μl per well and 100 μl of heated serum are added to the same wells in 3 repetitions, and after incubation, supernatant is taken from each well and 200 μl of a mixture of versene with chymopsin is added to the well; after creeping of the cells, the supernatant and medium up to 1000 μl are added.

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