Method for preparing interleukin-8 from donor blood neutrophiles

FIELD: medicine, immunology, cytokines.

SUBSTANCE: method for preparing interleukin-8 (L-8) from donor blood neutrophiles involves isolating neutrophiles from peripheral blood in double gradient ficol-verografin, bringing about the neutrophile content in cell suspension to the concentration 5 x 106 cells/ml, activation at temperature 37°C for 1 h followed by centrifugation and separation of supernatant containing the end product. For activation the cell suspension of neutrophiles in the concentration 5 x 106/ml of Henx's solution is poured in plastic Petri dishes. Invention provides the development of a simple and available method providing the high level of interleukin-8 (IL-8) from neutrophiles of donor blood not requiring using expensive preparations.

EFFECT: improved preparing method.

1 tbl

 

The invention relates to medicine, namely to immunology.

Immunocytokine are in the focus of modern immunology. One of the most important cytokines inflammation believe interleukin-8 (IL-8)pertaining to α-the superfamily of chemokines that regulate the activation and motility of cells in the inflammatory focus (Simbirtsev A.S., 1999; Smirnov, V.S., Freidlin, I.S., 2000). The main cells producing IL-8 are monocytes/macrophages and endothelial cells, but it can be produced and many other types of cells: lymphocytes, neutrophilic granulocytes, various epithelial cells, fibroblasts, hepatocytes and other (cetlinski S.A., Kalinin N.M., 1998; Simbirtsev A.S., 1999; Smirnov, V.S., Freidlin, I.S., 2000; Cassatella, M., Bazzoni f, Ceska, M., 1992). Production of IL-8 starts in response to the activation of cells of various biologically active substances, including components of the cell wall of bacteria, viruses, and many cytokines, lectins and other (cetlinski S.A., Kalinin N.M., 1998; Simbirtsev A.S., 1999; Smirnov, V.S., Freidlin, I.S., 2000).

Development of methods for" cytokines including IL-8 is the actual problem in immunology.

A known method for production of IL-8 from peripheral blood leukocytes. It includes a five-fold dilution medium Needle fresh heparinised blood, preparation of solution range of complete the ora of synthesis and secretion of IL-8 (prodigiozana). In a 96-well plate for culturing cells contribute 100 ál of working solution prodigiozana add to the wells, 100 ál of prepared peripheral blood and cultured in the CO2-the incubator for 24 hours at a temperature of 37°With, then carefully gather supernatant and investigated for the presence of IL-8 (cetlinski S.A., Kalinin NM Immunology for the doctor. - St. Petersburg: Hippocrates, 1998. - S-121).

The disadvantage of the described method for production of IL-8 is the use of different populations of cells, secreting cytokines with divergent spectrum of activity that contributes to the inhibition of the activity of the cells and reduce the secretion of cytokines. This method requires the use of expensive drugs and duration takes a lot of time.

The closest to the essential features of the claimed invention is a method for the secretory products of neutrophils (Dolgushin I.I., Bukharin O.V. Neutrophils and homeostasis, Ekaterinburg, 2001. - P.75), consisting in the allocation of leukocyte suspensions from heparinised peripheral blood donors by sedimentation of red blood cells by adding 10% solution of gelatin in a ratio of 10:1 at a temperature of 37°C for 30 minutes, the allocation of neutrophils from the leukocyte suspension on the double gradient is lotnosti ficoll-urografin, bringing neutrophil concentrations up to 5×10 cells/ml of medium 199 with subsequent activation of particles of monodisperse polystyrene latex with a diameter of 1.7 μm, 50 particles on one neutrophil. Then cells and latex particles were removed by centrifugation at a speed of 3000 rpm for 15 minutes and then filtering through Millipore filters with a pore size of 0.24 μm ("Millipore, USA).

The prototype of the invention has the following essential features: to highlight cytokines use the neutrophils of blood donors, which are isolated from the leukocyte suspension, bring neutrophils to a certain concentration and activate these cells.

The disadvantages of the prototype are:

1. Low secretion of IL-8;

2. The use of expensive drugs (particles of monodisperse polystyrene latex, milliprobe filters).

The claimed invention is directed to solution of the problem consisting in the development of a method for production of IL-8 from neutrophils of blood donors.

The solution of this problem allows us to develop a new simple and accessible way, providing a high level of secretion of IL-8 by neutrophils of blood donors that do not require the use of expensive drugs.

To achieve this result the invention "Method of production of interleukin-8 from neutrophils is rovi donors" includes the use of peripheral blood leukocytes donors, the selection of neutrophils from the leukocyte suspension, breeding them up to a certain concentration and activation of these cells. While neutrophils activate adhesion on polystyrene plastic for 1 hour at a temperature of 37°in Hanks solution without subsequent filtration supernatant through Millipore filters.

In relation to the prototype of the claimed invention has the following distinctive features: activation of neutrophils carry out their adhesion to polystyrene plastic for 1 hour at a temperature of 37°in Hanks solution that provides a high level of secretion of IL-8 and is economically advantageous because it eliminates the use of expensive drugs.

Between the features and the technical result there is a causal relationship: the method provides a high level of secretion of preformed IL-8 and is cost-effective.

For the method of obtaining the IL-8 is used by neutrophils in blood donors, which can activate their adhesion to polystyrene plastic for 1 hour at a temperature of 37°in Hanks solution that provides a high level of secretion of the cytokine in a short period of time without the use of expensive drugs, which is economically advantageous.

According to the author is in the information set of essential features, characterizing the essence of the claimed invention, is not known from the prior art, which allows to make a conclusion about conformity of the invention, the criterion of "novelty".

According to the authors of the essence of the invention is not necessary for the expert in the obvious way from the prior art, since it cannot be detected by the above-mentioned influence on the resulting technical result

- a new property object a set of attributes that differ from the prototype of the claimed invention, which allows to make a conclusion about its compliance with the criterion of "inventive step",

The set of essential features that characterize the invention, in principle, can be repeatedly used in medicine to obtain a technical result, which consists in receiving IL-8 from neutrophils of blood donors, which allows to make a conclusion about conformity of the invention, the criterion of "industrial applicability".

This method is as follows.

Donors make a fence of 20.0 ml of heparinized peripheral venous blood, which is the purpose of sedimentation of red blood cells and obtain a leukocyte suspension defend with the addition of a 10% solution of gelatin in a ratio of 10:1 at a temperature of 37°C for 30 minutes. Selected leukocyte suspension washed once in Hanks solution, centrif is the response at 1500 rpm for 10 minutes. Neutrophils isolated from the leukocyte suspension on a double density gradient sterile solutions ficoll-urografin (Pharmacia, Sweden; Spofa, CSSR). The density of the upper layer of the gradient is 1,075-1,077, and the lower - 1,093-1,095. The volume of each layer of the gradient is equal to 1,5 ml After 30 minutes centrifugation at 1500 rpm at the boundary between the plasma and the upper layer of the gradient is formed a ring, consisting mainly of mononuclear cells (lymphocytes - 45-50%, monocytes - 15-20%, granulocytes 10-15%). In the interphase between two layers of density gradients is a layer of granulocytes with a purity of 96-98%, 2-4% of mononuclear cells. Cells are gently harvested, transferred into sterile centrifuge tubes, washed three times with sterile Hanks solution and diluted with the same solution to a concentration of 5×106cells/ml Poured into 1.0 ml of cell suspension in sterile plastic Petri dishes with a diameter of 40 mm Cultured for 1 hour at a temperature of 37°C. Merge adosados. Neadgezivnye cells are removed from the supernatant by centrifugation at 3000 rpm for 10 minutes. In the selected supernatant of neutrophils donors determine the content of Pro-inflammatory cytokines: IL-1α, IL-1β, IL-8, TNF-α. For this purpose use the appropriate test systems LLP "Cytokine" (Saint-Petersburg). These test systems are based on the "sandwich"method is e solid-phase ELISA using horseradish peroxidase as indicator enzyme (cetlinski S. p., Kalinin K.M. Immunology for the doctor. - St. Petersburg: Hippocrates, 1998. - 156 S.).

To determine the effectiveness of the proposed method for production of IL-8 was used supernatant unactivated and activated particles monodisperse polystyrene latex with a diameter of 1.7 μm (obtained from the research Institute of synthetic rubber, Saint-Petersburg) neutrophils blood donors. Cultivation of neutrophils without inductor secreting cells and in his presence were carried out in the same mode, i.e. at a temperature of 37°C for 1 hour in Hanks solution.

The results showed that activated adhesion on plastic neutrophils blood donors significantly more compared with nonactivated and activated latex neutrophils secrete preformed Pro-inflammatory cytokines: IL-1β, IL-8, TNF-α. Probably the adhesion of neutrophils to plastic is the most annoying factor that leads to significantly more pronounced secretion of preformed cytokines compared with nonactivated and activated latex neutrophils blood donors. Most high level secretion was noted in relation to IL-8 (table 1).

Table 1

The content of cytokines in supernatant unactivated and activated n is Trofimov blood donors (M± m).
IL-1α (PG/ml)IL-1β (PG/ml)IL-8 (PG/ml)TNF-α (PG/ml)
Supernatant unactivated neutrophils donors(n=10)51,3±3,512,6±1,448,8±and 23.44,6±0,27
Supernatant activated latex particles neutrophil donors (n=10)53,5±6,124,8±14,1101,7±42,36,2±0,85
Supernatant activated adhesion on plastic neutrophil donors (n=10)63,1±5,363,5±16,6*, **889,5±28,3*, **to 163.1±43,3*, **
Note: * - reliability of differences of performance in relation to the group "Supernatant unactivated neutrophils donors";
** - reliability of differences of performance in relation to the group "Supernatant activated latex particles neutrophil donors." Used the Wilcoxon criterion.

Presents tabular data suggests that the proposed "Method of production of interleukin-8 from neutrophils of blood donors" is the most effective in comparison with used in modern immunology.

The method receiving the Oia interleukin-8 from neutrophils of blood donors, including the allocation of neutrophils from the peripheral blood of donors on the double gradient ficoll-urografin, bringing cell suspension of neutrophils to a concentration of 5·106cells/ml, activating at 37°C for 1 h followed by centrifugation and separation of the supernatant containing the target product, wherein to activate cell suspension of neutrophils at a concentration of 5·106cells/ml Hanks solution is poured into plastic Petri dishes.



 

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