RU2607041C2 - Method for evaluating pyorrhoea on paradontium based on level of human interleukin-8 (il-8) gene mrna - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, dentistry, molecular biology and clinical-laboratory diagnostics. Described is a method of determining intensity of suppuration (pyorrhoea) of periodontal tissues. Method is based on measuring in scrapes from periodontium level interleukin-8 (IL-8) gene RNA relative to representation of reference RNA or DNA. Obtained parameters are used to calculate ratio of levels for each specific sample. Value of obtained parameter is interpreted as a marker of suppuration. Invention can be used to determine pyorrhoea in individual patients with aggressive and chronic paradontium, including after loss of tooth, in cases when intensity of said process does not allow visual detection of pus, as well as in tissues in contact with implants and other orthodontic structures.

EFFECT: method provides highly sensitive and objective quantitative characteristic of degree of suppuration of analysed area of periodontium, which enables to evaluate risk of acute local inflammation and periodontium destruction in future, establish implants and other dental structures, evaluate effectiveness of therapeutic measures.

3 cl, 4 tbl

Description

FIELD OF THE INVENTION

The invention relates to the field of medicine, dentistry, molecular biology and clinical laboratory diagnostics and can be used to determine the severity of suppuration (suppuration) of periodontal tissues by the presence of interleukin-8 (IL-8) RNA in the scraping from the periodontal pocket. The invention can be used to identify the degree of suppuration in patients with various forms of periodontitis (including aggressive and chronic), including after tooth loss, in cases where the intensity of this process does not allow visual detection of pus, as well as in tissues in contact with implants and other orthodontic constructions. Using the method provides a highly sensitive and objective quantitative characteristic of the degree of suppuration of the studied periodontal area, which allows us to assess the risk of acute local inflammation and destruction of the periodontium in the future, the survival rate of implants and other orthodontic structures, and evaluate the effectiveness of treatment measures.

State of the art

The data of epidemiological studies indicate that the prevalence of inflammatory periodontal diseases in the adult population is up to 90% [Grudyanov A.I. et al., 1998; Dmitrieva L.A., 2001]. Against such a high background, there is a tendency toward an additional increase in the prevalence of atypical forms of periodontal disease, which include aggressive forms of periodontitis.

Most authors consider infection of the periodontal surface with pathogenic bacteria as the main cause of chronic periodontitis [Page R.C., 1985, 1991; Tanner A.C.R. et al., 2005]. At least 700 species of microorganisms have been identified in the oral cavity, however, the majority of authors name true periodontopathogens only Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythensis, Treponema denticola and Candida albicans [Finoti L.S. et al., 2013]. As a result of the reaction of the body to a bacterial infection, tissue destruction occurs, accompanied by progressive destruction of the bone of the alveolar process, and, ultimately, tooth loss [Dong Y. et al., 2015].

The occurrence and course of any disease of an infectious nature is determined by the strength of the body's response [Dosseva-Panova V.T. et al., 2014]. The nature of this response depends on the antigenic specificity of previously acquired immunity and is largely determined by the human genotype [Gilowski L. et al., 2012]. However, only part of the observed reactions of the body to infection can be considered protective [Gemmell E. et al., 2007]. In addition, neutral reactions often arise - useless from the point of view of protecting the body, but not self-absorbing immune system resources [Graswinckel J.E.M. et al., 2004], as well as autotoxic reactions that cause damage and death of their own cells and tissues. Autotoxic reactions often determine the pathophysiological picture of the disease as a whole. In addition, when considering bacterial infections that occur on the human mucous membranes, which include periodontal disease, it is necessary to take into account the symbiotic and antagonistic relationships between bacteria.

A. et al., 2007]. Особенно существенно - в сотни и тысячи раз, при этом возрастает активность нейтрофильной коллагеназы ММР8 и желатиназы ММР9: ферментов, участвующих в конечных стадиях деградации коллагена [Javed F. et al., 2014]. На этом основании большинство исследователей делают вывод об аутопротеолитической природе деградации соединительнотканного матрикса пародонта, лежащего в основе пародонтита [Jayaprakash К. et al., 2014]. При этом роль бактериальных протеиназ в качестве разрушителей коллагена и других компонентов матрикса рассматривается как сигнальная, хотя иногда ей также приписывается существенное значение. Однако этой точке зрения во многом противоречит тот факт, что подъем активности коллагеназ, непосредственно способных атаковать нативный коллаген, например ММР1 и ММР13, ни в одном исследовании не удалось связать с поражением пародонта [Kinney J.S., 2014]. Между тем, активация одних только терминальных матриксинов, даже достигающая нескольких порядков по сравнению с базовым уровнем, не способна влиять на целостность коллагеновой основы пародонтальной связки.Studies in the etiology of both aggressive and chronic periodontitis have led to the discovery of a sharp increase in the activity of matrixins (matrix proteinases synthesized, in particular, periodontal fibroblasts and macrophages) in the event of periodontal inflammation of any nature [

A. et al., 2007]. Especially significant - hundreds and thousands of times, while the activity of neutrophilic collagenase MMP8 and gelatinase MMP9: enzymes involved in the final stages of collagen degradation increases [Javed F. et al., 2014]. On this basis, most researchers conclude about the autoproteolytic nature of the degradation of the connective tissue periodontal matrix underlying periodontitis [Jayaprakash K. et al., 2014]. At the same time, the role of bacterial proteinases as destroyers of collagen and other matrix components is considered as a signal, although sometimes significant significance is also attributed to it. However, this point of view is largely contradicted by the fact that an increase in the activity of collagenases directly capable of attacking native collagen, such as MMP1 and MMP13, could not be associated with periodontal lesions [Kinney JS, 2014]. Meanwhile, the activation of terminal matrixins alone, even reaching several orders of magnitude compared to the base level, is not able to affect the integrity of the collagen base of the periodontal ligament.

О. et al., 2014].When considering the effects of MMP8 and MMP9 on the degradation of the periodontal matrix, an important issue is the regulatory mechanism for increasing their activity [Moutsopoulos NM et al., 2015]. Studies performed by real-time PCR and enzyme-linked immunosorbent assay show a significant increase in the levels of MMP8 and MMP9 mRNA [Noack B. et al., 2009], as well as the corresponding protein products [Ram VS et al., 2015]. However, these indicators only indirectly reflect the level of activity of the corresponding enzymes [Salminen A. et al., 2014]. The fact is that most of the matrixins of any normal tissue are in the form of precursors lacking enzymatic activity. The activation of the precursors occurs according to the cascade mechanism, and the activation of terminal MMPs (gelatinases and stromelysins) is achieved mainly due to the proteolytic activity of the matrixins located at the beginning of the signaling pathway. Thus, the hyperproduction of the precursors of MMP8 and MMP9, which undoubtedly occurs during periodontal inflammation, is not a sufficient condition for increasing the activity of these proteases: this also requires the preliminary activation of MMP1, MMP2, and MMP3 [Trivedi S. et al., 2015 ]. Moreover, the presence in the tissues of MMP inhibitors - TIMP1, TIMP2, TIMP3 and TIMP4, the total concentration of which is several times higher than the concentration of any MMP, can completely suppress the activity of even those MMP molecules that have undergone proteolytic activation and do not have a propeptide (as a rule , the propeptide exhibits high inhibitory activity against its own MMP) [

O. et al., 2014].

But the published experimental data do not provide any evidence that such processes occur with periodontal inflammation. In this case, however, it must be borne in mind that the PCR-RV, ELISA, and FA methods used in modern studies practically do not allow one to distinguish the active form of MMP from its predecessor [Schaumann T. et al., 2013]. This task is all the more difficult in relation to primary MMP (MMP1, MMP2 and MMP3), the concentration of which in tissues is significantly lower than terminal MMP (MMP8 and MMP9).

Summarizing the above, it should be noted that the role of MMP8 and MMP9 in the degradation of the periodontal ligament and alveolar bone needs to be specified, although the statistical relationship between the overproduction of the precursors of these matrixins and the severity of periodontitis can be considered proven.

Another well-publicized point regarding the role of MMP8 and MMP9 in the development of periodontitis is the activation mechanism of their synthesis by lymphokines. There is irrefutable evidence that the production of MMP8 and MMP9 in the affected areas of the periodontium correlates with an increase in the concentration of lymphokines there of an acute non-specific response to invasion (Th1 type): IL-1β, IL-12, IL-18, TNFα, NF-activator ligand κB (RANKL) and osteoprotegerin (OPG). In contrast, an increase in the concentration of lymphokines stimulating an antigen-dependent response (Th2 type): IL-10 and IL-4 correlates with a decrease in the production of MMP8 and MMP9. At the same time, there is an increase in the production of IL-17, a factor that stimulates wound healing after elimination of the infection [Finoti L.S. et al., 2014]. In the study of periodontal flushing, this process can be observed mainly at the stage of remission of periodontitis. There is also direct evidence that the stimulation of in vitro cultured fibroblasts with Th1 lymphokines: IL-1β, IL-12, IL-18, TNFα and RANKL increases the accumulation of metalloproteinase precursors by 2-3 orders of magnitude, primarily, MMP8 and MMP9 (results the definitions of mRNA and the precursor proteins encoded by them are the same) [Finoti LS et al., 2013]. Thus, lymphokines should be considered as one of the key factors in the activation of the production of MMP precursors in response to invasion. When summarizing the above facts, the following signal chain of response to bacterial invasion emerges, which under certain circumstances can cause the breakdown of the periodontal ligament [Fine D.H. et al., 2000]:

1) bacteria, their specific signaling factors or decay products (lipopolysaccharide and DNA) act on TLR receptors (TLR1, TLR4, TLR9) of the surface of periodontal resident cells (fibroblasts or macrophages);

2) periodontal cells receiving the stimulus eject lymphokines of an acute non-specific response (Th1): IL-1β, IL-12, IL-18, TNFα and RANKL;

3) Th1 response lymphokines cause overproduction of MMP8 and MMP9 metalloproteinases. This reaction is probably necessary to facilitate the migration of lymphocytes from the circulation into the focus of infection, as well as to facilitate the penetration of complement components and other bactericidal blood factors into the focus of infection. IL-6, formally related to the lymphokines of the Th2 response, is responsible for increasing the permeability of the walls of vascular capillaries. At the same time, all these factors stimulate positive chemotaxis of T-lymphocytes and NK cells from peripheral blood, which, in turn, are mobilized from bone marrow and regional lymph nodes;

4) in the case of a rapid suppression of the development of infection due to factors of the Th1 response, the stimulus that caused it disappears and all of the above reactions die out;

5) in case of infection insensitivity to the Th1 response due to IL-12-dependent development of the inhibitory mediator IL-10 and the activity of NK and T cells attracted to the focus after a short time (1-2 days or less), the Th1 response is suppressed and Th2 response is stimulated, which mediates the production of specific antibodies to antigens of bacteria that have penetrated the internal environment. The accumulation of antibodies stimulates a sharp increase in the shock power of complement and cytotoxic cells against the infectious agent, the opsonization of macrophages, which leads to the suppression of infection. It is advisable to consider IL-2, which cannot be produced by other types of cells, as a marker of lymphocyte periodontal accumulation;

6) in the event of a switching disorder of the Th1 / Th2 response, uncontrolled accumulation of MMP occurs, leading to chronic inflammation, decomposition of the collagen matrix of the periodontal ligament and gradual bone resorption. These phenomena underlie the well-known clinical picture of both aggressive and chronic periodontitis.

The main chemotactic factor for attracting neutrophils to the infection site is IL-8 [Anovazzi G. et al., 2013]. There are numerous experimental data on a local increase in the level of IL-8 in the foci of inflammation, including in case of chronic periodontitis [Microbiology and immunology for dentists], which forces some authors to classify it as a Th1 response factor. According to other authors, this point of view is not entirely accurate, since the accumulation of IL-8 in infected tissues occurs, as a rule, over a long period (several days), which is not comparable with the duration of the protective phase of the Th1 response [Bai D. et al. , 2015]. Rather, the IL-8-dependent neutrophilic response can be characterized as a reaction designed to cover the gap between the protective phases of the Th1 (antigen-independent) and Th2 (antigen-dependent) responses. In the case of periodontitis of any form, the overproduction of IL-8 can be considered as a marker, indicating the failure of the immune system's attempts to contain the infection due to the Th1 response. Moreover, the cell type, which is the main source of IL-8, remains unknown [Borilova-Linhartova P. et al., 2013]. There is also no doubt that a prolonged and massive infiltration of excess neutrophils into the periodontal tissue cannot ensure the protective response, although it is possible that when neutrophils come into contact with bacteria for the first time, they can inhibit the development of infection and even completely sterilize small foci of invasion [de Goncalves LS et al., 2006].

Most of the experimental studies of the mechanisms of induction of IL-8 synthesis in periodontitis have been performed on a model of human gingivial epithelium cells, in particular, on an immortalized OVA-9 cell line or on primary gingivial fibroblasts. An example of such a work is an article by Savitri et al., 2014, “Irzogladine maleate inhibits the induction of synthesis of type 2 toll receptors and IL-8 induced by Porphyromonas gingivalis in human gingivial epithelium cells”. It is postulated that irzogladine maleate (MI) is a well-known regulator of inflammation and the state of tight intercellular contacts. The authors set out to study the effect of this biologically active substance on the interaction of gingival epithelial cells with the periodontopathogen P. gingivalis. The choice of this bacterium, which is part of the red complex of periodontopathogens according to Sokransky, was not argued as a model by the authors, but it can be assumed that the reason for this is the relative ease of cultivation of this species in comparison with other known periodontopathogens. As a technical tool, the authors used in vitro stimulation of human cells with whole cells of P. gingivalis or its purified polysaccharide. The level of production of IL-8 and TLR2 cells (a component of the antigen-independent (innate) immunity system) was determined by real-time PCR. In order to study the relationship between the level of IL-8 synthesis and TLR2, expression of the TLR2 gene in culture was suppressed by transfection of cells with small interfering RNAs (siRNA). It was shown that MI removes the effect of inducing the synthesis of IL-8 by P. gingivalis or its purified polysaccharide on OVA-9 cells (P <0.01). Exactly the same effect is observed with respect to TLR2, both on the OVA-9 culture and on primary fibroblasts (P <0.01). The results of PCR-PB were confirmed by immunohistochemical staining. It was shown that stimulation with peptidoglycan (a typical ligand TLR2) in combination with MI leads to a decrease in the level of production of another toll receptor TLR4 (P <0.01). In contrast, the lipopolysaccharide of Escherichia coli, the main ligand of TLR4, did not affect the level of TLR2 mRNA synthesis. The inducing effect of the polysaccharide P. gingivalis on the production of TLR4 mRNA was removed by the addition of MI. Violation of the synthesis of TLR2 using siRNA led to a decrease in the level of synthesis of IL-8 mRNA under the influence of P. gingivalis or its polysaccharide on OVA-9 cells.

Swedish authors Palm et al., 2013, “Porphyromonas gingivalis lowers the immune response of fibroblasts” states that P. gingivalis is the main cause of the development of chronic periodontitis. Moreover, as a main factor of the pathogenicity of this bacterium, which also provides its protection and survival on the periodontium, gingvipaines are called proteinases of the papain family. Based on this, the authors investigated the behavior of P. gingivalis in the culture of primary fibroblasts of the gingival mucosa and human skin. The level of accumulation of chemokines and cytokines, in particular TNFα and CXCL8 (IL-8), served as a determined parameter. The production level of these factors was measured using ELISA and a cytokine test. The authors concluded that TNFα-dependent synthesis of CXCL8 is completely suppressed by living P. gingivalis cells, while P. gingivalis cells killed by heating do not affect the rate of production of CXCL8 fibroblasts. The addition of arginine-specific protease inhibitors partially removed the effect of suppressing the stimulating effect of TNFα on the synthesis of CXCL8. In addition, in the presence of an arginine-specific protease inhibitor, the synthesis of a number of other pro-inflammatory mediators was suppressed, suppressed by a live culture of P. gingivalis. On this basis, the authors make an assumption about the key role of the arginine-specific protease P. gingivalis in suppressing the immune response to periodontal microflora.

Schaumann et al., 2013, “Possible immunomodulatory activity of glycine in gum disease” describes experiments on the effects of glycine on gum epithelial cells. The authors suggested the existence of specific glycine receptors and performed experiments on their search by histochemical methods. Primary fibroblasts of the gingival epithelium in the culture were treated with IL-1β in the presence or absence of glycine, after which the dynamics of the synthesis of IL-6 and IL-8 were studied using real-time PCR. In addition, immunofluorescence microscopy was used to study the transport of regulatory factor NFκB into the nucleus. Glycine stimulation has been shown to enhance the effectiveness of IL-1β as an inducer of IL-6 and IL-8 mRNA synthesis, and also intensifies NFκB transport into the nucleus. Thus, glycine helps to reduce inflammation of the gum epithelium. According to the authors, there is the prospect of using glycine as an anti-inflammatory agent for periodontitis.

Swedish authors Salminen A et al., 2014, “Cytokines and chemokines are expressed differently in periodontitis patients: the possible role of TGFβ1 as a marker of the disease” states that severe periodontitis increases the risk of developing systemic diseases of an inflammatory nature, primarily atherosclerosis. With this in mind, they conducted a comprehensive study of the presence of various cytokines and lymphokines in blood serum, saliva and periodontal rinses of patients with periodontitis and a control sample of individuals with healthy periodontal disease. The most significant difference in the sample of patients was a reduced level of IL-6 in the blood and saliva. On the contrary, the level of CXCL8 (IL-8) in the blood did not differ, in saliva it was reduced, and in periodontal swabs it was increased. The lymphokine of the T-cell origin of IL-2 in the blood and saliva of patients of both samples was kept at the same concentration, but its content in periodontal washings of patients was significantly reduced. An unexpected result was a significant increase in the level of TGFβ1 in the blood, saliva and periodontal washes of patients with CP. Studying this observation, the authors found that in vitro stimulation of fibroblasts by living wild-type P. gingivalis cells or their protease-free mutants leads to a decrease in the production of CXCL8 and IL-6 and an increase in the expression of TGFJ31. According to the authors, the main factor responsible for this effect is the lysine-specific cysteine proteinase P. gingivalis gingvipain Kgp. The authors consider TGFβ1 to be one of the main candidates for the role of a factor mediating the systemic effects of periodontal inflammation.

Known RF patent No. 2381746 "Method for diagnosing the severity of periodontitis." The invention relates to dentistry and is used to diagnose the severity of periodontitis. The method consists in measuring the length of the roots of the teeth on the lower and upper jaw, namely one of the same teeth in the jaw: canine, premolar, first and second molars. The method allows to increase the diagnostic capabilities with a smoothed clinical picture of periodontitis. According to the authors, the claimed method for diagnosing the severity of periodontitis solves the problem of creating an appropriate method, the implementation of which ensures the achievement of a technical result, consisting in the possibility of diagnosing the severity of periodontitis, when the stage of exacerbation does not manifest itself explicitly; in increasing the reliability of the method; in simplifying and improving the security of the method; in increasing efficiency.

The authors explain the effectiveness of the proposed method for the diagnosis of periodontitis by their observations of the mechanism of periodontal functioning as a system of teeth attachment and chewing function. As a result, the authors established a correlation between the severity of the inflammatory process in periodontal disease, the degree of destruction of the bone tissue of the interalveolar septum, and the anatomical structure of the teeth, namely, the length of the roots of the teeth. Based on the statistical data obtained during the examination of patients, the authors proved that the metric characteristics of the teeth, namely the root length, can be used to predict the severity of periodontitis even in cases when the exacerbation of the disease does not manifest itself in an explicit form, and the clinical picture is smoothed.

Known RF patent No. 2451937 "Method for the diagnosis of breast cancer by the level of RNA of interleukins IL-8 and / or IL-18 in blood plasma." The invention relates to the field of medicine, oncology and molecular biology and can be used to diagnose breast cancer. The method is based on measuring in plasma the level of RNA of interleukins IL-8 and / or IL-18 and comparing the level of representation of RNA of IL-8 and / or IL-18 with the level of representation of reference transcripts. To implement the method using reverse transcription and polymerase chain reaction in "real time". ABL and / or HPRT and / or IL-1b RNAs are used as reference transcripts. The result of the study is considered positive when the difference in the levels of RNA representation of IL-8 and HPRT more than ten times. Using the claimed method allows for early diagnosis of breast cancer. The patent does not describe the use of real-time PCR for the diagnosis of periodontitis; it does not describe the method for determining IL-8 mRNA in periodontal swabs and sequences of primers and probes. Thus, the claimed invention does not violate the intellectual property rights described in RF patent No. 2451937.

Known RF patent No. 2180119 "Method for the early diagnosis of sepsis in newborns." The invention relates to methods for early laboratory diagnosis of septic infection in newborns. The method consists in examining the level of cytokines: interleukin-8 (IL8) and tumor necrosis factor (TNF), and when the concentration of IL8 is more than 5 pg / ml and TNF more than 2 pg / ml, the development of septic infection is diagnosed. The technical result is to obtain a highly sensitive method for the early diagnosis of septic infection in newborns, which allows you to make a clinical diagnosis with the greatest degree of probability and as early as possible. The method does not use PCR, including real-time PCR, and is not applicable in periodontics, since it does not describe the calibration and sample preparation for the analysis of periodontal swabs.

Thus, at present in the Russian Federation there are no active analogues of the claimed invention.

Description (disclosure) of the invention

A method is proposed for determining the degree of suppuration at the periodontium by assessing the level of RNA of the human interleukin-8 (IL-8) gene in scrapings from the periodontal pocket.

In samples of biological material (scrapings) of patients with suppuration (suppuration) compared with the control group, the relative amount of RNA of the IL-8 gene significantly increases in comparison with the reference marker (transcript).

To assess the level of IL-8 RNA present in scrapings, reverse transcription and polymerase chain reaction (RT-PCR) methods can be used to record the accumulation of reaction products in real-time mode, as well as any other standard methods for assessing the level of transcript representation (NASBA, hybridization on microchips, NGS, etc.).

Based on experimental data, when comparing patients with suppuration (suppuration) and conditionally healthy patients without signs of inflammatory periodontal tissue diseases by real-time RT-PCR, the results of determining the level of IL-8 mRNA representation relative to the reference β2 gene RNA were obtained and analyzed β-microglobulin of a person and a relatively unique (non-repeating) fragment of the human genome.

The main criterion for the diagnosis of chronic generalized periodontitis (CGP) was the destruction of the periodontal attachment. The severity was determined based on the depth of periodontal pockets and the degree of destruction of bone tissue.

The following clinical characteristics of patients were entered into the database using digital codes:

1) Age: up to 40 years - 1, from 41 to 55 - 2, over 56 - 3;

2) Gender: male - 1, female - 2;

3) The degree of periodontal disease: 0 - without pathology, 1 - gingivitis (were not present in the sample), 2 - mild degree of CGP, 3 - moderate degree of CGP, 4 - severe degree of CGP, 5 - aggressive periodontitis;

4) Bleeding: 0 - no, 1 - there is;

5) Suppuration: 0 - no, 1 - there is;

6) Exposure of the roots of the teeth: 0 - no, 1 - eat;

7) Pathological tooth mobility: 0 - no, 1 - eat;

8) Loss of teeth: 0 - no, 1 - eat;

9) Depth of periodontal pocket: 0 - 0-1 mm, 1 - 1.1-5.0 mm, 2 - 5.1-10 mm, 3 - more than 10 mm;

10) SL index;

11) GW index;

12) Mulleman Index;

13) Tobacco: 0 - no, 1 - eat;

14) History of unfavorable heredity: 0 - no; 1 - on the maternal side; 2 - on the paternal side; 3 - on the maternal and paternal lines; 4 - other relatives.

Flushing of periodontal pockets was removed from the deepest areas using sterile paper endodontic pins. Material sampling was performed in duplicate from each patient.

To isolate RNA, the pins were preserved in 1.5 ml Eppendorf tubes containing 0.5 ml of fixative for stabilization of RNA in IntactRNA biological samples (CJSC Evrogen, Russia, catalog number No. BC031).

Immediately prior to isolation, flush material was precipitated by centrifugation on a benchtop centrifuge for 5 minutes. The supernatant, which is an IntactRNA fixative solution, was carefully removed from the tube. RNA purification was performed using ExtractRNA reagent (Eurogen CJSC, Russia, catalog number BC032) in accordance with the manufacturer's instructions. The resulting RNA was dissolved in 10 μl of deionized water. RNA preparations were stored at a temperature of -70 ° C for no more than 6 months. No genomic DNA was purified.

For the synthesis of cDNA, a Mint reagent kit was used (ZAO Evrogen, Russia, catalog number SK001) in accordance with the manufacturer's instructions. The resulting cDNA preparation was used for real-time PCR.

The level of transcript presentation was assessed using commercial reagent kits: to analyze the interleukin-8 RNA level, the Immuno-Genetic RNA IL-8 reagent kit was used (NPO DNA-Technology LLC, Russia, catalog number R1-R180-N3 / 4), for analysis The reference marker used a set of reagents to detect β2-microglobulin B2M mRNA (NPO DNA-Technology LLC, Russia, catalog number R1-P805-23 / 9).

The threshold cycle value (Ct) for the corresponding marker in the sample was detected automatically by the detecting amplifier.

The results of the analysis are presented in Table 1.

The content of the specific transcript of the pro-inflammatory lymphokine interleukin-8 (IL-8) in periodontal swabs was analyzed. In addition, the content of total human genomic RNA was estimated by the KVM marker. The results of the comparative analysis of the samples are presented in the Tables.

To assess the degree of difference in parameters in the samples, the Mann-Whitney test was used. When describing the result, the median of the attribute, quantiles, and the direct value of the degree of reliability (p) were calculated. The results of the comparative analysis are presented in table 4.

The conducted statistical analysis allowed us to draw the following conclusions:

When analyzing patients with suppuration and without suppuration, a significant difference in the content of IL-8 mRNA on the periodontium was revealed. In the study of total human RNA on periodontal disease, a similar pattern is not revealed, which indicates the specificity of the reaction of hyperproduction of IL-8 on periodontal during suppuration. This observation suggests that, when analyzing the periodontal transcriptome, IL-8 mRNA should be considered as a marker of the chemotactic factor of neutrophils, causing them to infiltrate onto the periodontal surface.

The ratio of levels of representation in the sample of the studied markers is determined by the formula:

[IL-8] / [Ref] = 2 ^{(CtRef-CtIL-4)} (formula 1),

where IL-8 is mRNA of interleukin-8;

Ref — mRNA of a reference marker, for example, B2M;

Ct is the value of the threshold cycle for the corresponding marker in the sample, which is determined automatically by the detecting amplifier.

Moreover, if the ratio of expression levels is ≤16, it is necessary to diagnose the presence of suppuration (suppuration) at the periodontal period under study, and if the ratio of expression levels is> 16, the absence of suppuration (suppuration).

The implementation (implementation) of the invention

Clinical material was collected on the basis of TsNISiChLH MH RF. 288 patients aged 21 to 63 years without severe somatic pathology were examined. In the study sample, there were 204 patients diagnosed with periodontitis and 84 patients without periodontal pathology.

Rinses of periodontal pockets were obtained by taking the contents from the deepest areas using sterile paper endodontic pins (size 25), which were then placed in a test tube with 1.5 ml of RNA-Later reagent (preservative for RNA) (Life technologies - Invitrogen, catalog number No. 7020) and transported to the laboratory in a refrigerated state for no more than 6 hours.

The fence was carried out in duplicate from each patient. At the stage of RNA isolation and PCR analysis, each repetition was processed separately.

Immediately prior to isolation, flush material was precipitated by centrifugation on a benchtop centrifuge for 5 minutes. The supernatant, which is an RNA-later fixative solution, was carefully removed from the tube and 200 μl of lysis solution (4 M guanidine isothiocyanate) was added, the sample was thoroughly mixed and kept for 1 min at room temperature. Then, 100 μl of water-saturated phenol (the organic phase of the two-phase phenol – Tris-HCl 40 mM buffer, pH 8.0) and 100 μl of a mixture of chloroform and isoamyl alcohol (24: 1) were added to the sample tube. The contents were thoroughly mixed on a vortex and incubated for 5 min at room temperature, repeating stirring periodically. The tubes were centrifuged in a benchtop centrifuge at 13000g for 10 min. Carefully avoiding capture of the organic phase and interphase at the interface, the upper (aqueous) phase was taken and transferred to a clean tube, 200 μl of a mixture of chloroform and isoamyl alcohol (24: 1) was added to it. Centrifuged on a benchtop centrifuge at 13000g for 5 minutes. The upper (aqueous) phase was taken and transferred to a clean 1.5 ml polypropylene tube. Pure isopropyl alcohol was added to the aqueous phase with a volume of 200 μl (or in a ratio of 1: 1 relative to the collected aqueous phase), the contents were mixed with 5-6-fold inverting the tubes. The tubes were centrifuged in a benchtop centrifuge at 13000g for 10 min. The supernatant was carefully removed using a water jet pump, without touching the sediment containing nucleic acids. The precipitate was washed by adding 100 μl of 70% aqueous ethanol to the tube and turning the tube 5-6 times.

The tubes were centrifuged in a benchtop centrifuge at 13000g for 10 min. The supernatant was carefully removed using a water jet pump, without touching the sediment containing nucleic acids. The precipitate was dried at a temperature of 50 ° C with the tube open for 5 min.

16.5 μl of distilled water was added to the precipitate, the caps of the tubes were closed and incubated at a temperature of 50 ° С for 5 min. The tubes were shaken on a tabletop vortex to achieve complete dissolution. RNA preparations were stored at a temperature of -20 ° C for no more than two months.

Preparations isolated by the method described above were purified from genomic DNA impurities by treatment with RNase-free DNase. For this, a preparation of total nucleic acids with a volume of 15.5 μl was added to the preparation of DNase-free RNase with a volume of 1 μl containing 1-5 units. enzymatic activity. The mixture was incubated at 37 ° C for 30 minutes. After this, the enzyme was inactivated by heating the mixture at 95 ° C for 5 min. Drops from the walls of the tubes were collected by centrifugation for 3-5 seconds.

For reverse transcription, a commercial set of reagents manufactured by NPO DNA-Technology LLC (Russia) was used. The ingredients of the RT-buffer kit, OT-IL-8 primers from the reverse transcription reagent kit were thawed at room temperature for 10 minutes. Drops from the walls of the tubes were collected by centrifugation for 3-5 seconds.

The reaction mixture for reverse transcription was prepared according to the manufacturer's instructions for the following recipe in a sterile 1.5 ml tube:

1. RT buffer-2 × (N + 1) μl;

2. "Primers + dNTP" - (N + 1) μl;

3. Reverse transcriptase MuMLV 2 units / μl - 0.5 × (N + 1) μl,

where N is the number of analyzed samples taking into account the negative control. Drops from the walls of the tubes were collected by centrifugation for 3-5 seconds.

In tubes containing 16.5 μl of the prepared RNA preparation, 3.5 μl of the reverse transcription mixture was added. When setting the negative control, a tube containing 16.5 μl of purified water was used. The reaction mixtures were mixed with 5-7-fold pipetting. The tubes were incubated at 40 ° C for 30 min, then the reaction was stopped by heating at 95 ° C for 5 min. Drops from the walls of the tubes were collected by centrifugation for 3-5 seconds. A DNA preparation corresponding to 1/10 of the volume of one washout (50 μl) was dissolved in 50 μl of an eluting solution (from the Proba-GS reagent kit). As a matrix for one PCR reaction, 5 μl of the obtained preparation was used.

PCR was performed as follows:

1. The mixture was prepared in Scintific Specialties Inc. strip tubes (SSI) Part # 3135-00 UltraFlux Flat Cap PCR Tubes 200 μL, unit 1 × 125 tube strips with caps.

2. Labeled the rows of tubes for each test sample, taking into account the negative control sample (K (-)) and the positive control sample (K (+)) and 2 tubes to control the background fluorescence.

3. 20 μl buffer “H” was added to the tubes, which contains all the components of the mixture for PCR reaction, with the exception of DNA polymerase and DNA matrix.

The composition of the buffer "N" (to obtain 20 μl of the mixture), μl:

18 - NDP buffer (Set of reagents for PCR amplification, LLC NPO DNA-Technology, Russia),

1,695 - water,

0.27 - dNTP 25 mm,

0.014 - primer 1, 100 mm,

0.014 - primer 2, 100 mm,

0.007 probe (Fam), 50 mM.

4. A drop of molten paraffin was added to each tube, separating the reaction mixture.

5. A solution of Tag polymerase (Set of reagents for PCR amplification, NPO DNA-Technology LLC, Russia) was prepared by shaking a tube on a vortex and dropping drops from the walls.

6. Pipette 10 μl of Tag-polymerase solution into each labeled tube (except background control tubes) without damaging the paraffin layer.

7. Add a drop of mineral oil to each tube without damaging the paraffin layer.

8. 5 μl of the DNA preparation was added to the test tube with the test sample without damaging the paraffin layer.

9. 5 μl of a positive control DNA sample was added to a test tube with a positive control (K (+)), 5 μl of a negative control sample or water was added to test tubes with a negative control (K (-)) and background control, respectively, without damaging the paraffin layer .

10. Centrifuged tubes in a microcentrifuge for 3-5 seconds.

11. Set the tubes in the detecting amplifier.

The following oligonucleotide primers and probes were used for PCR:

Test tubes (prepared as described above) were installed in a DT-Prime detection thermal cycler (NPO DNA-Technology LLC, Moscow) and the reaction was carried out with the following cycling parameters:

Amplification program (50 cycles):

1. 94.0 ° C-01'30ʺ

2. 94.0 ° C-00'10ʺ

3.64.0 ° С-00'15ʺ (+ fluorescence reading)

Analysis settings installed on the device:

Method - Geometric (Cf) (BF), cr = 9, vt = 10, tp = 30, tv = 5

For each reaction, the thermal cycler software automatically calculated the threshold cycle Ct. The obtained values were averaged over two samples of the same series.

If the ratio of marker levels was ≤16, the clinical diagnosis of suppuration (suppuration) at the studied periodontal point was confirmed, and with a ratio of marker levels> 16, there was no suppuration (suppuration).

Information sources

1. Anovazzi G, Finoti LS, Corbi SC, Kim YJ, Marcaccini AM, Gerlach RF, Capela MV, Orrico SR, Cirelli JA, Scarel-Caminaga RM. Interleukin 4 haplotypes of susceptibility to chronic periodontitis are associated with IL-4 protein levels but not with clinical outcomes of periodontal therapy. // Hum Immunol. - 2013 .-- Vol. 74, No. 12, doi: 10.1016 / j.humimm. 2013.08.286.

2. Bai D, Nakao R, Ito A, Uematsu H, Senpuku H. Immunoreactive antigens recognized in serum samples from mice intranasally immunized with Porphyromonas gingivalis outer membrane vesicles. // Pathog Dis. - 2015. Vol. 73. No. 3.

3. Berdeli A., Emingil G., Gurkan A. et al. Association of the IL-1RN2 allele with periodontal diseases. // Clin. Biochem. - 2006. - Vol. 39. - P. 357-362.

4. Borilova-Linhartova P, Vokurka J, Poskerova H, Fassmann A, Izakovicova Holla L. Haplotype analysis of interleukin-8 gene polymorphisms in chronic and aggressive periodontitis. // Mediators Inflamm. - 2013; 2013: 342351.

5. de Goncalves L.S., Ferreira S.M., Souza C.O. et al. IL-1 gene polymorphism and periodontal status of HIV Brazilians on highly active antiretroviral therapy. // Acq. Immun. Defic. Syndr. - 2006. - Vol. 20. - P. 1779-1781.

6. Dosseva-Panova VT, Popova CL, Panov VE. Subgingival microbial profile and production of proinflammatory cytokines in chronic periodontitis. // Folia Med (Plovdiv). - 2014 .-- Jul. - Vol. 56, No. 3. - P. 152-60.

7. Fine D.H., Kaplan J.B., Kachlany S.C. et al. How we got attached to Actinobacillus actinomycetemcomitans: A model for infectious diseases. // Periodontol. - 2000. - 2006. - Vol. 42. - P. 114-157.

8. Finoti L.S., Anovazzi G., Pigossi S.C., Corbi S.C., Teixeira S.R., Braido G.V., Kim Y.J., Orrico S.R., Cirelli J.A., Mayer M.P., Scarel-Caminaga R.M. Periodontopathogens levels and clinical response to periodontal therapy in individuals with the interleukin-4 haplotype associated with susceptibility to chronic periodontitis. // Eur. J. Clin. Microbiol. Infect. Dis. - 2013 .-- Vol. 32, No. 12. - P. 1501-9.

9. Finoti L.S., Anovazzi G., Pigossi S.C., Corbi S.C., Teixeira S.R., Braido G.V., Kim Y.J., Orrico S.R., Cirelli J.A., Mayer M.P., Scarel-Caminaga R.M. Periodontopathogens levels and clinical response to periodontal therapy in individuals with the interleukin-4 haplotype associated with susceptibility to chronic periodontitis. // Eur. J. Clin. Microbiol. Infect. Dis. - 2013 .-- Vol. 32, No. 12. - P. 1501-9.

10. Fujise O., Miura M., Hamachi, T. et al. Risk of Porphyromonas gingivalis recolonization during the early period of periodontal maintenance in initially severe periodontitis sites. // J. Periodontol. - 2006. - Vol. 77, No. 8. - P. 1333-1339.

11. Gemmell E., Yamazaki K., Seymour G.J. The role of T cells in periodontal disease: homeostasis and autoimmunity. // Periodontol. - 2000. - 2007. - Vol. 43. - P. 14-40.

T.F. Efficacy of short-term adjunctive subantimicrobial dose doxycycline in diabetic patients-randomized study. // Oral. Dis. - 2012. - Vol. 18, №8. - P. 763-70.12. Gilowski L., Kondzielnik P., Wiench R., Plocica I., Strojek K.,

TF Efficacy of short-term adjunctive subantimicrobial dose doxycycline in diabetic patients-randomized study. // Oral. Dis. - 2012. - Vol. 18, No. 8. - P. 763-70.

P.F., Huang H., McAninley S., Alfant В., Harrison P., Aukhil I., Walker C., Shaddox L.M. Periodontal treatment reduces matrix metalloproteinase levels in localized aggressive periodontitis. // J. Periodontol. - 2013. - Vol. 84, №12. - P. 1801-8.13.

PF, Huang H., McAninley S., Alfant B., Harrison P., Aukhil I., Walker C., Shaddox LM Periodontal treatment reduces matrix metalloproteinase levels in localized aggressive periodontitis. // J. Periodontol. - 2013 .-- Vol. 84, No. 12. - P. 1801-8.

14. Graswinckel J.E.M., van der Velden U., van Winkelhoff A.J. et al. Plasma antibody levels in periodontitis patients and controls // J. Clin. Periodontol. - 2004. - Vol. 31. -P. 562-568.

A., Emingil G., Saygan B.H. et al. Matrix metalloproteinase-2, -9, and -12 gene polymorphisms in generalized aggressive periodontitis. // J. Periodontol. - 2007. - Vol. 78, №12. - P. 2338-2347.fifteen.

A., Emingil G., Saygan BH et al. Matrix metalloproteinase-2, -9, and -12 gene polymorphisms in generalized aggressive periodontitis. // J. Periodontol. - 2007. - Vol. 78, No. 12. - P. 2338-2347.

16. Guzeldemir E., Gunhan M., Ozcelik O. et al. Interleukin-1 and tumor necrosis factoralpha gene polymorphisms in Turkish patients with localized aggressive periodontitis. // J. Oral Sci. - 2008. - Vol. 50. - P. 151-159.

17. Javed F., Ahmed H. B., Mikami T., Almas K., Romanos G. E., Al-Hezaimi K. Cytokine profile in the gingival crevicular fluid of rheumatoid arthritis patients with chronic periodontitis. // J. Investig. Clin. Dent. - 2014. - Vol.5, No. 1. - P. 1-8.

18. Jayaprakash K., Khalaf H., Bengtsson T. Gingipains from Porphyromonas gingivalis play a significant role in induction and regulation of CXCL8 in THP-1 cells. // BMC Microbiol. - 2014 .-- Vol. 18. - P. 14-193.

19. Khosropanah H., Sarvestani E.K., Mahmoodi A., Golshah M. Association of IL-8 (-251A / T) gene polymorphism with clinical parameters and chronic periodontitis. // J. Dent (Tehran). - 2013 .-- Vol. 10 (4). - P. 312-8.

20. Kinney J.S., Morelli T., Oh M., Braun T.M., Ramseier C.A., Sugai J.V., Giannobile W.V. Crevicular fluid biomarkers and periodontal disease progression. // J. Clin. Periodontol. - 2014 .-- Vol. 41 (2). - P. 113-20.

21. Moutsopoulos NM, Chalmers NI, Barb JJ, Abusleme L, Greenwell-Wild T, Dutzan N, Paster BJ, Munson PJ, Fine DH, Uzel G, Holland SM. Subgingival microbial communities in leukocyte adhesion deficiency and their relationship with local immunopathology. // PLoS Pathog. - 2015 .-- Vol.5. - P. 11 (3).

22. Noack B., Gorgens H., Lorenz K. et al. TLR4 and IL-18 gene variants in chronic periodontitis: impact on disease susceptibility and severity. // Immunol. Invest. -2009. - Vol. 38, No. 3-4. - P. 297-310.

23. Page R.C. Critical issues in periodontal research. // J. Dent. Res. - 1995. - Vol. 74. - P. 1118-1128.

24. Palm E, Khalaf H, Bengtsson T. Porphyromonas gingivalis downregulates the immune response of fibroblasts. // BMC Microbiol. - 2013 .-- Vol. 10. - P. 13-155.

25. Ram VS, Parthiban, Sudhakar U, Mithradas N, Prabhakar R. Bonebiomarkers in periodontal disease: a review article. // J Clin. Diagn Res. - 2015. Vol. 9 (1). - P. 07-10.

K.,

P., Buhlin К.,

E., Nieminen M.S., Sorsa T., Sinisalo J., Pussinen P.J. Salivary biomarkers of bacterial burden, inflammatory response, and tissue destruction in periodontitis. // J Clin Periodontol. - 2014. - Vol. 41(5). - P. 442-50.26. Salminen A., Gursoy UK, Paju S.,

K.,

P., Buhlin K.,

E., Nieminen MS, Sorsa T., Sinisalo J., Pussinen PJ Salivary biomarkers of bacterial burden, inflammatory response, and tissue destruction in periodontitis. // J Clin Periodontol. - 2014 .-- Vol. 41 (5). - P. 442-50.

A. Potential immune modularly role of glycine in oral gingival inflammation. // Clin Dev Immunol. - 2013. - PubMed PMID: 24348681.27. Schaumann T, Kraus D, Winter J, Wolf M, Deschner J,

A. Potential immune modularly role of glycine in oral gingival inflammation. // Clin Dev Immunol. - 2013. - PubMed PMID: 24348681.

28. Tanner A.C.R., Kent R. Jr., Dyke Van T. et al. Clinical and other risk indicators for early periodontitis in adults. // J. Periodontal. - 2005. -Vol. 76, No. 4. - P. 573-581.

29. Trivedi S, Lai N, Mahdi AA, Singh B, Pandey S. Association of salivary lipid peroxidation levels, antioxidant enzymes, and chronic periodontitis. // Int J Periodontics Restorative Dent. - 2015. - Vol. 35 (2). - P. 14-9.

O, Becerik S, Tervahartiala T, Sorsa T, Atilla G, Emingil G. The effect of adjunctive chlorhexidine mouthrinse on GCF MMP-8 and TIMP-1 levels in gingivitis: a randomized placebo-controlled study. // BMC Oral Health. - 2014. - Vol. 20. - P. 14:55.thirty.

O, Becerik S, Tervahartiala T, Sorsa T, Atilla G, Emingil G. The effect of adjunctive chlorhexidine mouthrinse on GCF MMP-8 and TIMP-1 levels in gingivitis: a randomized placebo-controlled study. // BMC Oral Health. - 2014 .-- Vol. 20. - P. 14:55.

31. Grudyanov A.I., Dmitrieva L.A., Maksimovsky Yu.M. Periodontology: current status, issues and directions of scientific developments. // Periodontology. - 1998. - No. 3 (9). - S. 5-7.

32. Rebrikov D.V. RF patent No. 2451937. A method for the diagnosis of breast cancer by the level of RNA of interleukins IL-8 and / or IL-18 in blood plasma.

33. Uvarova L.V., Elovikova T.M. RF patent No. 2381746. A method for diagnosing the severity of periodontitis. Application dated 11/17/2008. Publ. 02/20/2010 (terminated).

34. Estrin V.V., Efanova E.A., Pukhtinskaya M.G. RF patent No. 2180119. A method for the early diagnosis of sepsis in newborns. Application dated 12/21/1998. Publ. 02/27/2002.

Claims (3)

1. A method for the diagnosis of suppuration (suppuration) of periodontal tissues, characterized in that in scrapings from the lesion area, the mRNA level of the interleukin-8 gene (IL-8) is measured and compared with the mRNA level of reference transcripts; based on the obtained levels of genetic markers, their ratio is calculated, and if the ratio of marker levels is ≤16, the presence of suppuration (suppuration) at the studied periodontal point should be diagnosed, and with the ratio of marker levels> 16, the absence of suppuration (suppuration). 2. The method according to p. 1, characterized in that as a method for determining the level of mRNA using reverse transcription and polymerase chain reaction "in real time". 3. The method according to p. 1, characterized in that the mRNA of the human B2M gene is used as reference markers.