RU2387384C1 - Method of obtaining biopsy material from atherosclerosis-affected vessels - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine, namely to cardiology. In order to obtain postmortal material from arterial vessels affected by atherosclerosis arteries of medium and/or large size of muscular-elastic or elastic types with signs of atherosclerosis are used. Section of vessel wall is opened along, cut of vessel intimal layer not more than 0.1 mm deep, 1 cm long, 2 cm wide is made in order to obtain sample mass not less than 10 mg. Near cut edge isolated section of intimal layer is caught with movement perpendicular to direction of cut line along vessel surface and separated from underlying layers. Obtained sample is washed in 0.9% solution of sodium chloride, weighed and frozen in liquid nitrogen. Time of tissue sampling, including opening and freezing constitutes not more than 15 minutes.

EFFECT: method allows to obtain samples of postmortal material, including only vessel intima, optimally necessary for high output of RNA.

2 ex

Description

The invention relates to medicine, namely to cardiology, and can be used to study the pathogenetic mechanisms of the development of atherosclerosis.

The etiology and pathogenesis of atherosclerosis are still poorly understood. The lack of sufficient and accurate information about specific pathogenetic links of the atherosclerotic process makes it difficult to study pathogenesis and develop adequate treatment methods.

To study the atherosclerotic process, there are various research methods. Some of them relate to invasive intravital studies, others are post-mortal diagnostics. They serve to determine the lipid composition of the blood, to study transverse and longitudinal sections of arterial vessels, on the basis of which an idea of the processes occurring in their wall is created.

A targeted study of the vascular wall using currently known methods provides information on changes in its microstructure: places of lipid deposition, the location in the lipid core of cellular elements - macrophages, foam cells.

A known method of studying a biopsy of a vascular wall affected by an atherosclerotic process, by immunohistochemical analysis (A.N. Voskanyants, V.A. Nagornev. Proliferation of human artery wall cells during atherogenesis as a factor in the manifestation of immune inflammation. // Cytokines and inflammation, 2004, No. 4 , p.10-11).

For research using longitudinal or transverse sections of the vessel wall, including all layers.

The study of the obtained biopsies includes the use of murine monoclonal antibodies against the proliferating cell nuclear antigen (Anti-Proliferating Cell Nuclear Antigen (PCNA)) labeled with horseradish peroxidase (HRP). Antibodies react with epitopes of nuclear antigens in proliferating cells. Maximum PCNA expression is characteristic of cells in the S phase.

The disadvantage of this method is that it allows you to get information only about the presence or absence of increased expression in the studied cells. Quantitative assessment of changes remains subjective. This is due to the fact that when working with material that includes all layers, it is difficult to establish in the nuclei of which cells and which layer that was able to be fixed, there is an increase or decrease in PCNA expression.

This type of research methods based on immunohistochemical analysis of the vascular wall makes it possible to diagnose atherosclerosis, but does not allow to identify the pathogenetic mechanisms of its development at the level of molecular genetics.

As the closest analogue, the method of obtaining biopsy material for the diagnosis of pathological changes in the vascular wall in case of atherosclerotic lesion is accepted (Avtandilov G.G. Fundamentals of pathoanatomical practice. Guide. 2nd ed. - M .: RMPO, 1998. - 505 p.). The method includes taking a biopsy sample of the test vessel, followed by processing and preparation for microscopic examination.

A biopsy specimen, which is a transverse or longitudinal section of the vessel wall, allows you to study its structure, evaluate changes caused by atherosclerotic lesions.

The method is informative in cases where it is necessary to make a clinical or pathoanatomical diagnosis, answer the question about the presence or absence of lesions of the vessel wall, and also describe their location.

However, the known method cannot be used for genetic analysis. This is due to the fact that in the process of preparing the material for biopsy, all layers of the vessel are captured into the slice, namely:

intima, media and adventitia. The resulting sample contains a variety of genetic material that does not allow one to judge changes only in the inner layer of the vascular wall. The sample contains endothelial, smooth muscle cells, T-lymphocytes, macrophages, a large number of lipids.

In addition, during the preparation of samples for histological examination, the material is exposed to various chemicals - formalin, alcohols, paraffin. For the differentiation of the layers using various painting methods. After such manipulations, the genetic apparatus of the cell and proteins are not suitable for molecular research.

Currently, a large number of domestic and foreign works are devoted to genetic research in multifactorial diseases, in particular with atherosclerosis.

Identification and study of the pathological links of the atherosclerotic process, namely the genes responsible for the development of atherosclerosis, will help to study the pathogenesis of this disease. An effective method in developing approaches for diagnosing, treating, and creating drugs for atherosclerosis is to search for critical links in the pathological process - candidate genes responsible for the development of atherosclerosis and the corresponding protein products isolated from autopsy material of vascular intima.

The objective of the invention is to provide a method for producing biopsy material from arterial vessels affected by atherosclerosis, allowing correct genetic studies to assess the level of gene expression.

The essence of the invention lies in the fact that in the method of obtaining biopsy material from arterial vessels affected by atherosclerosis, comprising opening a predetermined portion of the vessel wall and taking a sample of post-mortal biological material with its subsequent processing, medium and / or large-caliber muscular arteries are used to take post-mortal biological material -elastic or elastic types with signs of atherosclerosis, a predetermined section of the vessel wall is opened along, an intimal incision is made with a vessel with a depth of not more than 0.1 mm, a length of 1 cm, a width of 2 cm to achieve a sample mass of not less than 10 mg, at the edge of the notch, a selected portion of the intimal layer is captured by movement perpendicular to the direction of the notch line along the surface of the vessel and it is separated from the underlying layers , the resulting sample is washed in a 0.9% sodium chloride solution, weighed and frozen in liquid nitrogen, while the time of tissue collection, including opening and freezing, is no more than 15 minutes.

Using the invention allows to obtain the following technical result.

The method allows to extract samples of biopsy material affected by the atherosclerotic process, including only intima, free from the underlying tissues and products of the atherosclerotic process and having a mass of at least 10 mg, which is optimally necessary for high RNA yield.

The biopsy material obtained in this way allows an objective correct assessment of the level of gene expression, which makes it possible to identify and study the pathological links of the atherosclerotic process in order to develop effective methods of diagnosis and treatment.

The technical result is achieved due to the fact that the authors for the first time developed a method for producing biopsy material, which allows to extract the tissue of the intima of the vessel for genetic research in atherosclerotic lesions at various stages of its development: a visually healthy area, lipid spot and atherosclerotic plaque in order to obtain the maximum possible RNA yield.

In solving this problem, the authors proceeded from the fact that atherosclerosis is a chronic disease, manifested by focal thickening of the intima of the arteries of elastic and muscular-elastic types due to the deposition of lipids (lipoproteins) and reactive proliferation of connective tissue.

The morphogenesis of atherosclerosis includes several stages, among which dolipid, lipoidosis, liposclerosis, atheromatosis, ulceration, atherocalcinosis can be distinguished. The appearance of a clear microscopic picture of changes in the vascular wall is preceded by changes at the molecular level.

Since atherosclerotic lesions are formed inside the intima, a genetic analysis requires a method with which you can get the correct results for evaluating the level of gene expression based on the analysis of RNA isolated from the intima of vessels affected by atherosclerosis, freed from tissues of various types: muscle fibers, blood clots, fragments blood, the contents of atherosclerotic plaques that impede the study. That is why the separation of the intima of a visually healthy part of the vessel from the lipid spot, atherosclerotic plaque is necessary for the correct assessment of the results of gene expression.

The method was developed taking into account the fact that pathological changes that occur with atherosclerosis occur in the intima of medium and large-caliber arteries.

Biopsy material obtained by the proposed method allows for the first time to compare the level of gene expression in different parts of the vessel in the same patient. The intima of the vessel includes the endothelium, the basement membrane, the subendothelial layer and the inner elastic membrane. Comparison of the level of gene expression in different parts of the vessels - a visually healthy area, a site with a lipid stain and a site with an atherosclerotic plaque in the same patient, allows us to identify changes in the expression of certain genes at a genetic level for a more detailed study and determination of the pathogenetic mechanisms of the occurrence of atherosclerosis.

The technology for obtaining biopsy material developed by the authors ensures accurate extraction of intima, free of the products of the atherosclerotic process, eliminates the capture of the middle and outer shell in the test sample, having a mass of at least 10 mg, which is essential for RNA isolation. The entire process of taking tissue, including opening and freezing, is no more than 15 minutes. This is due to the fact that later in the tissues oxidative and other biochemical processes begin, leading to RNA degradation and a change in the necessary parameters, which does not allow for correct measurements.

Based on the proposed method, for the first time, a search, identification and comparative analysis of putative genes responsible for the occurrence and development of atherosclerosis, as well as a comparative analysis of RNA will be performed in the affected and unaffected areas of vascular intima taken from the same patient. Using molecular genetic research methods, it will also be possible to identify candidate genes responsible for the atherosclerotic process, which allows us to approach the pathogenetic processes in atherosclerosis at the genetic level and, in the future, to identify protein products of these genes and use them to search for new drugs affecting target proteins of pathological processes.

Thus, based on and taking into account the above factors, the authors developed a method for isolating the vascular intima for genetic research in atherosclerotic lesions at various stages of its development in order to obtain the maximum possible RNA yield, which allows one to obtain statistically reliable and reproducible results of polymerase chain reaction, coupled with the reverse transcription.

The method is as follows.

For taking post-mortal biological material, medium and large-caliber arteries of muscular-elastic or elastic types with signs of atherosclerosis, such as the abdominal aorta, are used.

A predetermined portion of the vessel wall is opened lengthwise for maximum access to the inner surface. Using a sharp tool (for example, a lancet), an incision is made on the inner surface of the vessel wall with a depth of not more than 0.1 mm, that is, not more than the thickness of the intimal layer of the vessel, 1 cm long and 2 cm wide, which ensures a sample weight of at least 10 mg .

Using serrated-forceps forceps at the edge of the incision, the surface layer is captured - the intima, by moving in the direction perpendicular to the direction of the incision line, along the surface of the vessel, the intima are separated from the underlying, medial layers. This technique allows the separation of the intimal layer from the middle layer of the vessel and to obtain a sample without additional layers of the middle and adventitious (outer) layers. The presence in the sample of additional layers of the vessel (middle and adventitious) can change the expression of the gene.

The resulting sample is washed in a 0.9% solution of sodium chloride, which is isotonic with respect to blood, in direct contact with which is the intimal layer of the vessel and allows you to separate it from tissues of another type, such as muscle elements of the middle layer of the vessel, the contents of the atherosclerotic plaque ( granules of cholesterol), from blood elements (white blood cells, platelets, erythrocytes), which can affect the process of RNA isolation. The solution is used once, since the used solution can contaminate the following sample with the above tissue elements. The resulting sample was weighed on an OHAUS electronic balance.

Tissue collection, including autopsy, is performed within no more than 15 minutes.

Then the sample is frozen in liquid nitrogen to prevent RNA degradation and sent for further RNA isolation.

The method was tested in the laboratory of functional genomics of the Institute of General Genetics. N.I. Vavilova on 20 patients, including 8 women and 12 men aged 58 to 73 years. Samples were used to analyze the level of gene expression of the AP-1 (Activating protein 1) transcriptional complex by real-time polymerase chain reaction. They made it possible to conduct correct genetic studies at the level of gene expression. In all cases, a high yield of RNA from the samples was achieved. The expression profiles of the studied genes were stably high in all samples affected by atherosclerosis.

Example 1. The sampling of post-mortal biological material was carried out 12 hours after biological death, while the abdominal aorta 12 cm long was opened lengthwise. Using a lancet in an area of a visually healthy vessel that was not affected by atherosclerosis, an incision was made 1 cm long, 2 cm wide and 0.1 mm deep, which guarantees the achievement of tissue mass of at least 10 mg. With the help of serrated-forceps, the surface layer - intima - is captured at the edge of the incision. By movement perpendicular to the direction of the incision, along the surface of the vessel, the intima is separated from the underlying medial layer with an area of about 2 square meters. see. The isolated fragment was washed in a 0.9% sodium chloride solution and weighed on an OHAUS electronic balance, then frozen in liquid nitrogen and sent for subsequent RNA isolation and evaluation of gene expression level.

Example 2. A sample of post-mortal biological material was taken 12 hours after biological death. The abdominal aorta is 12 cm long. Using a lancet in the area of the lipid stain, an incision was made in the vessel, the size of a lipid stain, 0.1 mm deep. With the help of serrated-tweezers, the surface layer - the intima, which is separated from the underlying, medial layers, with a total area of about 2 square meters, is captured at the edge of the incision. cm, weighing 11 mg. The isolated fragment was washed in 0.9% sodium chloride solution and frozen in liquid nitrogen for subsequent RNA isolation and assessment of gene expression level.

Claims (1)

A method of obtaining biopsy material from arterial vessels affected by atherosclerosis, including opening a predetermined portion of the vessel wall and taking a sample of post-mortal biological material with its subsequent processing, characterized in that medium and / or large caliber muscular-elastic or elastic arteries are used to take post-mortal biological material types with signs of atherosclerosis, a predetermined portion of the vessel wall is opened along, an incision of the intimal layer of the vessel is not performed more than 0.1 mm, 1 cm long, 2 cm wide to achieve a sample weight of at least 10 mg, at the edge of the notch, the selected portion of the intimal layer is captured by movement perpendicular to the direction of the notch line along the surface of the vessel, and it is separated from the underlying layers, the resulting sample is washed in a 0.9% sodium chloride solution, weighed and frozen in liquid nitrogen, while the time of tissue collection, including opening and freezing, is no more than 15 minutes.