Method of hystamine rate assessment in epithelial cells of rats endometrium within sex cycle

FIELD: medicine.

SUBSTANCE: invention belongs to medicine, notably to morphology and hystochemistry. In this method of histamine rate assessment in epithelial cells of rats endometrium within sex cycle, sample acquisition and following fluorescent-histochemical analysis with orthophtalic aldehide is made. In each phase of sex cycle histamine rate assessment in vaginal content is made. Assessment's result is compared with rate of histamine which is 0.50±0.07 at early estrus stage, 0.34±0.04 at late estrus stage, 0.68±0.08 at metaestrus stage, 0.72±0.12 at early diestrus stage, 0.75±0.13 at late diestrus stage, 0.87±0.11 at proestrus stage. Measurement unit is conventional unit of measuring device. When rate of histamine in vaginal content is below control value the rate of histamine in epithelial cells of endometrium is assessed as reduced, if it exceeds control value same does the histamine rate in endometrial cells.

EFFECT: improvement of evaluation accuracy.

2 tbl, 2 ex

 

The invention relates to medicine, namely to morphology and histochemistry.

Epithelial and glandular epithelial cells of the endometrium of the uterus contain histamine (Gunin A.G. Localization of histamine in the structures of the uterus. // Archive FFA. - 1988. No. 12. - P.66-68), which occupies one of leading places in the regulation of vital processes of the body. It is a tissue hormone and mediator, facilitator and inhibitor of cell and tissue metabolism (weisfeld I.L., Kassil GN. Histamine in biochemistry and physiology. - M.: Nauka, 1981). In recent years significantly expanded view of the important role of histamine in the implementation of reproductive function of women, in particular it is found to be involved in regulation of the processes of fertilization and implantation (tsyrkin VI, Hlybova S.V. Role of histamine in reproduction. // Vyatka medical Bulletin. - 2006. - No. 3-4). It is established that histamine participates in stimulated estrogen proliferation of epithelial cells of the endometrium (weisfeld I.L., Kassil G.N., 1981; Gunin A.G. Estrogen-induced histogenesis in the uterus in endocrine imbalance. Autorepair. of thesis ... Dr. med. Sciences. - M., 1996). There is evidence (X. Zhao et al. The Blastocyst H(2) receptor is the target for uterine histamine in implantation in the mouse // Development. - 2000. - Vol.127, No. 12. - P.2643-2651)that the epithelial cells of the endometrium can be a source of histamine. Our studies revealed the presence of this bioamine in the fleet of the uterine cavity (Dendauw SV, Vinogradov HE Siamenoside structure of the uterus of rats during the sexual cycle. // The Bulletin of the Ivanovo medical Academy. - 2006. - T, No. 3-4. - S-30). In connection with the foregoing determining the level of histamine in the epithelial cells has important clinical value.

One of the common ways histochemical detection of histamine is the method of Cross-Member Growth with ortho-phthalaldehyde (Cross S.W.D. et al. A study of the methods available for the cytochemical localization of histamine by fluorescence induced with o-phthalaldehyde or acetaldehyde // Histochem. J. - 1971. - Vol.3. - P.471-476).

Fence material for fluorescently-histochemical study of the level of histamine in the structures of the endometrium experimental animals is carried out after killing them (Gunin A.G. the Influence of estradiol on the distribution of histamine in the structures of the uterus of rats in normal and experimental introduction of the hormone. // Problems of endocrinology. - 1991. - V.37, №4. 49 - 51). Further, the research process includes the following steps:

1. Manufacturer cryostatic sections and mount them on top of the glasses.

2. Keeping slices in pairs of ortho-phthalic aldehyde in a thermostat at 100°C for 10 seconds. Glass vessel containing ortho-phthalic aldehyde and tightly closed lid, previously kept in the incubator for 10 minutes.

3. The space of sections in a humid chamber (preliminary what about the warmed up for 10 minutes, the vessel, containing at the bottom of the water) in a thermostat at 100°C for 1 minute.

4. Drying the slices in thermostat with a temperature of 70-80°C for 5 minutes.

5. Study of drugs under fluorescent microscope. Under the terms of this reaction has the highest concentration of histamine produces a yellow luminescence at a wavelength of 515 nm (Sergeeva VE Fluorescent-histochemical methods and fluorometry in morphological studies. - Cheboksary, 1985. - Dept. in VINITI. No. 6358-85).

For final identification and quantification of the expression level of histamine in the epithelial cells of the endometrium, the drug undergoes cytotechnologists using photometric fluorescent electronic attachment PMAL-1A to determine in units of the recording instrument of the concentration of histamine in biological structures. To determine the luminescence intensity of histamine in the studied objects according to traditional methods used interference filter No. 7 photometric luminous baits PMAL-1A (maximum length of transmitted waves 503±14 nm). Luminous flux luminescence from the studied area of the field of view is cut off automatically by the hardware probe and converted PMT into force of the current, which causes a deflection of the arrow a standard measuring device, which is proportional to the end of the ation of histamine under the probe.

The disadvantages of this method of determining the level of histamine in the epithelial cells of the endometrium of the uterus include:

1) neohumanist and financial costs associated with the killing of an animal,

2) the need for the cryostat.

The technical result of our proposed method is that in each phase of the sexual cycle determine the content of histamine in the content of the vagina experimental animal compared to the level of histamine, components in the early stage of oestrus 0,50±0,07, late estrus - 0,34±0,04, metestrus 0,68±0,08, early dioestrus - 0,72±0,12, late dioestrus - 0,75±0,13, proestrus - 0,87±0,11 used recording device, and when the values of the content of histamine below the calculated estimate the level of histamine in the epithelial cells of the endometrium as reduced, but values above as high.

Description of the essence of the proposed method

Each phase during the sexual cycle in the morning (9.00) with metal loop carrying out sampling of vaginal contents rats. The received content is smeared on 2 slides (stroke 1 and stroke No. 2).

The sequence further preparation

1. Keeping smear No. 1 in a glass jar with pairs of ortho-phthalic aldehyde, and smear No. 2 in the same vessel without ortoft the left aldehyde in a thermostat at 100°C for 10 seconds.

2. The location of the strokes in separate wet camera, pre-heated for 10 minutes in a thermostat at 100°C for 1 minute.

3. Drying of smears in thermostat with a temperature of 70-80°C for 5 minutes.

The study of drugs is carried out under fluorescent microscope LUMAM-2A. Using the interference filter No. 7 photometric luminous baits PMAL-1A defines the average levels of specific luminescence smear No. 1 (value A) and autoluminescence smear No. 2 (value) in units of the recording device.

The concentration of histamine in the content of the vagina is determined by the difference between the values a and B. the Results are presented in table 1.

These same rats during ether anesthesia were extracted from the womb, from the body which was made cryostate slices. Received cryostate sections were processed in accordance with the method of Cross-Member Growth. Microspectrophotometric in epithelial and glandular epithelial cells of the endometrium average concentration of histamine in every phase of the sexual cycle (table 2).

In the rank correlation analysis Spearman installed a high positive correlation (R=0,829) changes in concentrations of histamine in the content of the vagina with the fluctuations of the concentration bioamine in top and VC is Sistah epithelial cells of the endometrium.

Below are 2 examples of determining the level of histamine in the epithelial cells of the endometrium according to a study in the vaginal smears.

Example 1

Rat No. 1

Field of viewearly estruslate estrusmetestrusearly dioestruslate dioestrusproestrus
10,400,350,700,750,801,10
20,550,350,600,850,700,80
30,450,400,650,750,650,80
40,400,350,550,65 0,700,75
50,550,300,600,600,600,85
60,550,300,700,600,800,80
70,550,250,650,800,800,80
80,450,350,600,800,800,80
90,600,300,750,600,601,00
100,500,300,700,800,70The average concentration0,500±0,06710,325±0,650±0,72±0,715±0,855±
0,04030,05920,09230,07760,1036

The comparison of the obtained results with the data presented in table 1, shows the similarity that allows us to estimate the level of histamine in the epithelial cells of the uterine endometrium of rats No. 1 as the corresponding normal maintenance during the sexual cycle.

Example 2

Rat No. 2

Field of viewearly estruslate estrusmetestrusearly dioestruslate dioestrusproestrus
10,600,500,901,050,95 1,10
20,750,500,800,851,001,00
30,650,600,851,001,001,20
40,600,500,800,950,951,10
50,550,300,600,600,600,85
60,750,500,800,901,001,20
70,750,650,750,800,800,90
8 0,650,550,850,800,801,00
90,600,500,900,901,001,00
100,700,500,901,101,051,00
The average concentration0,680±
0,064
0,530±0,825±0,925±0,945±1,050±
0,0510,0640,0960,0820,092

The results of this study show during all stages of the sexual cycle higher content of histamine content in the vagina compared with control data.

Table 1
The content of histamines in smears the contents of the vagina of rats in different phases of the sexual cycle (used)
early estruslate estrusmetestrusearly dioestruslate dioestrusproestrus
Histamine0,507±0,0690,341±being 0.0360,683±0,0800,722±0,1210,749±0,1300,865±0,114

Table 2
The content of histamine in the epithelial cells of the endometrium of the uterus of rats in different phases of the sexual cycle (used)
early estruslate estrusmetestrusearly dioestruslate dioestrusproestrus
EP3,198±0,472,765±0,362,710±0,42 3,672±0,456,060±0,446,945±0,94
EOL3,348±0,483,058±0,332,945±0,413,769±0,925,963±0,607,616±0,77
EP - epithelial cells cover, EJ - glandular epithelial cells

The method of assessing the level of histamine in the epithelial cells of the uterine endometrium of rats during the sexual cycle, including extraction of the material with subsequent fluorescently-histochemical study using ortho-phthalic aldehyde, characterized in that in each phase of the sexual cycle determine the content of histamine in the content of the vagina rats, compare it with the level of histamine, components in the early stage of oestrus 0,50±0,07, late estrus -0,34±0,04, metestrus 0,68±0,08, early dioestrus - 0,72±0,12, late dioestrus - 0,75±0,13, proestrus - 0,87±0,11 used recording device, and when the values of the content of histamine in the vaginal content below the control assess the level of histamine in the epithelial cells of the endometrium as reduced, but values above as high.



 

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