Method of single-step telomere length and population division quantity measurement of proliferative cells in vitro

FIELD: medicine.

SUBSTANCE: method involves telomere length measurement by flow-FISH method in cells differentiated by a number of population divisions with a vital stain without preliminary recovery of proliferative cells with using Bis(sulfosuccinimidyl)suberat (BS3) amino group cross-linker.

EFFECT: invention provides the single-step telomere length measurement in cells with a common division quantity.

3 dwg, 1 ex

 

The invention relates to the study of biological material, in particular to the determination of telomere length.

Telomeric regions of DNA (telomeres) are the ends of chromosomes that do not carry the semantic load, which consist of repeated hexaprotodon TTAGGG having a tandem arrangement and identical for all vertebrates [1, 8]. During cell division in the end naturalisatie DNA newly synthesized sister chromatid becomes shorter, and this happens due to the loss of telomeric repeats [9]. Telomere shortening is a limitation to continuous cell division, therefore, telomere length determines the replicative potential of cells, and is an indirect marker of its replicative history. Short telomeres are a sign of "senescent" cells.

Telomere length is one of the main dynamic features of cell activity, and the study of such changes is an important fundamental and clinical significance. The change in telomere length with age and the possibility of its regulation are of great importance in studying the biology of aging and gerontology [2, 6]. Widely used measurement of telomere length in the diagnosis, evaluation of treatment efficacy and prognosis of cancer and conditions associated with disorders of the immune system [4, 13, 5]. The study of telomeres at the cellular level, changes associated with cell division, are of particular importance for understanding the biology of aging, as it affects the processes that determine the lifespan of the cell.

Measurement of telomere length has become a routine method by developing methods Flow-FISH-in situ hybridization analysis on a flow cytometer. This method allows to determine telomere length in single cells of the target population [11]. Measurement of telomere in proliferating cells is possible in several ways. The most simple is the selection of proliferating cells using DNA dyes, when cells in phase S-G2-M have a greater number of DNA that allows them to differentiate at a higher level of fluorescence channel luminosity DNA dye [3]. This technique included in method of Flow-FISH to measure telomere, so there is no need for separate sorting of proliferating cells. However, this method allows to determine the length of telomeres only in cells that were shared at the time of the study, and does not take into account cells that have shared earlier.

Another method is incubation of living cells in a solution containing bromodeoxyuridine (BrdU), which is incorporated into the newly synthesized DNA chain. Then add anti-BrdU monoclonal antibodies labeled with fluorochromes and analyze the on flow cytometer. This allows you to precisely select all shared cells, because they contain built-labeled nucleotide regardless of the time of the study.

Another method taken by the authors for the prototype that we can identify proliferating cells using vital fluorescent dye. The principle of the method lies in staining cells with the vital dye, which is strongly associated with blood cells, and after division is distributed between daughter cells [14]. Nepodelishsya cells have a maximum luminance channel fluorescence of the vital dye, and cells, making at least one division will have the level of fluorescence is much lower. To determine telomere length in proliferating cells, it is necessary first to distinguish from the General population, i.e. sort, for example, at the cellular case, because the fluorochromes can not withstand heat up to 80°C, necessary for carrying out the method of in situ hybridization [3, 5, 14]. This method, like the previous one, a very expensive and time-consuming, requiring considerable time and, apart from the special cell sorting device, it is necessary to prepare a large number of cells.

Staining cells with the vital dye also allows you to determine the exact number of committed cell divisions in culture. After division of the mother cell dye R is Vomero distributed between daughter cells, who have the level of fluorescence in two times lower than that repeats itself with each new division. The number of peaks of fluorescence of the vital dye, you can determine the number of cell divisions [7].

The authors conducted a series of experiments in which it was shown that the vital dye CFSE not withstand heat up to 80°C in formamide, and after in situ hybridization is impossible to determine the number of divisions by the number of separate peaks on the channel fluorescence of CFSE. There are ways to save fluorochrome on the cell when heated up to 80°C, one of which is the use of special chemicals BS3 (Bis(sulfosuccinimidyl)suberat), which is used in the method Flow-FISH for the differentiation of cell surface antigens [12]. Based on this method, it was hypothesized that the use of BS3 will contribute to the preservation of the vital dye in the cell after heating up to 80°C, which will allow researchers to simultaneously determine telomere length and the number of executed divisions in cultures of proliferating cells.

The aim of the invention is to provide a method of simultaneous determination of telomere length in cells with a known quantity of committed divisions.

The method consists in the fact that the cells differentiated by the number of completed divisions using the vital dye gradualism lower level is th fluorescence. Simultaneously with this, without preliminary separation of target proliferating cell population from the General population, use the flow-FISH (in situ hybridization) to determine the length of telomeres in cells, differentiated by the number of completed divisions and surface marker. Procedures to produce involving crosslinker amino Bis(sulfosuccinimidyl)suberat(BS3).

The method allows to determine the length of telomeres at the level of single cells using in situ hybridization, as well as to determine the number of divisions made this cell in vitro with subsequent analysis on a flow cytometer.

This makes the method more accessible, greatly increases the information content and performance, reduces research time and costs.

Description of the method with examples of specific implementations

The investigated cells stained with the vital dye CFSE (Molecular probes, USA) with a final concentration of 2µm in RPMI 1640 15 minutes, then washed in RPMI with 10% FCS (HyClone, UK). After that, cells are cultivated in vitro.

After cultivation, the cells washed in SFR with 0.1% BSA (Sigma, USA) (SR-BSA) and mark biotinylating antibodies against the target antigen. Incubated for 20 minutes, washed SFR-BSA and add streptavidin labeled with fluorochromes, and incubated for 20 minutes. After washing add a solution BS34 mm (Bis(sulfosuccnimidyl)suberat, Pierce, US) for 30 minutes at 37°C [12]. Then add Tris (Sigma, USA) at a final concentration of 20 mm and incubated for 15 minutes and washed SFR-BSA. Sediment resuspended in 300 μl of hybridization solution consisting of 70% formamide (Sigma, USA), 20 mm Tris and 1% BSA. The PNA probe complementary telomeric DNA sequences and labeled fluorochromes (Bio-Synthesis, Inc., USA), added at a concentration of 0.3 ág/ml [10]. In parallel, put the sample of control cells, which is used as a standard hybridization. The sample is heated for 10 minutes at 80°C and hybridized 3 hours at +20°C. the cells are Then transferred into tubes for cytometry, washed twice with a solution of 70% formamide and once SFR-BSA + 0.1% of Tween. Sediment resuspended in 0.5 ml SPR-BSA. Sample analysis is performed on a flow cytometer. According to the parameters of forward and side light scattering allocate region lymphocytes, which determine positive cells in the CD4 antigen on channel fluorescence used fluorochrome. From the positive region to build a histogram of the fluorescence signal of the vital dye CFSE, which will be visible peaks in the distribution of cells by the number of past divisions. This histogram peak with the highest fluorescence determines nepodelishsya cells, and with minimal fluorescence of cells committed to the maximum number of divisions. After selecting a population of cells with the correct number on the lines build a histogram of fluorescence fluorochrome SS3, which Machen PNA probe complementary telomeric DNA.

Telomere length in cells is defined as the average fluorescence of the cells on the channel flurochrome SS3 after hybridization in the presence of PNA probe minus background (autofluorescence) of cells that have passed the same conditions in the absence of PNA probe. The ratio of the average length of telomeres in cells to the indicator control cells reflects the average telomere length relative to control cells.

The following is an example illustrating this method of determining the relative average length of telomeres in proliferating cells.

Example. Determination of changes in telomere length with each new division in subpopulations of CD4+ and CD8+ human lymphocytes in vitro culture for 7 days in response to the activation of anti-D3 monoclonal antibodies.

From 6 donors 5 ml of peripheral blood stabilized with heparin, sterile centrifuged in the gradient ficoll-urografin for 20 minutes at 3 thousand rpm, after which the ring mononuclear cells are collected and washed twice with 5 ml of sterile buffered saline solution (SFR) for 5 minutes at 1 Tyson/min. the Precipitate resuspended in 1 ml SPR and count the number of cells in the cell Goryaeva.

The lymphocytes are stained with the vital dye CFSE and cultured in 24-hole tablets in CO2incubator with 5% CO27 with the current number of 2 million to 1 ml of complete medium (RPMI, FCS 10%), glutamine 0.3 mg/ml, gentamicin 50 μg/ml, Tienam 25 μg/ml) with the addition of anti-D3 antibody 1 μg/ml (Medispectra, Russia) and IL-2 100 u/ml (biotech, Russia). After 7 days the cells are harvested and carried out the in situ hybridization using mouse anti-CD4 and anti-CD8 biotinylated antibodies (Becton Dickinson, USA). The in situ hybridization is carried out with a PNA probe complementary telomeric DNA sequence (SSTA)3marked fluorochroman SS3 (Bio-Synthesis, Inc., USA) with a final concentration of 0.3 ág/ml [10]. For the control cells were taken murine splenocytes line C57B L/6 (Kennel "Dawn", Tomsk). Analysis was performed on a flow cytometer FACS Calibur (Becton Dickinson, USA) using the software Cell QuestPRO(Becton Dickinson, USA). After the separation of the gate of lymphocytes by forward and side svetorasseyanie was isolated cells positive for CD4+ (or CD8+) antigen, which determined the number of peaks on the channel fluorescence of the vital dye CFSE (figure 1). It was determined that 7 days the cells had committed a maximum of 7 divisions. From each gate corresponding to a certain number of committed lymphocytes divisions, was determined average telomere length by determining the level of fluorescence channel luminosity probe PNA-SS3; was additionally measured the average telomere length in splenocytes, as control cells, using the same probe (figure 2). Were determined relative telomere length in nepodelishsya cells and cells held from 1 to 7 levels for subpopulations of CD4+ (CD8+) lymphocytes. From Figure 3 it can be seen that nepodelishsya cells have short telomeres, and at the division of the gradual lengthening of telomeres with the peak on the 3rd division for CD4+ lymphocytes and 4-5 divisions for CD8+ lymphocytes. It was determined that under cultivation in vitro after activation of lymphocytes anti-CD3 antibody is an lengthening of telomeres, with great value for CD8+ cells. The data obtained indicate a significant influence of activation and in vitro cultivation on the molecular changes in lymphocytes.

Thus, the obtained results indicate the possibility of applying the method for measuring the average length of telomeres in cells depending on the number of traversed them tick. A significant advantage of this method is the simultaneous separation of cells by the number of committed divisions, surface marker and define them in average telomere length.

Sources of information

The method of measuring the length of telomeres in cells, differentiated by the number of completed divisions using the vital dye gradualism lower level of fluorescence, using the method flowFISH (in situ hybridization) and subsequent analysis on a flow cytometer, characterized in that telomere length measured in proliferating cells the General population simultaneously with their differentiation according to the number of divisions and differentiation by surface marker, which is used crosslinkers amino Bis(sulfosuccinimidyl)suberat(BS3).



 

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