JPS5913955A - Method for measuring lectin juvenilization - Google Patents

Abstract

PURPOSE:To determine simply and exactly juvenilization, by culturing lymphocyte with the addition of lectin, adding ethidium bromide to the cultured liquid to allow both to react, and measuring the intensity of emitted fluorescent. CONSTITUTION:Heparin-added peripheral blood is diluted with a phosphoric acid buffer solution. Lumphocyte is prepared by collecting a mononuclear layer with a specific-gravity centrifugal method, etc., and phytohemagglutinin, peanut agglutinin, etc. are added to said lymphocyte. Culturing is carried out at about 37 deg.C for 72hr by the use of modified RPMI-1640, MEM, etc. as a medium. Obtained lectin-treated lymphocyte is dissolved by using sodium lauryl sulfate (SDB), etc., and ethidium bromide (EB) is added thereto to allow both to react. The intensity of fluorescence I0 of EB plus SDS, the intensity of fluorescence I1 of EB plus untreated lumphocytes, and the intensity of fluorescence of EB plus SDS plus lectin-treated lymphocytes are measured. Then the increasing ratio of DNA is obtained according to the equation of (I2-I0)/(I1-I0) to determine lectin- induced junvenilization. Since this method is based on the measurement of intensity of fluorescence of EB and DNA complex, no errors due to different conductors occur, and no effect of the number of lymphocytes appears within a range from 0.5X10<6> to 1.5X10<6> per ml. In addition, the procedure is simple.

Description

[Detailed description of the invention] The present invention relates to a method for measuring lectin rejuvenation.

Nou, = c /l/ (Novell), 1
In 1960, it was reported that kidney bean extract had the effect of stimulating lymphocytes and promoting cell division (0ancer R
θS.

20.462-466.1960), many plant cell agglutinins (lectins) have been discovered since then. These lectins are said to non-specifically activate lymphocytes, and when lectins are added to lymphocytes, changes in cellular metabolic activity occur within an hour, followed by protein synthesis and DNA synthesis.
An increase in A synthesis occurs.

On the other hand, since the immaturity of lymphocytes (lymphocyte reaction) against lectins is reduced by cancer diseases, etc., cancer diseases and their progression can be examined by measuring the immaturity caused by lectins.

Currently, methods for measuring juvenile development using this lectin include morphological observation and measurement of radioactive substance uptake using a liquid scintillation counter. The former requires skill, and the latter requires special equipment to handle radioactive materials, and it is still difficult to judge the results. has not been standardized.

Therefore, the inventors of the present invention set out to overcome this drawback. Ethidium bromide (hereinafter referred to as lf BJ) has weak fluorescence by itself, but when combined with nucleic acids, the fluorescence intensity increases specifically. Focusing on this, as a result of intensive research, we discovered that juvenile development can be easily and accurately measured by reacting KB-i with DNA (deokine liponucleic acid), which increases during juvenile development, and measuring the resulting fluorescence intensity. The present invention has been completed.

Therefore, the present invention provides a method for measuring lectin rejuvenation, which is characterized by adding lectin to lymphocytes and culturing them, then adding F B-Q to this culture solution to cause a reaction, and measuring the resulting fluorescence intensity. be.

The lymphocytes used as a sample are prepared by a conventional method, for example, by diluting heparinized peripheral blood with phosphate buffered saline (PBS) or the like, and collecting a mononuclear cell layer by specific gravity centrifugation or the like.

As the lectin, any commonly used lectin can be used, such as phytohemagglutinin (PHA),
Peanut agglutinin (PNA), soyabin agglutinin (SBA), Hawkelyde mite-diene (PWM)
), concanavalin A (conA), wheat germ agglutinin (WGA), and the like.

Culture of lymphocytes and lectin is performed by a conventional method. For example, as a medium, F (PM any 64 Q
, MEM ticket, 15 q6 Fe2 and HEPES (
A culture medium modified with PHA-P (DI) is used, and the lymphocyte concentration is 0.5 to 1.5
(manufactured by FOO), the ratio is preferably about 0.1 to 0.2.

The i@culture is preferably carried out at about 67°C for about 72 hours.

The lectin-treated lymphocytes thus obtained were lysed using sodium lauryl sulfate (SDS) 'J, and F
Add B and react. The results of examining the reaction conditions between the lectin-treated lymphocytes (ie, the DNA contained therein) and FB are as follows.

■ Reaction time of KB and DNA EB (6 μg/m/PBS) DNA [
: Ca1f Thymus (Sigma)] Suspension liquid (5
ttg,/ml PBS) 1tnl was added and mixed, and the spectral fluorescence intensity meter (Hitachi 650
-10), the fluorescence intensity was measured. The results are shown in Figure 1. As is clear from this, 2 to 6
H in 0 minutes! The B-DNA 6 complex showed stable fluorescence intensity. Therefore, the reaction time is 2 minutes or more, especially 10 minutes or more.
20 minutes is a good time.

■ K B,! r Reaction temperature to DN E B (6μ
g/ml) DNA (2, 5, 5 and 1
Add 1 ml (0 μg/ml), mix, and shake at 4° (
, 'The reaction was carried out at IK temperature and 67°C for 15 minutes, and the fluorescence intensity was measured in the same manner as in (2). The results are shown in Figure 2. As is clear from this, the fluorescence intensity is 4°C〉room temperature〉67°'C, but in all six cases, the fluorescence intensity increases linearly as the number of DNA hearts increases, so the temperature was kept constant during the operation. Although any temperature may be used as long as the temperature is maintained, it is generally preferable to carry out the reaction at room temperature.

DNA and EB concentration 1 uB (10, 7, 5 and 5 μg/mi) DNA (5-20 μg/me) was added to 1 w, reacted for 15 minutes at room temperature, and the fluorescence intensity was measured in the same manner as in ①. The results are shown in Figure 3.As is clear from this,
The lower the EIJII degree, the narrower the range that can be quantitatively measured.
It is preferable to increase the A degree. Generally, it is preferable to use 1 μg of FBI per 1 μg of DNA.

■ 5DS once SDS (0, [l 15~0.5 m9/m1.PB
S) KB (6μg/1nl) or PB in 11nl
Sllnl was added and the mixture was left in room 7MK for 15 minutes, and the fluorescence intensity was measured in the same manner as in (2).

The results are shown in Figure 4. From now on, it is clear that SDS 0.015~0.125"Gl/m
e Te has no problem if the difference from the fluorescence of Chicken B alone is 0.5 or less, but if it exceeds this, light scattering etc. will appear, which is not preferable. Therefore, 5DS should be less than 0.125+ψ/rnl.

■ F, B and lymphocyte concentration lymphocytes (0.15-1.5 x 106 cells) in 5DS
(0,125m97ml) was dissolved in 2 d, 1 ni of FB (6 μg/mz) was added thereto, and the solution was dissolved at room temperature for 15 ml.
The reaction was allowed to proceed for a minute, and the fluorescence intensity was measured in the same manner as in ①. The results are shown in Figure 5. As is clear from this, even when the lymphocytes were treated with SDS, they showed the same tendency as shown in FIG. 3, and from this, it was possible to conduct constant DNA analysis.

■ Examination of cell number during culture Lymphocyte concentration 0-4. [] X 106 pieces/m/(I(
PMI-1640), and each suspension was incubated at 67°C at 172 hours in the presence or absence of Q and 125% PHA-P (manufactured by DIFOO).
5QI) Culture = k I in a CO2 incubator. Below, after S
e In the same manner as in Example (3), E B +SDS+fluorescence intensity of untreated lymphocytes (E), li': B+SDS+
Fluorescence intensity (E) and EB+S of PHA-treated lymphocytes
The fluorescence intensity (work 0) of Df3 was measured. The result is the 6th
As shown in the figure. Seedling, solid line in the figure (Work 2 - Work o)
The slope represents the DNA Jli addition rate in the wrinkles, which can be seen from the paralysis equation described later, but as is clear from the nuclear map, the lymphocyte concentration is preferably 0.5 to 1.5×1061/η21.

Fluorescence intensity (Io) of KB+SDS was determined as described above.
, F, B + SDS + fluorescence intensity of untreated lymphocytes (Eng. 1)
The fluorescence intensity (■2) of the lymphocytes treated with E B + SDS + lectin is measured, and the DNA increase rate can be determined according to the following formula, and lectin blastogenesis can be measured.

DNA increase rate = I2-Io/Eq1-■.

As the method of the present invention measures the fluorescence intensity of KB and DNA1j summer coalescence, there is no error caused by the measuring person, and the influence of the number of lymphocytes is 0.5 to 1.
．． This method has divine advantages over conventional methods, such as not occurring within the range of 5 x 106 cells/ml and being easy to operate.

Next, an example will be given and explained.

Example ()) An equal amount of PBS was added to 6 volumes of heparinized peripheral blood and mixed, and a mononuclear cell layer was collected by specific centrifugation using a lymphocyte separation solution and washed 6 times with PBS. Add this to RPM Nee 1640 medium and get 1xio6 lymphocytes/m7!
A lymphocyte suspension was obtained.

(2) 0.254 to 0.5yni of lymphocyte suspension
PHA-P (DIFOO M) 0.5 m F'B
Add 5 ml of the above medium Q (control) and add 5 ml of the above medium
Dispense into the bottom plate (manufactured by Nunc) and add 54 CO2,
It was incubated at 67°C for 72 hours.

(3) Using a pipette, transfer the cultured lymphocytes in the plate pole to test a while washing with PBB, and incubate at 160 Or
Centrifugation was performed for 10 minutes at pm, and the supernatant was removed. SDB on residue
Lymphocytes were lysed by adding 2mef, and WS (7) C6fig/m1PBs) 2mA! was added and reacted for 15 minutes at room temperature. Regarding this reaction solution, λ]li,
Fluorescence intensity was measured at x525 nm and x600 nm. FB 2d+ SDS 2+ as the same blank
Measure the fluorescence intensity of 111J and calculate D according to the above formula.
The NA increase rate was determined.

(41+1l-(Healthy person 21 measured as in 31)
The DNA increase rates of the 21 cases and patients were as shown in Figure 7. The patient's disease name is as follows.

(11 Acute sieroma, (2) Primary liver cancer, (3) Acute leukemia, (4) Psychoidosis, (5) Malignant lymphoma/diabetes, (6) Tumor cancer/liver cirrhosis, (7) Pneumonia/respiratory failure, (8), (9) Lung cancer, 001, aa esophageal cancer, 0] 1.03 prostate cancer, 021 lung cancer/pleural cancer, Q51. (16) Pulmonary tuberculosis or pneumonia, OD chronic rheumatism, 08), (21 gastric cancer, a1胛mm、QυHerpes Carcinoma

[Brief explanation of drawings]

Figure 1 shows the relationship (8) between the reaction time of FB and DNA and the fluorescence intensity. Figure 4 shows the relationship between SDS protection and fluorescence intensity, Figure 5 shows the influence of SDS treatment of lymphocytes, and Figure 6 shows the influence of lymphocyte concentration during culture. , FIG. 7 shows the DNA increase rate of healthy subjects and patients measured by the method of the present invention. Figure 1 Time (minutes) Figure 2 I) NA conformity (μg/g) Figure 3 DNA concentration (μP//rnz) Figure 4 SDS concentration (~/ml) Figure 6 Figure → (I-1) 2 0 263- DNA increase rate healthy patients

Claims (1)

[Claims] 1. A method for measuring lectin blastogenesis, which comprises adding lectin to lymphocytes and culturing them, then adding ethidium bromide to the culture solution to allow a reaction, and measuring the resulting fluorescence intensity.